Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

PubMed Central

Mitchell, Angela M.; Hirsch, Matthew L.; Li, Chengwen

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses. PMID:24198403

Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication.

PubMed

Mitchell, Angela M; Hirsch, Matthew L; Li, Chengwen; Samulski, R Jude

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses.

[Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma].

PubMed

Li, Bai-Ling; Zhang, Guan-Xin; Hou, Xiao-Lei; Tan, Meng-Wei; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Huang, Sheng-Dong

2009-03-01

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. AAV-mediated expression of siRNA could reduce the expression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.

Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket

USDA-ARS?s Scientific Manuscript database

Male ejaculate proteins, including both sperm and seminal fluid proteins, play an important role in mediating reproductive biology. The function of ejaculate proteins can include enabling sperm-egg interactions, enhancing sperm storage, mediating female attractiveness, and even regulating female lif...

Microinjection-based RNA interference knockdown of ecdysteroid biosynthetic genes in a non-model hemipteran pest, Lygus hesperus (western tarnished plant bug)

USDA-ARS?s Scientific Manuscript database

RNAi-mediated knockdown of target transcripts offers great potential, both in terms of insect functional genomics and the development of novel insect pest management strategies. Frequently, dsRNAs targeting transcripts of interest are introduced orally to the target organism via feeding. This delive...

Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis.

PubMed

Wang, Hongyan; Yang, Bin; Qiu, Linghua; Yang, Chunxing; Kramer, Joshua; Su, Qin; Guo, Yansu; Brown, Robert H; Gao, Guangping; Xu, Zuoshang

2014-02-01

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.

Silencing Genes in the Heart.

PubMed

Fechner, Henry; Vetter, Roland; Kurreck, Jens; Poller, Wolfgang

2017-01-01

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

Clathrin Heavy Chain Is Important for Viability, Oviposition, Embryogenesis and, Possibly, Systemic RNAi Response in the Predatory Mite Metaseiulus occidentalis

PubMed Central

Wu, Ke; Hoy, Marjorie A.

2014-01-01

Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods such as Drosophila melanogaster. However, the functional roles of clathrin heavy chain in chelicerate arthropods, such as the predatory mite Metaseiulus occidentalis, remain unknown. We previously showed that dsRNA ingestion, followed by feeding on spider mites, induced systemic and robust RNAi in M. occidentalis females. In the current study, we performed a loss-of-function analysis of the clathrin heavy chain gene in M. occidentalis using RNAi. We showed that ingestion of clathrin heavy chain dsRNA by M. occidentalis females resulted in gene knockdown and reduced longevity. In addition, clathrin heavy chain dsRNA treatment almost completely abolished oviposition by M. occidentalis females and the few eggs produced did not hatch. Finally, we demonstrated that clathrin heavy chain gene knockdown in M. occidentalis females significantly reduced a subsequent RNAi response induced by ingestion of cathepsin L dsRNA. The last finding suggests that clathrin heavy chain may be involved in systemic RNAi responses mediated by orally delivered dsRNAs in M. occidentalis. PMID:25329675

RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation.

PubMed

Griffith, Elen; Coutts, Amanda S; Black, Donald M

2005-03-01

TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Copyright 2005 Wiley-Liss, Inc.

Recombinant adeno-associated virus-delivered hypoxia-inducible stanniocalcin-1 expression effectively inhibits hypoxia-induced cell apoptosis in cardiomyocytes.

PubMed

Shi, Xin; Wang, Jianzhong; Qin, Yan

2014-12-01

Ischemia/hypoxia-induced oxidative stress is detrimental for the survival of cardiomyocytes and cardiac function. Stanniocalcin-1 (STC-1), a glycoprotein, has been found to play an inhibitory role in the production of reactive oxygen species (ROS). Here, we speculated that the overexpression of STC-1 might alleviate oxidative damage in cardiomyocytes under conditions of hypoxia. To control the expression of STC-1 in hypoxia, we constructed a recombinant adeno-associated virus (AAV) carrying the hypoxia-responsive element (HRE) to mediate hypoxia induction. Cardiomyocytes were infected with AAV-HRE-STC-1 and cultured in normoxic or hypoxic conditions, and STC-1 overexpression was only detected in hypoxic cultured cardiomyocytes by using quantitative real-time polymerase chain reaction and Western blot analysis. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, AAV-HRE-STC-1 infection was shown to significantly enhance cell survival under hypoxia. Hypoxia-induced cell apoptosis was inhibited by AAV-HRE-STC-1 infection by using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis assay. Moreover, the proapoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2, which were dysregulated by hypoxia, were reversed by AAV-HRE-STC-1 infection. AAV-HRE-STC-1-mediated STC-1 overexpression markedly inhibited ROS production in cardiomyocytes cultured under hypoxic conditions. AAV-HRE-STC-1 infection significantly upregulated uncoupled protein 3 (UCP3), whereas silencing of UCP3 blocked the inhibitory effect of AAV-HRE-STC-1 on ROS production. In contrast, AAV-HRE-STC-1 infection had no effect on UCP2, and knockdown of UCP2 did not block the inhibitory effect of AAV-HRE-STC-1 on ROS production in the cardiomyocytes cultured under hypoxic conditions. Taken together, STC1 activates antioxidant pathway in cardiomyocytes through the induction of UCP3, implying that AAV-HRE-STC-1 has potential in the treatment of ischemic-related heart disease.

Suppression of OsKu80 results in defects in developmental growth and increased telomere length in rice (Oryza sativa L.).

PubMed

Byun, Mi Young; Cui, Li Hua; Kim, Woo Taek

2015-12-25

The Ku70-Ku80 heterodimer plays a critical role in the maintenance of genomic stability in humans and yeasts. In this report, we identified and characterized OsKu80 in rice, a model monocot crop. OsKu80 forms a heterodimer with OsKu70 in yeast and plant cells, as demonstrated by yeast two-hybrid, in vivo co-immunoprecipitation, and bimolecular fluorescence complementation assays. RNAi-mediated knock-down T3 transgenic rice plants (Ubi:RNAi-OsKu80) displayed a retarded growth phenotype at the post-germination stage. In addition, the Ubi:RNAi-OsKu80 knock-down progeny exhibited noticeably increased telomere length as compared to wild-type rice. These results are discussed with the idea that OsKu80 plays a role in developmental growth and telomere length regulation in rice plants. Copyright © 2015 Elsevier Inc. All rights reserved.

A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

PubMed

Friedrich, Michael; Meier, Doreen; Schuster, Isabelle; Nellen, Wolfgang

2015-01-01

We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver.

PubMed

Piras, Bryan A; O'Connor, Daniel M; French, Brent A

2013-01-01

AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

PubMed Central

Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

2014-01-01

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

Symbiont-mediated RNA interference in insects

PubMed Central

Whitten, Miranda M. A.; Facey, Paul D.; Del Sol, Ricardo; Fernández-Martínez, Lorena T.; Evans, Meirwyn C.; Mitchell, Jacob J.; Bodger, Owen G.

2016-01-01

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. PMID:26911963

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

PubMed

Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

2016-03-20

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei*

PubMed Central

Dewar, Simon; Sienkiewicz, Natasha; Ong, Han B.; Wall, Richard J.; Horn, David

2016-01-01

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance. PMID:27703008

FlyPrimerBank: An Online Database for Drosophila melanogaster Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents

PubMed Central

Hu, Yanhui; Sopko, Richelle; Foos, Marianna; Kelley, Colleen; Flockhart, Ian; Ammeux, Noemie; Wang, Xiaowei; Perkins, Lizabeth; Perrimon, Norbert; Mohr, Stephanie E.

2013-01-01

The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo. PMID:23893746

RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

PubMed

Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

2016-07-14

The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD.

PubMed

Wallace, Lindsay M; Saad, Nizar Y; Pyne, Nettie K; Fowler, Allison M; Eidahl, Jocelyn O; Domire, Jacqueline S; Griffin, Danielle A; Herman, Adam C; Sahenk, Zarife; Rodino-Klapac, Louise R; Harper, Scott Q

2018-03-16

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.

Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

PubMed Central

Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

2011-01-01

Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

Adeno-associated virus-RNAi of GlyRα1 and characterization of its synapse-specific inhibition in OFF alpha transient retinal ganglion cells

PubMed Central

Zhang, C.; Rompani, S. B.; Roska, B.

2014-01-01

In the central nervous system, inhibition shapes neuronal excitation. In spinal cord glycinergic inhibition predominates, whereas GABAergic inhibition predominates in the brain. The retina uses GABA and glycine in approximately equal proportions. Glycinergic crossover inhibition, initiated in the On retinal pathway, controls glutamate release from presynaptic OFF cone bipolar cells (CBCs) and directly shapes temporal response properties of OFF retinal ganglion cells (RGCs). In the retina, four glycine receptor (GlyR) α-subunit isoforms are expressed in different sublaminae and their synaptic currents differ in decay kinetics. GlyRα1, expressed in both On and Off sublaminae of the inner plexiform layer, could be the glycinergic isoform that mediates On-to-Off crossover inhibition. However, subunit-selective glycine contributions remain unknown because we lack selective antagonists or cell class-specific subunit knockouts. To examine the role of GlyRα1 in direct inhibition in mature RGCs, we used retrogradely transported adeno-associated virus (AAV) that performed RNAi and eliminated almost all glycinergic spontaneous and visually evoked responses in PV5 (OFFαTransient) RGCs. Comparisons of responses in PV5 RGCs infected with AAV-scrambled-short hairpin RNA (shRNA) or AAV-Glra1-shRNA confirm a role for GlyRα1 in crossover inhibition in cone-driven circuits. Our results also define a role for direct GlyRα1 inhibition in setting the resting membrane potential of PV5 RGCs. The absence of GlyRα1 input unmasked a serial and a direct feedforward GABAAergic modulation in PV5 RGCs, reflecting a complex interaction between glycinergic and GABAAergic inhibition. PMID:25231618

Suppression of RND3 activity by AES downregulation promotes cancer cell proliferation and invasion.

PubMed

Xia, Hongwei; Li, Mingxing; Chen, Liang; Leng, Weibing; Yuan, Dandan; Pang, Xiaohui; Chen, Liu; Li, Ronghui; Tang, Qiulin; Bi, Feng

2013-05-01

Amino-terminal enhancer of split (AES) is a member of the Groucho/TLE family. Although it has no DNA-binding site, AES can regulate transcriptional activity by interacting with transcriptional factors. Emerging evidence indicates that AES may play an important role in tumor metastasis, but the molecular mechanism is still poorly understood. In this study, we found that knockdown of AES by RNA interference (RNAi) downregulated RND3 expression at the mRNA and protein levels in MDA-MB-231 and HepG2, two cancer cell lines. Furthermore, luciferase assays showed that overexpression of AES significantly enhanced RND3 promoter activity. Moreover, inhibition of AES both in MDA-MB-231 and HepG2 cells by RNAi significantly promoted cell proliferation, cell cycle progression and invasion, consistent with the effects of RNAi-mediated RND3 knockdown in these cells. For the first time, data are presented showing that alteration of the malignant behavior of cancer cells by AES is related to RND3 regulation, and these findings also provide new insights into the mechanism of AES action in regulating tumor malignancy.

Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice

PubMed Central

De Souza, Angus T.; Dai, Xudong; Spencer, Andrew G.; Reppen, Tom; Menzie, Ann; Roesch, Paula L.; He, Yudong; Caguyong, Michelle J.; Bloomer, Sherri; Herweijer, Hans; Wolff, Jon A.; Hagstrom, James E.; Lewis, David L.; Linsley, Peter S.; Ulrich, Roger G.

2006-01-01

RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara−/− mice. Combining the profiles from mice treated with the PPARα agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARα regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara−/− mice. In contrast to Ppara−/− mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies. PMID:16945951

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

PubMed Central

Ding, Jian; Lin, Zhi-Qiang; Jiang, Jian-Ming; Seidman, Christine E.; Seidman, Jonathan G.; Pu, William T.; Wang, Da-Zhi

2016-01-01

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimalimmunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations. PMID:28060283

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.

PubMed

Ding, Jian; Lin, Zhi-Qiang; Jiang, Jian-Ming; Seidman, Christine E; Seidman, Jonathan G; Pu, William T; Wang, Da-Zhi

2016-12-17

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

PubMed

Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

2010-08-18

Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

Advance of RNA interference technique in Hemipteran insects.

PubMed

Li, Jie; Wang, Xiaoping; Wang, Manqun; Ma, Weihua; Hua, Hongxia

2012-07-24

RNA interference (RNAi) suppressed the expression of the target genes by post transcriptional regulation and the double-stranded RNA (dsRNA) mediated gene silencing has been a conserved mechanism in many eukaryotes, which prompted RNAi to become a valuable tool for unveiling the gene function in many model insects. Recent research attested that RNAi technique can be also effective in downregulation target genes in Hemipteran insects. In this review, we collected the researches of utilizing RNAi technique in gene functional analysis in Hemipteran insects, highlighted the methods of dsRNA/siRNA uptake by insects and discussed the knock-down efficiency of these techniques. Although the RNA interference technique has drawbacks and obscure points, our primary goal of this review is try to exploit it for further discovering gene functions and pest control tactic in the Hemipteran insects. © 2012 The Societies and Blackwell Publishing Asia Pty Ltd.

Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins.

PubMed

Li, Chengwen; Xiao, Pingjie; Gray, Steven James; Weinberg, Marc Scott; Samulski, R Jude

2011-08-23

Molecular knockdown of disease proteins and restoration of wild-type activity represent a promising but challenging strategy for the treatment of diseases that result from the accumulation of misfolded proteins (i.e., Huntington disease, amyotrophic lateral sclerosis, and α-1 antitrypsin deficiency). In this study we used alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model system to evaluate the efficiency of gene-delivery approaches that both silence the piZZ transcript (e.g., shRNA) and restore circulating wild-type AAT expression from resistant codon-optimized AAT (AAT-opt) transgene cassette using adeno-associated virus (AAV) vector delivery. After systemic injection of a self-complimentary AAV serotype 8 (scAAV8) vector encoding shRNA in piZZ transgenic mice, both mutant AAT mRNA in the liver and defected serum protein level were inhibited by 95%, whereas liver pathology, as monitored by dPAS and fibrosis staining, reversed. To restore blood AAT levels in AAV8/shRNA-treated mice, several strategies to restore functional AAT levels were tested, including using AAV AAT-opt transgene cassettes targeted to muscle and liver, or combination vectors carrying piZZ shRNA and AAT-opt transgenes separately, or a single bicistronic AAV vector. With these molecular approaches, we observed over 90% knockdown of mutant AAT with a 13- to 30-fold increase of circulating wild-type AAT protein from the shRNA-resistant AAT-opt cassette. The molecular approaches applied in this study can simultaneously prevent liver pathology and restore blood AAT concentration in AAT deficiencies. Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins.

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål)

PubMed Central

Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

2016-01-01

The “target of rapamycin” (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens. PMID:27043527

TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

PubMed

Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

2016-03-28

The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics.

PubMed

Suzuki, Hiroshi I; Spengler, Ryan M; Grigelioniene, Giedre; Kobayashi, Tatsuya; Sharp, Phillip A

2018-05-01

RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as crosstalk with endogenous RBPs' (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

PubMed

Nicholson, Linda J; Philippe, Marie; Paine, Alan J; Mann, Derek A; Dolphin, Colin T

2005-04-01

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A.

PubMed

Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G S; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

2014-01-01

Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

PubMed Central

Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

2015-01-01

Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency. PMID:26305933

Establishment of a tissue-specific RNAi system in C. elegans.

PubMed

Qadota, Hiroshi; Inoue, Makiko; Hikita, Takao; Köppen, Mathias; Hardin, Jeffrey D; Amano, Mutsuki; Moerman, Donald G; Kaibuchi, Kozo

2007-10-01

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.

Establishment of a tissue-specific RNAi system in C. elegans

PubMed Central

Qadota, Hiroshi; Inoue, Makiko; Hikita, Takao; Köppen, Mathias; Hardin, Jeffrey D.; Amano, Mutsuki; Moerman, Donald G.; Kaibuchi, Kozo

2011-01-01

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal- and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues. PMID:17681718

Nanoparticle-mediated RNA interference of angiotensinogen decreases blood pressure and improves myocardial remodeling in spontaneously hypertensive rats.

PubMed

Yuan, Li-Fen; Sheng, Jing; Lu, Ping; Wang, Yu-Qiang; Jin, Tuo; Du, Qin

2015-09-01

Angiotensinogen (AGT) has been shown to have a role in cardiac hypertrophy, while depletion of the AGT gene in spontaneously hypertensive rats (SHR) has not been investigated. The present study investigated the effect of AGT knockdown on cardiac hypertrophy in SHR. For this, small hairpin (sh)RNAs were intravenously injected into SHRs, using a nanoparticle‑mediated transfection system. The experimental rats were divided into the following groups: a) Blank control with water treatment only, b) negative control with biscarbamate‑crosslinked Gal‑polyethylene glycol polyethylenimine nanoparticles (GPE)/negative shRNA, c) AGT‑RNA interference (RNAi) group with GPE/AGT‑shRNA, and 4) normotensive control using Wistar‑Kyoto rats (WKY) with water treatment. Three and five days following the first injection, the levels of hepatic AGT mRNA and AGT protein as well as plasma levels of AGT were markedly decreased in the AGT‑RNAi group (P<0.05). Furthermore, a significant decrease in systolic blood pressure (SBP), left ventricular weight to body weight ratio and heart weight to body weight ratio were observed in the AGT‑RNAi group compared with those in the control groups. The depletion of AGT in SHR led to a reduction in SBP by 30±4 mmHg, which was retained for >10 days. Cardiac hypertrophy was also significantly improved in AGT‑knockdown rats. In conclusion, the present study showed that AGT‑silencing had a significant inhibitory effect on hypertension and hypertensive‑induced cardiac hypertrophy in SHRs.

Delivery of RNAi reagents in murine models of obesity and diabetes.

PubMed

Wilcox, Denise M; Yang, Ruojing; Morgan, Sherry J; Nguyen, Phong T; Voorbach, Martin J; Jung, Paul M; Haasch, Deanna L; Lin, Emily; Bush, Eugene N; Opgenorth, Terry J; Jacobson, Peer B; Collins, Christine A; Rondinone, Cristina M; Surowy, Terry; Landschulz, Katherine T

2006-11-29

RNA interference (RNAi) is an exciting new tool to effect acute in vivo knockdown of genes for pharmacological target validation. Testing the application of this technology to metabolic disease targets, three RNAi delivery methods were compared in two frequently utilized preclinical models of obesity and diabetes, the diet-induced obese (DIO) and B6.V-Lep/J (ob/ob) mouse. Intraperitoneal (i.p.) and high pressure hydrodynamic intravenous (i.v.) administration of naked siRNA, and low pressure i.v. administration of shRNA-expressing adenovirus were assessed for both safety and gene knockdown efficacy using constructs targeting cJun N-terminal kinase 1 (JNK1). Hydrodynamic delivery of siRNA lowered liver JNK1 protein levels 40% in DIO mice, but was accompanied by iatrogenic liver damage. The ob/ob model proved even more intolerant of this technique, with hydrodynamic delivery resulting in severe liver damage and death of most animals. While well-tolerated, i.p. injections of siRNA in DIO mice did not result in any knockdown or phenotypic changes in the mice. On the other hand, i.v. injected adenovirus expressing shRNA potently reduced expression of JNK1 in vivo by 95% without liver toxicity. In conclusion, i.p. and hydrodynamic injections of siRNA were ineffective and/or inappropriate for in vivo gene targeting in DIO and ob/ob mice, while adenovirus-mediated delivery of shRNA provided a relatively benign and effective method for exploring liver target silencing.

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

PubMed

Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

2015-09-01

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

PubMed Central

Koch, Jan Christoph; Tönges, Lars; Michel, Uwe; Bähr, Mathias; Lingor, Paul

2014-01-01

The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush. PMID:25249936

Imbalanced Hemolymph Lipid Levels Affect Feeding Motivation in the Two-Spotted Cricket, Gryllus bimaculatus.

PubMed

Konuma, Takahiro; Tsukamoto, Yusuke; Nagasawa, Hiromichi; Nagata, Shinji

2016-01-01

Insect feeding behavior is regulated by many intrinsic factors, including hemolymph nutrient levels. Adipokinetic hormone (AKH) is a peptide factor that modulates hemolymph nutrient levels and regulates the nutritional state of insects by triggering the transfer of lipids into the hemolymph. We recently demonstrated that RNA interference (RNAi)-mediated knockdown of the AKH receptor (AKHR) reduces hemolymph lipid levels, causing an increase in the feeding frequency of the two-spotted cricket, Gryllus bimaculatus. This result indicated that reduced hemolymph lipid levels might motivate crickets to feed. In the present study, to elucidate whether hemolymph lipid levels contribute to insect feeding behavior, we attempted to manipulate hemolymph lipid levels via the lipophorin (Lp)-mediated lipid transferring system in G. bimaculatus. Of the constituent proteins in Lp, we focused on apolipophorin-III (GrybiApoLp-III) because of its possible role in facilitating lipid mobilization. First, we used RNAi to reduce the expression of GrybiApoLp-III. RNAi-mediated knockdown of GrybiApoLp-III had little effect on basal hemolymph lipid levels and the amount of food intake. In addition, hemolymph lipid levels remained static even after injecting AKH into GrybiApoLp-IIIRNAi crickets. These observations indicated that ApoLp-III does not maintain basal hemolymph lipid levels in crickets fed ad libitum, but is necessary for mobilizing lipid transfer into the hemolymph following AKH stimulation. Second, Lp (containing lipids) was injected into the hemolymph to induce a temporary increase in hemolymph lipid levels. Consequently, the initiation of feeding was delayed in a dose-dependent manner, indicating that increased hemolymph lipid levels reduced the motivation to feed. Taken together, these data validate the importance of basal hemolymph lipid levels in the control of energy homeostasis and for regulating feeding behavior in crickets.

Imbalanced Hemolymph Lipid Levels Affect Feeding Motivation in the Two-Spotted Cricket, Gryllus bimaculatus

PubMed Central

Konuma, Takahiro; Tsukamoto, Yusuke; Nagasawa, Hiromichi; Nagata, Shinji

2016-01-01

Insect feeding behavior is regulated by many intrinsic factors, including hemolymph nutrient levels. Adipokinetic hormone (AKH) is a peptide factor that modulates hemolymph nutrient levels and regulates the nutritional state of insects by triggering the transfer of lipids into the hemolymph. We recently demonstrated that RNA interference (RNAi)-mediated knockdown of the AKH receptor (AKHR) reduces hemolymph lipid levels, causing an increase in the feeding frequency of the two-spotted cricket, Gryllus bimaculatus. This result indicated that reduced hemolymph lipid levels might motivate crickets to feed. In the present study, to elucidate whether hemolymph lipid levels contribute to insect feeding behavior, we attempted to manipulate hemolymph lipid levels via the lipophorin (Lp)-mediated lipid transferring system in G. bimaculatus. Of the constituent proteins in Lp, we focused on apolipophorin-III (GrybiApoLp-III) because of its possible role in facilitating lipid mobilization. First, we used RNAi to reduce the expression of GrybiApoLp-III. RNAi-mediated knockdown of GrybiApoLp-III had little effect on basal hemolymph lipid levels and the amount of food intake. In addition, hemolymph lipid levels remained static even after injecting AKH into GrybiApoLp-IIIRNAi crickets. These observations indicated that ApoLp-III does not maintain basal hemolymph lipid levels in crickets fed ad libitum, but is necessary for mobilizing lipid transfer into the hemolymph following AKH stimulation. Second, Lp (containing lipids) was injected into the hemolymph to induce a temporary increase in hemolymph lipid levels. Consequently, the initiation of feeding was delayed in a dose-dependent manner, indicating that increased hemolymph lipid levels reduced the motivation to feed. Taken together, these data validate the importance of basal hemolymph lipid levels in the control of energy homeostasis and for regulating feeding behavior in crickets. PMID:27144650

Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function.

PubMed

Firnhaber, Christopher; Hammarlund, Marc

2013-11-01

Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.

DNA replication machinery is required for development in Drosophila.

PubMed

Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

2018-01-01

 In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

RNA therapeutics targeting osteoclast-mediated excessive bone resorption

PubMed Central

Wang, Yuwei; Grainger, David W

2011-01-01

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique developed with dramatically increasing utility for both scientific and therapeutic purposes. Short interfering RNA (siRNA) is currently exploited to regulate protein expression relevant to many therapeutic applications, and commonly used as a tool for elucidating disease-associated genes. Osteoporosis and their associated osteoporotic fragility fractures in both men and women are rapidly becoming a global healthcare crisis as average life expectancy increases worldwide. New therapeutics are needed for this increasing patient population. This review describes the diversity of molecular targets suitable for RNAi-based gene knock-down in osteoclasts to control osteoclast-mediated excessive bone resorption. We identify strategies for developing targeted siRNA delivery and efficient gene silencing, and describe opportunities and challenges of introducing siRNA as a therapeutic approach to hard and connective tissue disorders. PMID:21945356

Orexin signaling via the orexin 1 receptor mediates operant responding for food reinforcement.

PubMed

Sharf, Ruth; Sarhan, Maysa; Brayton, Catherine E; Guarnieri, Douglas J; Taylor, Jane R; DiLeone, Ralph J

2010-04-15

Orexin (hypocretin) signaling is implicated in drug addiction and reward, but its role in feeding and food-motivated behavior remains unclear. We investigated orexin's contribution to food-reinforced instrumental responding using an orexin 1 receptor (Ox1r) antagonist, orexin -/- (OKO) and littermate wildtype (WT) mice, and RNAi-mediated knockdown of orexin. C57BL/6J (n = 76) and OKO (n = 39) mice were trained to nose poke for food under a variable ratio schedule of reinforcement. After responding stabilized, a progressive ratio schedule was initiated to evaluate motivation to obtain food reinforcement. Blockade of Ox1r in C57BL/6J mice impaired performance under both the variable ratio and progressive ratio schedules of reinforcement, indicating impaired motivational processes. In contrast, OKO mice initially demonstrated a delay in acquisition but eventually achieved levels of responding similar to those observed in WT animals. Moreover, OKO mice did not differ from WT mice under a progressive ratio schedule, indicating delayed learning processes but no motivational impairments. Considering the differences between pharmacologic blockade of Ox1r and the OKO mice, animals with RNAi mediated knockdown of orexin were then generated and analyzed to eliminate possible developmental effects of missing orexin. Orexin gene knockdown in the lateral hypothalamus in C57BL/6J mice resulted in blunted performance under both the variable ratio and progressive ratio schedules, resembling data obtained following Ox1r antagonism. The behavior seen in OKO mice likely reflects developmental compensation often seen in mutant animals. These data suggest that activation of the Ox1r is a necessary component of food-reinforced responding, motivation, or both in normal mice. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Orexin signaling via the orexin 1 receptor mediates operant responding for food reinforcement

PubMed Central

Sharf, Ruth; Sarhan, Maysa; Brayton, Catherine E.; Guarnieri, Douglas J.; Taylor, Jane R.; DiLeone, Ralph J.

2010-01-01

Background Orexin (hypocretin) signaling is implicated in drug addiction and reward, but its role in feeding and food-motivated behavior remains unclear. Methods We investigated orexin’s contribution to food-reinforced instrumental responding using an orexin 1 receptor (Ox1r) antagonist, orexin −/− (OKO) and littermate wild-type (WT) mice, and RNAi-mediated knockdown of orexin. C57BL/6J (n=76) and OKO (n=39) mice were trained to nose poke for food under a variable ratio (VR) schedule of reinforcement. Once responding stabilized, a progressive ratio (PR) schedule was initiated to evaluate motivation to obtain food reinforcement. Results Blockade of Ox1r in C57BL/6J mice impaired performance under both the VR and PR schedules of reinforcement, indicating impaired motivational processes. In contrast, OKO mice initially demonstrated a delay in acquisition, but eventually achieved levels of responding similar to those observed in WT animals. Moreover, OKO mice did not differ from WT mice under a PR schedule, indicating delayed learning processes but no motivational impairments. Considering the differences between pharmacological blockade of Ox1r and the OKO mice, animals with RNAi mediated knockdown of orexin were then generated and analyzed to eliminate possible developmental effects of missing orexin. Orexin gene knockdown in the lateral hypothalamus (LH) in C57BL/6J mice resulted in blunted performance under both the VR and PR schedules, resembling data obtained following Ox1r antagonism. Conclusions The behavior seen in OKO mice likely reflects developmental compensation often seen in mutant animals. These data suggest that activation of the Ox1r is a necessary component of food-reinforced responding and/or motivation in normal mice. PMID:20189166

RNAi-Mediated Knockdown of vATPase Subunits Affects Survival and Reproduction of Bed Bugs (Hemiptera: Cimicidae).

PubMed

Basnet, Sanjay; Kamble, Shripat T

2018-05-04

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has resurged as one of the most troublesome household pests affecting people across the globe. Bed bug infestations have increased in recent years primarily due to the evolution of insecticide resistance and the insect's ability to hitchhike with travelers. vATPases are one of the most evolutionarily conserved holoenzymes in eukaryotes, which are mainly involved in proton transport across the plasma membranes and intracellular organelles. RNA interference (RNAi) has been developed as a promising tool for insect control. In this study, we used RNAi as an approach to knock down subunits A and E of the vATPase gene of bed bugs. Delivery of 0.2 µg/insect of dsRNA specific to vATPase-A and vATPase-E into female bed bugs dramatically impaired the laying and viability of eggs over time. Injection of the vATPase-E dsRNA decreased survival of the bed bugs over 30 d. Our results also showed that the knockdown of mRNA is highly effective and persistent up to 30 d post injection. This research demonstrated that silencing of the two vATPase subunits A and E offers a potential strategy to suppress bed bug populations.

RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts.

PubMed

Pierson, Lisa; Mousley, Angela; Devine, Lynda; Marks, Nikki J; Day, Tim A; Maule, Aaron G

2010-04-01

Evolving RNA interference (RNAi) platforms are providing opportunities to probe gene function in parasitic helminths using reverse genetics. Although relatively robust methods for the application of RNAi in parasitic flatworms have been established, reports of successful RNAi are confined to three genera and there are no known reports of the application of RNAi to the class Cestoda. Here we report the successful application of RNAi to a cestode. Our target species was the common ruminant tapeworm, Moniezia expansa which can significantly impact the health/productivity of cattle, sheep and goats. Initial efforts aimed to silence the neuronally expressed neuropeptide F gene (Me-npf-1), which encodes one of the most abundant neuropeptides in flatworms and a homologue of vertebrate neuropeptide Y (NPY). Double stranded (ds)RNAs, delivered by electroporation and soaking (4-8h), failed to trigger consistent Me-npf-1 transcript knock-down in adult worms; small interfering RNAs (siRNAs) were also ineffective. Identical approaches resulted in significant and consistent transcript knock-down of actin transcript (71+/-4%) following soaking in Me-act-1 dsRNA. Similar successes were seen with hydrophobic lipid-binding protein (Me-lbp-1), with a dsRNA inducing significant target transcript reduction (72+/-5%). To confirm the validity of the observed transcript knock-downs we further investigated Me-act-1 RNAi worms for associated changes in protein levels, morphology and phenotype. Me-act-1 RNAi worms displayed significant reductions in both filamentous actin immunostaining (62+/-3%) and the amount of actin detected in Western blots (54+/-13%). Morphologically, Me-act-1 RNAi worms displayed profound tegumental disruption/blebbing. Further, muscle tension recordings from Me-act-1 RNAi worms revealed a significant reduction in both the number of worms contracting in response to praziquantel (20+/-12%) and in their contractile ability. These data demonstrate, to our knowledge for the first time, a functional RNAi pathway in a cestode and show that the robust knock-down of abundant gene transcripts is achievable using long dsRNAs following short exposure times. 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

VPS36-Mediated plasma membrane protein turnover is critical for Arabidopsis root gravitropism.

PubMed

Hsu, Ya-Wen; Jauh, Guang-Yuh

2017-04-03

The gravitropic response is an evolutionary adaptation for plants to cope with the altered gravitational field. It involves reestablishing the distribution of the phytohormone auxin by differential degradation of auxin influx and efflux carriers. This process includes the endosomal sorting complexes required for transport (ESCRT) machinery to recognize ubiquitinated proteins and deliver them to vacuoles for degradation, as evidenced by vps36-1 mutants. Here, we generated RNAi knockdown plants of Vacuolar Protein Sorting 36 (VPS36) that could survive to adulthood. VPS36-induced RNAi plants showed PIN FORMED1 (PIN1) accumulation in the intracellular compartment, reduced root length and small stature, as observed in vps36-1 mutants. After gravistimulation, the roots of VPS36-induced RNAi plants did not show the bending observed in wild-type plants. The VPS36-containing ESCRT machinery may have a role in the gravitropic response possibly associated with the degradation of auxin transporters.

RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology.

PubMed

Raut, Sandeep; Mallik, Bhagaban; Parichha, Arpan; Amrutha, Valsakumar; Sahi, Chandan; Kumar, Vimlesh

2017-07-05

Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C -Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development. Copyright © 2017 Raut et al.

Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

PubMed Central

Awan, Aashir; Bell, Aaron J.; Satir, Peter

2009-01-01

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality. PMID:19290045

Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

PubMed Central

Crofton, Elizabeth J.; Nenov, Miroslav N.; Zhang, Yafang; Scala, Federico; Page, Sean A.; McCue, David L.; Li, Dingge; Hommel, Jonathan D.; Laezza, Fernanda; Green, Thomas A.

