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Sample records for adenoviral e1a gene

  1. Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

    PubMed

    Akbulut, Hakan; Zhang, Lixin; Tang, Yucheng; Deisseroth, Albert

    2003-05-01

    Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the

  2. CREB (cAMP response element binding protein) and C/EBPalpha (CCAAT/enhancer binding protein) are required for the superstimulation of phosphoenolpyruvate carboxykinase gene transcription by adenoviral E1a and cAMP.

    PubMed Central

    Routes, J M; Colton, L A; Ryan, S; Klemm, D J

    2000-01-01

    In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha. PMID:11085926

  3. A novel CRM1-dependent nuclear export signal in adenoviral E1A protein regulated by phosphorylation.

    PubMed

    Jiang, Hong; Olson, Melissa V; Medrano, Diana R; Lee, Ok-Hee; Xu, Jing; Piao, Yuji; Alonso, Marta M; Gomez-Manzano, Candelaria; Hung, Mien-Chie; Yung, W K Alfred; Fueyo, Juan

    2006-12-01

    Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication. The nuclear import of E1A is mediated by a nuclear localization signal; however, whether E1A can be actively exported from the nucleus is unknown. We first reported a CRM1-dependent nuclear export signal (NES) in E1A that is conserved in the group C adenoviruses. We showed that CRM1 and E1A coimmunoprecipitated and that blockage of CRM1 function by leptomycin B or small interfering RNA resulted in the nuclear localization of E1A. Through mutational analyses, we identified an active canonical NES element within the E1A protein spanning amino acids 70-80. We further demonstrated that phosphorylation of adjacent serine (S)89 resulted in the cytoplasmic accumulation of E1A. Interestingly, coincident with the accumulation of cells in the S/G2/M phase and histone H1 phosphorylation, E1A was relocated to the cytoplasm at the late stage of the viral cycle, which was blocked by the CDC2/CDK2 inhibitor roscovitine. Importantly, titration of the progenies of the viruses in infected cells showed that the replication efficiency of the NES mutant adenovirus was up to 500-fold lower than that of the wild-type adenovirus. Collectively, our data demonstrate the existence of a NES in E1A that is modulated by the phosphorylation of the S89 residue and the NES plays a role for an efficient viral replication in the host cells.

  4. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  5. Adenoviral vector-mediated gene transfer for human gene therapy.

    PubMed

    Breyer, B; Jiang, W; Cheng, H; Zhou, L; Paul, R; Feng, T; He, T C

    2001-07-01

    Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.

  6. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. PMID:27382434

  7. Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing.

    PubMed

    Himmelspach, M; Cavaloc, Y; Chebli, K; Stévenin, J; Gattoni, R

    1995-10-01

    Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

  8. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  9. Readministration of adenoviral gene delivery to dopamine neurons.

    PubMed

    Gonzalez, Sarah C; McMenamin, Margaret M; Charlton, Harry M; Goodman, James; Lantos, Tibor; Simpson, Christine; Wood, Matthew J A

    2007-10-01

    An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited. PMID:17885611

  10. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

  11. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

  12. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  13. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

    PubMed Central

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M.; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A. F. V.

    2013-01-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 ‘safe harbor’ locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform. PMID:23275534

  14. Transcription of interferon-stimulated genes is induced by adenovirus particles but is suppressed by E1A gene products.

    PubMed Central

    Reich, N; Pine, R; Levy, D; Darnell, J E

    1988-01-01

    Interferon treatment of cell cultures results in the rapid transcriptional induction of a specific set of genes. In this paper we explore the effect of cellular infection by several adenoviruses, both wild type and mutant, on the expression of these genes. Infection with adenovirus induces the transcription of the interferon-stimulated genes in the absence of any protein synthesis. In fact, the inhibition of protein synthesis during a wild-type infection produces enhanced stimulation of transcription of these genes. Experiments with viral mutants indicate the ability to specifically suppress this transcription maps to the E1A gene. In addition, the E1A gene products are capable of suppressing the specific transcriptional induction of interferon-stimulated promoters during cotransfection experiments and therefore presumably during viral infection. The dual effect of adenovirus on the expression of interferon-stimulated genes may represent an example of action and evolutionary reaction between virus and host. Images PMID:2446013