2017-01-01

Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico-limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction-related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen-synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno-associated viral vector (AAV2) and assessed changes in anxiety- and depression-like behavior and cocaine self-administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety-like behavior while increasing depression-like behavior and cocaine self-administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV-shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward-related behaviors. Loose-patch recordings showed that TANs transduced by AAV-shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole-cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization-induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression- and addiction-related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub-types. PMID:28126496

Pathogen and Pest Responses Are Altered Due to RNAi-Mediated Knockdown of GLYCOALKALOID METABOLISM 4 in Solanum tuberosum.

PubMed

Paudel, Jamuna Risal; Davidson, Charlotte; Song, Jun; Maxim, Itkin; Aharoni, Asaph; Tai, Helen H

2017-11-01

Steroidal glycoalkaloids (SGAs) are major secondary metabolites constitutively produced in cultivated potato Solanum tuberosum, and α-solanine and α-chaconine are the most abundant SGAs. SGAs are toxic to humans at high levels but their role in plant protection against pests and pathogens is yet to be established. In this study, levels of SGAs in potato were reduced by RNA interference (RNAi)-mediated silencing of GLYCOALKALOID METABOLISM 4 (GAME4)-a gene encoding cytochrome P450, involved in an oxidation step in the conversion of cholesterol to SGA aglycones. Two GAME4 RNAi lines, T8 and T9, were used to investigate the effects of manipulation of the SGA biosynthetic pathway in potato. Growth and development of an insect pest, Colorado potato beetle (CPB), were affected in these lines. While no effect on CPB leaf consumption or weight gain was observed, early instar larval death and accelerated development of the insect was found while feeding on leaves of GAME4 RNAi lines. Modulation of SGA biosynthetic pathway in GAME4 RNAi plants was associated with a larger alteration to the metabolite profile, including increased levels of one or both the steroidal saponins or phytoecdysteroids, which could affect insect mortality as well as development time. Colonization by Verticillium dahliae on GAME4 RNAi plants was also tested. There were increased pathogen levels in the T8 GAME4 RNAi line but not in the T9. Metabolite differences between T8 and T9 were found and may have contributed to differences in V. dahliae infection. Drought responses created by osmotic stress were not affected by modulation of SGA biosynthetic pathway in potato.

RNAi-Mediated Knockdown of Serine Protease Inhibitor Genes Increases the Mortality of Plutella xylostella Challenged by Destruxin A

PubMed Central

Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G. S.; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

2014-01-01

Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides. PMID:24837592

Importins α and β signaling mediates endothelial cell inflammation and barrier disruption.

PubMed

Leonard, Antony; Rahman, Arshad; Fazal, Fabeha

2018-04-01

Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and β importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser 536 , showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin β1, but not β2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or β1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or β1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and β1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa).

PubMed

Du, Yiwei; He, Wei; Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23-26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

PubMed Central

Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes. PMID:26934377

Slo1 regulates ethanol-induced scrunching in freshwater planarians

NASA Astrophysics Data System (ADS)

Cochet-Escartin, Olivier; Carter, Jason A.; Chakraverti-Wuerthwein, Milena; Sinha, Joydeb; Collins, Eva-Maria S.

2016-10-01

When freshwater planarians are exposed to a low-percentage (0.5%-1%) alcohol solution, they display a characteristic ‘drunken’ phenotype. Here we show that this drunken phenotype is a mixture of cilia-mediated gliding and scrunching, a muscular-based planarian gait which we recently demonstrated to be triggered by adverse environmental stimuli. At exogenous ethanol concentrations ≥2% (v/v), planarians become gradually immobilized and ultimately die. Using RNA interference (RNAi) for targeted gene knockdown, we elucidate the molecular basis for ethanol sensing and show that the big potassium ion channel SLO1 is necessary for ethanol sensitivity in planarians. Because slo1(RNAi) animals maintain their ability to scrunch in response to other adverse triggers, these results suggest that slo1 specifically regulates ethanol sensitivity and not the scrunching gait per se. Furthermore, this study demonstrates the ease of performing pharmacological studies in planarians. Combined with the worms’ amenability to quantitative behavioral assays and targeted gene knockdown, planarians are a valuable model organism for studying the effect of neuroactive compounds on brain function and behavior.

Slo1 regulates ethanol-induced scrunching in freshwater planarians.

PubMed

Cochet-Escartin, Olivier; Carter, Jason A; Chakraverti-Wuerthwein, Milena; Sinha, Joydeb; Collins, Eva-Maria S

2016-09-09

When freshwater planarians are exposed to a low-percentage (0.5%-1%) alcohol solution, they display a characteristic 'drunken' phenotype. Here we show that this drunken phenotype is a mixture of cilia-mediated gliding and scrunching, a muscular-based planarian gait which we recently demonstrated to be triggered by adverse environmental stimuli. At exogenous ethanol concentrations ≥2% (v/v), planarians become gradually immobilized and ultimately die. Using RNA interference (RNAi) for targeted gene knockdown, we elucidate the molecular basis for ethanol sensing and show that the big potassium ion channel SLO1 is necessary for ethanol sensitivity in planarians. Because slo1(RNAi) animals maintain their ability to scrunch in response to other adverse triggers, these results suggest that slo1 specifically regulates ethanol sensitivity and not the scrunching gait per se. Furthermore, this study demonstrates the ease of performing pharmacological studies in planarians. Combined with the worms' amenability to quantitative behavioral assays and targeted gene knockdown, planarians are a valuable model organism for studying the effect of neuroactive compounds on brain function and behavior.

An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio.

PubMed

Lin, Wei-Hsiang; He, Miaomiao; Fan, Yuen Ngan; Baines, Richard A

2018-05-02

Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, para bss . We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.

D-beta-hydroxybutyrate extends lifespan in C. elegans

PubMed Central

Edwards, Clare; Canfield, John; Copes, Neil; Rehan, Muhammad; Lipps, David; Bradshaw, Patrick C.

2014-01-01

The ketone body beta-hydroxybutyrate (βHB) is a histone deacetylase (HDAC) inhibitor and has been shown to be protective in many disease models, but its effects on aging are not well studied. Therefore we determined the effect of βHB supplementation on the lifespan of C. elegans nematodes. βHB supplementation extended mean lifespan by approximately 20%. RNAi knockdown of HDACs hda-2 or hda-3 also increased lifespan and further prevented βHB-mediated lifespan extension. βHB-mediated lifespan extension required the DAF-16/FOXO and SKN-1/Nrf longevity pathways, the sirtuin SIR-2.1, and the AMP kinase subunit AAK-2. βHB did not extend lifespan in a genetic model of dietary restriction indicating that βHB is likely functioning through a similar mechanism. βHB addition also upregulated βHB dehydrogenase activity and increased oxygen consumption in the worms. RNAi knockdown of F55E10.6, a short chain dehydrogenase and SKN-1 target gene, prevented the increased lifespan and βHB dehydrogenase activity induced by βHB addition, suggesting that F55E10.6 functions as an inducible βHB dehydrogenase. Furthermore, βHB supplementation increased worm thermotolerance and partially prevented glucose toxicity. It also delayed Alzheimer's amyloid-beta toxicity and decreased Parkinson's alpha-synuclein aggregation. The results indicate that D-βHB extends lifespan through inhibiting HDACs and through the activation of conserved stress response pathways. PMID:25127866

Variable laterality of corticospinal tract axons that regenerate after spinal cord injury as a result of PTEN deletion or knock-down

PubMed Central

Willenberg, Rafer; Zukor, Katherine; Liu, Kai; He, Zhigang; Steward, Oswald

2016-01-01

Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting PTEN in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the “wrong” side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical AAV-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral BDA injections in PTEN-deleted mice with spinal cord injury and in non-injured PTEN-floxed mice that had not received AAV-Cre. In non-injured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in grey matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side, in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using shRNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the non-injured CST, highlighting one aspect for how resultant circuitry from regenerating axons may differ from that of the uninjured CST. PMID:26878190

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

DOE PAGES

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

2015-03-31

Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

Reduction of Cav1.3 channels in dorsal hippocampus impairs the development of dentate gyrus newborn neurons and hippocampal-dependent memory tasks.

PubMed

Kim, Su-Hyun; Park, Ye-Ryoung; Lee, Boyoung; Choi, Byungil; Kim, Hyun; Kim, Chong-Hyun

2017-01-01

Cav1.3 has been suggested to mediate hippocampal neurogenesis of adult mice and contribute to hippocampal-dependent learning and memory processes. However, the mechanism of Cav1.3 contribution in these processes is unclear. Here, roles of Cav1.3 of mouse dorsal hippocampus during newborn cell development were examined. We find that knock-out (KO) of Cav1.3 resulted in the reduction of survival of newborn neurons at 28 days old after mitosis. The retroviral eGFP expression showed that both dendritic complexity and the number and length of mossy fiber bouton (MFB) filopodia of newborn neurons at ≥ 14 days old were significantly reduced in KO mice. Both contextual fear conditioning (CFC) and object-location recognition tasks were impaired in recent (1 day) memory test while passive avoidance task was impaired only in remote (≥ 20 days) memory in KO mice. Results using adeno-associated virus (AAV)-mediated Cav1.3 knock-down (KD) or retrovirus-mediated KD in dorsal hippocampal DG area showed that the recent memory of CFC was impaired in both KD mice but the remote memory was impaired only in AAV KD mice, suggesting that Cav1.3 of mature neurons play important roles in both recent and remote CFC memory while Cav1.3 in newborn neurons is selectively involved in the recent CFC memory process. Meanwhile, AAV KD of Cav1.3 in ventral hippocampal area has no effect on the recent CFC memory. In conclusion, the results suggest that Cav1.3 in newborn neurons of dorsal hippocampus is involved in the survival of newborn neurons while mediating developments of dendritic and axonal processes of newborn cells and plays a role in the memory process differentially depending on the stage of maturation and the type of learning task.

Reduction of Cav1.3 channels in dorsal hippocampus impairs the development of dentate gyrus newborn neurons and hippocampal-dependent memory tasks

PubMed Central

Kim, Su-Hyun; Park, Ye-Ryoung; Lee, Boyoung; Choi, Byungil; Kim, Hyun

2017-01-01

Cav1.3 has been suggested to mediate hippocampal neurogenesis of adult mice and contribute to hippocampal-dependent learning and memory processes. However, the mechanism of Cav1.3 contribution in these processes is unclear. Here, roles of Cav1.3 of mouse dorsal hippocampus during newborn cell development were examined. We find that knock-out (KO) of Cav1.3 resulted in the reduction of survival of newborn neurons at 28 days old after mitosis. The retroviral eGFP expression showed that both dendritic complexity and the number and length of mossy fiber bouton (MFB) filopodia of newborn neurons at ≥ 14 days old were significantly reduced in KO mice. Both contextual fear conditioning (CFC) and object-location recognition tasks were impaired in recent (1 day) memory test while passive avoidance task was impaired only in remote (≥ 20 days) memory in KO mice. Results using adeno-associated virus (AAV)-mediated Cav1.3 knock-down (KD) or retrovirus-mediated KD in dorsal hippocampal DG area showed that the recent memory of CFC was impaired in both KD mice but the remote memory was impaired only in AAV KD mice, suggesting that Cav1.3 of mature neurons play important roles in both recent and remote CFC memory while Cav1.3 in newborn neurons is selectively involved in the recent CFC memory process. Meanwhile, AAV KD of Cav1.3 in ventral hippocampal area has no effect on the recent CFC memory. In conclusion, the results suggest that Cav1.3 in newborn neurons of dorsal hippocampus is involved in the survival of newborn neurons while mediating developments of dendritic and axonal processes of newborn cells and plays a role in the memory process differentially depending on the stage of maturation and the type of learning task. PMID:28715454

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

PubMed

Mayra, Azat; Tomimitsu, Hiroyuki; Kubodera, Takayuki; Kobayashi, Masaki; Piao, Wenying; Sunaga, Fumiko; Hirai, Yukihiko; Shimada, Takashi; Mizusawa, Hidehiro; Yokota, Takanori

2011-02-11

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes. Copyright © 2011 Elsevier Inc. All rights reserved.

Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.

Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

DOE PAGES

Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; ...

2016-02-17

Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

PubMed Central

Zhang, Wenli; Solanki, Manish; Müther, Nadine; Ebel, Melanie; Wang, Jichang; Sun, Chuanbo; Izsvak, Zsuzsanna; Ehrhardt, Anja

2013-01-01

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells. PMID:24116154

Using RNAi in C. "elegans" to Demonstrate Gene Knockdown Phenotypes in the Undergraduate Biology Lab Setting

ERIC Educational Resources Information Center

Roy, Nicole M.

2013-01-01

RNA interference (RNAi) is a powerful technology used to knock down genes in basic research and medicine. In 2006 RNAi technology using "Caenorhabditis elegans" ("C. elegans") was awarded the Nobel Prize in medicine and thus students graduating in the biological sciences should have experience with this technology. However,…

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. Conclusion This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme. PMID:15193153

Knockdown of RRS1 by lentiviral-mediated RNAi promotes apoptosis and suppresses proliferation of human hepatocellular carcinoma cells.

PubMed

Wang, Jitao; Li, Zhi; Zuo, Changzeng; Xie, Qingfan; Li, Hui; Jia, Junhong; Zhen, Zhongguang; Qi, Ruizhao; Li, Zhiwei; Liu, Dengxiang; Sun, Baijun

2017-10-01

In recent years it was found that the synthesis and biological activity of ribosomes are closely associated with tumor cell growth, tumorigenesis, and malignant transformation. However, the role of regulator of ribosome synthesis 1 (RRS1) in hepatocellular carcinoma (HCC) has not yet been reported. In the present study, we aimed to examine the potential role of RRS1 in tumor cell growth by using a lentivirus-mediated RNA interference (RNAi) system in the HCC cell line SMMC-7721 in vitro. Compared with that of the negative control group (Lv-shCon), the mRNA and protein expression levels of RRS1 in SMMC-7721 cells transfected with Lv-shRRS1 were significantly decreased. Further experiments found that silencing of RRS1 gene expression in SMMC-7721 cells significantly suppressed cell proliferation, inhibited colony formation capacity, increased apoptosis and arrested cells in the G1 phase. These results suggest that the RRS1 gene plays a critical role in cell proliferation, colony formation, cell apoptosis and cell cycle distribution in human HCC cells, and that silencing of RRS1 by RNAi is a promising therapeutic approach for the treatment of HCC, and should be further developed.

Unsupervised automated high throughput phenotyping of RNAi time-lapse movies.

PubMed

Failmezger, Henrik; Fröhlich, Holger; Tresch, Achim

2013-10-04

Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens. We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene's function. Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.

Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior.

PubMed

Luo, Jiangnan; Lushchak, Oleh V; Goergen, Philip; Williams, Michael J; Nässel, Dick R

2014-01-01

A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Weihong; Wu Jianqing; Zhong Li

2006-09-30

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency ofmore » conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

PubMed Central

Tomoyasu, Yoshinori

2014-01-01

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting. PMID:25350485

Knockdown of genes in the Toll pathway reveals new lethal RNA interference targets for insect pest control.

PubMed

Bingsohn, L; Knorr, E; Billion, A; Narva, K E; Vilcinskas, A

2017-02-01

RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well-established model, with a strong and robust RNAi response, which can be used as a high-throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large-scale screening, to encompass others identified using targeted knowledge-based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double-stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal-related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies. © 2016 The Royal Entomological Society.

Gene Therapy for Posttraumatic Osteoarthritis

DTIC Science & Technology

2017-10-01

are currently no useful treatments. To provide a clear assessment of the clinical potential of this technology we are testing the following hypothesis...efficacy of scAAV-mediated gene delivery of IL-1Ra for treatment of OA. We will test the hypothesis that scAAV-mediated gene delivery of IL-1Ra to...1Ra) Post -traumatic OA (PTOA) Self-complimentary AAV (scAAV) Cartilage Synovium Gene Transfer Large animal model 6 2. ACCOMPLISHMENTS

ROCK and PRK-2 Mediate the Inhibitory Effect of Y-27632 on Polyglutamine Aggregation

PubMed Central

Shao, Jieya; Welch, William J.; Diamond, Marc I.

2009-01-01

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, H-1077, HA-1152), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. PMID:18423405

Insights into the Sigma-1 receptor chaperone’s cellular functions: a microarray report

PubMed Central

Tsai, Shang-Yi; Rothman, Richard Kyle; Su, Tsung-Ping

2013-01-01

We previously demonstrated that Sig-1Rs are critical regulators in neuronal morphogenesis and development via the regulation of oxidative stress and mitochondrial functions. In the present study, we sought to identify pathways and genes that are affected by Sig-1R. Gene expression profiles were examined in rat hippocampal neurons that had been cultured for18 days in vitro (DIV). The cells were transduced with AAV siRNA targeting Sig-1R on DIV 10 for 7 days, followed by gene expression analysis using a rat genome cDNA array. The gene array results indicated that Sig-1R knockdown hampered cellular functions including steroid biogenesis, protein ubiquitination, actin cytoskeleton network, and Nrf-2 mediated oxidative stress. Many of the cellular components important for actin polymerization and synapse plasticity, including F-actin capping protein and neurofilaments, were significantly changed in AAV-siSig-1R neurons. Further, cytochrome c was reduced in AAV-Sig-1R neurons whereas free-radical generating enzymes including cytochrome p450 and cytochrome b-245 were increased. The microarray results also suggest that Sig-1Rs may regulate genes that are involved in the pathogenesis of many CNS diseases including Alzheimer’s disease and Parkinson’s disease. These data further confirmed that Sig-1Rs play critical roles in the CNS and thus these findings may aid in future development of therapeutic treatments targeting neurodegenerative disorders. PMID:21905129

Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle

PubMed Central

Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

2013-01-01

Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×1010 –1 × 1011 particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 109 AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike. PMID:23591809

Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.

PubMed

Nash, Kevin R; Gordon, Marcia N

2016-01-01

Recombinant adeno-associated virus (rAAV) has become an extremely useful tool for the study of gene over expression or knockdown in the central nervous system of experimental animals. One disadvantage of intracranial injections of rAAV vectors into the brain parenchyma has been restricted distribution to relatively small volumes of the brain. Convection enhanced delivery (CED) is a method for delivery of clinically relevant amounts of therapeutic agents to large areas of the brain in a direct intracranial injection procedure. CED uses bulk flow to increase the hydrostatic pressure and thus improve volume distribution. The CED method has shown robust gene transfer and increased distribution within the CNS and can be successfully used for different serotypes of rAAV for increased transduction of the mouse CNS. This chapter details the surgical injection of rAAV by CED into a mouse brain.

Acute tau knockdown in the hippocampus of adult mice causes learning and memory deficits.

PubMed

Velazquez, Ramon; Ferreira, Eric; Tran, An; Turner, Emily C; Belfiore, Ramona; Branca, Caterina; Oddo, Salvatore

2018-05-10

Misfolded and hyperphosphorylated tau accumulates in several neurodegenerative disorders including Alzheimer's disease, frontotemporal dementia with Parkinsonism, corticobasal degeneration, progressive supranuclear palsy, Down syndrome, and Pick's disease. Tau is a microtubule-binding protein, and its role in microtubule stabilization is well defined. In contrast, while growing evidence suggests that tau is also involved in synaptic physiology, a complete assessment of tau function in the adult brain has been hampered by robust developmental compensation of other microtubule-binding proteins in tau knockout mice. To circumvent these developmental compensations and assess the role of tau in the adult brain, we generated an adeno-associated virus (AAV) expressing a doxycycline-inducible short-hairpin (Sh) RNA targeted to tau, herein referred to as AAV-ShRNATau. We performed bilateral stereotaxic injections in 7-month-old C57Bl6/SJL wild-type mice with either the AAV-ShRNATau or a control AAV. We found that acute knockdown of tau in the adult hippocampus significantly impaired motor coordination and spatial memory. Blocking the expression of the AAV-ShRNATau, thereby allowing tau levels to return to control levels, restored motor coordination and spatial memory. Mechanistically, the reduced tau levels were associated with lower BDNF levels, reduced levels of synaptic proteins associated with learning, and decreased spine density. We provide compelling evidence that tau is necessary for motor and cognitive function in the adult brain, thereby firmly supporting that tau loss-of-function may contribute to the clinical manifestations of many tauopathies. These findings have profound clinical implications given that anti-tau therapies are in clinical trials for Alzheimer's disease. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen; Yang, Xi-fei

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we exploremore » the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.« less

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

PubMed

van Lieshout, Laura P; Soule, Geoff; Sorensen, Debra; Frost, Kathy L; He, Shihua; Tierney, Kevin; Safronetz, David; Booth, Stephanie A; Kobinger, Gary P; Qiu, Xiangguo; Wootton, Sarah K

2018-03-05

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs.

PubMed

Vyas, Meenal; Raza, Amir; Ali, Muhammad Yousaf; Ashraf, Muhammad Aleem; Mansoor, Shahid; Shahid, Ahmad Ali; Brown, Judith K

2017-01-01

Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly 'gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.

Recombinant AAV-directed gene therapy for type I glycogen storage diseases

PubMed Central

Chou, JY; Mansfield, BC

2011-01-01

Introduction Glycogen storage disease (GSD) type Ia and Ib are disorders of impaired glucose homeostasis affecting the liver and kidney. GSD-Ib also affects neutrophils. Current dietary therapies cannot prevent long-term complications. In animal studies, recombinant adeno-associated virus (rAAV) vector-mediated gene therapy can correct or minimize multiple aspects of the disorders, offering hope for human gene therapy. Areas covered A summary of recent progress in rAAV-mediated gene therapy for GSD-I; strategies to improve rAAV-mediated gene delivery, transduction efficiency and immune avoidance; and vector refinements that improve expression. Expert opinion rAAV-mediated gene delivery to the liver can restore glucose homeostasis in preclinical models of GSD-I, but some long-term complications of the liver and kidney remain. Gene therapy for GSD-Ib is less advanced than for GSD-Ia and only transient correction of myeloid dysfunction has been achieved. A question remains whether a single rAAV vector can meet the expression efficiency and tropism required to treat all aspects of GSD-I, or if a multi-prong approach is needed. An understanding of the strengths and weaknesses of rAAV vectors in the context of strategies to achieve efficient transduction of the liver, kidney, and hematopoietic stem cells is required for treating GSD-I. PMID:21504389

Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1

PubMed Central

Kucharewicz, Weronika; Distelfeld, Assaf; Bilger, Wolfgang; Müller, Maren; Munné-Bosch, Sergi; Hensel, Götz

2017-01-01

Abstract WHIRLY1 in barley was isolated as a potential regulator of the senescence-associated gene HvS40. In order to investigate whether the plastid–nucleus-located DNA/RNA-binding protein WHIRLY1 plays a role in regulation of leaf senescence, primary foliage leaves from transgenic barley plants with an RNAi-mediated knockdown of the WHIRLY1 gene were characterized by typical senescence parameters, namely pigment contents, function and composition of the photosynthetic apparatus, as well as expression of selected genes known to be either down- or up-regulated during leaf senescence. When the plants were grown at low light intensity, senescence progression was similar between wild-type and RNAi-W1 plants. Likewise, dark-induced senescence of detached leaves was not affected by reduction of WHIRLY1. When plants were grown at high light intensity, however, senescence was induced prematurely in wild-type plants but was delayed in RNAi-W1 plants. This result suggests that WHIRLY1 plays a role in light sensing and/or stress communication between chloroplasts and the nucleus. PMID:28338757

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

PubMed Central

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

2015-01-01

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

PubMed Central

Lew, Carolyn Ritterson; Tolan, Dean R.

2012-01-01

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer. PMID:23093405

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong Li; Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL; Genetics Institute, University of Florida College of Medicine, Gainesville, FL

2008-11-25

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, theirmore » transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange

PubMed Central

Premsrirut, Prem K.; Dow, Lukas E.; Park, Youngkyu; Hannon, Gregory J.; Lowe, Scott W.

2014-01-01

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells. The activities of promoters that are commonly used for shRNA expression in cell culture can vary enormously in different tissues, and founder lines also typically vary in transgene expression due to the effects of their single integration sites. There are many ways to produce mice carrying shRNA transgenes and the method described here uses recombinase-mediated cassette exchange (RMCE). RMCE permits insertion of the shRNA transgene into a well-characterized locus that gives reproducible and predictable expression in each founder and enhances the probability of potent expression in many cell types. This procedure is more involved and complex than simple pronuclear injection, but if even a few shRNA mice are envisioned, for example, to probe the functions of several genes, the effort of setting up the processes outlined below are well worthwhile. Note that when creating a transgenic mouse, one should take care to use the most potent shRNA possible. As a rule of thumb, the sequence chosen should provide >90% knockdown when introduced into cultured cells at single copy (e.g., on retroviral infection at a multiplicity of ≤0.3). PMID:24003198

Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes

PubMed Central

Halbert, Christine L.; Rutledge, Elizabeth A.; Allen, James M.; Russell, David W.; Miller, A. Dusty

2000-01-01

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors. PMID:10627564

A Re-Examination of Global Suppression of RNA Interference by HIV-1

PubMed Central

Sanghvi, Viraj R.; Steel, Laura F.

2011-01-01

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

MK-STYX, a Catalytically Inactive Phosphatase Regulating Mitochondrially Dependent Apoptosis ▿

PubMed Central

Niemi, Natalie M.; Lanning, Nathan J.; Klomp, Jeff A.; Tait, Stephen W.; Xu, Yong; Dykema, Karl J.; Murphy, Leon O.; Gaither, L. Alex; Xu, H. Eric; Furge, Kyle A.; Green, Douglas R.; MacKeigan, Jeffrey P.

2011-01-01

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism. PMID:21262771

BCL-2 Modifying Factor (BMF) Is a Central Regulator of Anoikis in Human Intestinal Epithelial Cells*

PubMed Central

Hausmann, Martin; Leucht, Katharina; Ploner, Christian; Kiessling, Stephan; Villunger, Andreas; Becker, Helen; Hofmann, Claudia; Falk, Werner; Krebs, Michaela; Kellermeier, Silvia; Fried, Michael; Schölmerich, Jürgen; Obermeier, Florian; Rogler, Gerhard

2011-01-01

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf−/−) with dextran sulfate sodium. Colonic crypt length in Bmf−/− mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf−/− mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival. PMID:21673109

Ubiquitin-Specific Protease 2 Regulates Hepatic Gluconeogenesis and Diurnal Glucose Metabolism Through 11β-Hydroxysteroid Dehydrogenase 1

PubMed Central

Molusky, Matthew M.; Li, Siming; Ma, Di; Yu, Lei; Lin, Jiandie D.

2012-01-01

Hepatic gluconeogenesis is important for maintaining steady blood glucose levels during starvation and through light/dark cycles. The regulatory network that transduces hormonal and circadian signals serves to integrate these physiological cues and adjust glucose synthesis and secretion by the liver. In this study, we identified ubiquitin-specific protease 2 (USP2) as an inducible regulator of hepatic gluconeogenesis that responds to nutritional status and clock. Adenoviral-mediated expression of USP2 in the liver promotes hepatic glucose production and exacerbates glucose intolerance in diet-induced obese mice. In contrast, in vivo RNA interference (RNAi) knockdown of this factor improves systemic glycemic control. USP2 is a target gene of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), a coactivator that integrates clock and energy metabolism, and is required for maintaining diurnal glucose homeostasis during restricted feeding. At the mechanistic level, USP2 regulates hepatic glucose metabolism through its induction of 11β-hydroxysteroid dehydrogenase 1 (HSD1) and glucocorticoid signaling in the liver. Pharmacological inhibition and liver-specific RNAi knockdown of HSD1 significantly impair the stimulation of hepatic gluconeogenesis by USP2. Together, these studies delineate a novel pathway that links hormonal and circadian signals to gluconeogenesis and glucose homeostasis. PMID:22447855

Using adenovirus armed short hairpin RNA targeting transforming growth factor β1 inhibits melanoma growth and metastasis in an ex vivo animal model.

PubMed

Tai, Kuo-Feng; Wang, Chien-Hsing

2013-12-01

The transforming growth factor β (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-β can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-β1-RNA interference (RNAi) which mediated the RNAi for TGF-β1 gene silencing. We examined the effects of TGF-β1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-β1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFβ1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-β1-RNAi tumors. The blood vessel density of the tumors markedly reduced in B16F0/TGF-β1-RNAi tumors. Our results showed that Ad/TGF-β1-RNAi could induce silencing of the TGF-β1 gene effectively. Silencing of TGF-β1 expression in B16F0 cells by RNAi technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. This kind of adenoviral vector based on RNAi might be a promising vector for cancer therapy.

PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration.

PubMed

Kang, Hojin; Jo, Areum; Kim, Hyein; Khang, Rin; Lee, Ji-Yeong; Kim, Hanna; Park, Chi-Hu; Choi, Jeong-Yun; Lee, Yunjong; Shin, Joo-Ho

2018-01-22

Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H 2 O 2 , and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS. Copyright © 2017 Elsevier Inc. All rights reserved.

AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro.

PubMed

Wu, Wenyi; Duan, Yajian; Ma, Gaoen; Zhou, Guohong; Park-Windhol, Cindy; D'Amore, Patricia A; Lei, Hetian

2017-12-01

Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

RNA interference: learning gene knock-down from cell physiology

PubMed Central

Mocellin, Simone; Provenzano, Maurizio

2004-01-01

Over the past decade RNA interference (RNAi) has emerged as a natural mechanism for silencing gene expression. This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene. RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design. PMID:15555080

A neuropeptide modulates sensory perception in the entomopathogenic nematode Steinernema carpocapsae.

PubMed

Morris, Robert; Wilson, Leonie; Sturrock, Matthew; Warnock, Neil D; Carrizo, Daniel; Cox, Deborah; Maule, Aaron G; Dalzell, Johnathan J

2017-03-01

Entomopathogenic nematodes (EPNs) employ a sophisticated chemosensory apparatus to detect potential hosts. Understanding the molecular basis of relevant host-finding behaviours could facilitate improved EPN biocontrol approaches, and could lend insight to similar behaviours in economically important mammalian parasites. FMRFamide-like peptides are enriched and conserved across the Phylum Nematoda, and have been linked with motor and sensory function, including dispersal and aggregating behaviours in the free living nematode Caenorhabditis elegans. The RNA interference (RNAi) pathway of Steinernema carpocapsae was characterised in silico, and employed to knockdown the expression of the FMRFamide-like peptide 21 (GLGPRPLRFamide) gene (flp-21) in S. carpocapsae infective juveniles; a first instance of RNAi in this genus, and a first in an infective juvenile of any EPN species. Our data show that 5 mg/ml dsRNA and 50 mM serotonin triggers statistically significant flp-21 knockdown (-84%***) over a 48 h timecourse, which inhibits host-finding (chemosensory), dispersal, hyperactive nictation and jumping behaviours. However, whilst 1 mg/ml dsRNA and 50 mM serotonin also triggers statistically significant flp-21 knockdown (-51%**) over a 48 h timecourse, it does not trigger the null sensory phenotypes; statistically significant target knockdown can still lead to false negative results, necessitating appropriate experimental design. SPME GC-MS volatile profiles of two EPN hosts, Galleria mellonella and Tenebrio molitor reveal an array of shared and unique compounds; these differences had no impact on null flp-21 RNAi phenotypes for the behaviours assayed. Localisation of flp-21 / FLP-21 to paired anterior neurons by whole mount in situ hybridisation and immunocytochemistry corroborates the RNAi data, further suggesting a role in sensory modulation. These data can underpin efforts to study these behaviours in other economically important parasites, and could facilitate molecular approaches to EPN strain improvement for biocontrol.

Conditional RNAi: towards a silent gene therapy.

PubMed

Lee, Sang-Kyung; Kumar, Priti

2009-07-02

RNA interference (RNAi) has the potential to permit the downregulation of virtually any gene. While transgenic RNAi enables stable propagation of the resulting phenotype to progeny, the dominant nature of RNAi limits its use to applications where the continued suppression of gene expression does not disturb normal cell functioning. This is of particular importance when the target gene product is essential for cell survival, development or differentiation. It is therefore desirable that knockdown be externally regulatable. This review is aimed at providing an overview of the approaches for conditional RNAi in mammalian systems, with a special mention of studies employing these approaches to target therapeutically/biologically relevant molecules, their advantages and disadvantages, and a pointer towards approaches best suited for RNAi-based gene therapy.

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

PubMed Central

Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

2013-01-01

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

PubMed Central

Ellis, BL; Hirsch, ML; Porter, SN; Samulski, RJ; Porteus, MH

2016-01-01

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy. PMID:22257934

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

Treesearch

Chaoyang Zhao; Miguel A. Alvarez Gonzales; Therese M. Poland; Omprakash Mittapalli

2015-01-01

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong...

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

Using RNA interference to knock down the adhesion protein TES.

PubMed

Griffith, Elen

2007-01-01

RNA interference (RNAi) is a specific and efficient method to knock down protein levels using small interfering RNAs (siRNAs), which target mRNA degradation. RNAi can be used in mammalian cell culture systems to target any protein of interest, and several studies have used this method to knock down adhesion proteins. We used siRNAs to knock down the levels of TES, a focal adhesion protein, in HeLa cells. We demonstrated knockdown of both TES mRNA and TES protein. Although total knockdown of TES was not achieved, the observed reduction in TES protein was sufficient to result in a cellular phenotype of reduced actin stress fibers.

G-protein-coupled receptors participate in cytokinesis

PubMed Central

Zhang, Xin; Bedigian, Anne V.; Wang, Wenchao; Eggert, Ulrike S.

2014-01-01

Cytokinesis, the last step during cell division, is a highly coordinated process that involves the relay of signals from both the outside and inside of the cell. We have a basic understanding of how cells regulate internal events, but how cells respond to extracellular cues is less explored. In a systematic RNAi screen of G-protein-coupled receptors (GPCRs) and their effectors, we found that some GPCRs are involved in cytokinesis. RNAi knockdown of these GPCRs caused increased binucleated cell formation, and live cell imaging showed that most formed midbodies but failed at the abscission stage. OR2A4 localized to cytokinetic structures in cells and its knockdown caused cytokinesis failure at an earlier stage, likely due to effects on the actin cytoskeleton. Identifying the downstream components that transmit GPCR signals during cytokinesis will be the next step and we show that GIPC1, an adaptor protein for GPCRs, may play a part. RNAi knockdown of GIPC1 significantly increased binucleated cell formation. Understanding the molecular details of GPCRs and their interaction proteins in cytokinesis regulation will give us important clues about GPCRs signaling as well as how cells communicate with their environment during division. PMID:22888021

RNA interference of insulin-related peptide and neuroparsins affects vitellogenesis in the desert locust Schistocerca gregaria.

PubMed

Badisco, Liesbeth; Marchal, Elisabeth; Van Wielendaele, Pieter; Verlinden, Heleen; Vleugels, Rut; Vanden Broeck, Jozef

2011-03-01

The 'classic' insect hormones, juvenile hormone and 20-hydroxyecdysone, can stimulate vitellogenesis and/or ovarian development in adult females of several insect species. Accumulating evidence also indicates a crucial role in female reproductive physiology for peptide hormones, such as insulin-related peptides (IRPs) and neuroparsins (NPs). Especially in dipteran species, IRP signaling has been shown to regulate female reproductive events. The first NP was originally identified from the migratory locust (Locusta migratoria) as an antigonadotropic factor that delayed vitellogenesis. Moreover, NP family members display sequence similarities with the N-terminal domain of vertebrate insulin-like growth factor binding proteins (IGFBPs). In the current study, RNA interference (RNAi) was employed to investigate the possible involvement of IRP and NPs in the control of the female desert locust (Schistocerca gregaria) reproductive system. The cDNAs encoding an IRP (Scg-IRP) and four NPs (Scg-NPs) had previously been cloned from S. gregaria. An RNAi-mediated knock-down of either Scg-NP or Scg-IRP transcript levels was induced in adult female desert locusts and the subsequent effects were analyzed. Knock-down of the Scg-NPs or Scg-IRP affected vitellogenin transcript levels and oocyte growth in a positive and negative way, respectively. The current findings are indicative for a role of Scg-NPs and Scg-IRP in the control of vitellogenin synthesis. A plausible hypothesis is that Scg-IRP may act as a sensor of the nutritional and metabolic status that determines whether vitellogenesis can occur. That the same processes were affected in opposite ways in both RNAi experiments offers an extra argument for antagonizing roles of Scg-NPs and Scg-IRP. Copyright © 2010 Elsevier Inc. All rights reserved.

Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1.

PubMed

Kucharewicz, Weronika; Distelfeld, Assaf; Bilger, Wolfgang; Müller, Maren; Munné-Bosch, Sergi; Hensel, Götz; Krupinska, Karin

2017-02-01

WHIRLY1 in barley was isolated as a potential regulator of the senescence-associated gene HvS40. In order to investigate whether the plastid-nucleus-located DNA/RNA-binding protein WHIRLY1 plays a role in regulation of leaf senescence, primary foliage leaves from transgenic barley plants with an RNAi-mediated knockdown of the WHIRLY1 gene were characterized by typical senescence parameters, namely pigment contents, function and composition of the photosynthetic apparatus, as well as expression of selected genes known to be either down- or up-regulated during leaf senescence. When the plants were grown at low light intensity, senescence progression was similar between wild-type and RNAi-W1 plants. Likewise, dark-induced senescence of detached leaves was not affected by reduction of WHIRLY1. When plants were grown at high light intensity, however, senescence was induced prematurely in wild-type plants but was delayed in RNAi-W1 plants. This result suggests that WHIRLY1 plays a role in light sensing and/or stress communication between chloroplasts and the nucleus. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

NHR-23 dependent collagen and hedgehog-related genes required for molting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouns, Nathaniel A.; Nakielna, Johana; Behensky, Frantisek

2011-10-07

Highlights: {yields} NHR-23 is a critical regulator of nematode development and molting. {yields} The manuscript characterizes the loss-of-function phenotype of an nhr-23 mutant. {yields} Whole genome expression analysis identifies new potential targets of NHR-23. {yields} Hedgehog-related genes are identified as NHR-23 dependent genes. {yields} New link between sterol mediated signaling and regulation by NHR-23 is found. -- Abstract: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparativemore » expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.« less

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

2016-03-01

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

PubMed

Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

2009-11-01

Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

Corazonin Signaling Is Required in the Male for Sperm Transfer in the Oriental Fruit Fly Bactrocera dorsalis

PubMed Central

Hou, Qiu-Li; Chen, Er-Hu; Jiang, Hong-Bo; Yu, Shuai-Feng; Yang, Pei-Jin; Liu, Xiao-Qiang; Park, Yoonseong; Wang, Jin-Jun; Smagghe, Guy

2018-01-01

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis. Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Corazonin Signaling Is Required in the Male for Sperm Transfer in the Oriental Fruit Fly Bactrocera dorsalis.

PubMed

Hou, Qiu-Li; Chen, Er-Hu; Jiang, Hong-Bo; Yu, Shuai-Feng; Yang, Pei-Jin; Liu, Xiao-Qiang; Park, Yoonseong; Wang, Jin-Jun; Smagghe, Guy

2018-01-01

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis . Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.

PubMed

Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

2013-04-16

Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013.

AAV-mediated netrin-1 overexpression increases peri-infarct blood vessel density and improves motor function recovery after experimental stroke.

PubMed

Sun, Hui; Le, Thang; Chang, Tiffany T J; Habib, Aisha; Wu, Steven; Shen, Fanxia; Young, William L; Su, Hua; Liu, Jialing

2011-10-01

Apart from its role in axon guidance, netrin-1 is also known to be pro-angiogenic. The aim of this study is to determine whether adeno-associated viral (AAV) mediated overexpression of netrin-1 improves post-stroke neurovascular structure and recovery of function. AAV-Netrin-1 or AAV-LacZ of 1×10(10) genome copies each was injected medial and posterior to ischemic lesion at one hour following reperfusion using the distal middle cerebral artery occlusion (MCAO) method. Quantitative RT-PCR revealed that the expression of netrin-1 transgene began as early as one day and increased dramatically about 3 weeks following vector injection. Western blot analysis and confocal microscopy suggested that both the endogenous and transduced netrin-1 were expressed in the neurons of the peri-infarct cortex after MCAO. AAV-mediated netrin-1 overexpression significantly increased vascular density in the peri-infarct cortex and promoted the migration of immature neurons into the peri-infarct white matter, but it did not significantly reduce infarct size. Netrin-1 overexpression also enhanced post-stroke locomotor activity, improved exploratory behavior, and reduced ischemia-induced motor asymmetry in forelimb usage. However, it had little effect on post-stroke spatial learning and memory. Our results suggest that AAV mediated netrin-1 overexpression improves peri-infarct vascular density and post stroke motor function. Published by Elsevier Inc.

Caspase Inhibition with XIAP as an Adjunct to AAV Vector Gene-Replacement Therapy: Improving Efficacy and Prolonging the Treatment Window

PubMed Central

Yao, Jingyu; Jia, Lin; Khan, Naheed; Zheng, Qiong-Duan; Moncrief, Ashley; Hauswirth, William W.; Thompson, Debra A.; Zacks, David N.

2012-01-01

Purpose AAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase β-subunit (PDEβ) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEβ gene-replacement therapy. Methods Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received sub-retinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEβ, AAV5-GFP plus AAV5- PDEβ, or AAV- PDEβ alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEβ two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light. Results Injection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEβ resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEβ alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEβ when injected two weeks after moving to a light-cycling environment. Conclusions Adjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to gene-replacement therapy with AAV5-PDEβ in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy remains effective, thus effectively prolonging the window of opportunity for therapeutic intervention. PMID:22615940

Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition

PubMed Central

Lykke-Andersen, Karin; Gilchrist, Michael J.; Grabarek, Joanna B.; Das, Partha; Miska, Eric

2008-01-01

Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA “slicer” activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery. PMID:18701707

RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

PubMed

Sugahara, Ryohei; Tanaka, Seiji; Shiotsuki, Takahiro

2017-09-01

The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria. Copyright © 2017 Elsevier Inc. All rights reserved.

Survivin safeguards chromosome numbers and protects from aneuploidy independently from p53

PubMed Central

2014-01-01

Background Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has a dual role in mitosis and in apoptosis. It is abundantly expressed in every human tumor, compared with normal tissues. During mitosis Survivin assembles with the chromosomal passenger complex and regulates chromosomal segregation. Here, we aim to explore whether interference with the mitotic function of Survivin is linked to p53-mediated G1 cell cycle arrest and affects chromosomal stability. Methods In this study, we used HCT116, SBC-2, and U87-MG and generated corresponding isogenic p53-deficient cells. Retroviral vectors were used to stably knockdown Survivin. The resulting phenotype, in particular the mechanisms of cell cycle arrest and of initiation of aneuploidy, were investigated by Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and RNAi. Results In all cell lines Survivin-RNAi did not induce instant apoptosis but caused polyplodization irrespective of p53 status. Strikingly, polyploidization after knockdown of Survivin resulted in merotelic kinetochore spindle assemblies, γH2AX-foci, and DNA damage response (DDR), which was accompanied by a transient p53-mediated G1-arrest. That p53 wild type cells specifically arrest due to DNA damage was shown by simultaneous inhibition of ATM and DNA-PK, which abolished induction of p21waf/cip. Cytogenetic analysis revealed chromosomal aberrations indicative for DNA double strand break repair by the mechanism of non-homologous end joining (NHEJ), only in Survivin-depleted cells. Conclusion Our findings suggest that Survivin plays an essential role in proper amphitelic kinetochore-spindle assembly and that constraining Survivin’s mitotic function results in polyploidy and aneuploidy which cannot be controlled by p53. Therefore, Survivin critically safeguards chromosomal stability independently from p53. PMID:24886358

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.

PubMed

Li, Hang; Jiang, Weihua; Zhang, Zan; Xing, Yanru; Li, Fei

2013-01-01

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited "half-ecdysis". In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua

PubMed Central

Zhang, Zan; Xing, Yanru; Li, Fei

2013-01-01

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1st to 5th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control. PMID:23823756

The Influence of Endocrine Disrupting Chemicals on the Proliferation of ERα Knockdown-Human Breast Cancer Cell Line MCF-7; New Attempts by RNAi Technology

PubMed Central

Miyakoshi, Takashi; Miyajima, Katsuhiro; Takekoshi, Susumu; Osamura, Robert Yoshiyuki

2009-01-01

Bisphenol A (BPA) is a monomer use in manufacturing a wide range of chemical products which include epoxy resins and polycarbonate. It has been reported that BPA increases the cell proliferation activity of human breast cancer MCF-7 cells as well as 17-β estradiol (E2) and diethylstilbestrol (DES). However, BPA induces target genes through ER-dependent and ER-independent manners which are different from the actions induced by E2. Therefore, BPA may be unique in estrogen-dependent cell proliferation compared to other endocrine disrupting chemicals (EDCs). In the present study, to test whether ERα is essential to the BPA-induced proliferation on MCF-7 cells, we suppressed the ERα expression of MCF-7 cells by RNA interference (RNAi). Proliferation effects in the presence of E2, DES and BPA were not observed in ERα-knockdown MCF-7 cells in comparison with control MCF-7. In addition, a marker of proliferative potential, MIB-1 labeling index (LI), showed no change in BPA-treated groups compared with vehicle-treated groups on ERα-knockdown MCF-7 cells. In conclusion, we demonstrated that ERα has a role in BPA-induced cell proliferation as well as E2 and DES. Moreover, this study indicated that the direct knockdown of ERα using RNAi serves as an additional tool to evaluate, in parallel with MCF-7 cell proliferation assay, for potential EDCs. PMID:19492024

Long-term effects of systemic gene therapy in a canine model of myotubular myopathy.

PubMed

Elverman, Matthew; Goddard, Melissa A; Mack, David; Snyder, Jessica M; Lawlor, Michael W; Meng, Hui; Beggs, Alan H; Buj-Bello, Ana; Poulard, Karine; Marsh, Anthony P; Grange, Robert W; Kelly, Valerie E; Childers, Martin K

2017-11-01

X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years. We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. Four years following AAV-mediated gene therapy, gait, respiratory performance, neurological function and pathology in AAV-infused XLMTM dogs remained comparable to their healthy littermate controls despite a decline in VCN and muscle strength. AAV-mediated gene transfer of MTM1 in young XLMTM dogs results in long-term expression of myotubularin transgene with normal muscular performance and neurological function in the absence of muscle pathology. These findings support a clinical trial in patients. Muscle Nerve 56: 943-953, 2017. © 2017 Wiley Periodicals, Inc.

Search and Neutralize Factors (Cspgs) that Induce Decline in Transmission to Motoneurons from Spared Fibers after Chronic Spinal Cord Injury

DTIC Science & Technology

2014-04-01

conducted experiments using intraspinal injections of AAV10-NG2Ab combined with AAV10 vector expressing neurotrophin 3 (AAV10-NT3-gfp) in adult rat...mediated delivery of NG2-Ab and neurotrophin NT3 expressing units significantly improved locomotor function following SCI. These behavioral improvements...mediated delivery of NG2-Ab combined with neurotrophin NT3 in rats that received either hemisection or contusion SCI. We found that rats that

Matrix metalloproteinase-3 causes dopaminergic neuronal death through Nox1-regenerated oxidative stress.

PubMed

Choi, Dong-Hee; Kim, Ji-Hye; Seo, Joo-Ha; Lee, Jongmin; Choi, Wahn Soo; Kim, Yoon-Seong

2014-01-01

In the present study we investigated the interplay between matrix metalloproteinase 3 (MMP3) and NADPH oxidase 1 (Nox1) in the process of dopamine (DA) neuronal death. We found that MMP3 activation causes the induction of Nox1 via mitochondrial reactive oxygen species (ROS) production and subsequently Rac1 activation, eventually leading to Nox1-derived superoxide generation in a rat DA neuronal N27 cells exposed to 6-OHDA. While a MMP3 inhibitor, NNGH, largely attenuated mitochondrial ROS and subsequent Nox1 induction, both apocynin, a putative Nox inhibitor and GKT137831, a Nox1 selective inhibitor failed to reduce 6-OHDA-induced mitochondrial ROS. However, both inhibitors for MMP3 and Nox1 similarly attenuated 6-OHDA-induced N27 cell death. RNAi-mediated selective inhibition of MMP3 or Nox1 showed that knockdown of either MMP3 or Nox1 significantly reduced 6-OHDA-induced ROS generation in N27 cells. While 6-OHDA-induced Nox1 was abolished by MMP3 knockdown, Nox1 knockdown did not alter MMP3 expression. Direct overexpression of autoactivated MMP3 (actMMP3) in N27 cells or in rat substantia nigra (SN) increased expression of Nox1. Selective knockdown of Nox1 in the SN achieved by adeno-associated virus-mediated overexpression of Nox1-specific shRNA largely attenuated the actMMP3-mediated dopaminergic neuronal loss. Furthermore, Nox1 expression was significantly attenuated in Mmp3 null mice treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Together we established novel molecular mechanisms underlying oxidative stress-mediated dopaminergic neuronal death in which MMP3 activation is a key upstream event that leads to mitochondrial ROS, Nox1 induction and eventual dopaminergic neuronal death. Our findings may lead to the development of novel therapeutic approach.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

PubMed

Shao, Wenwei; Earley, Lauriel F; Chai, Zheng; Chen, Xiaojing; Sun, Junjiang; He, Ting; Deng, Meng; Hirsch, Matthew L; Ting, Jenny; Samulski, R Jude; Li, Chengwen

2018-06-21

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease

PubMed Central

2017-01-01

Background Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. We knocked down BI-1 in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila melanogaster to investigate its neuroprotective functions. We additionally sought to rescue the BI-1-induced phenotypes by co-expression with the pro-survival Buffy and determined the effect of BI-1 knockdown on the neurodegenerative α-synuclein-induced Parkinson disease (PD) model. Methods We used organismal assays to assess longevity of the flies to determine the effect of the altered expression of BI-1 in the Ddc-Gal4-expressing neurons by employing two RNAi transgenic fly lines. We measured the locomotor ability of these RNAi lines by computing the climbing indices of the climbing ability and compared them to a control line that expresses the lacZ transgene. Finally, we performed biometric analysis of the developing eye, where we counted the number of ommatidia and calculated the area of ommatidial disruption. Results The knockdown of BI-1 in these neurons was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. The co-expression of Buffy, the Drosophila anti-apoptotic Bcl-2 homologue, with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human α-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. We exploited this neurotoxic system to investigate possible BI-1 neuroprotective function. The co-expression of α-synuclein with BI-1-RNAi results in a slight decrease in lifespan coupled with an impairment in climbing ability. In supportive experiments, we employed the neuron-rich Drosophila compound eye to investigate subtle phenotypes that result from altered gene expression. The knockdown of BI-1 in the Drosophila developing eye under the direction of the GMR-Gal4 transgene results in reduced ommatidia number and increased disruption of the ommatidial array. Similarly, the co-expression of BI-1-RNAi with Buffy results in the suppression of the eye phenotypes. The expression of α-synuclein along with the knockdown of BI-1 resulted in reduction of ommatidia number and more disruption of the ommatidial array. Conclusion Knockdown of BI-1 in the dopaminergic neurons of Drosophila results in a shortened lifespan and premature loss in climbing ability, phenotypes that appear to be strongly associated with models of PD in Drosophila, and which are suppressed upon overexpression of Buffy and worsened by co-expression with α-synuclein. This suggests that BI-1 is neuroprotective and its knockdown can be counteracted by the overexpression of the pro-survival Bcl-2 homologue. PMID:28243526

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance

PubMed Central

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L.; Abbas, Tarek; Larner, James M.

2017-01-01

Lung cancer resists radiation therapy, making it one of the deadliest forms of cancer. Here we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor p21 protein is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, over-expression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone heat shock protein 70 (HSP70). These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity; thus, suggesting a novel strategy for the treatment of lung cancer. Implications The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. PMID:28232384

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

PubMed

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

2017-06-01

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

PubMed Central

Curtis, Helen J.; Wood, Matthew J.A.

2017-01-01

Abstract We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach. PMID:28575281

Multiple effects of the special AT-rich binding protein 1 (SATB1) in colon carcinoma.

PubMed

Frömberg, Anja; Rabe, Michael; Aigner, Achim

2014-12-01

SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, β-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival. © 2014 UICC.

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

PubMed

Li, Ting; Liu, Nannan

2017-12-01

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

PubMed Central

Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

2014-01-01

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer

PubMed Central

Shukla, Jayendra Nath; Palli, Subba Reddy

2012-01-01

Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods. PMID:22924109

A neuropeptide modulates sensory perception in the entomopathogenic nematode Steinernema carpocapsae

PubMed Central

Morris, Robert; Wilson, Leonie; Warnock, Neil D.; Maule, Aaron G.

2017-01-01

Entomopathogenic nematodes (EPNs) employ a sophisticated chemosensory apparatus to detect potential hosts. Understanding the molecular basis of relevant host-finding behaviours could facilitate improved EPN biocontrol approaches, and could lend insight to similar behaviours in economically important mammalian parasites. FMRFamide-like peptides are enriched and conserved across the Phylum Nematoda, and have been linked with motor and sensory function, including dispersal and aggregating behaviours in the free living nematode Caenorhabditis elegans. The RNA interference (RNAi) pathway of Steinernema carpocapsae was characterised in silico, and employed to knockdown the expression of the FMRFamide-like peptide 21 (GLGPRPLRFamide) gene (flp-21) in S. carpocapsae infective juveniles; a first instance of RNAi in this genus, and a first in an infective juvenile of any EPN species. Our data show that 5 mg/ml dsRNA and 50 mM serotonin triggers statistically significant flp-21 knockdown (-84%***) over a 48 h timecourse, which inhibits host-finding (chemosensory), dispersal, hyperactive nictation and jumping behaviours. However, whilst 1 mg/ml dsRNA and 50 mM serotonin also triggers statistically significant flp-21 knockdown (-51%**) over a 48 h timecourse, it does not trigger the null sensory phenotypes; statistically significant target knockdown can still lead to false negative results, necessitating appropriate experimental design. SPME GC-MS volatile profiles of two EPN hosts, Galleria mellonella and Tenebrio molitor reveal an array of shared and unique compounds; these differences had no impact on null flp-21 RNAi phenotypes for the behaviours assayed. Localisation of flp-21 / FLP-21 to paired anterior neurons by whole mount in situ hybridisation and immunocytochemistry corroborates the RNAi data, further suggesting a role in sensory modulation. These data can underpin efforts to study these behaviours in other economically important parasites, and could facilitate molecular approaches to EPN strain improvement for biocontrol. PMID:28253355

Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

PubMed

Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie

2016-08-15

In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

PubMed Central

Phatak, Nitasha R.; Stankowska, Dorota L.

2016-01-01

Purpose Brn3b is a class IV POU domain transcription factor that plays an important role in the development of retinal ganglion cells (RGCs), RGC survival, and particularly axon growth and pathfinding. Our previous study demonstrated that recombinant adenoassociated virus serotype 2 (rAAV-2)–mediated overexpression of Brn3b in RGCs promoted neuroprotection in a rodent model of glaucoma. However, the mechanisms underlying neuroprotection of RGCs in rats overexpressing Brn3b in animal models of glaucoma remain largely unknown. The goal of this study was to understand some of the mechanisms underlying the neuroprotection of RGCs overexpressing Brn3b during intraocular pressure (IOP) elevation in Brown Norway rats. Methods One eye of Brown Norway rats (Rattus norvegicus) was injected with an AAV construct encoding either green fluorescent protein (GFP; recombinant adenoassociated virus–green fluorescent protein, rAAV-hSyn-GFP) or Brn3b (rAAV-hSyn-Brn3b). Expression of antiapoptotic proteins, including B cell lymphoma/leukemia-2 (Bcl-2) family proteins (Bcl-2 and Bcl-xL), and p-AKT, was observed following immunostaining of rat retinas that overexpress Brn3b. In a different set of experiments, intraocular pressure was elevated in one eye of Brown Norway rats, which was followed by intravitreal injection with AAV constructs encoding either GFP (rAAV-CMV-GFP) or Brn3b (rAAV-CMV-Brn3b). Retinal sections were stained for prosurvival factors, including Bcl-2, Bcl-XL, and p-AKT. Results AAV-mediated expression of transcription factor Brn3b promoted statistically significant upregulation of the Bcl-2 protein and increased expression of p-AKT in RGCs of Brown Norway rats. In addition, following IOP elevation, AAV-mediated Brn3b expression also statistically significantly increased levels of Bcl-2 in the RGC layer in Brown Norway rats. Conclusions Adenoassociated virus–mediated Brn3b protein overexpression may promote neuroprotection by upregulating key antiapoptotic proteins, including Bcl-2, Bcl-xL, and p-AKT, in animal models of glaucoma. PMID:27587945

The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

PubMed

Chen, W; Gao, B; Hao, L; Zhu, G; Jules, J; MacDougall, M J; Wang, J; Han, X; Zhou, X; Li, Y-P

2016-10-01

Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

PubMed

Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Gimenez Y Ribotta, Minerva; Privat, Alain; Mallet, Jacques

2009-07-14

The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole livermore » cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.« less

Aldolase promotes the development of cardiac hypertrophy by targeting AMPK signaling.

PubMed

Li, Yapeng; Zhang, Dianhong; Kong, Lingyao; Shi, Huiting; Tian, Xinyu; Gao, Lu; Liu, Yuzhou; Wu, Leiming; Du, Binbin; Huang, Zhen; Liang, Cui; Wang, Zheng; Yao, Rui; Zhang, Yanzhou

2018-06-11

Metabolic dysfunction is a hallmark of cardiac hypertrophy and heart failure. During cardiac failure, the metabolism of cardiomyocyte switches from fatty acid oxidation to glycolysis. However, the roles of key metabolic enzymes in cardiac hypertrophy are not understood fully. Here in the present work, we identified Aldolase A (AldoA) as a core regulator of cardiac hypertrophy. The mRNA and protein levels of AldoA were significantly up-regulated in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced hypertrophic mouse hearts. Overexpression of AldoA in cardiomyocytes promoted ISO-induced cardiomyocyte hypertrophy, whereas AldoA knockdown repressed cardiomyocyte hypertrophy. In addition, adeno-associated virus 9 (AAV9)-mediated in vivo knockdown of AldoA in the hearts rescued ISO-induced decrease in cardiac ejection fraction and fractional shortening and repressed cardiac hypertrophy. Mechanism study revealed that AldoA repressed the activation of AMP-dependent protein kinase (AMPK) signaling in a liver kinase B1 (LKB1)-dependent and AMP-independent manner. Inactivation of AMPK is a core mechanism underlying AldoA-mediated promotion of ISO-induced cardiomyocyte hypertrophy. By contrast, activation of AMPK with metformin and AICAR blocked AldoA function during cardiomyocyte hypertrophy. In summary, our data support the notion that AldoA-AMPK axis is a core regulatory signaling sensing energetic status and participates in cardiac hypertrophy. Copyright © 2018 Elsevier Inc. All rights reserved.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

PubMed

Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

2015-01-01

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

PubMed Central

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling

PubMed Central

Mleczko-Sanecka, Katarzyna; Roche, Franziska; Rita da Silva, Ana; Call, Debora; D’Alessio, Flavia; Ragab, Anan; Lapinski, Philip E.; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D’Alessandro, Lorenza A.; Klingmüller, Ursula; King, Philip D.; Boutros, Michael; Hentze, Matthias W.

2014-01-01

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the “iron-regulated” bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6–triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism. PMID:24385536

A simple and robust vector-based shRNA expression system used for RNA interference.

PubMed

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

OsMYC2 mediates numerous defence-related transcriptional changes via jasmonic acid signalling in rice.

PubMed

Ogawa, Satoshi; Kawahara-Miki, Ryouka; Miyamoto, Koji; Yamane, Hisakazu; Nojiri, Hideaki; Tsujii, Yoshimasa; Okada, Kazunori

2017-05-06

Jasmonic acid (JA) plays central roles in various events in plants, especially defence against pathogens and insects. The basic helix-loop-helix (bHLH) transcription factor MYC2 has attracted attention as a master regulator of JA signalling in dicotyledonous plants. However, how MYC2 functions in monocotyledonous plants, including agriculturally important crops such as cultivated rice, has been poorly understood. To elucidate the comprehensive effects of rice MYC2 (OsMYC2) on the JA-inducible transcriptional modifications, we performed RNA-sequencing by using OsMYC2-knockdown plants (osmyc2RNAi). In osmyc2RNAi, JA-inducible expression of many defence-related genes, for example chitinases and proteinase inhibitors, was compromised. Decrease in JA-dependent activation of the biosynthetic pathways of specialised metabolites, especially defence compounds, was also evident in the osmyc2RNAi line. Furthermore, a substantial change was noted in the expression of distinct types of transcription factors, such as MYB-type factors, likely depicting the importance of OsMYC2 in not only defence responses but also other morphogenetic events. Our findings provide fundamental information to understand the overall functions of MYC2 in JA signalling in monocotyledonous plants, which might yield agricultural benefits. Copyright © 2017 Elsevier Inc. All rights reserved.

RNAi or overexpression: Alternative therapies for Spinocerebellar Ataxia Type 1

PubMed Central

Keiser, Megan S.; Geoghegan, James C.; Boudreau, Ryan L.; Lennox, Kim A.; Davidson, Beverly L.

2014-01-01

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant late onset neurodegenerative disease caused by an expanded polyglutamine tract in ataxin-1. Here, we compared the protective effects of overexpressing ataxin-1-like using recombinant AAVs, or reducing expression of mutant ataxin-1 using virally delivered RNA interference (RNAi), in a transgenic mouse model of SCA1. For the latter, we used an artificial microRNA (miR) design that optimizes potency, efficacy and safety to suppress ataxin-1 expression (miS1). Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mice cerebella resulted in widespread cerebellar Purkinje cell transduction and improved behavioral and histological phenotypes. Our data indicate the utility of either approach as a possible therapy for SCA1 patients. PMID:23583610

Neuroprotective Effects of Transcription Factor Brn3b in an Ocular Hypertension Rat Model of Glaucoma

PubMed Central

Stankowska, Dorota L.; Minton, Alena Z.; Rutledge, Margaret A.; Mueller, Brett H.; Phatak, Nitasha R.; He, Shaoqing; Ma, Hai-Ying; Forster, Michael J.; Yorio, Thomas; Krishnamoorthy, Raghu R.

2015-01-01

Purpose. Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. Methods. Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. Results. Adeno-associated virus–mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b–injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP– injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. Conclusions. Adeno-associated virus–mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma. PMID:25587060

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

PubMed

Haga, K; Lemp, N A; Logg, C R; Nagashima, J; Faure-Kumar, E; Gomez, G G; Kruse, C A; Mendez, R; Stripecke, R; Kasahara, N; Kasahara, N A; Cicciarelli, J C

2006-12-01

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

PubMed

Ferreira, Flávia Viana; Aguiar, Eric Roberto Guimarães Rocha; Olmo, Roenick Proveti; de Oliveira, Karla Pollyanna Vieira; Silva, Emanuele Guimarães; Sant'Anna, Maurício Roberto Viana; Gontijo, Nelder de Figueiredo; Kroon, Erna Geessien; Imler, Jean Luc; Marques, João Trindade

2018-06-01

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Adeno-associated virus-mediated expression of myostatin propeptide improves the growth of skeletal muscle and attenuates hyperglycemia in db/db mice.

PubMed

Jiang, J G; Shen, G F; Li, J; Qiao, C; Xiao, B; Yan, H; Wang, D W; Xiao, X

2017-03-01

Inhibition of myostatin, a negative growth modulator for muscle, can functionally enhance muscle mass and improve glucose and fat metabolism in myostatin propeptide (MPRO) transgenic mice. This study was to investigate whether myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of MPRO could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in the db/db mice and the mechanisms involved in that process. Eight-week-old male db/db mice were administered saline, AAV-GFP and AAV-MPRO/Fc vectors and monitored random blood glucose levels and body weight for 36 weeks. Body weight gain was not different during follow-up among the groups, but AAV-MPRO/Fc vectors resulted high level of MPRO in the blood companied by an increase in skeletal muscle mass and muscle hypertrophy. In addition, AAV-MPRO/Fc-treated db/db mice showed significantly lower blood glucose and insulin levels and significantly increased glucose tolerance and insulin sensitivity compared with the control groups (P<0.05). Moreover, these mice exhibited lower triglyceride (TG) and free fatty acid (FFA) content in the skeletal muscle, although no difference was observed in fat pad weights and serum TG and FFA levels. Finally, AAV-MPRO/Fc-treated mice had enhanced insulin signaling in the skeletal muscle. These data suggest that AAV-mediated MPRO therapy may provide an important clue for potential clinical applications to prevent type II diabetes, and these studies confirm that MPRO is a therapeutic target for type II diabetes.

Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype.

PubMed

Calhoun, Colonya C; Lu, Ying-Chun; Song, Jun; Chiu, Robert

2009-01-01

Cyclophilin A (CypA) was originally identified as a cytosolic protein possessing peptidyl-prolyl isomerase activity. CypA has been shown to play a pivotal role in the immune response, but little is known about other molecular mechanisms of CypA-mediated biologic events. In our present study, we demonstrate that knockdown CypA expression using RNAi in U2OS cells resulted in disruption of the F-actin structure, as well as decreased anchorage-independent growth, proliferation, and migration. Wild-type U2OS cells treated with cyclosporine A (CsA), a peptidyl-prolyl isomerase inhibitor, displayed the same phenotype as knockdown CypA cells, suggesting that the isomerase activity of CypA is required to maintain a normal phenotype. In vitro and in vivo binding assays revealed that CypA binds to N-WASP, which functions in the nucleation of actin via the Arp2/3 complex. Pulse-chase labeling study indicated an enhanced degradation of N-WASP in cell lacking CypA, suggesting that CypA is required for stabilizing N-WASP to form a N-WASP/Arp2/3 complex for the nucleation/initiation of F-actin polymerization.

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

PubMed Central

Jabed, Anower; Wagner, Stefan; McCracken, Judi; Wells, David N.; Laible, Goetz

2012-01-01

Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA–mediated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits. PMID:23027958

Mitoferrin-1 is involved in the progression of alzheimer's disease through targeting mitochondrial iron metabolism in a caenorhabditis elegans model of alzheimers disease.