  15. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  16. Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

    PubMed Central

    Vetrini, Francesco; Ng, Philip

    2010-01-01

    Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd) vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application. PMID:21994713

  17. Adenovirus E1A Targets the DREF Nuclear Factor To Regulate Virus Gene Expression, DNA Replication, and Growth

    PubMed Central

    Radko, Sandi; Koleva, Maria; James, Kris M. D.; Jung, Richard; Mymryk, Joe S.

    2014-01-01

    ABSTRACT The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication. IMPORTANCE This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to

  18. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

    PubMed Central

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-01-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models. PMID:27274908

  19. Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer.

    PubMed

    Kim, Soo-Yeon; Lee, Sang-Jin; Lim, Soo-Jeong

    2014-11-20

    Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(poly-ethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus-emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus-emulsion complex may act as a lung-targeting adenoviral gene delivery system.

  20. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  1. Adenoviral-vector-mediated gene transfer to dendritic cells.

    PubMed

    Song, W; Crystal, R G

    2001-01-01

    Dendritic cells (DC) are the most potent antigen presenting cells capable of initiating T-cell-dependent immune responses (1-5). This biologic potential can be harnessed to elicit effective antigen-specific immune responses by transferring the relevant antigens to the DC. Once the DC have been mobilized and purified, the relevant antigens can be transferred to the DC as intact proteins, or as peptides representing specific epitopes, or with gene transfer using sequences of DNA or RNA coding for the pertinent antigen(s) (6-15). Theoretically, genetically modifying DC with genes coding for specific antigens has potential advantages over pulsing the DC with peptides repeating the antigen or antigen fragment. First, the genetically modified DC may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DC ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted cytotoxic T lymphocytes (CTL), the effector arm of cell-mediated immune responses. Finally, in addition to genes coding for the antigen(s), genetic modification of the DC can induce genes coding for mediators relevant to generation of the immune response to the antigen(s), further boosting host responses to the antigens presented by the modified DC. Different gene transfer approaches have been explored to genetically modify DC, including retroviral vectors (16-18), recombinant vaccinia virus vectors (19), and recombinant adenovirus (Ad) vectors (19-23). The focus of this chapter is on using recombinant Ad vectors to transfer genes to murine DC. We have used a similar strategy to transfer genes to human DC (24). As an example of the power of this technology, we will describe the use of Ad-vector-modified DC to suppress the growth of tumor cells modified to express a specific antigen.

  2. Gene transfer into neural cells in vitro using adenoviral vectors.

    PubMed

    Southgate, T D; Kingston, P A; Castro, M G

    2001-05-01

    Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post-mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication-defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step-by-step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell-sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.

  3. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  4. Immune responses to adenoviral vectors during gene transfer in the brain.

    PubMed

    Kajiwara, K; Byrnes, A P; Charlton, H M; Wood, M J; Wood, K J

    1997-02-10

    We have investigated the immune response to E1-deleted adenovirus vectors encoding the lacZ gene introduced into the brains of adult mice. Injection of these nonreplicating vectors caused a marked inflammatory response in the brain as assessed by immunocytochemistry and flow cytometry of leukocytes. Infiltrating leukocytes were detectable within 2 days of injection and reached a maximum by 9 days. Thereafter, the number of infiltrating cells decreased, but a small number persisted in the brain until day 60. Between 2 and 4 days after injection, the percentage of CD8+ cells detectable increased whereas the percentage of CD4+ cells present in the infiltrating population did not significantly increase until day 6, peaking on day 15. Activated CD25+ T cells were detectable between days 6 and 15. beta-Galactosidase (beta-Gal), the product of the lacZ gene encoded by the vector, was also detected, both at the injection site in the striatum and also in the substantia nigra. Expression peaked between 4 and 6 days but a small number of beta-Gal+ cells was still seen at 60 days after injection. This study demonstrates that a quantitative analysis of the immune responses caused by a nonreplicating adenovirus vector is possible in the brain. E1-deleted adenoviral vectors trigger a strong inflammatory response in the brain, but this immune response is not sufficient to eliminate completely expression of genes encoded by the adenoviral construct. PMID:9048192