PubMed

Huang, Jiatao; Chen, Sixi; Hu, Li; Niu, Huan; Sun, Qianqian; Li, Wenna; Tan, Guoqian; Li, Jianghui; Jin, LongJin; Lyu, Jianxin; Zhou, Huaibin

2018-06-13

In mammals, mitoferrin-1and mitoferrin-2, two homologous proteins of the mitochondrial solute carrier family are required for iron delivery into mitochondria. However, there is only one kind, called W02B12 (mitoferrin-1 or mfn-1), in Caenorhabditis elegans and its regulatory mechanism is unknown. In this study, we used Caenorhabditis elegans strains CL2006 and GMC101 as models to investigate what role mitoferrin-1 played in Alzheimer's disease (AD). We found that knockdown of mitoferrin-1 by feeding-RNAi treatment extended lifespans of both strains of C. elegans. In addition, it reduced the paralysis rate in the GMC101 strain. These results suggest that mitoferrin-1 may be involved in the progression of Alzheimer's disease. Knockdown of mitoferrin-1 was seen to disturb mitochondrial morphology in the CB5600 strain. We tested whether knockdown of mitoferrin-1 could influence mitochondrial metabolism. Analysis of mitochondrial iron metabolism and mitochondrial ROS showed that knockdown of mitoferrin-1 could reduce mitochondrial iron content and reduce the level of mitochondrial ROS in the CL2006 and GMC101 strains. These results confirm that knockdown of mitoferrin-1 can slow the progress of disease in Alzheimer model of C. elegans and suggest that mitoferrin-1 plays a major role in mediating mitochondrial iron metabolism in this process. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

γ-Secretase Inhibition of Murine Choroidal Neovascularization Is Associated with Reduction of Superoxide and Proinflammatory Cytokines

PubMed Central

Qi, Xiaoping; Cai, Jun; Ruan, Qing; Liu, Li; Boye, Sanford L.; Chen, Zhijuan; Hauswirth, William W.; Ryals, Renee C.; Shaw, Lynn; Caballero, Sergio; Grant, Maria B.

2012-01-01

Purpose. This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines. Methods. γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA. Results. γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis. Conclusions. γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV. PMID:22205609

Prolonged Expression of an Anti-HIV-1 gp120 Minibody to the Female Rhesus Macaque Lower Genital Tract by AAV Gene Transfer

PubMed Central

Abdel-Motal, Ussama M.; Harbison, Carole; Han, Thomas; Pudney, Jeffrey; Anderson, Deborah J.; Zhu, Quan; Westmoreland, Susan; Marasco, Wayne A.

2014-01-01

Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women, however, numerous pharmacokinetic limitations associated with coitally-related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various GFP-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intra-vaginally, including p63+ epithelial stem cells. Moreover, intra-vaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79 day study period. These data provide proof-of-principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection. PMID:24965083

Prolonged expression of an anti-HIV-1 gp120 minibody to the female rhesus macaque lower genital tract by AAV gene transfer.

PubMed

Abdel-Motal, U M; Harbison, C; Han, T; Pudney, J; Anderson, D J; Zhu, Q; Westmoreland, S; Marasco, W A

2014-09-01

Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women; however, numerous pharmacokinetic limitations associated with coitally related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various green fluorescent protein (GFP)-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intravaginally, including p63+ epithelial stem cells. Moreover, intravaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79-day study period. These data provide proof of principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection.

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus (AAV) Types 2, 5, and 6 in Cystic Fibrosis and Normal Populations: Implications for Gene Therapy using AAV Vectors

PubMed Central

Halbert, Christine L.; Miller, A. Dusty; McNamara, Sharon; Emerson, Julia; Gibson, Ronald L.; Ramsey, Bonnie; Aitken, Moira L.

2014-01-01

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared to that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20–30% were seropositive for AAV6, and 10–20% were seropositive for AAV5. CF children were seropositive for AAV types 2, 5, or 6 at rates of 4–15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared to the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children. PMID:16610931

Conditional knockdown of BCL2A1 reveals rate-limiting roles in BCR-dependent B-cell survival

PubMed Central

Sochalska, M; Ottina, E; Tuzlak, S; Herzog, S; Herold, M; Villunger, A

2016-01-01

Bcl2 family proteins control mitochondrial apoptosis and its members exert critical cell type and differentiation stage-specific functions, acting as barriers against autoimmunity or transformation. Anti-apoptotic Bcl2a1/Bfl1/A1 is frequently deregulated in different types of blood cancers in humans but its physiological role is poorly understood as quadruplication of the Bcl2a1 gene locus in mice hampers conventional gene targeting strategies. Transgenic overexpression of A1, deletion of the A1-a paralogue or constitutive knockdown in the hematopoietic compartment of mice by RNAi suggested rate-limiting roles in lymphocyte development, granulopoiesis and mast cell activation. Here we report on the consequences of conditional knockdown of A1 protein expression using a reverse transactivator (rtTA)-driven approach that highlights a critical role for this Bcl2 family member in the maintenance of mature B-cell homeostasis. Furthermore, we define the A1/Bim (Bcl-2 interacting mediator of cell death) axis as a target of key kinases mediating B-cell receptor (BCR)-dependent survival signals, such as, spleen tyrosine kinase (Syk) and Brutons tyrosine kinase (Btk). As such, A1 represents a putative target for the treatment of B-cell-related pathologies depending on hyperactivation of BCR-emanating survival signals and loss of A1 expression accounts, in part, for the pro-apoptotic effects of Syk- or Btk inhibitors that rely on the ‘BH3-only' protein Bim for cell killing. PMID:26450454

Establishment of an AAV Reverse Infection-Based Array

PubMed Central

Wang, Gang; Dong, Zheyue; Shen, Wei; Zheng, Gang; Wu, Xiaobing; Xue, Jinglun; Wang, Yue; Chen, Jinzhong

2010-01-01

Background The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays. PMID:20976058

Comparative biology of rAAV transduction in ferret, pig and human airway epithelia.

PubMed

Liu, X; Luo, M; Guo, C; Yan, Z; Wang, Y; Engelhardt, J F

2007-11-01

Differences between rodent and human airway cell biology have made it difficult to translate recombinant adeno-associated virus (rAAV)-mediated gene therapies to the lung for cystic fibrosis (CF). As new ferret and pig models for CF become available, knowledge about host cell/vector interactions in these species will become increasingly important for testing potential gene therapies. To this end, we have compared the transduction biology of three rAAV serotypes (AAV1, 2 and 5) in human, ferret, pig and mouse-polarized airway epithelia. Our results indicate that apical transduction of ferret and pig airway epithelia with these rAAV serotypes closely mirrors that observed in human epithelia (rAAV1>rAAV2 congruent withrAAV5), while transduction of mouse epithelia was significantly different (rAAV1>rAAV5>rAAV2). Similarly, ferret, pig and human epithelia also shared serotype-specific differences in the polarity (apical vs basolateral) and proteasome dependence of rAAV transduction. Despite these parallels, N-linked sialic acid receptors were required for rAAV1 and rAAV5 transduction of human and mouse airway epithelia, but not ferret or pig airway epithelia. Hence, although the airway tropisms of rAAV serotypes 1, 2 and 5 are conserved better among ferret, pig and human as compared to mouse, viral receptors/co-receptors appear to maintain considerable species diversity.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

PubMed Central

Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

2009-01-01

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

PubMed Central

Halbert, Christine L.; Allen, James M.; Miller, A. Dusty

2001-01-01

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis. PMID:11413329

AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

PubMed

Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

2016-01-01

Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates | Center for Cancer Research

Cancer.gov

Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yi; Li, Dechun; Zhao, Xin

Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level ofmore » DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ{sup 2} = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells. - Highlights: • We investigated the role of DcR3 in FasL resistance in pancreatic cancer. • Knockdown of DcR3 in SW1990 cells reduced resistance to FasL-induced apoptosis. • DcR3 knockdown also elevated caspase expression, and reduced ERK1/2 phosphorylation. • Tumor and non-tumor tissues were collected from 66 pancreatic carcinoma patients. • 47 tumor tissue specimens, but only 15 matched non-tumor specimens contained DcR3.« less

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

PubMed

Smendziuk, Christopher M; Messenberg, Anat; Vogl, A Wayne; Tanentzapf, Guy

2015-08-01

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues. © 2015. Published by The Company of Biologists Ltd.

An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

PubMed

Burrows, Jeffrey T A; Pearson, Bret J; Scott, Ian C

2015-04-14

The Mediator complex has recently been shown to be a key player in the maintenance of embryonic and induced pluripotent stem cells. However, the in vivo consequences of loss of many Mediator subunits are unknown. We identified med14 as the gene affected in the zebrafish logelei (log) mutant, which displayed a morphological arrest by 2 days of development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Indeed, log cells transplanted into a wild-type environment were able to survive into adulthood. In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos. Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of insecticides chlorpyrifos, emamectin benzoate and fipronil on Spodoptera litura might be mediated by OBPs and CSPs.

PubMed

Lin, X; Jiang, Y; Zhang, L; Cai, Y

2017-12-04

Spodoptera litura is a widespread polyphagous insect pest that can develop resistance and cross-resistance to insecticides, making it difficult to control. Insecticide exposure has previously been linked with induction of specific olfactory-related proteins, including some chemosensory proteins (CSPs) and odorant-binding proteins (OPBs), which may disrupt detection of environmental factors and reduce fitness. However, functional evidence supporting insecticide and OBPs/CSPs mediation remains unknown. Here we fed male S. litura moths with sucrose water containing one of three insecticides, chlorpyrifos, emamectin benzoate or fipronil, and used real-time quantitative polymerase chain reaction and RNAi to investigate OBPs and CSPs expression and their correlations with survival. Chlorpyrifos and emamectin benzoate increased expression of 78% of OBPs, plus 63 and 56% of CSP genes, respectively, indicating a major impact on these gene families. RNAi knockdown of SlituCSP18, followed by feeding with chlorpyrifos or fipronil, decreased survival rates of male moths significantly compared with controls. Survival rate also decreased significantly with the downregulation of SlituOBP9 followed by feeding with chlorpyrifos. Thus, although these three insecticides had different effects on OBP and CSP gene expression, we hypothesize that SlituOBPs and SlituCSPs might mediate their effects by increasing their expression levels to improve survival. Moreover, the differential response of S. litura male moths to the three insecticides indicated the potential specificity of chlorpyrifos affect SlituCSP18 and SlituOBP9 expression.

Contributions of 3'-overhang to the dissociation of small interfering RNAs from the PAZ domain: molecular dynamics simulation study.

PubMed

Lee, Hui Sun; Lee, Soo Nam; Joo, Chul Hyun; Lee, Heuiran; Lee, Han Saem; Yoon, Seung Yong; Kim, Yoo Kyum; Choe, Han

2007-03-01

RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.

Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

PubMed Central

Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

2016-01-01

In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

Photoinduced RNA interference.

PubMed

Matsushita-Ishiodori, Yuka; Ohtsuki, Takashi

2012-07-17

Because RNA interference (RNAi) can be applied to any gene, this technique has been widely used for studying gene functions. In addition, many researchers are attempting to use RNAi technology in RNAi-based therapies. However, several challenging and controversial issues have arisen during the widespread application of RNAi including target gene specificity, target cell specificity, and spatiotemporal control of gene silencing. To address these issues, several groups have utilized photochemistry to control the RNA release, both spatially and temporally. In this Account, we focus on recent studies using photocleavable protecting groups, photosensitizers, Hand gold nanoparticles for photoinduced RNAi. In 2005 the first report of photoinduced RNAi used a caged short interfering RNA (siRNA), an siRNA carrying a photocleavable protecting group. Caging groups block the bioactivities of target molecules, but allow for complete recovery of these functions via photoactivation. However, some RNAi activity can occur in these caged siRNAs, so it will be necessary to decrease this "leakage" and raise the RNAi activity restored after irradiation. This technique also uses UV light around 350 nm, which is cytotoxic, but in the near future we expect that it will be possible to use visible and near-infrared light We also examine the application of photochemical internalization (PCI) to RNAi technology, which involves a combination of photosensitizers and light. Instead of inducing RNAi using light, the strategy behind this method was to enhance RNAi using RNA carriers. Many wellknown RNA carriers deliver siRNAs into cells by endocytosis. The siRNAs are trapped in endocytic vesicles and have to be released into the cytoplasm in order to express their activity. To achieve the endosomal escape of siRNAs, PCI technology employed photosensitizers to generate light-dependent reactive oxygen species (ROS) that disrupted the endocytic vesicles. In most studies, RNAi-mediated knockdown of the target gene was detected even without PCI. Recently, a polymer capable of trapping the siRNA in endocytic vesicles controlled RNAi almost entirely by light. CLIP-RNAi uses photosensitizing carrier proteins that can be activated over a wide range of visible light wavelengths. With this method RNA carrier/siRNA complexes are completely trapped within endosomes, and RNAi is controlled strictly by light. Such precise, light-dependent control will open up new possibilities for cellular and molecular biology and therapy. Most recently, gold nanoparticles (AuNPs) conjugated to siRNA have provided temporal and spatial control of RNAi. The light-dependent melting of AuNPs accompanied by a shape transformation induces the release of thiolated siRNAs from AuNPs. In this method, the unique optical properties of the AuNP enable deep penetration of the excitation light into tissues at nearinfrared wavelengths. The development of photoinduced RNAi technology will lead to novel insights into gene functions and selective drug delivery, and many other scientific fields will continue to influence its progress.

RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

2015-09-01

RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

PubMed

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation.

PubMed

Jensen, Victor L; Simonsen, Karina T; Lee, Yu-Hui; Park, Donha; Riddle, Donald L

2010-12-31

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.

RNAi-Mediated Knock-Down of transformer and transformer 2 to Generate Male-Only Progeny in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

PubMed Central

Li, Jianwei; Zhang, Guifen; Wan, Fanghao

2015-01-01

The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest. PMID:26057559

RNAi-Mediated Knock-Down of transformer and transformer 2 to Generate Male-Only Progeny in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel).

PubMed

Liu, Guiqing; Wu, Qiang; Li, Jianwei; Zhang, Guifen; Wan, Fanghao

2015-01-01

The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest.

Role of FAT/CD36 in fatty acid sensing, energy, and glucose homeostasis regulation in DIO and DR rats.

PubMed

Le Foll, Christelle; Dunn-Meynell, Ambrose A; Levin, Barry E

2015-02-01

Hypothalamic fatty acid (FA) sensing neurons alter their activity utilizing the FA translocator/receptor, FAT/CD36. Depletion of ventromedial hypothalamus (VMH) CD36 with adeno-associated viral vector expressing CD36 shRNA (AAV CD36 shRNA) leads to redistribution of adipose stores and insulin resistance in outbred rats. This study assessed the requirement of VMH CD36-mediated FA sensing for the regulation of energy and glucose homeostasis in postnatal day 5 (P5) and P21 selectively bred diet-induced obese (DIO) and diet-resistant (DR) rats using VMH AAV CD36 shRNA injections. P5 CD36 depletion altered VMH neuronal FA sensing predominantly in DIO rats. After 10 wk on a 45% fat diet, DIO rats injected with VMH AAV CD36 shRNA at P21 ate more and gained more weight than DIO AAV controls, while DR AAV CD36 shRNA-injected rats gained less weight than DR AAV controls. VMH CD36 depletion increased inguinal fat pad weights and leptin levels in DIO and DR rats. Although DR AAV CD36 shRNA-injected rats became as obese as DIO AAV controls, only DIO control and CD36 depleted rats became insulin-resistant on a 45% fat diet. VMH CD36 depletion stunted linear growth in DIO and DR rats. DIO rats injected with AAV CD36 shRNA at P5 had increased fat mass, mostly due to a 45% increase in subcutaneous fat. They were also insulin-resistant with an associated 71% increase of liver triglycerides. These results demonstrate that VMH CD36-mediated FA sensing is a critical factor in the regulation of energy and glucose homeostasis and fat deposition in DIO and DR rats.

Intramyocardial Injection of siRNAs Can Efficiently Establish Myocardial Tissue-Specific Renalase Knockdown Mouse Model.

PubMed

Huang, Kun; Liu, Ju; Zhang, Hui; Wang, Jiliang; Li, Huili

2016-01-01

Ischaemia/reperfusion (I/R) injury will cause additional death of cardiomyocytes in ischaemic heart disease. Recent studies revealed that renalase was involved in the I/R injury. So, the myocardial tissue-specific knockdown mouse models were needed for the investigations of renalase. To establish the mouse models, intramyocardial injection of siRNAs targeting renalase was performed in mice. The wild distribution and high transfection efficiency of the siRNAs were approved. And the renalase expression was efficiently suppressed in myocardial tissue. Compared with the high cost, time consumption, and genetic compensation risk of the Cre/loxP technology, RNA interference (RNAi) technology is much cheaper and less time-consuming. Among the RNAi technologies, injection of siRNAs is safer than virus. And considering the properties of the I/R injury mouse models, the efficiency and durability of injection with siRNAs are acceptable for the studies. Altogether, intramyocardial injection of siRNAs targeting renalase is an economical, safe, and efficient method to establish myocardial tissue-specific renalase knockdown mouse models.

Decoy receptor 3 suppresses FasL-induced apoptosis via ERK1/2 activation in pancreatic cancer cells.

PubMed

Zhang, Yi; Li, Dechun; Zhao, Xin; Song, Shiduo; Zhang, Lifeng; Zhu, Dongming; Wang, Zhenxin; Chen, Xiaochen; Zhou, Jian

2015-08-07

Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level of DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ(2) = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

PubMed

Galdeano, Diogo Manzano; Breton, Michèle Claire; Lopes, João Roberto Spotti; Falk, Bryce W; Machado, Marcos Antonio

2017-01-01

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

PubMed Central

EL Andaloussi, Samir; Lehto, Taavi; Mäger, Imre; Rosenthal-Aizman, Katri; Oprea, Iulian I.; Simonson, Oscar E.; Sork, Helena; Ezzat, Kariem; Copolovici, Dana M.; Kurrikoff, Kaido; Viola, Joana R.; Zaghloul, Eman M.; Sillard, Rannar; Johansson, Henrik J.; Said Hassane, Fatouma; Guterstam, Peter; Suhorutšenko, Julia; Moreno, Pedro M. D.; Oskolkov, Nikita; Hälldin, Jonas; Tedebark, Ulf; Metspalu, Andres; Lebleu, Bernard; Lehtiö, Janne; Smith, C. I. Edvard; Langel, Ülo

2011-01-01

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential. PMID:21245043

Harnessing RNA interference to develop neonatal therapies: from Nobel Prize winning discovery to proof of concept clinical trials.

PubMed

DeVincenzo, John P

2009-10-01

A revolution in the understanding of RNA biological processing and control is leading to revolutionary new concepts in human therapeutics. It has become increasingly clear that the so called "non-coding RNA" exerts specific and profound functional control on regulation of protein production and indeed controls the expression of all genes. Harnessing this naturally-occurring RNA-mediated regulation of protein production has immense human therapeutic potential. These processes are collectively known as RNA interference (RNAi). RNAi is a recently discovered, naturally-occurring intracellular process that regulates gene expression through the silencing of specific mRNAs. Methods of harnessing this natural pathway are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small inhibitory RNAs (siRNA). siRNAs are being chemically modified to acquire drug-like properties. Numerous recent high profile publications have provided proofs of concept that RNA interference may be useful therapeutically. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. This makes this approach very attractive for developing therapies targeting orphan diseases including neonatal diseases. Theoretically, any disease that can be ameliorated through knockdown of any endogenous or exogenous protein is a potential therapeutic target for RNAi-based therapeutics. Lung diseases are particularly attractive targets for RNAi therapeutics since the affected cells' location increases their accessibility to topical administration of siRNA, for example by aerosol. Respiratory viral infections and chronic lung disease are examples of such diseases. RNAi therapeutics have been shown to be active against RSV, parainfluenza and human metapneumoviruses in vitro and in vivo resulting in profound antiviral effects. The first proof of concept test of efficacy of an RNAi-based therapeutic in man has been initiated. A discussion of the science behind RNA interference is followed by a presentation of the potential practical issues in applying this technology to neonatal respiratory viral diseases. RNAi may offer new strategies for the treatment of a variety of orphan diseases including neonatal diseases, RSV infections, and other respiratory viruses.

Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective autophagy.

PubMed

Chi, Hsiang-Cheng; Chen, Shen-Liang; Tsai, Chung-Ying; Chuang, Wen-Yu; Huang, Ya-Hui; Tsai, Ming-Ming; Wu, Sheng-Ming; Sun, Cheng-Pu; Yeh, Chau-Ting; Lin, Kwang-Huei

2016-12-01

Recent studies have demonstrated a critical association between disruption of cellular thyroid hormone (TH) signaling and the incidence of hepatocellular carcinoma (HCC), but the underlying mechanisms remain largely elusive. Here, we showed that disruption of TH production results in a marked increase in progression of diethylnitrosamine (DEN)-induced HCC in a murine model, and conversely, TH administration suppresses the carcinogenic process via activation of autophagy. Inhibition of autophagy via treatment with chloroquine (CQ) or knockdown of ATG7 (autophagy-related 7) via adeno-associated virus (AAV) vectors, suppressed the protective effects of TH against DEN-induced hepatic damage and development of HCC. The involvement of autophagy in TH-mediated protection was further supported by data showing transcriptional activation of DAPK2 (death-associated protein kinase 2; a serine/threonine protein kinase), which enhanced the phosphorylation of SQSTM1/p62 (sequestosome 1) to promote selective autophagic clearance of protein aggregates. Ectopic expression of DAPK2 further attenuated DEN-induced hepatoxicity and DNA damage though enhanced autophagy, whereas, knockdown of DAPK2 displayed the opposite effect. The pathological significance of the TH-mediated hepatoprotective effect by DAPK2 was confirmed by the concomitant decrease in the expression of THRs and DAPK2 in matched HCC tumor tissues. Taken together, these findings indicate that TH promotes selective autophagy via induction of DAPK2-SQSTM1 cascade, which in turn protects hepatocytes from DEN-induced hepatotoxicity or carcinogenesis.

Megalin-mediated specific uptake of chitosan/siRNA nanoparticles in mouse kidney proximal tubule epithelial cells enables AQP1 gene silencing.

PubMed

Gao, Shan; Hein, San; Dagnæs-Hansen, Frederik; Weyer, Kathrin; Yang, Chuanxu; Nielsen, Rikke; Christensen, Erik I; Fenton, Robert A; Kjems, Jørgen

2014-01-01

RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.

Megalin-Mediated Specific Uptake of Chitosan/siRNA Nanoparticles in Mouse Kidney Proximal Tubule Epithelial Cells Enables AQP1 Gene Silencing

PubMed Central

Gao, Shan; Hein, San; Dagnæs-Hansen, Frederik; Weyer, Kathrin; Yang, Chuanxu; Nielsen, Rikke; Christensen, Erik I; Fenton, Robert A; Kjems, Jørgen

2014-01-01

RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases. PMID:25157280

Adeno-associated virus type 8 vector–mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model

PubMed Central

Igarashi, Tsutomu; Miyake, Noriko; Fujimoto, Chiaki; Yaguchi, Chiemi; Iijima, Osamu; Shimada, Takashi; Takahashi, Hiroshi

2014-01-01

Purpose To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated an adeno-associated virus type 8 vector (AAV2/8) encoding an siRNA targeting vascular endothelial growth factor (VEGF), and determined the AAV2/8 vector’s ability to inhibit angiogenesis. Methods We initially transfected 3T3 cells expressing VEGF with the AAV2/8 plasmid vector psiRNA-VEGF using the H1 promoter and found that VEGF expression was significantly diminished in the transfectants. We next injected 1 μl (3 × 1014 vg/ml) of AAV2/8 vector encoding siRNA targeting VEGF (AAV2/8/SmVEGF-2; n = 12) or control vector encoding green fluorescent protein (GFP) (AAV2/8/GFP; n = 14) into the subretinal space in C57BL/6 mice. One week later, CNV was induced by using a diode laser to make four separate choroidal burns around the optic nerve in each eye. After an additional 2 weeks, the eyes were removed for flat mount analysis of the CNV surface area. Results Subretinal delivery of AAV2/8/SmVEGF-2 significantly diminished CNV at the laser lesions, compared to AAV8/GFP (1597.3±2077.2 versus 5039.5±4055.9 µm2; p<0.05). Using an enzyme-linked immunosorbent assay, we found that VEGF levels were reduced by approximately half in the AAV2/8/SmVEGF-2 treated eyes. Conclusions These results suggest that siRNA-VEGF can be expressed across the retina and that long-term suppression of CNV is possible through the use of stable AAV2/8-mediated siRNA-VEGF expression. In vivo gene therapy may thus be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration. PMID:24744609

Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy.

PubMed

Zygmunt, Deborah A; Crowe, Kelly E; Flanigan, Kevin M; Martin, Paul T

2017-09-01

Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (<18 years old) had measurable rAAV titers to all serotypes tested, and this percentage increased to almost 50% in adult normal subjects (>18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences.

PubMed

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccas e 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like ( Sil1 ), scavenger receptor class C ( Src ), and scavenger receptor class B ( Srb3 and Srb4 ) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences

PubMed Central

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean. PMID:29773992

Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation.

PubMed

Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S; Naash, Muna I

2016-01-01

The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

PubMed Central

Strillacci, A; Griffoni, C; Spisni, E; Manara, M C; Tomasi, V

2006-01-01

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity. PMID:16622456

Early-life inflammation, immune response and ageing.

PubMed

Khan, Imroze; Agashe, Deepa; Rolff, Jens

2017-03-15

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response. © 2017 The Author(s).

RNAi-mediated disruption of neuropeptide genes, nlp-3 and nlp-12, cause multiple behavioral defects in Meloidogyne incognita.

PubMed

Dash, Manoranjan; Dutta, Tushar K; Phani, Victor; Papolu, Pradeep K; Shivakumara, Tagginahalli N; Rao, Uma

2017-08-26

Owing to the current deficiencies in chemical control options and unavailability of novel management strategies, root-knot nematode (M. incognita) infections remain widespread with significant socio-economic impacts. Helminth nervous systems are peptide-rich and appear to be putative drug targets that could be exploited by antihelmintic chemotherapy. Herein, to characterize the novel peptidergic neurotransmitters, in silico mining of M. incognita genomic and transciptomic datasets revealed the presence of 16 neuropeptide-like protein (nlp) genes with structural hallmarks of neuropeptide preproproteins; among which 13 nlps were PCR-amplified and sequenced. Two key nlp genes (Mi-nlp-3 and Mi-nlp-12) were localized to the basal bulb and tail region of nematode body via in situ hybridization assay. Mi-nlp-3 and Mi-nlp-12 were greatly expressed (in qRT-PCR assay) in the pre-parasitic juveniles and adult females, suggesting the association of these genes in host recognition, development and reproduction of M. incognita. In vitro knockdown of Mi-nlp-3 and Mi-nlp-12 via RNAi demonstrated the significant reduction in attraction and penetration of M. incognita in tomato root in Pluronic gel medium. A pronounced perturbation in development and reproduction of NLP-silenced worms was also documented in adzuki beans in CYG growth pouches. The deleterious phenotypes obtained due to NLP knockdown suggests that transgenic plants engineered to express RNA constructs targeting nlp genes may emerge as an environmentally viable option to manage nematode problems in crop plants. Copyright © 2017 Elsevier Inc. All rights reserved.

Early-life inflammation, immune response and ageing

PubMed Central

2017-01-01

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response. PMID:28275145

Convection-enhanced delivery of AAV2 in white matter--a novel method for gene delivery to cerebral cortex.

PubMed

Barua, N U; Woolley, M; Bienemann, A S; Johnson, D; Wyatt, M J; Irving, C; Lewis, O; Castrique, E; Gill, S S

2013-10-30

Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

PubMed Central

Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

2015-01-01

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

PubMed

Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

2014-10-30

For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

Life-span extension by dietary restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C. elegans.

PubMed

Park, Sang-Kyu; Link, Christopher D; Johnson, Thomas E

2010-02-01

Recent studies have shown that the rate of aging can be modulated by diverse interventions. Dietary restriction is the most widely used intervention to promote longevity; however, the mechanisms underlying the effect of dietary restriction remain elusive. In a previous study, we identified two novel genes, nlp-7 and cup-4, required for normal longevity in Caenorhabditis elegans. nlp-7 is one of a set of neuropeptide-like protein genes; cup-4 encodes an ion-channel involved in endocytosis by coelomocytes. Here, we assess whether nlp-7 and cup-4 mediate longevity increases by dietary restriction. RNAi of nlp-7 or cup-4 significantly reduces the life span of the eat-2 mutant, a genetic model of dietary restriction, but has no effect on the life span of long-lived mutants resulting from reduced insulin/IGF-1 signaling or dysfunction of the mitochondrial electron transport chain. The life-span extension observed in wild-type N2 worms by dietary restriction using bacterial dilution is prevented significantly in nlp-7 and cup-4 mutants. RNAi knockdown of genes encoding candidate receptors of NLP-7 and genes involved in endocytosis by coelomocytes also specifically shorten the life span of the eat-2 mutant. We conclude that two novel pathways, NLP-7 signaling and endocytosis by coelomocytes, are required for life extension under dietary restriction in C. elegans.

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

PubMed Central

Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

2015-01-01

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight.

PubMed

Benzon, C R; Johnson, S B; McCue, D L; Li, D; Green, T A; Hommel, J D

2014-01-31

Neuromedin U (NMU) is a highly conserved neuropeptide which regulates food intake and body weight. Transgenic mice lacking NMU are hyperphagic and obese, making NMU a novel target for understanding and treating obesity. Neuromedin U receptor 2 (NMUR2) is a high-affinity receptor for NMU found in discrete regions of the central nervous system, in particular the paraventricular nucleus of the hypothalamus (PVN), where it may be responsible for mediating the anorectic effects of NMU. We hypothesized that selective knock down of NMUR2 in the PVN of rats would increase their sensitivity to the reinforcing properties of food resulting in increased intake and preference for high-fat obesogenic food. To this end, we used viral-mediated RNAi to selectively knock down NMUR2 gene expression in the PVN. In rats fed a standard chow, NMUR2 knockdown produced no significant effect on food intake or body weight. However, when the same rats were fed a high-fat diet (45% fat), they consumed significantly more food, gained more body weight, and had increased feed efficiency relative to controls. Furthermore, NMUR2 knockdown rats demonstrated significantly greater binge-type food consumption of the high-fat diet and showed a greater preference for higher-fat food. These results demonstrate that NMUR2 signaling in the PVN regulates consumption and preference for high-fat foods without disrupting feeding behavior associated with non-obesogenic standard chow. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Adeno-associated virus-mediated gene transfer

PubMed Central

Srivastava, Arun

2008-01-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors in a murine serial bone marrow transplantation model in vivo, where stable integration of the proviral AAV genome does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. PMID:18500727

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

PubMed

Matsushima, Yuichi; Adán, Cristina; Garesse, Rafael; Kaguni, Laurie S

2005-04-29

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

High-Throughput Dissection of AAV-Host Interactions: The Fast and the Curious.

PubMed

Herrmann, Anne-Kathrin; Grimm, Dirk

2018-05-18

Over fifty years after its initial description, Adeno-associated virus (AAV) remains a most exciting but also most elusive study object in basic or applied virology. On the one hand, its simple structure not only facilitates investigations into virus biology, but combined with the availability of numerous natural AAV variants with distinct infection efficiency and specificity also makes AAV a preferred substrate for engineering of gene delivery vectors. On the other hand, it is striking to witness a recent flurry of reports that highlight and partially close persistent gaps in our understanding of AAV virus and vector biology. This is all the more perplexing considering that recombinant AAVs have already been used in >160 clinical trials and recently been commercialized as gene therapeutics. Here, we discuss a reason for these advances in AAV research, namely, the advent and application of powerful high-throughput technology for dissection of AAV-host interactions and optimization of AAV gene therapy vectors. As relevant examples, we focus on the discovery of (i) a "new" cellular AAV receptor, AAVR, (ii) host restriction factors for AAV entry, and (iii) AAV capsid determinants that mediate trafficking through the blood-brain barrier. While (i)/(ii) are prototypes of extra- or intracellular AAV host factors that were identified via high-throughput screenings, (iii) exemplifies the power of molecular evolution to investigate the virus itself. In the future, we anticipate that these and other key technologies will continue to accelerate the dissection of AAV biology and will yield a wealth of new designer viruses for clinical use. Copyright © 2018. Published by Elsevier Ltd.

Adeno-Associated Virus-Mediated Correction of a Canine Model of Glycogen Storage Disease Type Ia

PubMed Central

Weinstein, David A.; Correia, Catherine E.; Conlon, Thomas; Specht, Andrew; Verstegen, John; Onclin-Verstegen, Karine; Campbell-Thompson, Martha; Dhaliwal, Gurmeet; Mirian, Layla; Cossette, Holly; Falk, Darin J.; Germain, Sean; Clement, Nathalie; Porvasnik, Stacy; Fiske, Laurie; Struck, Maggie; Ramirez, Harvey E.; Jordan, Juan; Andrutis, Karl; Chou, Janice Y.; Byrne, Barry J.

2010-01-01

Abstract Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-α. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver. PMID:20163245

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice.

PubMed

Mallol, Cristina; Casana, Estefania; Jimenez, Veronica; Casellas, Alba; Haurigot, Virginia; Jambrina, Claudia; Sacristan, Victor; Morró, Meritxell; Agudo, Judith; Vilà, Laia; Bosch, Fatima

2017-07-01

Type 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. Transgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28-30 weeks. In transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. Local expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses ▿ †

PubMed Central

Grimm, Dirk; Lee, Joyce S.; Wang, Lora; Desai, Tushar; Akache, Bassel; Storm, Theresa A.; Kay, Mark A.

2008-01-01

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications. PMID:18400866

Long-term safety and efficacy of AAV gene therapy in the canine model of glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Conlon, Thomas J; Specht, Andrew; Coleman, Kirsten E; Brown, Laurie M; Estrella, Ana M; Dambska, Monika; Dahlberg, Kathryn R; Weinstein, David A

2018-05-25

Viral mediated gene therapy has progressed after overcoming early failures, and gene therapy has now been approved for several conditions in Europe and the USA. Glycogen storage disease (GSD) type Ia, caused by a deficiency of glucose-6-phosphatase-α, has been viewed as an outstanding candidate for gene therapy. This follow-up report describes the long-term outcome for the naturally occurring GSD-Ia dogs treated with rAAV-GPE-hG6PC-mediated gene therapy. A total of seven dogs were treated with rAAV-GPE-hG6PC-mediated gene therapy. The first four dogs were treated at birth, and three dogs were treated between 2 and 6 months of age to assess the efficacy and safety in animals with mature livers. Blood and urine samples, radiographic studies, histological evaluation, and biodistribution were assessed. Gene therapy improved survival in the GSD-Ia dogs. With treatment, the biochemical studies normalized for the duration of the study (up to 7 years). None of the rAAV-GPE-hG6PC-treated dogs had focal hepatic lesions or renal abnormalities. Dogs treated at birth required a second dose of rAAV after 2-4 months; gene therapy after hepatic maturation resulted in improved efficacy after a single dose. rAAV-GPE-hG6PC treatment in GSD-Ia dogs was found to be safe and efficacious. GSD-Ia is an attractive target for human gene therapy since it is a monogenic disorder with limited tissue involvement. Blood glucose and lactate monitoring can be used to assess effectiveness and as a biomarker of success. GSD-Ia can also serve as a model for other hepatic monogenic disorders.

Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats.

PubMed

Yu, Hongwei; Fischer, Gregory; Ferhatovic, Lejla; Fan, Fan; Light, Alan R; Weihrauch, Dorothee; Sapunar, Damir; Nakai, Hiroyuki; Park, Frank; Hogan, Quinn H

2013-01-01

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

PubMed Central

Hammond, Sean L.; Leek, Ashley N.; Richman, Evan H.

2017-01-01

The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression in vivo and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined in vivo at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson’s Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain regions in both astrocytes and neurons. PMID:29244806

Regulation of Endothelial Cell Inflammation and Lung PMN Infiltration by Transglutaminase 2

PubMed Central

Bijli, Kaiser M.; Kanter, Bryce G.; Minhajuddin, Mohammad; Leonard, Antony; Xu, Lei; Fazal, Fabeha; Rahman, Arshad

2014-01-01

We addressed the role of transglutaminase2 (TG2), a calcium-dependent enzyme that catalyzes crosslinking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung PMN infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor NF-κB and its target genes, VCAM-1, MCP-1, and IL-6. RNAi knockdown of TG2 inhibited these responses. Analysis of NF-κB activation pathway showed that TG2 knockdown was associated with inhibition of thrombin-induced DNA binding as well as serine phosphorylation of RelA/p65, a crucial event that controls transcriptional capacity of the DNA-bound RelA/p65. These results implicate an important role for TG2 in mediating EC inflammation by promoting DNA binding and transcriptional activity of RelA/p65. Because thrombin is released in high amounts during sepsis and its concentration is elevated in plasma and lavage fluids of patients with Acute Respiratory Distress Syndrome (ARDS), we determined the in vivo relevance of TG2 in a mouse model of sepsis-induced lung PMN recruitment. A marked reduction in NF-κB activation, adhesion molecule expression, and lung PMN sequestration was observed in TG2 knockout mice compared to wild type mice exposed to endotoxemia. Together, these results identify TG2 as an important mediator of EC inflammation and lung PMN sequestration associated with intravascular coagulation and sepsis. PMID:25057925

Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis

PubMed Central

Wandke, Cornelia; Barisic, Marin; Sigl, Reinhard; Rauch, Veronika; Wolf, Frank; Amaro, Ana C.; Tan, Chia H.; Pereira, Antonio J.; Kutay, Ulrike; Maiato, Helder; Meraldi, Patrick

2012-01-01

Chromokinesins are microtubule plus end–directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively. PMID:22945934

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2.

PubMed

Camargo, Carolina; Wu, Ke; Fishilevich, Elane; Narva, Kenneth E; Siegfried, Blair D

2018-06-01

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA Interference in Infectious Tropical Diseases

PubMed Central

Hong, Young S.

2008-01-01

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi. PMID:18344671

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

PubMed Central

Delgado Tascón, Julia; Nyffenegger-Jann, Naja J.; Hauck, Christof R.

2012-01-01

Background CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment. PMID:22448228

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

PubMed

Pils, Stefan; Kopp, Kathrin; Peterson, Lisa; Delgado Tascón, Julia; Nyffenegger-Jann, Naja J; Hauck, Christof R

2012-01-01

CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System1[OPEN

PubMed Central

Okazaki, Yozo; Lithio, Andrew; Jin, Huanan

2017-01-01

We report the characterization of the Arabidopsis (Arabidopsis thaliana) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism. PMID:28202596

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

PubMed Central

Foda, Bardees M.; Singh, Upinder

2015-01-01

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

PubMed

Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

2016-01-01

Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

Brain ACE2 overexpression reduces DOCA-salt hypertension independently of endoplasmic reticulum stress

PubMed Central

de Queiroz, Thyago Moreira; Sriramula, Srinivas; Feng, Yumei; Johnson, Tanya; Mungrue, Imran N.; Lazartigues, Eric

2014-01-01

Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress. PMID:25519733

Insecticidal potency of RNAi-based catalase knockdown in Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae).

PubMed

Al-Ayedh, Hassan; Rizwan-Ul-Haq, Muhammad; Hussain, Abid; Aljabr, Ahmed M

2016-11-01

Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

2011-08-05

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less

Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster.

PubMed

Byrne, Dwayne J; Harmon, Mark J; Simpson, Jeremy C; Blackstone, Craig; O'Sullivan, Niamh C

2017-10-20

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Assessing the potential for AAV vector genotoxicity in a murine model

PubMed Central

Li, Hojun; Malani, Nirav; Hamilton, Shari R.; Schlachterman, Alexander; Bussadori, Giulio; Edmonson, Shyrie E.; Shah, Rachel; Arruda, Valder R.; Mingozzi, Federico; Fraser Wright, J.; Bushman, Frederic D.

2011-01-01

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cysteine-rich regions. Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation. PMID:21106988

Adeno-associated virus-mediated gene transfer.

PubMed

Srivastava, Arun

2008-09-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

PubMed Central

Li, Chengwen; He, Yi; Nicolson, Sarah; Hirsch, Matt; Weinberg, Marc S.; Zhang, Ping; Kafri, Tal; Samulski, R. Jude

2013-01-01

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials. PMID:23454772

Assaying the Stability and Inactivation of AAV Serotype 1 Vectors

PubMed Central

Howard, Douglas B.; Harvey, Brandon K.

2017-01-01

Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze–thaw cycles, the resulting transduction efficiency became variable at 60–120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments. PMID:28192678

Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors

PubMed Central

Qi, Yi-xiang; Huang, Jia; Li, Meng-qi; Wu, Ya-su; Xia, Ren-ying; Ye, Gong-yin

2016-01-01

Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution. DOI: http://dx.doi.org/10.7554/eLife.12241.001 PMID:26974346

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments.

PubMed

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Yan; McGraw, John; Woods, Matthew; Murray, Stuart; Eckert, Amy; Liu, Wei; Ryan, Terence E

2011-06-01

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.

Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X-Linked Muscular Dystrophy and May Induce Immune Tolerance

PubMed Central

Hayashita-Kinoh, Hiromi; Yugeta, Naoko; Okada, Hironori; Nitahara-Kasahara, Yuko; Chiyo, Tomoko; Okada, Takashi; Takeda, Shin'ichi

2015-01-01

Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype. PMID:25586688

RNAi-Based GluN3A Silencing Prevents and Reverses Disease Phenotypes Induced by Mutant huntingtin.

PubMed

Marco, Sonia; Murillo, Alvaro; Pérez-Otaño, Isabel

2018-06-15

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. HD symptoms include severe motor, cognitive, and psychiatric impairments that result from dysfunction and later degeneration of medium-sized spiny neurons (MSNs) in the striatum. A key early pathogenic mechanism is dysregulated synaptic transmission due to enhanced surface expression of juvenile NMDA-type glutamate receptors containing GluN3A subunits, which trigger the aberrant pruning of synapses formed by cortical afferents onto MSNs. Here, we tested the therapeutic potential of silencing GluN3A expression in YAC128 mice, a well-established HD model. Recombinant adeno-associated viruses encoding a short-hairpin RNA against GluN3A (rAAV-shGluN3A) were generated, and the ability of different serotypes to transduce MSNs was compared. A single injection of rAAV9-shGluN3A into the striatum of 1-month-old mice drove potent (>90%) and long-lasting reductions of GluN3A expression in MSNs, prevented dendritic spine loss and improved motor performance in YAC128 mice. Later delivery, when spine pathology is already apparent, was also effective. Our data provide proof-of-concept for GluN3A silencing as a beneficial strategy to prevent or reverse corticostriatal disconnectivity and motor impairment in HD and support the use of RNAi-based or small-molecule approaches for harnessing this therapeutic potential. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

77 FR 69866 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-21

... for Diabetes and Obesity Description of Technology: This invention is directed to adeno- associated virus (AAV) vector delivery of exendin-4 (Ex-4) to salivary glands as treatment for diabetes and obesity... and weight profile in two rat models of obesity and type 2 diabetes. Further, AAV-mediated delivery of...

Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice

PubMed Central

Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.

2005-01-01

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817

AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension.

PubMed

Chen, Peter Gin-Fu; Sun, Zhongjie

2017-02-01

Cold temperatures are associated with increased prevalence of hypertension. Cold exposure increases endothelin-1 (ET1) production. The purpose of this study is to determine whether upregulation of ET1 contributes to cold-induced hypertension (CIH). In vivo RNAi silencing of the ET1 gene was achieved by adeno-associated virus 2 (AAV2) delivery of ET1 short-hairpin small interfering RNA (ET1-shRNA). Four groups of male rats were used. Three groups were given AAV.ET1-shRNA, AAV.SC-shRNA (scrambled shRNA), and phosphate-buffered saline (PBS), respectively, before exposure to a moderately cold environment (6.7 ± 2°C), while the last group was given PBS and kept at room temperature (warm, 24 ± 2°C) and served as a control. We found that systolic blood pressure of the PBS-treated and SC-shRNA-treated groups increased significantly within 2 weeks of exposure to cold, reached a peak level (145 ± 4.8 mmHg) by 6 weeks, and remained elevated thereafter. By contrast, blood pressure of the ET1-shRNA-treated group did not increase, suggesting that silencing of ET1 prevented the development of CIH. Animals were euthanized after 10 weeks of exposure to cold. Cold exposure significantly increased the left ventricle (LV) surface area and LV weight in cold-exposed rats, suggesting LV hypertrophy. Superoxide production in the heart was increased by cold exposure. Interestingly, ET1-shRNA prevented cold-induced superoxide production and cardiac hypertrophy. ELISA assay indicated that ET1-shRNA abolished the cold-induced upregulation of ET1 levels, indicating effective silencing of ET1. In conclusion, upregulation of ET1 plays a critical role in the pathogenesis of CIH and cardiac hypertrophy. AAV delivery of ET1-shRNA is an effective therapeutic strategy for cold-related cardiovascular disease.

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

PubMed Central

van der Sanden, Sabine M. G.; Wu, Weilin; Dybdahl-Sissoko, Naomi; Weldon, William C.; Brooks, Paula; O'Donnell, Jason; Jones, Les P.; Brown, Cedric; Tompkins, S. Mark; Karpilow, Jon; Tripp, Ralph A.

2015-01-01

ABSTRACT Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases. PMID:26581994

Differential contributions of Caenorhabditis elegans histone deacetylases to huntingtin polyglutamine toxicity.

PubMed

Bates, Emily A; Victor, Martin; Jones, Adriana K; Shi, Yang; Hart, Anne C

2006-03-08

Expansion of a polyglutamine tract in the huntingtin protein causes neuronal degeneration and death in Huntington's disease patients, but the molecular mechanisms underlying polyglutamine-mediated cell death remain unclear. Previous studies suggest that expanded polyglutamine tracts alter transcription by sequestering glutamine rich transcriptional regulatory proteins, thereby perturbing their function. We tested this hypothesis in Caenorhabditis elegans neurons expressing a human huntingtin fragment with an expanded polyglutamine tract (Htn-Q150). Loss of function alleles and RNA interference (RNAi) were used to examine contributions of C. elegans cAMP response element-binding protein (CREB), CREB binding protein (CBP), and histone deacetylases (HDACs) to polyglutamine-induced neurodegeneration. Deletion of CREB (crh-1) or loss of one copy of CBP (cbp-1) enhanced polyglutamine toxicity in C. elegans neurons. Loss of function alleles and RNAi were then used to systematically reduce function of each C. elegans HDAC. Generally, knockdown of individual C. elegans HDACs enhanced Htn-Q150 toxicity, but knockdown of C. elegans hda-3 suppressed toxicity. Neuronal expression of hda-3 restored Htn-Q150 toxicity and suggested that C. elegans HDAC3 (HDA-3) acts within neurons to promote degeneration in response to Htn-Q150. Genetic epistasis experiments suggested that HDA-3 and CRH-1 (C. elegans CREB homolog) directly oppose each other in regulating transcription of genes involved in polyglutamine toxicity. hda-3 loss of function failed to suppress increased neurodegeneration in hda-1/+;Htn-Q150 animals, indicating that HDA-1 and HDA-3 have different targets with opposing effects on polyglutamine toxicity. Our results suggest that polyglutamine expansions perturb transcription of CREB/CBP targets and that specific targeting of HDACs will be useful in reducing associated neurodegeneration.

Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

PubMed Central

Lock, Martin; Alvira, Mauricio R.

2012-01-01

Abstract Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium. PMID:22428980

Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues

PubMed Central

Li, Jia; Chen, Shu; Cleary, Rachel A.; Wang, Ruping; Gannon, Olivia J.; Seto, Edward

2014-01-01

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction. PMID:24920679

Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

PubMed

Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

2018-04-18

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector.

PubMed

Arnett, Andrea L H; Garikipati, Dilip; Wang, Zejing; Tapscott, Stephen; Chamberlain, Jeffrey S

2011-01-01

Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector

PubMed Central

Arnett, Andrea L. H.; Garikipati, Dilip; Wang, Zejing; Tapscott, Stephen; Chamberlain, Jeffrey S.

2011-01-01

Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice. PMID:22065964

Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.

PubMed

Bish, Lawrence T; Sleeper, Meg M; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E; Buchlis, George; Hui, Daniel; High, Katherine A; Gao, Guangping; Wilson, James M; Sweeney, H Lee

2011-08-01

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.

Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

PubMed Central

Sleeper, Meg M.; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E.; Buchlis, George; Hui, Daniel; High, Katherine A.; Gao, Guangping; Wilson, James M.; Sweeney, H. Lee

2011-01-01

Abstract Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways. PMID:21542669

Transduction of Nonhuman Primate Brain with Adeno-Associated Virus Serotype 1: Vector Trafficking and Immune Response

PubMed Central

Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L.; Bankiewicz, Krystof S.

2009-01-01

Abstract We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohisto-chemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm3 coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4+ lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2. PMID:19292604

The flipflop orphan genes are required for limb bud eversion in the Tribolium embryo.

PubMed

Thümecke, Susanne; Beermann, Anke; Klingler, Martin; Schröder, Reinhard

2017-01-01

Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood. Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2 , a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop -like RNAi-phenotype. The similarity of the inverted appendage phenotype in both the flipflop - and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.

A DEMETER-like DNA demethylase governs tomato fruit ripening.

PubMed

Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H; Maucourt, Mickael; Hodgman, T Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B; Giovannoni, James J; Gallusci, Philippe

2015-08-25

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

PubMed

Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

2017-01-01

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Micheva-Viteva, Sofiya N.; Shou, Yulin; Ganguly, Kumkum

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signalingmore » as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. As a result, identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.« less

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis

DOE PAGES

Micheva-Viteva, Sofiya N.; Shou, Yulin; Ganguly, Kumkum; ...

2017-06-07

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signalingmore » as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. As a result, identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.« less

Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

PubMed

Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

2012-03-01

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Improvement of lactic acid production in Saccharomyces cerevisiae by a deletion of ssb1.

PubMed

Lee, Jinsuk J; Crook, Nathan; Sun, Jie; Alper, Hal S

2016-01-01

Polylactic acid (PLA) is an important renewable polymer, but current processes for producing its precursor, lactic acid, suffer from process inefficiencies related to the use of bacterial hosts. Therefore, improving the capacity of Saccharomyces cerevisiae to produce lactic acid is a promising approach to improve industrial production of lactic acid. As one such improvement required, the lactic acid tolerance of yeast must be significantly increased. To enable improved tolerance, we employed an RNAi-mediated genome-wide expression knockdown approach as a means to rapidly identify potential genetic targets. In this approach, several gene knockdown targets were identified which confer increased acid tolerance to S. cerevisiae BY4741, of which knockdown of the ribosome-associated chaperone SSB1 conferred the highest increase (52%). This target was then transferred into a lactic acid-overproducing strain of S. cerevisiae CEN.PK in the form of a knockout and the resulting strain demonstrated up to 33% increased cell growth, 58% increased glucose consumption, and 60% increased L-lactic acid production. As SSB1 contains a close functional homolog SSB2 in yeast, this result was counterintuitive and may point to as-yet-undefined functional differences between SSB1 and SSB2 related to lactic acid production. The final strain produced over 50 g/L of lactic acid in under 60 h of fermentation.

Involvement of Bim in Photofrin-mediated photodynamically induced apoptosis.

PubMed

Wang, Xianwang; He, Xiaobing; Hu, Shujuan; Sun, Anbang; Lu, Chengbiao

2015-01-01

Photodynamic therapy (PDT) is a promising noninvasive technique, which has been successfully applied to the treatment of human cancers. Studies have shown that the Bcl-2 family proteins play important roles in PDT-induced apoptosis. However, whether Bcl-2-interacting mediator of cell death (Bim) is involved in photodynamic treatment remains unknown. In this study, we attempt to determine the effect of Bim on Photofrin photodynamic treatment (PPT)-induced apoptosis in human lung adenocarcinoma ASTC-a-1 cells. The translocation of Bim/Bax of the cells were monitored by laser confocal scanning microscope. The levels of Bim protein and activated caspase-3 in cells were detected by western blot assay. Caspase-3 activities were measured by Caspase-3 Fluorogenic Substrate (Ac-DEVD-AFC) analysis. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. The effect of Bim on PPT-induced apoptosis was determined by RNAi. BimL translocated to mitochondria in response to PPT, similar to the downstream pro-apoptotic protein Bax activation. PPT increased the level of Bim and activated caspase-3 in cells and that knockdown of Bim by RNAi significantly protected against caspase-3 activity. PPT-induced apoptosis were suppressed in cells transfected with shRNA-Bim. We demonstrated the involvement of Bim in PPT-induced apoptosis in human ASTC-a-1 lung adenocarcinoma cells and suggested that enhancing Bim activity might be a potential strategy for treating human cancers. © 2015 S. Karger AG, Basel.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

PubMed

Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

2018-03-16

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

Pleiotrophin suppression of receptor protein tyrosine phosphatase-β/ζ maintains the self-renewal competence of fetal human oligodendrocyte progenitor cells.

PubMed

McClain, Crystal R; Sim, Fraser J; Goldman, Steven A

2012-10-24

Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16-22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a(+)-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-Renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells

PubMed Central

McClain, Crystal R.; Sim, Fraser J.

2012-01-01

Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16–22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a+-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription. PMID:23100427

AAV Vectorization of DSB-mediated Gene Editing Technologies.

PubMed

Moser, Rachel J; Hirsch, Matthew L

2016-01-01

Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

Bacterial delivery of RNAi effectors: transkingdom RNAi.

PubMed

Lage, Hermann; Krühn, Andrea

2010-08-18

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter beta1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

PubMed

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo

PubMed Central

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells. PMID:29725339

Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia B

PubMed Central

Nathwani, Amit C.; Tuddenham, Edward G.D.; Rangarajan, Savita; Rosales, Cecilia; McIntosh, Jenny; Linch, David C.; Chowdary, Pratima; Riddell, Anne; Pie, Arnulfo Jaquilmac; Harrington, Chris; O’Beirne, James; Smith, Keith; Pasi, John; Glader, Bertil; Rustagi, Pradip; Ng, Catherine Y.C.; Kay, Mark A.; Zhou, Junfang; Spence, Yunyu; Morton, Christopher L.; Allay, James; Coleman, John; Sleep, Susan; Cunningham, John M.; Srivastava, Deokumar; Basner-Tschakarjan, Etiena; Mingozzi, Federico; High, Katherine A.; Gray, John T.; Reiss, Ulrike M.; Nienhuis, Arthur W.; Davidoff, Andrew M.

2012-01-01

BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8–pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.) PMID:22149959

Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans.

PubMed

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

2013-05-02

Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively.

A Molecular Toolbox for Rapid Generation of Viral Vectors to Up- or Down-Regulate Neuronal Gene Expression in vivo

PubMed Central

White, Melanie D.; Milne, Ruth V. J.; Nolan, Matthew F.

2011-01-01

We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins, and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV) or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1, and Kir3.2) and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miRNA). We show that AAV assembled to express HCN1 miRNA produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miRNA with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience. PMID:21772812

Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

PubMed

Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

2015-07-01

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. © 2015 by the Society for the Study of Reproduction, Inc.

Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice1

PubMed Central

Welborn, Joshua P.; Davis, Matthew G.; Ebers, Steven D.; Stodden, Genna R.; Hayashi, Kanako; Cheatwood, Joseph L.; Rao, Manjeet K.; MacLean, James A.

2015-01-01

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

Transgene expression in target-defined neuron populations mediated by retrograde infection with adeno-associated viral vectors.

PubMed

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta; Wachowiak, Matt

2013-09-18

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

PubMed Central

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta

2013-01-01

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors—in particular, recombinant adeno-associated viral vectors (rAAVs)—have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested—in particular, though not exclusively, Cre-dependent vectors—showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal. PMID:24048849

Dendritic Degeneration, Neurovascular Defects, and Inflammation Precede Neuronal Loss in a Mouse Model for Tau-Mediated Neurodegeneration

PubMed Central

Jaworski, Tomasz; Lechat, Benoit; Demedts, David; Gielis, Lies; Devijver, Herman; Borghgraef, Peter; Duimel, Hans; Verheyen, Fons; Kügler, Sebastian; Van Leuven, Fred

2011-01-01

Adeno-associated virus (AAV)–mediated expression of wild-type or mutant P301L protein tau produces massive degeneration of pyramidal neurons without protein tau aggregation. We probed this novel model for genetic and structural factors and early parameters of pyramidal neurodegeneration. In yellow fluorescent protein–expressing transgenic mice, intracerebral injection of AAV-tauP301L revealed early damage to apical dendrites of CA1 pyramidal neurons, whereas their somata remained normal. Ultrastructurally, more and enlarged autophagic vacuoles were contained in degenerating dendrites and manifested as dark, discontinuous, vacuolated processes surrounded by activated astrocytes. Dendritic spines were lost in AAV-tauP301L–injected yellow fluorescent protein–expressing transgenic mice, and ultrastructurally, spines appeared dark and degenerating. In CX3CR1EGFP/EGFP-deficient mice, microglia were recruited early to neurons expressing human tau. The inflammatory response was accompanied by extravasation of plasma immunoglobulins. α2-Macroglobulin, but neither albumin nor transferrin, became lodged in the brain parenchyma. Large proteins, but not Evans blue, entered the brain of mice injected with AAV-tauP301L. Ultrastructurally, brain capillaries were constricted and surrounded by swollen astrocytes with extensions that contacted degenerating dendrites and axons. Together, these data corroborate the hypothesis that neuroinflammation participates essentially in tau-mediated neurodegeneration, and the model recapitulates early dendritic defects reminiscent of “dendritic amputation” in Alzheimer's disease. PMID:21839061

Direct comparison of administration routes for AAV8-mediated ocular gene therapy.

PubMed

Igarashi, Tsutomu; Miyake, Koichi; Asakawa, Nagisa; Miyake, Noriko; Shimada, Takashi; Takahashi, Hiroshi

2013-05-01

We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration. The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) - AAV8/Luc and AAV8/GFP, respectively - were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging. One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 10(5 )photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery. Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.

Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

PubMed Central

Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

2013-01-01

Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

Postentry processing of recombinant adeno-associated virus type 1 and transduction of the ferret lung are altered by a factor in airway secretions.

PubMed

Yan, Ziying; Sun, Xingshen; Evans, Idil A; Tyler, Scott R; Song, Yi; Liu, Xiaoming; Sui, Hongshu; Engelhardt, John F

2013-09-01

We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.

Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability.

PubMed

Buhl, Edgar; Bradlaugh, Adam; Ogueta, Maite; Chen, Ko-Fan; Stanewsky, Ralf; Hodge, James J L

2016-11-22

We have characterized a light-input pathway regulating Drosophila clock neuron excitability. The molecular clock drives rhythmic electrical excitability of clock neurons, and we show that the recently discovered light-input factor Quasimodo (Qsm) regulates this variation, presumably via an Na + , K + , Cl - cotransporter (NKCC) and the Shaw K + channel (dK V 3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression ( OX ) and knockdown ( RNAi ) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsm OX led to a constitutive less active, night-like state, and qsm RNAi led to a more active, day-like state. Qsm also affected daily changes in K + currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of Shaw OX and NKCC RNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. We propose that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

PubMed

Kim, Eunmi; Oh, Ji-Seon; Ahn, Ik-Sung; Park, Kook In; Jang, Jae-Hyung

2011-11-01

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis).

PubMed

Zhao, Chaoyang; Alvarez Gonzales, Miguel A; Poland, Therese M; Mittapalli, Omprakash

2015-01-01

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula.

PubMed

Gao, Yan Ru; Huang, Wen Ling; Tang, Chun Lian; Liu, Rong; Zhao, Qin Ping; Ming, Zhen Ping; Dong, Hui Fen

2018-01-18

Schistosomiasis caused by Schistosoma japonicum is among the most serious endemic zoonoses in China. To study interactions between schistosomula, the pre-adult juvenile stage, and hosts, it is important to study the functions of key genes involved in schistosomula growth and development. Programmed cell death protein 10 (pcdp10) is an important apoptosis-related gene with various biological functions. This study described the molecular characterization of S. japonicum PCDP10 (SjPCDP10) and evaluated its functions in schistosomula. Real-time quantitative polymerase chain reaction (qPCR) and western blot were used to detect Sjpcdp10 mRNA and protein levels, respectively, at different developmental stages. Immunolocalization was performed to determine SjPCDP10 expression in the parasite. RNA interference (RNAi) experiments were used to assess gene functions associated with SjPCDP10 in schistosomula growth and development. Real-time qPCR revealed that Sjpcdp10 was expressed during all investigated developmental stages and upregulated during schistosomula growth and development. Histochemical localization showed that SjPCDP10 was mainly distributed in the teguments of schistosomula in all investigated stages and part of the parenchymal area of 14-, 18-, and 21-day-old schistosomula. Following Sjpcdp10 knockdown by RNAi, the lengths, widths, areas, and volumes of schistosomula were significantly lower than those in the control group. Scanning electron microscopy showed that the body surfaces of schistosomula subjected to RNAi were seriously damaged, with few tegumental spines and sensory papillae. Transmission electron microscopy indicated that the teguments of Sjpcdp10-knockdown schistosomula were incomplete, the number of layers was reduced, and the thickness decreased significantly as compared with those in the control group. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labelling results showed that the rate of apoptosis in Sjpcdp10-knockdown schistosomula was significantly higher than that in the control group. Sjpcdp10-knockdown influenced the growth and development of schistosomula. Therefore, our results indicated that SjPCDP10 contributes to the regulation of cell apoptosis and is essential for schistosomula growth and development.

A DEMETER-like DNA demethylase governs tomato fruit ripening

PubMed Central

Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H.; Maucourt, Mickael; Hodgman, T. Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B.; Giovannoni, James J.; Gallusci, Philippe

2015-01-01

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato. PMID:26261318

NHR-23 dependent collagen and hedgehog-related genes required for molting.

PubMed

Kouns, Nathaniel A; Nakielna, Johana; Behensky, Frantisek; Krause, Michael W; Kostrouch, Zdenek; Kostrouchova, Marta

2011-10-07

NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa. Copyright © 2011 Elsevier Inc. All rights reserved.

Systemic Gene Delivery Transduces the Enteric Nervous System of Guinea Pigs and Cynomolgus Macaques

PubMed Central

Gombash, Sara E; Cowley, Christopher J; Fitzgerald, Julie A; Lepak, Christina A; Neides, Mitchell G; Hook, Kathryn; Todd, Levi J; Wang, Guo-Du; Mueller, Christian; Kaspar, Brian K; Bielefeld, Eric C; Fischer, Andrew J; Wood, Jackie D; Foust, Kevin D

2017-01-01

Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or PBS. Piglets were euthanized three weeks post-injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9 treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders. PMID:28771235

Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

PubMed

Gombash, S E; Cowley, C J; Fitzgerald, J A; Lepak, C A; Neides, M G; Hook, K; Todd, L J; Wang, G-D; Mueller, C; Kaspar, B K; Bielefeld, E C; Fischer, A J; Wood, J D; Foust, K D

2017-10-01

Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

PubMed

Parzych, Elizabeth M; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M; High, Katherine A; Levine, Matthew H; Ertl, Hildegund C J

2013-04-01

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Effects of Immunosuppression on Circulating Adeno-Associated Virus Capsid-Specific T cells in Humans

PubMed Central

Parzych, Elizabeth M.; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M.; High, Katherine A.; Levine, Matthew H.

2013-01-01

Abstract In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4+ T cells, whereas numbers of circulating CD8+ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients. PMID:23461589

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

PubMed Central

Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

2015-01-01

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle.

PubMed

Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

2013-06-07

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.

Lentivirus mediated RNA interference of EMMPRIN (CD147) gene inhibits the proliferation, matrigel invasion and tumor formation of breast cancer cells.

PubMed

Yang, Jing; Wang, Rong; Li, Hongjiang; Lv, Qing; Meng, Wentong; Yang, Xiaoqin

2016-07-08

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) or cluster of differentiation 147 (CD147), a glycoprotein enriched on the plasma membrane of tumor cells, promotes proliferation, invasion, metastasis, and survival of malignant tumor cells. In this study, we sought to examine the expression of EMMPRIN in breast tumors, and to identify the potential roles of EMMPRIN on breast cancer cells. EMMPRIN expression in breast cancer tissues was assessed by immunohistochemistry. We used a lentivirus vector-based RNA interference (RNAi) approach expressing short hairpin RNA (shRNA) to knockdown EMMPRIN gene in breast cancer cell lines MDA-MB-231 and MCF-7. In vitro, Cell proliferative, invasive potential were determined by Cell Counting Kit (CCK-8), cell cycle analysis and matrigel invasion assay, respectively. In vivo, tumorigenicity was monitored by inoculating tumor cells into breast fat pad of female nude mice. EMMPRIN was over-expressed in breast tumors and breast cancer cell lines. Down-regulation of EMMPRIN by lentivirus vector-based RNAi led to decreased cell proliferative, decreased matrigel invasion in vitro, and attenuated tumor formation in vivo. High expression of EMMPRIN plays a crucial role in breast cancer cell proliferation, matrigel invasion and tumor formation.

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes.

PubMed

Shi, Ji-Feng; Xu, Qing-Yu; Sun, Qiang-Kun; Meng, Qing-Wei; Mu, Li-Li; Guo, Wen-Chao; Li, Guo-Qing

2016-10-01

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae. Copyright © 2016 Elsevier Ltd. All rights reserved.

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive failure in dogs with pacing-induced dilated cardiomyopathy.

PubMed

Pepe, Martino; Mamdani, Mohammed; Zentilin, Lorena; Csiszar, Anna; Qanud, Khaled; Zacchigna, Serena; Ungvari, Zoltan; Puligadda, Uday; Moimas, Silvia; Xu, Xiaobin; Edwards, John G; Hintze, Thomas H; Giacca, Mauro; Recchia, Fabio A

2010-06-25

Vascular endothelial growth factor (VEGF)-B selectively binds VEGF receptor (VEGFR)-1, a receptor that does not mediate angiogenesis, and is emerging as a major cytoprotective factor. To test the hypothesis that VEGF-B exerts non-angiogenesis-related cardioprotective effects in nonischemic dilated cardiomyopathy. AAV-9-carried VEGF-B(167) cDNA (10(12) genome copies) was injected into the myocardium of chronically instrumented dogs developing tachypacing-induced dilated cardiomyopathy. After 4 weeks of pacing, green fluorescent protein-transduced dogs (AAV-control, n=8) were in overt congestive heart failure, whereas the VEGF-B-transduced (AAV-VEGF-B, n=8) were still in a well-compensated state, with physiological arterial Po(2). Left ventricular (LV) end-diastolic pressure in AAV-VEGF-B and AAV-control was, respectively, 15.0+/-1.5 versus 26.7+/-1.8 mm Hg and LV regional fractional shortening was 9.4+/-1.6% versus 3.0+/-0.6% (all P<0.05). VEGF-B prevented LV wall thinning but did not induce cardiac hypertrophy and did not affect the density of alpha-smooth muscle actin-positive microvessels, whereas it normalized TUNEL-positive cardiomyocytes and caspase-9 and -3 activation. Consistently, activated Akt, a major negative regulator of apoptosis, was superphysiological in AAV-VEGF-B, whereas the proapoptotic intracellular mediators glycogen synthase kinase (GSK)-3beta and FoxO3a (Akt targets) were activated in AAV-control, but not in AAV-VEGF-B. Cardiac VEGFR-1 expression was reduced 4-fold in all paced dogs, suggesting that exogenous VEGF-B(167) exerted a compensatory receptor stimulation. The cytoprotective effects of VEGF-B(167) were further elucidated in cultured rat neonatal cardiomyocytes exposed to 10(-8) mol/L angiotensin II: VEGF-B(167) prevented oxidative stress, loss of mitochondrial membrane potential, and, consequently, apoptosis. We determined a novel, angiogenesis-unrelated cardioprotective effect of VEGF-B(167) in nonischemic dilated cardiomyopathy, which limits apoptotic cell loss and delays the progression toward failure.