  5. Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers

    PubMed Central

    Lecollinet, S.; Gavard, F.; Havenga, M. J. E.; Spiller, O. B.; Lemckert, A.; Goudsmit, J.; Eloit, M.; Richardson, J.

    2006-01-01

    A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses. PMID:16501084

  6. Adenoviral vector-mediated insulin gene transfer in the mouse pancreas corrects streptozotocin-induced hyperglycemia.

    PubMed

    Shifrin, A L; Auricchio, A; Yu, Q C; Wilson, J; Raper, S E

    2001-10-01

    Therapy for type 1 diabetes consists of tight blood glucose (BG) control to minimize complications. Current treatment relies on multiple insulin injections or an insulin pump placement, beta-cell or whole pancreas transplantation. All approaches have significant limitations and have led to the realization that novel treatment strategies are needed. Pancreatic acinar cells have features that make them a good target for insulin gene transfer. They are not subject to autoimmune attack, a problem with pancreas or islets transplantation, they are avidly transduced by recombinant adenoviral vectors, and capable of exporting a variety of peptides into the portal circulation. Recombinant adenoviral vectors were engineered to express either wild-type or furin-modified human insulin cDNA (AdCMVhInsM). Immunodeficient mice were made diabetic with streptozotocin and injected intrapancreatically with the vectors. BG and blood insulin levels have normalized after administration of AdCMVhInsM. Immunohistochemistry and electron microscopy showed the presence of insulin in acinar cells throughout the pancreas and localization of insulin molecules to acinar cell vesicles. The data clearly establish a relationship between intrapancreatic vector administration, decreased BG and elevated blood insulin levels. The findings support the use of pancreatic acinar cells to express and secrete insulin into the blood stream. PMID:11593361

  7. Magnetically Responsive Biodegradable Nanoparticles Enhance Adenoviral Gene Transfer in Cultured Smooth Muscle and Endothelial Cells

    PubMed Central

    Chorny, Michael; Fishbein, Ilia; Alferiev, Ivan; Levy, Robert J.

    2012-01-01

    Replication-defective adenoviral (Ad) vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their clinical use is however limited by therapeutically suboptimal transduction levels in cell types expressing low levels of Coxsackie-Ad receptor (CAR), the primary receptor responsible for the cell entry of the virus, and by systemic adverse reactions. Targeted delivery achievable with Ad complexed with biodegradable magnetically responsive nanoparticles (MNP) may therefore be instrumental for improving both the safety and efficiency of these vectors. Our hypothesis was that magnetically driven delivery of Ad affinity-bound to biodegradable MNP can substantially increase transgene expression in CAR deficient vascular cells in culture. Fluorescently labeled MNP were formulated from polylactide with inclusion of iron oxide and surface-modified with the D1 domain of CAR as an affinity linker. MNP cellular uptake and GFP reporter transgene expression were assayed fluorimetrically in cultured endothelial and smooth muscle cells using λex/λem of 540 nm/575 nm and 485 nm/535 nm, respectively. Stable vector-specific association of Ad with MNP resulted in formation of MNP–Ad complexes displaying rapid cell binding kinetics following a brief exposure to a high gradient magnetic field with resultant gene transfer levels significantly increased compared to free vector or nonmagnetic control treatment. Multiple regression analysis suggested a mechanism of MNP–Ad mediated transduction distinct from that of free Ad, and confirmed the major contribution of the complexes to the gene transfer under magnetic conditions. The magnetically enhanced transduction was achieved without compromising the cell viability or growth kinetics. The enhancement of adenoviral gene delivery by affinity complexation with biodegradable MNP represents a promising approach with a potential to extend the applicability of the viral gene therapeutic strategies. PMID:19496618

  8. Lipid- and adenoviral-mediated gene transfer into AIDS-Kaposi's sarcoma cell lines.

    PubMed

    Campain, J A; Matassa, A A; Felgner, P L; Barnhart, K M; Curiel, D T; Harrison, G S

    1998-01-01

    Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.