Functions and substrates of NEDDylation during cell cycle in the silkworm, Bombyx mori.

PubMed

Li, Zhiqing; Cui, Qixin; Wang, Xiaoyan; Li, Bingqian; Zhao, Dongchao; Xia, Qingyou; Zhao, Ping

2017-11-01

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm. Copyright © 2017. Published by Elsevier Ltd.

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction*

PubMed Central

Wang, Ruping; Cleary, Rachel A.; Wang, Tao; Li, Jia; Tang, Dale D.

2014-01-01

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation. PMID:24700464

Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections

PubMed Central

Li, Wenfeng; Evans, Jay D.; Huang, Qiang; Rodríguez-García, Cristina; Liu, Jie; Hamilton, Michele; Grozinger, Christina M.; Webster, Thomas C.; Su, Songkun

2016-01-01

ABSTRACT Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. IMPORTANCE Given the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encoding nkd gene can suppress the reproduction of N. ceranae and improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices. PMID:27613683

Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections.

PubMed

Li, Wenfeng; Evans, Jay D; Huang, Qiang; Rodríguez-García, Cristina; Liu, Jie; Hamilton, Michele; Grozinger, Christina M; Webster, Thomas C; Su, Songkun; Chen, Yan Ping

2016-11-15

Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. Given the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encoding nkd gene can suppress the reproduction of N. ceranae and improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections.

PubMed

Robert, Marc-André; Nassoury, Nasha; Chahal, Parminder S; Venne, Marie-Hélène; Racine, Trina; Qiu, Xiangguo; Kobinger, Gary; Kamen, Amine; Gilbert, Rénald; Gaillet, Bruno

2018-04-01

Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 10 10 and 13.0 × 10 10 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 10 10 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.

Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy.

PubMed

Yi, Haiqing; Zhang, Quan; Brooks, Elizabeth D; Yang, Chunyu; Thurberg, Beth L; Kishnani, Priya S; Sun, Baodong

2017-03-01

Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1 ys/ys ). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1 ys/ys mice at a dose of 5 × 10 11 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1 ys/ys mice.

Adeno-Associated Viral Vector Serotype DJ-Mediated Overexpression of N171-82Q-Mutant Huntingtin in the Striatum of Juvenile Mice Is a New Model for Huntington's Disease.

PubMed

Jang, Minhee; Lee, Seung Eun; Cho, Ik-Hyun

2018-01-01

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. HD is caused by an expansion of CAG repeats in the huntingtin ( HTT ) gene in various areas of the brain including striatum. There are few suitable animal models to study the pathogenesis of HD and validate therapeutic strategies. Recombinant adeno-associated viral (AAV) vectors successfully transfer foreign genes to the brain of adult mammalians. In this article, we report a novel mouse model of HD generated by bilateral intrastriatal injection of AAV vector serotype DJ (AAV-DJ) containing N171-82Q mutant HTT (82Q) and N171-18Q wild type HTT (18Q; sham). The AAV-DJ-82Q model displayed motor dysfunctions in pole and rotarod tests beginning 4 weeks after viral infection in juvenile mice (8 weeks after birth). They showed behaviors reflecting neurodegeneration. They also showed increased apoptosis, robust glial activation and upregulated representative inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), mediators (cyclooxygenase-2 and inducible nitric oxide synthase) and signaling pathways (nuclear factor kappa B and signal transducer and activator of transcription 3 (STAT3)) in the striatum at 10 weeks after viral infection (14 weeks after birth) via successful transfection of mutant HTT into neurons, microglia, and astrocytes in the striatum. However, little evidence of any of these events was found in mice infected with the AAV-DJ-18Q expressing construct. Intrastriatal injection of AAV-DJ-82Q might be useful as a novel in vivo model to investigate the biology of truncated N-terminal fragment (N171) in the striatum and to explore the efficacy of therapeutic strategies for HD.

Knockdown of the Chromatin Remodeling Gene Brahma by RNA Interference Reduces Reproductive Fitness and Lifespan in Common Bed Bug (Hemiptera: Cimicidae).

PubMed

Basnet, Sanjay; Kamble, Shripat T

2018-05-04

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) is a nuisance household pest causing significant medical and economic impacts. RNA interference (RNAi) of genes that are involved in vital physiological processes can serve as potential RNAi targets for insect control. Brahma is an ATPase subunit of a chromatin-remodeling complex involved in transcription of several genes for cellular processes, most importantly the homeotic genes. In this study, we used a microinjection technique to deliver double stranded RNA into female bed bugs. Delivery of 0.05 and 0.5 µg/insect of brahma dsRNA directly into hemocele resulted substantial reduction in oviposition. Eggs laid by bed bugs receiving both doses of brahma dsRNA exhibited significantly lower hatching percentage as compared to controls. In addition, brahma RNAi in female bed bugs caused significant mortality. Our results disclosed the potential of brahma RNAi to suppress bed bug population through injection of specific dsRNA, suggesting a critical function of this gene in bed bugs' reproduction and survival. Based on our data, brahma can be a promising RNAi target for suppression of bed bug population.

Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella

PubMed Central

Hameed, Muhammad Salman; Wang, Zhengbing; Yang, Guang

2018-01-01

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella (PxAgo3) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella. PMID:29677157

Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella.

PubMed

Hameed, Muhammad Salman; Wang, Zhengbing; Vasseur, Liette; Yang, Guang

2018-04-20

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella ( PxAgo3 ) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella .

Homo sapiens Systemic RNA Interference-defective-1 Transmembrane Family Member 1 (SIDT1) Protein Mediates Contact-dependent Small RNA Transfer and MicroRNA-21-driven Chemoresistance*

PubMed Central

Elhassan, Mohamed O.; Christie, Jennifer; Duxbury, Mark S.

2012-01-01

Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents. PMID:22174421

Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae.

PubMed

Sigle, L T; Hillyer, J F

2018-03-09

Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. Here, we tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, we show that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems. © 2018 The Royal Entomological Society.

Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

PubMed Central

Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A.; Song, Juan

2016-01-01

Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design. PMID:27699236

Assessment of RNAi-induced silencing in banana (Musa spp.).

PubMed

Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

2014-09-18

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt). RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

Catalytic in vivo protein knockdown by small-molecule PROTACs

PubMed Central

Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

2015-01-01

The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

PubMed Central

Lang, Minglin; Braun, Caroline L.; Kanost, Michael R.; Gorman, Maureen J.

2012-01-01

Multicopper ferroxidases catalyze the oxidation of ferrous iron to ferric iron. In yeast and algae, they participate in cellular uptake of iron; in mammals, they facilitate cellular efflux. The mechanisms of iron metabolism in insects are still poorly understood, and insect multicopper ferroxidases have not been identified. In this paper, we present evidence that Drosophila melanogaster multicopper oxidase-1 (MCO1) is a functional ferroxidase. We identified candidate iron-binding residues in the MCO1 sequence and found that purified recombinant MCO1 oxidizes ferrous iron. An association between MCO1 function and iron homeostasis was confirmed by two observations: RNAi-mediated knockdown of MCO1 resulted in decreased iron accumulation in midguts and whole insects, and weak knockdown increased the longevity of flies fed a toxic concentration of iron. Strong knockdown of MCO1 resulted in pupal lethality, indicating that MCO1 is an essential gene. Immunohistochemistry experiments demonstrated that MCO1 is located on the basal surfaces of the digestive system and Malpighian tubules. We propose that MCO1 oxidizes ferrous iron in the hemolymph and that the resulting ferric iron is bound by transferrin or melanotransferrin, leading to iron storage, iron withholding from pathogens, regulation of oxidative stress, and/or epithelial maturation. These proposed functions are distinct from those of other known ferroxidases. Given that MCO1 orthologues are present in all insect genomes analyzed to date, this discovery is an important step toward understanding iron metabolism in insects. PMID:22847425

Effects of RNAi-mediated MUC4 gene silencing on the proliferation and migration of human pancreatic carcinoma BxPC-3 cells.

PubMed

Li, Yong; Wu, Changqiang; Chen, Tianwu; Zhang, Juanjuan; Liu, Gang; Pu, Yu; Zhu, Jiang; Shen, Chengyi; Zhang, Yang; Zeng, Nanlin; Zhang, Xiaoming

2016-12-01

It was previously demonstrated that mucin 4 (MUC4) is not expressed in normal pancreatic tissues or in chronic pancreatitis tissue but is highly expressed in pancreatic cancer (PC) tissue. Effective MUC4 gene knockdown in PC may contribute to the elucidation of pancreatic tumor development and metastasis, and may be valuable in new therapeutic approaches. Thus to confirm this, in the present study, the BxPC-3 cell line was transfected with eight pairs of shRNA lentiviral vectors for MUC4. The qPCR results showed that expression of MUC4 mRNA in the BxPC-3 cells was significantly decreased at 96 h after transfection. One of these shRNA lentiviral vectors (shRNA‑A141) had showed the strongest suppressive effect on MUC4 mRNA expression and was used for MUC4 knockdown in BxPC-3 cells. After stable transfection, BxPC-3 cells showed a significantly lower expression of MUC4 mRNA and MUC4 protein, and were suppressed on cell growth and migration. In vivo, lower tumor growth rates and tumor volume were observed in the tumors derived from the MUC4-knockdown cells, whereas the transplanted tumors derived from the control group cells, grew rapidly. Thus, inhibition of MUC4 expression may be an effective means for mitigating metastasis and invasion of PC.

IRS and TOR nutrient-signaling pathways act via juvenile hormone to influence honey bee caste fate.

PubMed

Mutti, Navdeep S; Dolezal, Adam G; Wolschin, Florian; Mutti, Jasdeep S; Gill, Kulvinder S; Amdam, Gro V

2011-12-01

Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, we suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. We discuss our results in the context of other recent findings on honey bee caste and development and propose that IRS is an alternative substrate for the Egfr (epidermal growth factor receptor) in honey bees. Overall, our study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes.

Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

PubMed

Jõers, Priit; Lewis, Samantha C; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J; Jacobs, Howard T

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

Mitochondrial Transcription Terminator Family Members mTTF and mTerf5 Have Opposing Roles in Coordination of mtDNA Synthesis

PubMed Central

Jõers, Priit; Lewis, Samantha C.; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J.; Jacobs, Howard T.

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis. PMID:24068965

IRS and TOR nutrient-signaling pathways act via juvenile hormone to influence honey bee caste fate

PubMed Central

Mutti, Navdeep S.; Dolezal, Adam G.; Wolschin, Florian; Mutti, Jasdeep S.; Gill, Kulvinder S.; Amdam, Gro V.

2011-01-01

SUMMARY Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, we suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. We discuss our results in the context of other recent findings on honey bee caste and development and propose that IRS is an alternative substrate for the Egfr (epidermal growth factor receptor) in honey bees. Overall, our study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes. PMID:22071189

Adeno-associated virus vector-mediated transduction in the cat brain.

PubMed

Vite, Charles H; Passini, Marco A; Haskins, Mark E; Wolfe, John H

2003-10-01

Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. In this study, we evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme beta-glucuronidase (GUSB) was used as a reporter gene, because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cortex, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of 8-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira

2012-01-06

Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors,more » therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.« less

Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

PubMed

Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

2016-01-01

Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

PubMed

Kyostio-Moore, Sirkka; Berthelette, Patricia; Cornell, Cathleen Sookdeo; Nambiar, Bindu; Figueiredo, Monica Dias

2018-05-01

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

RNA-Interference Pathways Display High Rates of Adaptive Protein Evolution in Multiple Invertebrates

PubMed Central

Palmer, William H.; Hadfield, Jarrod D.; Obbard, Darren J.

2018-01-01

Conflict between organisms can lead to a reciprocal adaptation that manifests as an increased evolutionary rate in genes mediating the conflict. This adaptive signature has been observed in RNA-interference (RNAi) pathway genes involved in the suppression of viruses and transposable elements in Drosophila melanogaster, suggesting that a subset of Drosophila RNAi genes may be locked in an arms race with these parasites. However, it is not known whether rapid evolution of RNAi genes is a general phenomenon across invertebrates, or which RNAi genes generally evolve adaptively. Here we use population genomic data from eight invertebrate species to infer rates of adaptive sequence evolution, and to test for past and ongoing selective sweeps in RNAi genes. We assess rates of adaptive protein evolution across species using a formal meta-analytic framework to combine data across species and by implementing a multispecies generalized linear mixed model of mutation counts. Across species, we find that RNAi genes display a greater rate of adaptive protein substitution than other genes, and that this is primarily mediated by positive selection acting on the genes most likely to defend against viruses and transposable elements. In contrast, evidence for recent selective sweeps is broadly spread across functional classes of RNAi genes and differs substantially among species. Finally, we identify genes that exhibit elevated adaptive evolution across the analyzed insect species, perhaps due to concurrent parasite-mediated arms races. PMID:29437826

Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

PubMed Central

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

2013-01-01

Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively. PMID:23640458

Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

PubMed

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell'Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

2008-03-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

PubMed Central

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell’Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

2010-01-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 × 1010 vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations. PMID:18209734

Adeno-associated virus vectors can be efficiently produced without helper virus.

PubMed

Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

1998-07-01

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Development of Intrathecal AAV9 Gene Therapy for Giant Axonal Neuropathy.

PubMed

Bailey, Rachel M; Armao, Diane; Nagabhushan Kalburgi, Sahana; Gray, Steven J

2018-06-15

An NIH-sponsored phase I clinical trial is underway to test a potential treatment for giant axonal neuropathy (GAN) using viral-mediated GAN gene replacement (https://clinicaltrials.gov/ct2/show/NCT02362438). This trial marks the first instance of intrathecal (IT) adeno-associated viral (AAV) gene transfer in humans. GAN is a rare pediatric neurodegenerative disorder caused by autosomal recessive loss-of-function mutations in the GAN gene, which encodes the gigaxonin protein. Gigaxonin is involved in the regulation, turnover, and degradation of intermediate filaments (IFs). The pathologic signature of GAN is giant axonal swellings filled with disorganized accumulations of IFs. Herein, we describe the development and characterization of the AAV vector carrying a normal copy of the human GAN transgene (AAV9/JeT-GAN) currently employed in the clinical trial. Treatment with AAV/JeT-GAN restored the normal configuration of IFs in patient fibroblasts within days in cell culture and by 4 weeks in GAN KO mice. IT delivery of AAV9/JeT-GAN in aged GAN KO mice preserved sciatic nerve ultrastructure, reduced neuronal IF accumulations and attenuated rotarod dysfunction. This strategy conferred sustained wild-type gigaxonin expression across the PNS and CNS for at least 1 year in mice. These results support the clinical evaluation of AAV9/JeT-GAN for potential therapeutic outcomes and treatment for GAN patients.

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

PubMed Central

Trubetskoy, Vladimir S.; Griffin, Jacob B.; Nicholas, Anthony L.; Nord, Eric M.; Xu, Zhao; Peterson, Ryan M.; Wooddell, Christine I.; Rozema, David B.; Wakefield, Darren H.; Lewis, David L.

2017-01-01

Abstract The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships. PMID:28180327

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.

PubMed

Grimm, Dirk; Wang, Lora; Lee, Joyce S; Schürmann, Nina; Gu, Shuo; Börner, Kathleen; Storm, Theresa A; Kay, Mark A

2010-09-01

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

PubMed

Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods.

Pathology Associated with AAV Mediated Expression of Beta Amyloid or C100 in Adult Mouse Hippocampus and Cerebellum

PubMed Central

Drummond, Eleanor S.; Muhling, Jill; Martins, Ralph N.; Wijaya, Linda K.; Ehlert, Erich M.; Harvey, Alan R.

2013-01-01

Accumulation of beta amyloid (Aβ) in the brain is a primary feature of Alzheimer’s disease (AD) but the exact molecular mechanisms by which Aβ exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Aβ40-GFP, Aβ42-GFP, C100-GFP or C100V717F-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Aβ protein after injection with AAV vectors, injection of rAAV2-Aβ42-GFP and rAAV2- C100V717F-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Aβ40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Aβ and C100 is a powerful technique with which to examine the direct in vivo consequences of Aβ expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse. PMID:23516609

Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis.

PubMed

Wen, Qing; Li, Nan; Xiao, Xiang; Lui, Wing-Yee; Chu, Darren S; Wong, Chris K C; Lian, Qingquan; Ge, Renshan; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

2018-02-12

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

Knockdown of a Rice Stelar Nitrate Transporter Alters Long-Distance Translocation But Not Root Influx1[W][OA

PubMed Central

Tang, Zhong; Fan, Xiaorong; Li, Qing; Feng, Huimin; Miller, Anthony J.; Shen, Qirong; Xu, Guohua

2012-01-01

Root nitrate uptake is well known to adjust to the plant’s nitrogen demand for growth. Long-distance transport and/or root storage pools are thought to provide negative feedback signals regulating root uptake. We have characterized a vascular specific nitrate transporter belonging to the high-affinity Nitrate Transporter2 (NRT2) family, OsNRT2.3a, in rice (Oryza sativa ssp. japonica ‘Nipponbare’). Localization analyses using protoplast expression, in planta promoter-β-glucuronidase assay, and in situ hybridization showed that OsNRT2.3a was located in the plasma membrane and mainly expressed in xylem parenchyma cells of the stele of nitrate-supplied roots. Knockdown expression of OsNRT2.3a by RNA interference (RNAi) had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mm) nitrate supply. In comparison with the wild type, the RNAi lines contained both nitrate and total nitrogen significantly higher in the roots and lower in the shoots. The short-term [15N]NO3− influx (5 min) in entire roots and NO3− fluxes in root surfaces showed that the knockdown of OsNRT2.3a in comparison with the wild type did not affect nitrate uptake by roots. The RNAi plants showed no significant changes in the expression of some root nitrate transporters (OsNRT2.3b, OsNRT2.4, and OsNAR2.1), but transcripts for nia1 (nitrate reductase) had increased and OsNRT2.1 and OsNRT2.2 had decreased when the plants were supplied with nitrate. Taken together, the data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice. PMID:23093362

Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase

PubMed Central

Puzzo, Francesco; Colella, Pasqualina; Biferi, Maria G.; Bali, Deeksha; Paulk, Nicole K.; Vidal, Patrice; Collaud, Fanny; Simon-Sola, Marcelo; Charles, Severine; Hardet, Romain; Leborgne, Christian; Meliani, Amine; Cohen-Tannoudji, Mathilde; Astord, Stephanie; Gjata, Bernard; Sellier, Pauline; van Wittenberghe, Laetitia; Vignaud, Alban; Boisgerault, Florence; Barkats, Martine; Laforet, Pascal; Kay, Mark A.; Koeberl, Dwight D.; Ronzitti, Giuseppe; Mingozzi, Federico

2018-01-01

Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa−/−) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa−/− mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector–mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease. PMID:29187643

Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

PubMed

Rey-Rico, Ana; Cucchiarini, Magali

2016-04-01

Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

PubMed Central

Whitford, Margaret H.; Arruda, Valder R.; Stedman, Hansell H.; Kay, Mark A.; High, Katherine A.

2015-01-01

Abstract Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

PubMed

Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

2015-03-01

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

The Neuron-Specific Protein TMEM59L Mediates Oxidative Stress-Induced Cell Death.

PubMed

Zheng, Qiuyang; Zheng, Xiaoyuan; Zhang, Lishan; Luo, Hong; Qian, Lingzhi; Fu, Xing; Liu, Yiqian; Gao, Yuehong; Niu, Mengxi; Meng, Jian; Zhang, Muxian; Bu, Guojun; Xu, Huaxi; Zhang, Yun-Wu

2017-08-01

TMEM59L is a newly identified brain-specific membrane-anchored protein with unknown functions. Herein we found that both TMEM59L and its homolog, TMEM59, are localized in Golgi and endosomes. However, in contrast to a ubiquitous and relatively stable temporal expression of TMEM59, TMEM59L expression was limited in neurons and increased during development. We also found that both TMEM59L and TMEM59 interacted with ATG5 and ATG16L1, and that overexpression of them triggered cell autophagy. However, overexpression of TMEM59L induced intrinsic caspase-dependent apoptosis more dramatically than TMEM59. In addition, downregulation of TMEM59L prevented neuronal cell death and caspase-3 activation caused by hydrogen peroxide insults and reduced the lipidation of LC3B. Finally, we found that AAV-mediated knockdown of TMEM59L in mice significantly ameliorated caspase-3 activation, increased mouse duration in the open arm during elevated plus maze test, reduced mouse immobility time during forced swim test, and enhanced mouse memory during Y-maze and Morris water maze tests. Together, our study indicates that TMEM59L is a pro-apoptotic neuronal protein involved in animal behaviors such as anxiety, depression, and memory, and that TMEM59L downregulation protects neurons against oxidative stress.

DNA end-processing enzyme polynucleotide kinase as a potential target in the treatment of cancer.

PubMed

Allinson, Sarah L

2010-06-01

Pharmacological inhibition of DNA-repair pathways as an approach for the potentiation of chemo- and radio-therapeutic cancer treatments has attracted increasing levels of interest in recent years. Inhibitors of several enzymes involved in the repair of DNA strand breaks are currently at various stages of the drug development process. Polynucleotide kinase (PNK), a bifunctional DNA-repair enzyme that possesses both 3'-phosphatase and 5'-kinase activities, plays an important role in the repair of both single strand and double strand breaks and as a result, RNAi-mediated knockdown of PNK sensitizes cells to a range of DNA-damaging agents. Recently, a small molecule inhibitor of PNK has been developed that is able to sensitize cells to ionizing radiation and the topoisomerase I poison, camptothecin. Although still in the early stages of development, PNK inhibition represents a promising means of enhancing the efficacy of existing cancer treatments.

Charting organellar importomes by quantitative mass spectrometry

PubMed Central

Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina

2017-01-01

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles. PMID:28485388

Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Aβ42-induced neurodegeneration

PubMed Central

Lin, Ran; Angelin, Alessia; Da Settimo, Federico; Martini, Claudia; Taliani, Sabrina; Zhu, Shigong; Wallace, Douglas C

2014-01-01

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target. PMID:24977274

BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori.

PubMed

Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

2016-02-15

Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of human olfactory receptor 10J5 in heart aorta, coronary artery, and endothelial cells and its functional role in angiogenesis.

PubMed

Kim, Sung-Hee; Yoon, Yeo Cho; Lee, Ae Sin; Kang, NaNa; Koo, JaeHyung; Rhyu, Mee-Ra; Park, Jae-Ho

2015-05-01

ORs are ectopically expressed in non-chemosensory tissues including muscle, kidney, and keratinocytes; however, their physiological roles are largely unknown. We found that human olfactory receptor 10J5 (OR10J5) is expressed in the human aorta, coronary artery, and umbilical vein endothelial cells (HUVEC). Lyral induces Ca(2+) and phosphorylation of AKT in HUVEC. A knockdown study showed the inhibition of the lyral-induced Ca(2+) and the phosphorylation AKT and implied that these processes are mediated by OR10J5. In addition, lyral enhanced migration of HUVEC, which were also inhibited by RNAi in a migration assay. In addition, matrigel plug assay showed that lyral enhanced angiogenesis in vivo. Together these data demonstrate the physiological role of OR10J5 in angiogenesis and represent roles of ORs in HUVEC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

RNAi and retroviruses: are they in RISC?

PubMed

Vasselon, Thierry; Bouttier, Manuella; Saumet, Anne; Lecellier, Charles-Henri

2013-02-01

RNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.

Functional kinomics identifies candidate therapeutic targets in head and neck cancer

PubMed Central

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo

2014-01-01

Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259

Functional kinomics identifies candidate therapeutic targets in head and neck cancer.

PubMed

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo

2014-08-15

To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

PubMed Central

2014-01-01

Background Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. Methods To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. Results Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. Conclusions Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma. PMID:24661910

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding Yun; Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37208; Zhang Li

2008-02-01

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, therebymore » suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.« less

Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.

PubMed

Gotoh, Hiroki; Zinna, Robert A; Warren, Ian; DeNieu, Michael; Niimi, Teruyuki; Dworkin, Ian; Emlen, Douglas J; Miura, Toru; Lavine, Laura C

2016-03-22

Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females. We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes--Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD. Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.

Phase I/II Trial of Adeno-Associated Virus–Mediated Alpha-Glucosidase Gene Therapy to the Diaphragm for Chronic Respiratory Failure in Pompe Disease: Initial Safety and Ventilatory Outcomes

PubMed Central

Smith, Barbara K.; Collins, Shelley W.; Conlon, Thomas J.; Mah, Cathryn S.; Lawson, Lee Ann; Martin, Anatole D.; Fuller, David D.; Cleaver, Brian D.; Clément, Nathalie; Phillips, Dawn; Islam, Saleem; Dobjia, Nicole

2013-01-01

Abstract Pompe disease is an inherited neuromuscular disease caused by deficiency of lysosomal acid alpha-glucosidase (GAA) leading to glycogen accumulation in muscle and motoneurons. Cardiopulmonary failure in infancy leads to early mortality, and GAA enzyme replacement therapy (ERT) results in improved survival, reduction of cardiac hypertrophy, and developmental gains. However, many children have progressive ventilatory insufficiency and need additional support. Preclinical work shows that gene transfer restores phrenic neural activity and corrects ventilatory deficits. Here we present 180-day safety and ventilatory outcomes for five ventilator-dependent children in a phase I/II clinical trial of AAV-mediated GAA gene therapy (rAAV1-hGAA) following intradiaphragmatic delivery. We assessed whether rAAV1-hGAA results in acceptable safety outcomes and detectable functional changes, using general safety measures, immunological studies, and pulmonary functional testing. All subjects required chronic, full-time mechanical ventilation because of respiratory failure that was unresponsive to both ERT and preoperative muscle-conditioning exercises. After receiving a dose of either 1×1012 vg (n=3) or 5×1012 vg (n=2) of rAAV1-hGAA, the subjects' unassisted tidal volume was significantly larger (median [interquartile range] 28.8% increase [15.2–35.2], p<0.05). Further, most patients tolerated appreciably longer periods of unassisted breathing (425% increase [103–851], p=0.08). Gene transfer did not improve maximal inspiratory pressure. Expected levels of circulating antibodies and no T-cell-mediated immune responses to the vector (capsids) were observed. One subject demonstrated a slight increase in anti-GAA antibody that was not considered clinically significant. These results indicate that rAAV1-hGAA was safe and may lead to modest improvements in volitional ventilatory performance measures. Evaluation of the next five patients will determine whether earlier intervention can further enhance the functional benefit. PMID:23570273

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game.

PubMed

van der Sanden, Sabine M G; Wu, Weilin; Dybdahl-Sissoko, Naomi; Weldon, William C; Brooks, Paula; O'Donnell, Jason; Jones, Les P; Brown, Cedric; Tompkins, S Mark; Oberste, M Steven; Karpilow, Jon; Tripp, Ralph A

2016-02-15

Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles

PubMed Central

Agrawal, Parul

2016-01-01

In Drosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns of Drosophila phosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons when Lar expression is knocked down during development, but not in adults. Loss of Lar function eliminates sLNv dorsal projections, but PDF expression persists in sLNv and large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF, Lar RNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate that Lar is required for sLNv dorsal projection development and suggest that PDF expression in LNv cell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle. SIGNIFICANCE STATEMENT In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather, Lar disrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lack Lar anticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles. PMID:27030770

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect

PubMed Central

Beermann, Anke; Prühs, Romy; Lutz, Rebekka; Schröder, Reinhard

2011-01-01

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. We analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). We show that simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. We identify Tc-Arr as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation. We discuss how Wnt signalling is regulated at the receptor and co-receptor levels in a dose-dependent fashion. PMID:21652652

MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

PubMed

Doose, Gero; Haake, Andrea; Bernhart, Stephan H; López, Cristina; Duggimpudi, Sujitha; Wojciech, Franziska; Bergmann, Anke K; Borkhardt, Arndt; Burkhardt, Birgit; Claviez, Alexander; Dimitrova, Lora; Haas, Siegfried; Hoell, Jessica I; Hummel, Michael; Karsch, Dennis; Klapper, Wolfram; Kleo, Karsten; Kretzmer, Helene; Kreuz, Markus; Küppers, Ralf; Lawerenz, Chris; Lenze, Dido; Loeffler, Markus; Mantovani-Löffler, Luisa; Möller, Peter; Ott, German; Richter, Julia; Rohde, Marius; Rosenstiel, Philip; Rosenwald, Andreas; Schilhabel, Markus; Schneider, Markus; Scholz, Ingrid; Stilgenbauer, Stephan; Stunnenberg, Hendrik G; Szczepanowski, Monika; Trümper, Lorenz; Weniger, Marc A; Hoffmann, Steve; Siebert, Reiner; Iaccarino, Ingram

2015-09-22

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

RNAi control of aflatoxins in peanut plants, a multifactorial system

USDA-ARS?s Scientific Manuscript database

RNA-interference (RNAi)-mediated control of aflatoxin contamination in peanut plants is a multifactorial and hyper variable system. The use of RNAi biotechnology to silence single genes in plants has inherently high-variability among transgenic events. Also the level of expression of small interfe...

RNA interference by feeding in vitro synthesized double-stranded RNA to planarians: methodology and dynamics

PubMed Central

Rouhana, Labib; Weiss, Jennifer A.; Forsthoefel, David J.; Lee, Hayoung; King, Ryan S.; Inoue, Takeshi; Shibata, Norito; Agata, Kiyokazu; Newmark, Phillip A.

2013-01-01

Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time, and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. PMID:23441014

Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis

PubMed Central

Fishbein, Ilia; Guerrero, David T.; Alferiev, Ivan S.; Foster, Jonathan B.; Minutolo, Nicholas G.; Chorny, Michael; Mas Monteys, Alejandro; Driesbaugh, Kathryn H.; Nagaswami, Chandrasekaran; Levy, Robert J.

2017-01-01

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty. PMID:28832561

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes

PubMed Central

Ram, Rashmi; Wescott, Andrew P.; Varandas, Katherine; Dirksen, Robert T.

2013-01-01

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1. PMID:24186093

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

PubMed

Ram, Rashmi; Wescott, Andrew P; Varandas, Katherine; Dirksen, Robert T; Blaxall, Burns C

2014-01-01

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.

Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system.

PubMed

Liu, Wendy W; Wilson, Rachel I

2013-06-18

Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. In this study, we investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, we found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition.

RNAi knockdown of acetyl-CoA gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparfata lugens

USDA-ARS?s Scientific Manuscript database

A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reprod...

A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro Dystrophin Vector

DTIC Science & Technology

2017-09-01

future experimental therapeutic studies in the canine model such as CRISPR -mediated gene editing, stem cell therapy, dystrophin-independent disease...There is no scientific/budget overlap with the current proposal.) CRISPR /Cas9-based gene editing for the correction of Duchenne muscular dystrophy...lab will perform in vivo gene delivery and functional outcome measurements in mice treated by AAV- CRISPR gene repair vectors and if needed will also

RNAi mediated, stable resistance to Triticum mosaic virus in wheat

USDA-ARS?s Scientific Manuscript database

Triticum mosaic virus (TriMV), discovered in 2006, affects wheat production systems in the Great Plains of the United States. There are no available TriMV resistant commercial varieties. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA...

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

PubMed Central

Maczuga, Piotr; Lubelski, Jacek; van Logtenstein, Richard; Borel, Florie; Blits, Bas; Fakkert, Erwin; Costessi, Adalberto; Butler, Derek; van Deventer, Sander; Petry, Harald; Koornneef, Annemart; Konstantinova, Pavlina

2013-01-01

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics. PMID:23089734

Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

PubMed

Li, Hua; Zhang, Feng-Lan; Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

2015-01-01

The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

PubMed

Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

2012-05-01

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

Possible role of PAPR-1 in protecting human HaCaT cells against cytotoxicity of SiO2 nanoparticles.

PubMed

Gong, Chunmei; Yang, Linqing; Zhou, Jichang; Guo, Xiang; Zhuang, Zhixiong

2017-10-05

Nano-SiO 2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO 2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO 2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO 2 induced cytotoxicity. We found that nano-SiO 2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO 2 -treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO 2 treatment. Nano-SiO 2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO 2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO 2 . Copyright © 2017 Elsevier B.V. All rights reserved.

The association of cortactin with profilin-1 is critical for smooth muscle contraction.

PubMed

Wang, Ruping; Cleary, Rachel A; Wang, Tao; Li, Jia; Tang, Dale D

2014-05-16

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Adeno-Associated Viral Vector Serotype DJ-Mediated Overexpression of N171-82Q-Mutant Huntingtin in the Striatum of Juvenile Mice Is a New Model for Huntington’s Disease

PubMed Central

Jang, Minhee; Lee, Seung Eun; Cho, Ik-Hyun

2018-01-01

Huntington’s disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. HD is caused by an expansion of CAG repeats in the huntingtin (HTT) gene in various areas of the brain including striatum. There are few suitable animal models to study the pathogenesis of HD and validate therapeutic strategies. Recombinant adeno-associated viral (AAV) vectors successfully transfer foreign genes to the brain of adult mammalians. In this article, we report a novel mouse model of HD generated by bilateral intrastriatal injection of AAV vector serotype DJ (AAV-DJ) containing N171-82Q mutant HTT (82Q) and N171-18Q wild type HTT (18Q; sham). The AAV-DJ-82Q model displayed motor dysfunctions in pole and rotarod tests beginning 4 weeks after viral infection in juvenile mice (8 weeks after birth). They showed behaviors reflecting neurodegeneration. They also showed increased apoptosis, robust glial activation and upregulated representative inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), mediators (cyclooxygenase-2 and inducible nitric oxide synthase) and signaling pathways (nuclear factor kappa B and signal transducer and activator of transcription 3 (STAT3)) in the striatum at 10 weeks after viral infection (14 weeks after birth) via successful transfection of mutant HTT into neurons, microglia, and astrocytes in the striatum. However, little evidence of any of these events was found in mice infected with the AAV-DJ-18Q expressing construct. Intrastriatal injection of AAV-DJ-82Q might be useful as a novel in vivo model to investigate the biology of truncated N-terminal fragment (N171) in the striatum and to explore the efficacy of therapeutic strategies for HD. PMID:29946240

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

PubMed

Cunningham, Sharon C; Siew, Susan M; Hallwirth, Claus V; Bolitho, Christine; Sasaki, Natsuki; Garg, Gagan; Michael, Iacovos P; Hetherington, Nicola A; Carpenter, Kevin; de Alencastro, Gustavo; Nagy, Andras; Alexander, Ian E

2015-08-01

Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. © 2015 by the American Association for the Study of Liver Diseases.

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

PubMed Central

Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

2016-01-01

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

PubMed

Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

2015-10-01

The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

Establishment of Functional Genomics Pipeline in Mouse Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

PubMed

Takata, Nozomu; Sakakura, Eriko; Kasukawa, Takeya; Sakuma, Tetsushi; Yamamoto, Takashi; Sasai, Yoshiki

2016-06-01

The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of mouse embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics.

Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences.

PubMed

Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N; Bolli, Roberto

2011-11-01

Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction").

[GPER silence inhibits the stimulation of growth and inhibition of apoptosis induced by tamoxifen in breast cancer-associated fibroblasts].

PubMed

Yan, Yuzhao; Yu, Tenghua; Tu, Gang; Liu, Manran; Yuan, Jie; Yang, Guanglun

2015-09-01

To construct a lentiviral vector (Lenti-GPER-shRNA) targeting G-protein coupled estrogen receptor (GPER) and explore the role of GPER in the effect of tamoxifen on cell proliferation and apoptosis in breast cancer associated fibroblasts (BCAFs). The target sequence of GPER gene and negative control were cloned into lentiviral vectors. The recombinant lentivirus and control were extracted after HEK293T cells were transfected with the recombinant vector and helper vectors. After infection of BCAFs with the GPER lentiviral vector under the best interfering condition, GPER expression was detected by real-time quantitative PCR and Western blotting. BCAFs were divided into negative control group, GPER-RNAi group, negative control combined with tamoxifen (10(-8) mmol/L) group and GPER-RNAi combined with tamoxifen (10(-8) mmol/L) group. CCK-8 assay was used to detect the proliferation and annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) combined with flow cytometry was used to detect the apoptosis of BCAFs after the treatment of tamoxifen. Lenti-GPER-shRNA significantly interfered the expression of GPER in BCAFs. Tamoxifen promoted the growth of BCAFs, which could be attenuated by knockdown of GPER. Moreover, the apoptosis of BCAFs was reduced by tamoxifen, which was also reversed by knockdown of GPER. Lenti-GPER-shRNA could effectively silence the GPER expression in BCAFs. The ability of tamoxifen to accelerate cell proliferation and decrease cell apoptosis could be weakened by knockdown of GPER.

Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

PubMed

Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

2014-05-16

Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals.

PubMed

Qiu, Yang; Xu, Yanpeng; Zhang, Yao; Zhou, Hui; Deng, Yong-Qiang; Li, Xiao-Feng; Miao, Meng; Zhang, Qiang; Zhong, Bo; Hu, Yuanyang; Zhang, Fu-Chun; Wu, Ligang; Qin, Cheng-Feng; Zhou, Xi

2017-06-20

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

Suppression of HLA Expression by Lentivirus-mediated Gene Transfer of siRNA Cassettes and In Vivo Chemoselection to Enhance Hematopoietic Stem Cell Transplantation

PubMed Central

Hacke, Katrin; Falahati, Rustom; Flebbe-Rehwaldt, Linda; Kasahara, Noriyuki; Gaensler, Karin M. L.

2010-01-01

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts. PMID:19048410

[Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro].

PubMed

Wang, Kai; Peng, Jian-Qiang; Chen, Fang-Ping; Wu, Xiao-Bin

2006-04-01

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

Prevention of autoimmune diabetes and islet allograft rejection by beta cell expression of XIAP: Insight into possible mechanisms of local immunomodulation.

PubMed

Obach, Mercè; Hosseini-Tabatabaei, Azadeh; Montane, Joel; Wind, Katarina; Soukhatcheva, Galina; Dai, Derek; Priatel, John J; Orban, Paul C; Verchere, C Bruce

2018-06-05

Overexpression of the X-linked inhibitor of apoptosis (XIAP) prevents islet allograft rejection. We constructed an adeno-associated virus expressing XIAP driven by the rat insulin promoter (dsAAV8-RIP-XIAP) for long-term beta-cell gene expression in vivo. Pancreatic delivery of dsAAV8-RIP-XIAP prevented autoimmune diabetes in 70% of non-obese diabetic (NOD) mice, associated with decreased insulitis. Islets from Balb/c mice transduced with dsAAV8-RIP-XIAP were protected following transplantation into streptozotocin (STZ)-diabetic Bl/6 recipients, associated with decreased graft infiltration. Interestingly, dsAAV8-RIP-XIAP transduction induced expression of lactate dehydrogenase (LDHA) and monocarboxylate transporter 1 (MCT1), two genes normally suppressed in beta cells and involved in production and release of lactate, a metabolite known to suppress local immune responses. Transduction of Balb/c islets with AAV8-RIP-LDHA-MCT1 tended to prolong allograft survival following transplant into STZ-diabetic Bl/6 recipients. These findings suggest that XIAP has therapeutic potential in autoimmune diabetes and raise the possibility that local lactate production may play a role in XIAP-mediated immunomodulation. Copyright © 2018. Published by Elsevier B.V.

AAV9-mediated engineering of autotransplanted kidney of non-human primates.

PubMed

Tomasoni, S; Trionfini, P; Azzollini, N; Zentilin, L; Giacca, M; Aiello, S; Longaretti, L; Cozzi, E; Baldan, N; Remuzzi, G; Benigni, A

2017-05-01

Ex vivo gene transfer to the graft before transplantation is an attractive option for circumventing systemic side effects of chronic antirejection therapy. Gene delivery of the immunomodulatory protein cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) prevented chronic kidney rejection in a rat model of allotransplantation without the need for systemic immunosuppression. Here we generated adeno-associated virus type 2 (AAV2) and AAV9 vectors encoding for LEA29Y, an optimized version of CTLA4-Ig. Both LEA29Y vectors were equally efficient for reducing T-cell proliferation in vitro. Serotype 9 was chosen for in vivo experiments owing to a lower frequency of preformed antibodies against the AAV9 capsid in 16 non-human primate tested sera. AAV9-LEA29Y was able to transduce the kidney of non-human primates in an autotransplantation model. Expression of LEA29Y mRNA by renal cells translated into the production of the corresponding protein, which was confined to the graft but not detected in serum. Results in non-human primates represent a step forward in maintaining the portability of this strategy into clinics.

A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer's disease.

PubMed

Rafii, Michael S; Baumann, Tiffany L; Bakay, Roy A E; Ostrove, Jeffrey M; Siffert, Joao; Fleisher, Adam S; Herzog, Christopher D; Barba, David; Pay, Mary; Salmon, David P; Chu, Yaping; Kordower, Jeffrey H; Bishop, Kathie; Keator, David; Potkin, Steven; Bartus, Raymond T

2014-09-01

Nerve growth factor (NGF) is an endogenous neurotrophic-factor protein with the potential to restore function and to protect degenerating cholinergic neurons in Alzheimer's disease (AD), but safe and effective delivery has proved unsuccessful. Gene transfer, combined with stereotactic surgery, offers a potential means to solve the long-standing delivery obstacles. An open-label clinical trial evaluated the safety and tolerability, and initial efficacy of three ascending doses of the genetically engineered gene-therapy vector adeno-associated virus serotype 2 delivering NGF (AAV2-NGF [CERE-110]). Ten subjects with AD received bilateral AAV2-NGF stereotactically into the nucleus basalis of Meynert. AAV2-NGF was safe and well-tolerated for 2 years. Positron emission tomographic imaging and neuropsychological testing showed no evidence of accelerated decline. Brain autopsy tissue confirmed long-term, targeted, gene-mediated NGF expression and bioactivity. This trial provides important evidence that bilateral stereotactic administration of AAV2-NGF to the nucleus basalis of Meynert is feasible, well-tolerated, and able to produce long-term, biologically active NGF expression, supporting the initiation of an ongoing multicenter, double-blind, sham-surgery-controlled trial. Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

The Skeletal Muscle Environment and Its Role in Immunity and Tolerance to AAV Vector-Mediated Gene Transfer

PubMed Central

Boisgérault, Florence; Mingozzi, Federico

2015-01-01

Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors. PMID:26122097

Isolating of Target Genes for NKX3.1 in Prostate Carcinogenesis

DTIC Science & Technology

2005-03-01

stainin on e te o tesr prostate hist log .(Band - . (C a .. ( 2 (M -P) Ki6 s shows . celula pr lf rto in c( ssi Fg1.prosthate cane r progresin is enh...prostate tumorigenicity. s{ LA Finally, to assess the significance of Cyclin D1 for prostatea ,. 0 tumorigenicity, we used RNAi to knock-down its expression

Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm.

PubMed

Davis-Vogel, Courtney; Ortiz, Angel; Procyk, Lisa; Robeson, Jonathan; Kassa, Adane; Wang, Yiwei; Huang, Emily; Walker, Carl; Sethi, Amit; Nelson, Mark E; Sashital, Dipali G

2018-05-18

Western corn rootworm (Diabrotica virgifera virgifera) is a serious agricultural pest known for its high adaptability to various management strategies, giving rise to a continual need for new control options. Transgenic maize expressing insecticidal RNAs represents a novel mode of action for rootworm management that is dependent on the RNA interference (RNAi) pathways of the insect for efficacy. Preliminary evidence suggests that western corn rootworm could develop broad resistance to all insecticidal RNAs through changes in RNAi pathway genes; however, the likelihood of field-evolved resistance occurring through this mechanism remains unclear. In the current study, eight key genes involved in facilitating interference in the microRNA and small interfering RNA pathways were targeted for knockdown in order to evaluate impact on fitness of western corn rootworm. These genes include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2. Depletion of targeted transcripts in rootworm larvae led to changes in microRNA expression, decreased ability to pupate, reduced adult beetle emergence, and diminished reproductive capacity. The observed effects do not support evolution of resistance through changes in expression of these eight genes due to reduced insect fitness.

Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors.

PubMed

Fraites, Thomas J; Schleissing, Mary R; Shanely, R Andrew; Walter, Glenn A; Cloutier, Denise A; Zolotukhin, Irene; Pauly, Daniel F; Raben, Nina; Plotz, Paul H; Powers, Scott K; Kessler, Paul D; Byrne, Barry J

2002-05-01

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Systemic injection of AAV9 carrying a periostin promoter targets gene expression to a myofibroblast-like lineage in mouse hearts after reperfused myocardial infarction.

PubMed

Piras, B A; Tian, Y; Xu, Y; Thomas, N A; O'Connor, D M; French, B A

2016-05-01

Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.

A Single Intravenous rAAV Injection as Late as P20 Achieves Efficacious and Sustained CNS Gene Therapy in Canavan Mice

PubMed Central

Ahmed, Seemin Seher; Li, Huapeng; Cao, Chunyan; Sikoglu, Elif M; Denninger, Andrew R; Su, Qin; Eaton, Samuel; Liso Navarro, Ana A; Xie, Jun; Szucs, Sylvia; Zhang, Hongwei; Moore, Constance; Kirschner, Daniel A; Seyfried, Thomas N; Flotte, Terence R; Matalon, Reuben; Gao, Guangping

2013-01-01

Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤1 month) AspA−/− mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20. PMID:23817205

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Gene Delivery to Adipose Tissue Using Transcriptionally Targeted rAAV8 Vectors

PubMed Central

Uhrig-Schmidt, Silke; Geiger, Matthias; Luippold, Gerd; Birk, Gerald; Mennerich, Detlev; Neubauer, Heike; Grimm, Dirk; Wolfrum, Christian; Kreuz, Sebastian

2014-01-01

In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A – a lipid-droplet-associated protein – resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo. PMID:25551639

Impact of age and vector construct on striatal and nigral transgene expression

PubMed Central

Polinski, Nicole K; Manfredsson, Fredric P; Benskey, Matthew J; Fischer, D Luke; Kemp, Christopher J; Steece-Collier, Kathy; Sandoval, Ivette M; Paumier, Katrina L; Sortwell, Caryl E

2016-01-01

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD. PMID:27933309

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

PubMed

Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

2014-02-04

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.

PubMed

Roche-Molina, Marta; Sanz-Rosa, David; Cruz, Francisco M; García-Prieto, Jaime; López, Sergio; Abia, Rocío; Muriana, Francisco J G; Fuster, Valentín; Ibáñez, Borja; Bernal, Juan A

2015-01-01

Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels. Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses. © 2014 American Heart Association, Inc.

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

PubMed Central

Bengtsson, Niclas E.; Hall, John K.; Odom, Guy L.; Phelps, Michael P.; Andrus, Colin R.; Hawkins, R. David; Hauschka, Stephen D.; Chamberlain, Joel R.; Chamberlain, Jeffrey S.

2017-01-01

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders. PMID:28195574

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.

PubMed

Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta; Hawk, Sarah M; Flake, Alan W; Mingozzi, Federico; High, Katherine A

2012-03-29

In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

PubMed Central

Buchlis, George; Podsakoff, Gregory M.; Radu, Antonetta; Hawk, Sarah M.; Flake, Alan W.; Mingozzi, Federico

2012-01-01

In previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer. PMID:22271447

Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

PubMed Central

Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

2011-01-01

Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

PubMed Central

Guo, Wen; Wong, Siu; Bhasin, Shalender

2013-01-01

Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

PubMed Central

Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

2013-01-01

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

On the role of PDZ domain-encoding genes in Drosophila border cell migration.

PubMed

Aranjuez, George; Kudlaty, Elizabeth; Longworth, Michelle S; McDonald, Jocelyn A

2012-11-01

Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.

RNAi-mediated silencing of MAP kinase signalling genes (Fmk1, Hog1, and Pbs2) in Fusarium oxysporum reduces pathogenesis on tomato plants.

PubMed

Pareek, Manish; Rajam, Manchikatla Venkat

2017-09-01

Fusarium oxysporum is a soil-borne plant fungal pathogen, and causes colossal losses in several crop plants including tomato. Effective control measures include the use of harmful fungicides and resistant cultivars, but these methods have shown limited success. Conventional methods to validate fungal pathogenic genes are labour intensive. Therefore, an alternative strategy is required to efficiently characterize unknown pathogenic genes. RNA interference (RNAi) has emerged as a potential tool to functionally characterize novel fungal pathogenic genes and also to control fungal diseases. Here, we report an efficient method to produce stable RNAi transformants of F. oxysporum using Agrobacterium-mediated transformation (AMT). We have transformed F. oxysporum spores using RNAi constructs of Fmk1, Hog1, and Pbs2 MAP kinase signalling genes. Fmk1 RNAi fungal transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings. Hog1 and Pbs2 RNAi transformants showed altered conidial size, and reduced invasive growth and pathogenesis. These results showed that AMT using RNAi constructs is an effective approach for dissecting the role of genes involved in pathogenesis in F. oxysporum and this could be extended for other fungal systems. The obtained knowledge can be easily translated for developing fungal resistant crops by RNAi. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

PubMed Central

Meerbrey, Kristen L.; Hu, Guang; Kessler, Jessica D.; Roarty, Kevin; Fang, Justin E.; Herschkowitz, Jason I.; Burrows, Anna E.; Ciccia, Alberto; Sun, Tingting; Schmitt, Earlene M.; Bernardi, Ronald J.; Fu, Xiaoyong; Bland, Christopher S.; Cooper, Thomas A.; Schiff, Rachel; Rosen, Jeffrey M.; Westbrook, Thomas F.; Elledge, Stephen J.

2011-01-01

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets. PMID:21307310

Smed-Evi/Wntless is required for beta-catenin-dependent and -independent processes during planarian regeneration.

PubMed

Adell, Teresa; Salò, Emili; Boutros, Michael; Bartscherer, Kerstin

2009-03-01

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-beta-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-beta-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through beta-catenin-dependent and -independent pathways.

Specific Jak3 Downregulation in Lymphocytes Impairs γc Cytokine Signal Transduction and Alleviates Antigen-driven Inflammation In Vivo

PubMed Central

Gómez-Valadés, Alicia G; Llamas, María; Blanch, Sílvia; Perales, José C; Román, Juan; Gómez-Casajús, Lluís; Mascaró, Cristina

2012-01-01

Jak3, one of the four members comprising the Jak family of cytosolic tyrosine kinases, has emerged as a promising target for nontoxic immunotherapies. Although a number of Jak inhibitors has already demonstrated efficacy, they suffer from secondary effects apparently associated to their pan-Jak activity. However, whether selective Jak3 inhibition would afford therapeutic efficacy remains unclear. To address this question we have investigated the immunosuppressive potential of selective Jak3 intervention in lymphocytes using RNA interference (RNAi) technology in vitro and in vivo. Using synthetic small interference RNA (siRNA) sequences we achieved successful transfections into human and mouse primary T lymphocytes. We found that Jak3 knockdown was sufficient to impair not only interleukin-2 (IL-2) and T cell receptor (TCR)-mediated cell activation in vitro, but also antigen-triggereds welling, inflammatory cell infiltration, and proinflammatory cytokine raise in vivo. Furthermore, Jak1 (which mediates γc cytokine signaling in conjunction with Jak3) cosilencing did not provide higher potency to the aforementioned immunosuppressant effects. Our data provides direct evidences indicating that Jak3 protein plays an important role in γc cytokine and antigen-mediated T cell activation and modulates Th1-mediated inflammatory disorders, all in all highlighting its potential as a target in immunosuppressive therapies. PMID:23344234

"Caenorhabditis Elegans" as an Undergraduate Educational Tool for Teaching RNAi

ERIC Educational Resources Information Center

Andersen, Janet; Krichevsky, Alexander; Leheste, Joerg R.; Moloney, Daniel J.

2008-01-01

Discovery of RNA-mediated interference (RNAi) is widely recognized as one of the most significant molecular biology breakthroughs in the past 10 years. There is a need for science educators to develop teaching tools and laboratory activities that demonstrate the power of this new technology and help students to better understand the RNAi process.…

Intraneural convection enhanced delivery of AAVrh20 for targeting primary sensory neurons.

PubMed

Pleticha, Josef; Jeng-Singh, Christian; Rezek, Rahaf; Zaibak, Manal; Beutler, Andreas S

2014-05-01

Gene therapy using adeno-associated virus (AAV) is an attractive strategy to treat disorders of the peripheral nervous system (PNS), such as chronic pain or peripheral neuropathies. Although intrathecal (IT) administration of AAV has been the standard in the field for targeting the PNS, it lacks anatomical specificity and results in wide rostro-caudal distribution of the vector. An alternative approach is to deliver AAV directly to the peripheral nerve axon. The present study employed convection-enhanced delivery (CED) of a novel AAV serotype, AAVrh20, expressing enhanced green fluorescent protein (EGFP) into rat sciatic nerve investigating its efficacy, anatomical selectivity, and safety, compared to the IT route. Intraneural CED resulted in transduction confined to the ipsilateral L4 and L5 DRG while IT administration led to promiscuous DRG transduction encompassing the entire lumbar region bilaterally. The transduction rate for intraneural AAV administration was similar to IT delivery (24% for L4 and 31.5% for L5 DRG versus 50% for L4 and 19.5% for L5 DRG). The use of hyperosmotic diluent did not further improve the transduction efficiency. AAVrh20 was superior to reference serotypes previously described to be most active for each route. Intraneural CED of AAV was associated with transient allodynia that resolved spontaneously. These findings establish intraneural CED as an alternative to IT administration for AAV mediated gene transfer to the PNS and, based on a reference rodent model, suggest AAVrh20 as a superior serotype for targeting the PNS. Copyright © 2014 Elsevier Inc. All rights reserved.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

PubMed Central

Doerfler, Phillip A.; Todd, Adrian G.; Clément, Nathalie; Falk, Darin J.; Nayak, Sushrusha; Herzog, Roland W.; Byrne, Barry J.

2016-01-01

Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa−/− mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders. PMID:26603344

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria.

PubMed

Spit, Jornt; Philips, Annelies; Wynant, Niels; Santos, Dulce; Plaetinck, Geert; Vanden Broeck, Jozef

2017-02-01

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

PubMed Central

Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

2013-01-01

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

Genomewide Screen for Synthetic Lethal Interactions with Mutant KRAS in Lung Cancer

DTIC Science & Technology

2017-11-01

proposed work uses the same concept but with a novel approach. 15. SUBJECT TERMS CRISPR /Cas9, synthetic lethality, KRAS 16. SECURITY CLASSIFICATION OF: 17...essential in cells bearing an activated RAS. We used CRISPR /Cas9-based screening approach for this purpose. Body The overall objective was to identify...of the inducible hCas9 into the AAVS1 site through CRISPR /Cas9-mediated knockin approach. Shown in the top is gRNA sequence for AAVS1. Shown in the

Two distinct roles of the yorkie/yap gene during homeostasis in the planarian Dugesia japonica.

PubMed

Hwang, Byulnim; An, Yang; Agata, Kiyokazu; Umesono, Yoshihiko

2015-04-01

Adult planarians possess somatic pluripotent stem cells called neoblasts that give rise to all missing cell types during regeneration and homeostasis. Recent studies revealed that the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family plays an important role in the regulation of tissue growth during development and regeneration, and therefore we investigated the role of a planarian yki-related gene (termed Djyki) during regeneration and homeostasis of the freshwater planarian Dugesia japonica. We found that knockdown of the function of Djyki by RNA interference (RNAi) downregulated neoblast proliferation and caused regeneration defects after amputation. In addition, Djyki RNAi caused edema during homeostasis. These seemingly distinct defects induced by Djyki RNAi were rescued by simultaneous RNAi of a planarian mats-related gene (termed Djmats), suggesting an important role of Djmats in the negative regulation of Djyki, in accordance with the conservation of the functional relationship of these two genes during the course of evolution. Interestingly, Djyki RNAi did not prevent normal protonephridial structure, suggesting that Djyki RNAi induced the edema phenotype without affecting the excretory system. Further analyses revealed that increased expression of the D. japonica gene DjaquaporinA (DjaqpA), which belongs to a large gene family that encodes a water channel protein for the regulation of transcellular water flow, promoted the induction of edema, but not defects in neoblast dynamics, in Djyki(RNAi) animals. Thus, we conclude that Djyki plays two distinct roles in the regulation of active proliferation of stem cells and in osmotic water transport across the body surface in D. japonica. © 2015 The Authors Development, Growth & Differentiation published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Society of Developmental Biologists.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture1[OPEN

PubMed Central

Kir, Gokhan; Ye, Huaxun; Nelissen, Hilde; Neelakandan, Anjanasree K.; Kusnandar, Andree S.; Luo, Anding; Inzé, Dirk; Sylvester, Anne W.; Yin, Yanhai; Becraft, Philip W.

2015-01-01

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling. PMID:26162429

The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis.

PubMed

Frost, Christopher J; Nyamdari, Batbayar; Tsai, Chung-Jui; Harding, Scott A

2012-01-01

The Populus sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of Populus, SUT4 is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in Populus. Here, we describe the effects of PtaSUT4-RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in Populus.

Adipokinetic hormone receptor gene identification and its role in triacylglycerol mobilization and sexual behavior in the oriental fruit fly (Bactrocera dorsalis).

PubMed

Hou, Qiu-Li; Chen, Er-Hu; Jiang, Hong-Bo; Wei, Dan-Dan; Gui, Shun-Hua; Wang, Jin-Jun; Smagghe, Guy

2017-11-01

Energy homeostasis requires continuous compensation for fluctuations in energy expenditure and availability of food resources. In insects, energy mobilization is under control of the adipokinetic hormone (AKH) where it is regulating the nutritional status by supporting the mobilization of lipids. In this study, we characterized the gene coding for the AKH receptor (AKHR) and investigated its function in the oriental fruit fly (Bactrocera dorsalis) that is economically one of the most important pest insects of tropical and subtropical fruit. Bacdo-AKHR is a typical G protein-coupled receptor (GPCR) and phylogenetic analysis confirmed that Bacdo-AKHR is closely related to insect AKHRs from other species. When expressed in Chinese hamster ovary (CHO) cells, Bacdo-AKHR exhibited a high sensitivity and selectivity for AKH peptide (EC 50  = 19.3 nM). Using qPCR, the developmental stage and tissue-specific expression profiles demonstrated that Bacdo-AKHR was highly expressed in both the larval and adult stages, and also specifically in the fat body and midgut of the adult with no difference in sex. To investigate the role of AKHR in B. dorsalis, RNAi assays were performed with dsRNA against Bacdo-AKHR in adult flies of both sexes and under starvation and feeding condition. As major results, the knockdown of this gene resulted in triacylglycerol (TAG) accumulation. With RNAi-males, we observed a severe decrease in their sexual courtship activity when starved, but there was a partial rescue in copulation when refed. Also in RNAi-males, the tethered-flight duration declined compared with the control group when starved, which is confirming the dependency on energy metabolism. In RNAi-females, the sexual behavior was not affected, but their fecundity was decreased. Our findings indicate an interesting role of AKHR in the sexual behavior of males specifically. The effects are associated with TAG accumulation, and we also reported that the conserved role of AKH-mediated system in B. dorsalis is nutritional state-dependent. Hence, we provided further understanding on the multiple functions of AKH/AKHR in B. dorsalis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Treatment with Trehalose Prevents Behavioral and Neurochemical Deficits Produced in an AAV α-Synuclein Rat Model of Parkinson's Disease.

PubMed

He, Qing; Koprich, James B; Wang, Ying; Yu, Wen-bo; Xiao, Bao-guo; Brotchie, Jonathan M; Wang, Jian

2016-05-01

The accumulation of misfolded α-synuclein in dopamine (DA) neurons is believed to be of major importance in the pathogenesis of Parkinson's disease (PD). Animal models of PD, based on viral-vector-mediated over-expression of α-synuclein, have been developed and show evidence of dopaminergic toxicity, providing us a good tool to investigate potential therapies to interfere with α-synuclein-mediated pathology. An efficient disease-modifying therapeutic molecule should be able to interfere with the neurotoxicity of α-synuclein aggregation. Our study highlighted the ability of an autophagy enhancer, trehalose (at concentrations of 5 and 2% in drinking water), to protect against A53T α-synuclein-mediated DA degeneration in an adeno-associated virus serotype 1/2 (AAV1/2)-based rat model of PD. Behavioral tests and neurochemical analysis demonstrated a significant attenuation in α-synuclein-mediated deficits in motor asymmetry and DA neurodegeneration including impaired DA neuronal survival and DA turnover, as well as α-synuclein accumulation and aggregation in the nigrostriatal system by commencing 5 and 2% trehalose at the same time as delivery of AAV. Trehalose (0.5%) was ineffective on the above behavioral and neurochemical deficits. Further investigation showed that trehalose enhanced autophagy in the striatum by increasing formation of LC3-II. This study supports the concept of using trehalose as a novel therapeutic strategy that might prevent/reverse α-synuclein aggregation for the treatment of PD.

RNAi-mediated resistance against Cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha.

PubMed

Khatoon, Sameena; Kumar, Abhinav; Sarin, Neera B; Khan, Jawaid A

2016-08-01

Cotton leaf curl disease (CLCuD) is caused by several distinct begomovirus species in association with disease-specific betasatellite essential for induction of disease symptoms. CLCuD is a serious threat for the cultivation of cotton (Gossypium sp.) and several species in the family Malvaceae. In this study, RNAi-based approach was applied to generate transgenic cotton (Gossypium hirsutum) plants resistant to Cotton leaf curl Rajasthan virus (CLCuRV). An intron hairpin (ihp) RNAi construct capable of expressing dsRNA homologous to the intergenic region (IR) of CLCuRV was designed and developed. Following Agrobacterium tumefaciens-mediated transformation of cotton (G. hirsutum cv. Narasimha) plants with the designed ihpRNAi construct, a total of 9 independent lines of transformed cotton were obtained. The presence of the potential stretch of IR in the transformed cotton was confirmed by PCR coupled with Southern hybridization. Upon inoculation with viruliferous whiteflies, the transgenic plants showed high degree of resistance. None of them displayed any CLCuD symptoms even after 90 days post inoculation. The transformed cotton plants showed the presence of siRNAs. The present study demonstrated that ihp dsRNA-mediated resistance strategy of RNAi is an effective means to combat the CLCuD infection in cotton.

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

PubMed Central

Maxwell, Michele M.; Pasinelli, Piera; Kazantsev, Aleksey G.; Brown, Robert H.

2004-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In ≈25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing in vitro and in vivo. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS. PMID:14981234

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

PubMed

Han, Jie; Goldstein, Leslie A; Gastman, Brian R; Rabinowich, Hannah

2006-04-14

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

IGF-1 deficiency in a critical period early in life influences the vascular aging phenotype in mice by altering miRNA-mediated post-transcriptional gene regulation: implications for the developmental origins of health and disease hypothesis.

PubMed

Tarantini, Stefano; Giles, Cory B; Wren, Jonathan D; Ashpole, Nicole M; Valcarcel-Ares, M Noa; Wei, Jeanne Y; Sonntag, William E; Ungvari, Zoltan; Csiszar, Anna

2016-08-01

Epidemiological findings support the concept of Developmental Origins of Health and Disease, suggesting that early-life hormonal influences during a sensitive period of development have a fundamental impact on vascular health later in life. The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in circulating IGF-1 levels during adolescence. The present study was designed to characterize the effect of developmental IGF-1 deficiency on the vascular aging phenotype. To achieve that goal, early-onset endocrine IGF-1 deficiency was induced in mice by knockdown of IGF-1 in the liver using Cre-lox technology (Igf1 f/f mice crossed with mice expressing albumin-driven Cre recombinase). This model exhibits low-circulating IGF-1 levels during the peripubertal phase of development, which is critical for the biology of aging. Due to the emergence of miRNAs as important regulators of the vascular aging phenotype, the effect of early-life IGF-1 deficiency on miRNA expression profile in the aorta was examined in animals at 27 months of age. We found that developmental IGF-1 deficiency elicits persisting late-life changes in miRNA expression in the vasculature, which significantly differed from those in mice with adult-onset IGF-1 deficiency (TBG-Cre-AAV8-mediated knockdown of IGF-1 at 5 month of age in Igf1 f/f mice). Using a novel computational approach, we identified miRNA target genes that are co-expressed with IGF-1 and associate with aging and vascular pathophysiology. We found that among the predicted targets, the expression of multiple extracellular matrix-related genes, including collagen-encoding genes, were downregulated in mice with developmental IGF-1 deficiency. Collectively, IGF-1 deficiency during a critical period during early in life results in persistent changes in post-transcriptional miRNA-mediated control of genes critical targets for vascular health, which likely contribute to the deleterious late-life cardiovascular effects known to occur with developmental IGF-1 deficiency.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

PubMed

Pompey, Justine M; Foda, Bardees; Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

PubMed Central

Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

FER mediated HGF-independent regulation of HGFR/MET activates RAC1-PAK1 pathway to potentiate metastasis in ovarian cancer.

PubMed

Fan, Gaofeng

2017-11-03

Uncontrolled metastasis significantly contributes to high lethality of patients suffering from ovarian cancer. To date, the detailed molecular mechanisms which account for ovarian tumor cell spreading and metastasis remain largely unknown. In a recent study, we have demonstrated that aberrantly high expression of the non-receptor tyrosine kinase FER is responsible for ovarian tumor cell metastasis both in vitro and in vivo. Mechanistically, we indentified Hepatocyte Growth Factor Receptor HGFR/MET as a novel substrate of FER, and through which the kinase FER modulates ovarian cancer cell motility and invasiveness in a ligand-independent manner. We also observed aberrantly high expression of PAK1 kinase in cancer cells, and RNAi-mediated knockdown of FER kinase inactivated the RAC1-PAK1 signaling pathway and decreased metastatic potential of CAOV4 ovarian cancer cells. Overall, our study revealed a previously uncharacterized, pro-metastatic role of the kinase FER in ovarian cancer through the MET-RAC1-PAK1 pathway. Further efforts are essential to investigating beneficial outcomes towards targeting the RAC1-PAK1 signaling pathway in reducing metastatic burden of this deadly disease.

A critical role for noncoding 5S rRNA in regulating Mdmx stability.

PubMed

Li, Muyang; Gu, Wei

2011-09-16

Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and, subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2, whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal a critical role for noncoding 5S rRNA in modulating the p53-Mdmx axis. Copyright © 2011 Elsevier Inc. All rights reserved.

Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night1[OPEN

PubMed Central

Ifuku, Kentaro; Ikeda, Ken-ichi; Inoue, Kanako Ikeda; Park, Pyoyun; Tamoi, Masahiro; Inoue, Hironori; Sakamoto, Katsuhiko; Saito, Ryota

2016-01-01

Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,β-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night. PMID:26884484

Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night.

PubMed

Takagi, Daisuke; Ifuku, Kentaro; Ikeda, Ken-Ichi; Inoue, Kanako Ikeda; Park, Pyoyun; Tamoi, Masahiro; Inoue, Hironori; Sakamoto, Katsuhiko; Saito, Ryota; Miyake, Chikahiro

2016-04-01

Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,β-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night. © 2016 American Society of Plant Biologists. All Rights Reserved.

The RNAi Inheritance Machinery of Caenorhabditis elegans.