  9. Modifications to the INSM1 promoter to preserve specificity and activity for use in adenoviral gene therapy of neuroendocrine carcinomas.

    PubMed

    Akerstrom, V; Chen, C; Lan, M S; Breslin, M B

    2012-12-01

    The INSM1 gene encodes a transcriptional repressor that is exclusively expressed in neuronal and neuroendocrine tissue during embryonic development that is re-activated in neuroendocrine tumors. Using the 1.7 kbp INSM1 promoter, an adenoviral HSV thymidine kinase gene therapy was tested for the treatment of neuroendocrine tumors. An unforeseen interference on the INSM1 promoter specificity from the adenoviral genome was observed. Attempts were made to protect the INSM1 promoter from the influence of essential adenoviral sequences and to further enhance the tissue specificity of the INSM1 promoter region. Using the chicken β-globin HS4 insulator sequence, we eliminated off-target tissue expression from the Ad-INSM1 promoter-luciferase2 constructs in vivo. In addition, inclusion of two copies of the mouse nicotinic acetylcholine receptor (n(AchR)) neuronal-restrictive silencer element (NRSE) reduced nonspecific activation of the INSM1 promoter both in vitro and in vivo. Further, inclusion of both the HS4 insulator with the n(AchR) 2 × NRSE modification showed a two log increase in luciferase activity measured from the NCI-H1155 xenograft tumors compared with the original adenovirus construct. The alterations increase the therapeutic potential of adenoviral INSM1 promoter-driven suicide gene therapy for the treatment of a variety of neuroendocrine tumors. PMID:23079673

  10. Effects of an adenoviral vector containing a suicide gene fusion on growth characteristics of breast cancer cells.

    PubMed

    Kong, Heng; Liu, Chunli; Zhu, Ting; Huang, Zonghai; Yang, Liucheng; Li, Qiang

    2014-12-01

    The herpes simplex virus thymidine kinase/ganciclovir (HSV‑TK/GCV) and the cytosine deaminase/5‑fluorocytosine (CD/5‑FC) systems have been widely applied in suicide gene therapy for cancer. Although suicide gene therapy has been successfully used in vitro and in vivo studies, the number of studies on the effects of recombinant adenoviruses (Ads) containing suicide genes on target cancer cells is limited. The aim of this study was to examine whether recombinant Ads containing the CD/TK fusion gene affect cell proliferation of breast cancer cells in vitro. In the present study, we explored the use of a recombinant adenoviral vector to deliver the CD/TK fusion gene to the breast cancer cell line MCF‑7. We found that the recombinant adenoviral vector efficiently infected MCF‑7 cells. Western blot analysis revealed that CD and TK proteins are expressed in the infected cells. The infected breast cancer cells did not show any significant changes in morphology, ultrastructure, cell growth, and cell‑cycle distribution compared to the uninfected cells. This study revealed that the Ad‑vascular endothelial growth factor promoter (VEGFp)‑CD/TK vector is non‑toxic to MCF‑7 cells at the appropriate titer. Our results indicate that it is feasible to use a recombinant adenoviral vector containing the CD/TK fusion gene in suicide gene therapy to target breast cancer cells. PMID:25323393