PubMed

Spracklin, George; Fields, Brandon; Wan, Gang; Becker, Diveena; Wallig, Ashley; Shukla, Aditi; Kennedy, Scott

2017-07-01

Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in Caenorhabditis elegans (termed RNAi inheritance). Here we describe the results of a forward genetic screen in C. elegans that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality. Copyright © 2017 by the Genetics Society of America.

Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy.

PubMed

Wang, Wenjun; Wang, Qing; Wan, Danyang; Sun, Yue; Wang, Lin; Chen, Hong; Liu, Chengyu; Petersen, Robert B; Li, Jianshuang; Xue, Weili; Zheng, Ling; Huang, Kun

2017-05-04

Autophagy plays critical and complex roles in many human diseases, including diabetes and its complications. However, the role of autophagy in the development of diabetic retinopathy remains uncertain. Core histone modifications have been reported involved in the development of diabetic retinopathy, but little is known about the histone variants. Here, we observed increased autophagy and histone HIST1H1C/H1.2, an important variant of the linker histone H1, in the retinas of type 1 diabetic rodents. Overexpression of histone HIST1H1C upregulates SIRT1 and HDAC1 to maintain the deacetylation status of H4K16, leads to upregulation of ATG proteins, then promotes autophagy in cultured retinal cell line. Histone HIST1H1C overexpression also promotes inflammation and cell toxicity in vitro. Knockdown of histone HIST1H1C reduces both the basal and stresses (including high glucose)-induced autophagy, and inhibits high glucose induced inflammation and cell toxicity. Importantly, AAV-mediated histone HIST1H1C overexpression in the retinas leads to increased autophagy, inflammation, glial activation and neuron loss, similar to the pathological changes identified in the early stage of diabetic retinopathy. Furthermore, knockdown of histone Hist1h1c by siRNA in the retinas of diabetic mice significantly attenuated the diabetes-induced autophagy, inflammation, glial activation and neuron loss. These results indicate that histone HIST1H1C may offer a novel therapeutic target for preventing diabetic retinopathy.

Small cell lung cancer growth is inhibited by miR-342 through its effect of the target gene IA-2.

PubMed

Xu, Huanyu; Cai, Tao; Carmona, Gilberto N; Abuhatzira, Liron; Notkins, Abner L

2016-09-26

Small cell lung cancers (SCLC) are tumors of neuroendocrine origin. Previous in vitro studies from our laboratory showed that SCLC expresses high levels of the transmembrane dense core vesicle protein IA-2 (islet cell antigen-2) as compared to normal lung cells. IA-2, through its effect on dense core vesicles (DCVs), is known to be involved in the secretion of hormones and neurotransmitters. It is believed that the dysregulated release of the neurotransmitter Acetylcholine (ACh) by DCVs has an autocrine effect on SCLC cell growth. Recently, we found that IA-2 is a target of the microRNA miR-342 and that miR-342 mimics suppress the expression of IA-2. The present experiments were initiated to see whether IA-2 and/or miR-342 affect the growth of SCLC. SCLC cell growth was evaluated following the knockdown of endogenous IA-2 with RNAi or by overexpressing miR-342 with a mimic. The secretion and content of ACh in SCLC cells was analyzed using a human acetylcholine ELISA (enzyme-linked immunosorbent assay) kit. The knockdown of endogenous IA-2 by RNAi reduced SCLC cell growth within 4 days by 40 % or more. Similar results were obtained when these cell lines were transfected with a miR-342 mimic. The knockdown of IA-2 by RNAi or miR-342 with a mimic also resulted in a significant decrease in the secretion of ACh, one of the autocrine hormones secreted by SCLC. Further studies revealed that the growth of SCLC cell lines that had been treated with the miR-342 mimic was restored to nearly normal levels by treatment with ACh. Our studies show for the first time that both miR-342 and its target gene IA-2 are involved in the growth process of SCLC cells and act by their effect on autocrine secretion. These findings point to possible new therapeutic approaches for the treatment of autocrine-induced tumor proliferation.

The Kinetics of G2 and M Transitions Regulated by B Cyclins

PubMed Central

Huang, Yehong; Sramkoski, R. Michael; Jacobberger, James W.

2013-01-01

B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary. PMID:24324638

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

PubMed Central

Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2016-01-01

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

Selective deletion of leptin receptors in adult hippocampus induces depression-related behaviors

PubMed Central

Guo, Ming; Huang, Tung-Yi; Garza, Jacob C.; Chua, Streamson C.; Lu, Xin-Yun

2013-01-01

Previous studies have demonstrated that leptin and its receptors (LepRb) in the central nervous system play an important role in regulating depression- and anxiety-related behaviors. However, the physiological functions of LepRb in specific brain regions for mediating different emotional behaviors remain to be defined. In this study, we examined the behavioral effects of LepRb ablation in the adult hippocampus using a series of behavioral paradigms for assessing depression- and anxiety-related behaviors. Targeted deletion of LepRb was achieved using the Cre/loxP site-specific recombination system through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the dentate gyrus of adult mice homozygous for a floxed leptin receptor allele. AAV-Cre-mediated deletion of the floxed region of LepRb was detected 2 weeks after injection. In accordance with this, leptin-stimulated phophorylation of Akt was attenuated in the hippocampus of AAV-Cre injected mice. Mice injected with AAV-Cre displayed normal locomotor activity and anxiety-like behavior, as determined in the elevated plus maze, light dark box and open field tests, but showed increased depression-like behaviors in the tail suspension, sucrose preference and learned helplessness tests. Taken together, this data suggests that deletion of LepRb in the adult hippocampus is sufficient to induce depression-like behaviors. Our results support the view that leptin signaling in the hippocampus may be essential for maintaining positive mood states and active coping to stress. PMID:22932068

Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

PubMed

Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

2015-12-01

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

RNAi-Mediated Knockdown of Catalase Causes Cell Cycle Arrest in SL-1 Cells and Results in Low Survival Rate of Spodoptera litura (Fabricius)

PubMed Central

Hu, Meiying; Chen, Shaohua; Muhammad, Rizwan-ul-Haq; Dong, Xiaolin; Gong, Liang

2013-01-01

Deregulated reactive oxygen species (ROS) production can lead to the disruption of structural and functional integrity of cells as a consequence of reactive interaction between ROS and various biological components. Catalase (CAT) is a common enzyme existing in nearly all organisms exposed to oxygen, which decomposes harmful hydrogen peroxide, into water and oxygen. In this study, the full length sequence that encodes CAT-like protein from Spodoptera litura named siltCAT (GenBank accession number: JQ_663444) was cloned and characterized. Amino acid sequence alignment showed siltCAT shared relatively high conservation with other insect, especially the conserved residues which defined heme and NADPH orientation. Expression pattern analysis showed that siltCAT mRNA was mainly expressed in the fat body, midgut, cuticle and malpighian tube, and as well as over last instar larvae, pupa and adult stages. RNA interference was used to silence CAT gene in SL-1 cells and the fourth-instar stage of S. litura larvae respectively. Our results provided evidence that CAT knockdown induced ROS generation, cell cycle arrest and apoptosis in SL-1 cells. It also confirmed the decrease in survival rate because of increased ROS production in experimental groups injected with double-stranded RNA of CAT (dsCAT). This study implied that ROS scavenging by CAT is important for S. litura survival. PMID:23555693

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

PubMed Central

Anastas, Jamie N.; Kulikauskas, Rima M.; Tamir, Tigist; Rizos, Helen; Long, Georgina V.; von Euw, Erika M.; Yang, Pei-Tzu; Chen, Hsiao-Wang; Haydu, Lauren; Toroni, Rachel A.; Lucero, Olivia M.; Chien, Andy J.; Moon, Randall T.

2014-01-01

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAFV600E/K) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance. PMID:24865425

AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

PubMed

Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

2014-04-30

As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished. Copyright © 2014 Elsevier B.V. All rights reserved.

Progranulin Gene Therapy Improves Lysosomal Dysfunction and Microglial Pathology Associated with Frontotemporal Dementia and Neuronal Ceroid Lipofuscinosis.

PubMed

Arrant, Andrew E; Onyilo, Vincent C; Unger, Daniel E; Roberson, Erik D

2018-02-28

Loss-of-function mutations in progranulin, a lysosomal glycoprotein, cause neurodegenerative disease. Progranulin haploinsufficiency causes frontotemporal dementia (FTD) and complete progranulin deficiency causes CLN11 neuronal ceroid lipofuscinosis (NCL). Progranulin replacement is a rational therapeutic strategy for these disorders, but there are critical unresolved mechanistic questions about a progranulin gene therapy approach, including its potential to reverse existing pathology. Here, we address these issues using an AAV vector (AAV- Grn ) to deliver progranulin in Grn -/- mice (both male and female), which model aspects of NCL and FTD pathology, developing lysosomal dysfunction, lipofuscinosis, and microgliosis. We first tested whether AAV- Grn could improve preexisting pathology. Even with treatment after onset of pathology, AAV- Grn reduced lipofuscinosis in several brain regions of Grn -/- mice. AAV- Grn also reduced microgliosis in brain regions distant from the injection site. AAV-expressed progranulin was only detected in neurons, not in microglia, indicating that the microglial activation in progranulin deficiency can be improved by targeting neurons and thus may be driven at least in part by neuronal dysfunction. Even areas with sparse transduction and almost undetectable progranulin showed improvement, indicating that low-level replacement may be sufficiently effective. The beneficial effects of AAV- Grn did not require progranulin binding to sortilin. Finally, we tested whether AAV- Grn improved lysosomal function. AAV-derived progranulin was delivered to the lysosome, ameliorated the accumulation of LAMP-1 in Grn -/- mice, and corrected abnormal cathepsin D activity. These data shed light on progranulin biology and support progranulin-boosting therapies for NCL and FTD due to GRN mutations. SIGNIFICANCE STATEMENT Heterozygous loss-of-function progranulin ( GRN ) mutations cause frontotemporal dementia (FTD) and homozygous mutations cause neuronal ceroid lipofuscinosis (NCL). Here, we address several mechanistic questions about the potential of progranulin gene therapy for these disorders. GRN mutation carriers with NCL or FTD exhibit lipofuscinosis and Grn -/- mouse models develop a similar pathology. AAV-mediated progranulin delivery reduced lipofuscinosis in Grn -/- mice even after the onset of pathology. AAV delivered progranulin only to neurons, not microglia, but improved microgliosis in several brain regions, indicating cross talk between neuronal and microglial pathology. Its beneficial effects were sortilin independent. AAV-derived progranulin was delivered to lysosomes and corrected lysosomal abnormalities. These data provide in vivo support for the efficacy of progranulin-boosting therapies for FTD and NCL. Copyright © 2018 the authors 0270-6474/18/382342-18$15.00/0.

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors.

PubMed

Ji, Guangchen; Zhang, Wei; Mahimainathan, Lenin; Narasimhan, Madhusudhanan; Kiritoshi, Takaki; Fan, Xiuzhen; Wang, Jigong; Green, Thomas A; Neugebauer, Volker

2017-02-08

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT 2C R in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT 2C R in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT 2C R knockdown in the BLA with stereotaxic injection of 5-HT 2C R shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT 2C R knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT 2C R knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT 2C R knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT 2C R in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT 2C R in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT 2C R knockdown in the BLA inhibits neuropathic-pain-related behaviors. SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT 2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT 2C R knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area. Copyright © 2017 the authors 0270-6474/17/371378-16$15.00/0.

Function of a STIM1 Homologue in C. elegans: Evidence that Store-operated Ca2+ Entry Is Not Essential for Oscillatory Ca2+ Signaling and ER Ca2+ Homeostasis

PubMed Central

Yan, Xiaohui; Xing, Juan; Lorin-Nebel, Catherine; Estevez, Ana Y.; Nehrke, Keith; Lamitina, Todd; Strange, Kevin

2006-01-01

1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530–amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)–tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1∷GFP expression, suppresses the EF-hand mutation–induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and IP3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions. PMID:16966474

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis.

PubMed

Yan, Xiaohui; Xing, Juan; Lorin-Nebel, Catherine; Estevez, Ana Y; Nehrke, Keith; Lamitina, Todd; Strange, Kevin

2006-10-01

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

PubMed

Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

2017-01-01

STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

PubMed

Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

2018-06-01

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

Gα-cAMP/PKA pathway positively regulates pigmentation, chaetoglobosin A biosynthesis and sexual development in Chaetomium globosum

PubMed Central

Hu, Yang; Chen, Longfei; Akhberdi, Oren; Yu, Xi; Liu, Yanjie; Zhu, Xudong

2018-01-01

Sensing the environmental signals, the canonical Gα-cAMP/PKA pathway modulates mycelial growth and development, and negatively regulates some secondary metabolism in filamentous fungi, e.g. aflatoxin in Aspergillus nidulans. Here we report the characterization of this signaling pathway in Chaetomium globosum, a widely spread fungus known for synthesizing abundant secondary metabolites, e.g. chaetoglobosin A (ChA). RNAi-mediated knockdown of a putative Gα-encoding gene gna-1, led to plural changes in phenotype, e.g. albino mycelium, significant restriction on perithecium development and decreased production of ChA. RNA-seq profiling and qRT-PCR verified significantly fall in expression of corresponding genes, e.g. pks-1 and CgcheA. These defects could be restored by simultaneous knock-down of the pkaR gene encoding a regulatory subunit of cAMP-dependent protein kinase A (PKA), suggesting that pkaR had a negative effect on the above mentioned traits. Confirmatively, the intracellular level of cAMP in wild-type strain was about 3.4-fold to that in gna-1 silenced mutant pG14, and addition of a cAMP analog, 8-Br-cAMP, restored the same defects, e.g., the expression of CgcheA. Furthermore, the intracellular cAMP in gna-1 and pkaR double silenced mutant was approaching the normal level. The following activity inhibition experiment proved that the expression of CgcheA was indeed regulated by PKA. Down-regulation of LaeA/VeA/SptJ expression in gna-1 mutant was also observed, implying that Gα signaling may crosstalk to other regulatory pathways. Taken together, this study proposes that the heterotrimeric Gα protein-cAMP/PKA signaling pathway positively mediates the sexual development, melanin biosynthesis, and secondary metabolism in C. globosum. PMID:29652900

Therapeutic effect of adeno-associated virus (AAV)-mediated ADNF-9 expression on cochlea of kanamycin-deafened guinea pigs.

PubMed

Zheng, Guoxi; Zhu, Zhu; Zhu, Kang; Wei, Junrong; Jing, Yang; Duan, Maoli

2013-10-01

rAAV-NT4-ADNF-9 could ameliorate the damage to auditory function and repair previous impairment of cochlear hair cell loss induced by kanamycin. To investigate the therapeutic effect of ADNF-9 on cochlear hair cells using the recombinant adeno-associated virus (AAV) carrying fusion gene NT4-ADNF-9 and the kanamycin-deafened guinea pig model. Forty white guinea pigs with normal auricle reflex and normal auditory brainstem responses (ABRs) were randomly divided into four groups. Kanamycin was administered to the animals in groups A, B, and C to establish the deafened guinea pig model. rAAV-NT4-ADNF-9, vector only, and artificial perilymph were then delivered to the cochlear tissue of animals in groups A, B, and C, respectively, through the round window membrane. Animals in group D did not receive any treatment and acted as normal controls. The hearing thresholds on the surgery side were recorded before and after the transfection treatment. Fourteen days after treatment, cochleae were removed for paraffin slide preparation and cochlear surface preparation. A phase contrast microscope was used to observe the protective effect of ADNF-9 on hair cells. Significant reduction of the ABR threshold was observed after rAAV-NT4-ADNF-9 treatment (p < 0.05). After 14 days of treatment, the ABR threshold was also significantly different between the rAAV-NT4-ADNF-9-infected group and the non-infected group. Moreover, phase contrast microscopy showed significantly less hair cell damage or hair cell loss in the group treated with rAAV-NT4-ADNF-9 than in the groups treated with vector only or artificial perilymph (p < 0.05).

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

PubMed

Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

2013-03-06

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

Role of the adapter protein Abi1 in actin-associated signaling and smooth muscle contraction.

PubMed

Wang, Tao; Cleary, Rachel A; Wang, Ruping; Tang, Dale D

2013-07-12

Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.

Role of the Adapter Protein Abi1 in Actin-associated Signaling and Smooth Muscle Contraction*

PubMed Central

Wang, Tao; Cleary, Rachel A.; Wang, Ruping; Tang, Dale D.

2013-01-01

Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl. PMID:23740246

Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia.

PubMed

Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D; Landau, Dustin J; Drake, Elizabeth J; Kozink, Daniel M; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R; Brown, Talmage T; Kemper, Alex R; Koeberl, Dwight D

2012-04-01

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector-mediated gene therapy in GSD-Ia.

AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.

PubMed

Huang, Xinping; Hartley, Antja-Voy; Yin, Yishi; Herskowitz, Jeremy H; Lah, James J; Ressler, Kerry J

2013-11-01

The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 μg DNA per ml of media (21 μg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer. Copyright © 2013 Elsevier B.V. All rights reserved.

Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

PubMed Central

Jans, Ralph; Mottram, Laura; Johnson, Darren L; Brown, Anna M; Sikkink, Stephen; Ross, Kehinde; Reynolds, Nick J

2013-01-01

Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway. PMID:23096711

R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4.

PubMed

Wobst, Ivonne; Ebert, Lisa; Birod, Kerstin; Wegner, Marthe-Susanna; Hoffmann, Marika; Thomas, Dominique; Angioni, Carlo; Parnham, Michael J; Steinhilber, Dieter; Tegeder, Irmgard; Geisslinger, Gerd; Grösch, Sabine

2016-12-30

R -flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S -flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E₂ (PGE₂) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R -flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA 2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R -flurbiprofen traps PGE₂ inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R -flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R -flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

Implication of SUMO E3 ligases in nucleotide excision repair.

PubMed

Tsuge, Maasa; Kaneoka, Hidenori; Masuda, Yusuke; Ito, Hiroki; Miyake, Katsuhide; Iijima, Shinji

2015-08-01

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

PubMed Central

Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

2013-01-01

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

PubMed Central

Su, Feifei; Myers, Valerie D.; Knezevic, Tijana; Wang, JuFang; Gao, Erhe; Madesh, Muniswamy; Tahrir, Farzaneh G.; Gupta, Manish K.; Gordon, Jennifer; Rabinowitz, Joseph; Tilley, Douglas G.; Khalili, Kamel; Cheung, Joseph Y.

2016-01-01

Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction. PMID:27882354

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

PubMed Central

White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

2013-01-01

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors.

PubMed

Vande Velde, G; Rangarajan, J R; Toelen, J; Dresselaers, T; Ibrahimi, A; Krylychkina, O; Vreys, R; Van der Linden, A; Maes, F; Debyser, Z; Himmelreich, U; Baekelandt, V

2011-06-01

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells.

PubMed

White, Pamela M; Serbus, Laura R; Debec, Alain; Codina, Adan; Bray, Walter; Guichet, Antoine; Lokey, R Scott; Sullivan, William

2017-04-01

Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia -infected Drosophila cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. Host gene knockdowns that reduced Wolbachia titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte. The presence of Wolbachia in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of Wolbachia titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that Wolbachia is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia ' s potent ability to prevent RNA virus replication. Copyright © 2017 by the Genetics Society of America.

Comparative analysis of RNAi screening technologies at genome-scale reveals an inherent processing inefficiency of the plasmid-based shRNA hairpin.

PubMed

Bhinder, Bhavneet; Shum, David; Djaballah, Hakim

2014-02-01

RNAi screening in combination with the genome-sequencing projects would constitute the Holy Grail of modern genetics; enabling discovery and validation towards a better understanding of fundamental biology leading to novel targets to combat disease. Hit discordance at inter-screen level together with the lack of reproducibility is emerging as the technology's main pitfalls. To examine some of the underlining factors leading to such discrepancies, we reasoned that perhaps there is an inherent difference in knockdown efficiency of the various RNAi technologies. For this purpose, we utilized the two most popular ones, chemically synthesized siRNA duplex and plasmid-based shRNA hairpin, in order to perform a head to head comparison. Using a previously developed gain-of-function assay probing modulators of the miRNA biogenesis pathway, we first executed on a siRNA screen against the Silencer Select V4.0 library (AMB) nominating 1,273, followed by an shRNA screen against the TRC1 library (TRC1) nominating 497 gene candidates. We observed a poor overlap of only 29 hits given that there are 15,068 overlapping genes between the two libraries; with DROSHA as the only common hit out of the seven known core miRNA biogenesis genes. Distinct genes interacting with the same biogenesis regulators were observed in both screens, with a dismal cross-network overlap of only 3 genes (DROSHA, TGFBR1, and DIS3). Taken together, our study demonstrates differential knockdown activities between the two technologies, possibly due to the inefficient intracellular processing and potential cell-type specificity determinants in generating intended targeting sequences for the plasmid-based shRNA hairpins; and suggests this observed inefficiency as potential culprit in addressing the lack of reproducibility.

Minocycline protects, rescues and prevents knockdown transgenic parkin Drosophila against paraquat/iron toxicity: Implications for autosomic recessive juvenile parkinsonism.

PubMed

Ortega-Arellano, Hector Flavio; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

2017-05-01

Autosomal recessive Juvenile Parkinsonism (AR-JP) is a chronic, progressive neurodegenerative disorder caused by mutation in the PARKIN gene, and invariably associated with dopaminergic (DAergic) neuronal loss and brain iron accumulation. Since current medical therapy is symptomatic and lacks significant disease-modifying effects, other treatment approaches are urgently needed it. In the present work, we investigate the role of minocycline (MC) in paraquat (PQ)/iron-induced neurotoxicity in the Drosophila TH>parkin-RNAi/+ (w[*]; UAS-parkin-RNAi; TH-GAL4) fly and have shown the following: (i) MC increased life span and restored the locomotor activity of knockdown (KD) transgenic parkin flies in comparison with the control (vehicle) group; (ii) MC at low (0.1 and 0.3mM) and middle (0.5mM) concentrations protected, rescued and prevented KD parkin Drosophila against PQ toxicity. However, MC at high (1mM) concentration aggravated the toxic effect of PQ; (iii) MC protected and rescued DAergic neurons against the PQ toxic effect according to tyrosine hydroxylase (TH)>green-fluorescent protein (GFP) reporter protein microscopy and anti-TH Western blotting analysis; (iv) MC protected DAergic neurons against PQ/iron toxicity; (v) MC significantly abridged lipid peroxidation (LPO) in the protection, rescue and prevention treatment in TH>parkin-RNAi/+ flies against PQ or iron alone or combined (PQ/iron)-induced neuronal oxidative stress (OS). Our results suggest that MC exerts neuroprotection against PQ/iron-induced OS in DAergic neurons most probably by the scavenging activity of reactive oxygen species (ROS), and by chelating iron. Therefore, MC might be a potential therapeutic drug to delay, revert, or prevent AR-JP. Copyright © 2017 Elsevier B.V. All rights reserved.

AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

PubMed

Kiyota, T; Ingraham, K L; Swan, R J; Jacobsen, M T; Andrews, S J; Ikezu, T

2012-07-01

Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

Multiple Neuropeptide-Coding Genes Involved in Planarian Pharynx Extension.

PubMed

Shimoyama, Seira; Inoue, Takeshi; Kashima, Makoto; Agata, Kiyokazu

2016-06-01

Planarian feeding behavior involves three steps: moving toward food, extending the pharynx from their planarian's ventral side after arriving at the food, and ingesting the food through the pharynx. Although pharynx extension is a remarkable behavior, it remains unknown what neuronal cell types are involved in its regulation. To identify neurons involved in regulating pharynx extension, we quantitatively analyzed pharynx extension and sought to identify these neurons by RNA interference (RNAi) and in situ hybridization. This assay, when performed using planarians with amputation of various body parts, clearly showed that the head portion is indispensable for inducing pharynx extension. We thus tested the effects of knockdown of brain neurons such as serotonergic, GABAergic, and dopaminergic neurons by RNAi, but did not observe any effects on pharynx extension behavior. However, animals with RNAi of the Prohormone Convertase 2 (PC2, a neuropeptide processing enzyme) gene did not perform the pharynx extension behavior, suggesting the possible involvement of neuropeptide(s in the regulation of pharynx extension. We screened 24 neuropeptide-coding genes, analyzed their functions by RNAi using the pharynx extension assay system, and identified at least five neuropeptide genes involved in pharynx extension. These was expressed in different cells or neurons, and some of them were expressed in the brain, suggesting complex regulation of planarian feeding behavior by the nervous system.

Potato plants with genetically engineered tropane alkaloid precursors.

PubMed

Küster, Nadine; Rosahl, Sabine; Dräger, Birgit

2017-02-01

Solanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation. Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

New genes often acquire male-specific functions but rarely become essential in Drosophila.

PubMed

Kondo, Shu; Vedanayagam, Jeffrey; Mohammed, Jaaved; Eizadshenass, Sogol; Kan, Lijuan; Pang, Nan; Aradhya, Rajaguru; Siepel, Adam; Steinhauer, Josefa; Lai, Eric C

2017-09-15

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function. © 2017 Kondo et al.; Published by Cold Spring Harbor Laboratory Press.

[Update Churg-Strauss syndrome].

PubMed

Moosig, F; Hellmich, B

2012-11-01

The Churg-Strauss syndrome (CSS) is the rarest subtype of the so-called anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) and has the lowest frequency of ANCA-positivity (around 30%). In addition to asthma and blood eosinophilia, CSS is characterized by end-organ damage, which can be caused by either vasculitis and/or tissue infiltration of eosinophilic granulocytes. The CSS shares many etiological and clinical features of other hypereosinophilic syndromes. Recently, a distinct genetic background could be demonstrated for both the ANCA-positive and ANCA-negative subtypes of CSS as compared to the other two forms of AAV. Among other cytokines, interleukin-5 (IL-5) could be identified as a key mediator of eosinophilia. Therefore, recent clinical trials in CSS aimed to target IL-5. Outside of clinical trials, treatment of CSS is adapted to disease stage and activity, as recommended for other types of AAV.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

PubMed

Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

2017-12-01

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na + sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H + -ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H + -PPase (V-PPase) and vacuolar Na + /H + antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H + -PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na + staining showed the reduced vacuolar Na + sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na + sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

PubMed

Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

2012-08-01

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori

PubMed Central

Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

2015-01-01

Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior. PMID:25630665

Silencing of Apis mellifera dorsal genes reveals their role in expression of the antimicrobial peptide defensin-1.

PubMed

Lourenço, Anete Pedro; Florecki, Mônica Mazzei; Simões, Zilá Luz Paulino; Evans, Jay Daniel

2018-04-17

Like all other insects, two key signaling pathways (Toll and Imd) regulate the induction of honey bee immune effectors that target microbial pathogens. Among these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the NF-κB factors Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin-1 was regulated by the Toll pathway, while hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin-1. Honey bees have two dorsal genes (dorsal-1 and dorsal-2) and two splicing isoforms of dorsal-1 (dorsal-1A and dorsal-1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down-regulation of defensin-1 was observed for dorsal-1A and dorsal-2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin-1 is regulated by Dorsal (Toll pathway). This article is protected by copyright. All rights reserved. © 2018 The Royal Entomological Society.

The dose can make the poison: lessons learned from adverse in vivo toxicities caused by RNAi overexpression.

PubMed

Grimm, Dirk

2011-10-26

For the past five years, evidence has accumulated that vector-mediated robust RNA interference (RNAi) expression can trigger severe side effects in small and large animals, from cytotoxicity and accelerated tumorigenesis to organ failure and death. The recurring notions in these studies that a critical parameter is the strength of RNAi expression and that Exportin-5 and the Argonaute proteins are rate-limiting mammalian RNAi, strongly imply dose-dependent saturation of the endogenous miRNA pathway as one of the underlying mechanisms. This minireview summarizes the relevant work and data leading to this intriguing model and highlights potential avenues by which to alleviate RNAi-induced toxicities in future clinical applications.

C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling

PubMed Central

Falin, Rebecca A.; Miyazaki, Hiroaki

2011-01-01

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl− cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes. PMID:21160027

Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.

PubMed

Tomita, Hiroshi; Sugano, Eriko; Yawo, Hiromu; Ishizuka, Toru; Isago, Hitomi; Narikawa, Satoko; Kügler, Sebastian; Tamai, Makoto

2007-08-01

To investigate whether the channelopsin-2 (Chop2) gene would restore visual responses in 10-month-old dystrophic Royal College of Surgeons (aged RCS; rdy/rdy) rats, the authors transferred the Chop2 gene into the retinal cells of aged RCS rats using the adenoassociated virus (AAV) vector. The N-terminal fragment (residues 1-315) of Chop2 was fused to a fluorescent protein, Venus, in frame at the end of the Chop2 coding fragment. The viral vector construct (AAV-Chop2V) for the expression of the Chop2V in the retina was made by subcloning into an adenoassociated virus vector, including the CAG promoter. To evaluate the expression profile of Chop2V in the retina, the rats were killed and the eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Retinal wholemount specimens and cryosections were made. Under anesthetized conditions, electrodes for the recording of visually evoked potentials (VEPs) were implanted onto the visual cortex in aged-RCS (rdy/rdy) rats. AAV-Chop2V vectors were then injected into the vitreous cavity of the left eyes. As a control, AAV-Venus vectors were applied to the right eyes. VEPs were evoked by the flash of a blue, white, or red light-emitting diode (LED) and were recorded from the visual cortex of the rats at various time points after the AAV vector injection. Chop2V fluorescence was predominantly observed in retinal ganglion cells (RGCs). Some fluorescence was observed in the inner nuclear layer and the inner plexiform layer neurites. A tendency of recovery was observed in the VEPs of aged RCS (rdy/rdy) rats after the AAV-Chop2V injection but not after the AAV-Venus injection. The visual response of AAV-Chop2V-injected aged RCS (rdy/rdy) rats was less sensitive to the blue LED flash than that of nondystrophic RCS (+/+) rats. The AAV-Chop2V-injected aged RCS (rdy/rdy) rats were insensitive to the red LED flash, which evoked a robust VEP in the RCS (+/+) rats. The visual response of aged RCS (rdy/rdy) rats was partially restored by transduction of the Chop2 gene through AAV into the inner retinal neurons, mainly RGCs. These results suggest that the transduction of Chop2 would provide a new strategy to treat some retinitis pigmentosa (RP) symptoms independent of their etiology.

Gene expression analysis upon lncRNA DDSR1 knockdown in human fibroblasts

PubMed Central

Jia, Li; Sun, Zhonghe; Wu, Xiaolin; Misteli, Tom; Sharma, Vivek

2015-01-01

Long non-coding RNAs (lncRNAs) play important roles in regulating diverse biological processes including DNA damage and repair. We have recently reported that the DNA damage inducible lncRNA DNA damage-sensitive RNA1 (DDSR1) regulates DNA repair by homologous recombination (HR). Since lncRNAs also modulate gene expression, we identified gene expression changes upon DDSR1 knockdown in human fibroblast cells. Gene expression analysis after RNAi treatment targeted against DDSR1 revealed 119 genes that show differential expression. Here we provide a detailed description of the microarray data (NCBI GEO accession number GSE67048) and the data analysis procedure associated with the publication by Sharma et al., 2015 in EMBO Reports [1]. PMID:26697398

High throughput RNAi assay optimization using adherent cell cytometry.

PubMed

Nabzdyk, Christoph S; Chun, Maggie; Pradhan, Leena; Logerfo, Frank W

2011-04-25

siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

High throughput RNAi assay optimization using adherent cell cytometry

PubMed Central

2011-01-01

Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs. PMID:21518450

Activity-Dependent Ubiquitination of GluA1 Mediates a Distinct AMPAR Endocytosis and Sorting Pathway

PubMed Central

Schwarz, Lindsay A.; Hall, Benjamin J.; Patrick, Gentry N.

2010-01-01

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, while dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer’s disease. Previous work has shown that ubiquitination of integral membrane proteins is a common post-translational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its carboxy-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA, but not for internalization of AMPARs in response to the NMDA receptor (NMDAR) agonist NMDA. Through over-expression or RNAi-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1, is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues, and suggest that changes to this pathway may occur as neurons mature. PMID:21148011

Graphene Oxide Dysregulates Neuroligin/NLG-1-Mediated Molecular Signaling in Interneurons in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Chen, He; Li, Huirong; Wang, Dayong

2017-01-01

Graphene oxide (GO) can be potentially used in many medical and industrial fields. Using assay system of Caenorhabditis elegans, we identified the NLG-1/Neuroligin-mediated neuronal signaling dysregulated by GO exposure. In nematodes, GO exposure significantly decreased the expression of NLG-1, a postsynaptic cell adhesion protein. Loss-of-function mutation of nlg-1 gene resulted in a susceptible property of nematodes to GO toxicity. Rescue experiments suggested that NLG-1 could act in AIY interneurons to regulate the response to GO exposure. In the AIY interneurons, PKC-1, a serine/threonine protein kinase C (PKC) protein, was identified as the downstream target for NLG-1 in the regulation of response to GO exposure. LIN-45, a Raf protein in ERK signaling pathway, was further identified as the downstream target for PKC-1 in the regulation of response to GO exposure. Therefore, GO may dysregulate NLG-1-mediated molecular signaling in the interneurons, and a neuronal signaling cascade of NLG-1-PKC-1-LIN-45 was raised to be required for the control of response to GO exposure. More importantly, intestinal RNAi knockdown of daf-16 gene encoding a FOXO transcriptional factor in insulin signaling pathway suppressed the resistant property of nematodes overexpressing NLG-1 to GO toxicity, suggesting the possible link between neuronal NLG-1 signaling and intestinal insulin signaling in the regulation of response to GO exposure.