  11. Robust Hepatic Gene Silencing for Functional Studies Using Helper-Dependent Adenoviral Vectors

    PubMed Central

    Ruiz, Rafaela; Witting, Scott R.; Saxena, Romil

    2009-01-01

    Abstract RNA interference is currently envisioned as the basis of gene function and drug target validation studies. This novel technology has the advantage of providing a remarkably faster tool for gene silencing than traditional transgenic animal methodologies. In vivo administration of short interfering RNA (siRNA) typically results in reduced target gene expression for approximately 1 week. Viral vectors offer the possibility to express constitutive levels of short hairpin RNA (shRNA) so that the effects of knocking down the target gene can be studied for a few weeks, rather than a few days. Helper-dependent vectors have a significant advantage over previous generations of adenoviral vectors because of their much higher cloning capacity, potential for long-term transgene expression, and enhanced safety profiles on administration in vivo. Therefore, this advanced type of vector is an excellent tool to carry out in vivo studies directed at constitutive expression of shRNA. Here we show it is possible to obtain more than 90% target gene knockdown in an animal model of type 2 diabetes for several weeks, thereby consolidating this technology as an alternative to generating liver-specific knockout animals. PMID:18828727

  12. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

  13. Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer

    NASA Astrophysics Data System (ADS)

    Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.

    1997-10-01

    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.

  14. Inhibition of IFN-stimulated gene expression and IFN induction of cytolytic resistance to natural killer cell lysis correlate with E1A-p300 binding.

    PubMed

    Routes, J M; Li, H; Bayley, S T; Ryan, S; Klemm, D J

    1996-02-01

    Treatment of target cells with IFN induces resistance to NK cell lysis. This process is blocked by expression of E1A gene products in adenovirus (Ad)-infected and Ad-transformed cells. We compared the ability of adenovirus serotype 5 (Ad5) E1A exon 1 mutants to inhibit the induction of cytolytic resistance by IFN and block IFN-stimulated gene expression with their capacity to bind the cellular proteins p105 (retinoblastoma gene product), p107, and p300. E1A mutants that did not express conserved region 3 (CR3; residues 138-184) or contained deletions in the nonconserved regions between residues 26-35 or 86-120, bound p105, p107, and p300 and were not impaired in their capacity to block IFN-stimulated gene expression or IFN's induction of cytolytic resistance. E1A mutants with deletions in CR2 (residues 121-138) could not bind p105 or p107, but blocked IFN-stimulated gene expression and IFN's induction of cytolytic resistance. In contrast, mutants in CR1 or the N-terminal nonconserved region (residues 2, 4-25, and 48-60), which define E1A's binding site for p300, were unable to block either IFN-stimulated gene expression or IFN's induction of cytolytic resistance. We conclude that E1A's capacity to block both IFN-stimulated gene expression and IFN's induction of cytolytic resistance appears to be transduced through a pathway that involves E1A-p300 binding. The capacity of E1A to block IFN's induction of cytolytic resistance is probably secondary to E1A's more general ability to inhibit IFN-stimulated gene expression. PMID:8557979

  15. Progress and prospects: gene therapy for genetic diseases with helper-dependent adenoviral vectors

    PubMed Central

    Brunetti-Pierri, N; Ng, P

    2013-01-01

    Preclinical studies in small and large animal models using helper-dependent adenoviral vectors (HDAds) have generated promising results for the treatment of genetic diseases. However, clinical translation is complicated by the dose-dependent, capsid-mediated acute toxic response following systemic vector injection. With the advancements in vectorology, a better understanding of vector-mediated toxicity, and improved delivery methods, HDAds may emerge as an important vector for gene therapy of genetic diseases and this report highlights recent progress and prospects in this field. In briefProgressHDAds provide stable, long-term transgene expression in small and large animal models without chronic toxicity for liver-directed gene therapy.High vector doses are required for efficient hepatocyte transduction by systemic administration.Strategies to improve the therapeutic index of HDAd are available or currently under investigation for liver-directed gene therapy.High-efficiency pulmonary transduction and clinically relevant end points can be achieved delivering HDAd in conjunction with tight junction opening agents for CF gene therapy.HDAd delivered with an intracorporeal nebulizing catheter results in high-efficiency transduction of the respiratory epithelium in large animals.Encouraging results have been obtained with HDAd for brain- and muscle-directed gene therapy in animal models.ProspectsA better understanding of the acute innate response will provide new targets for pharmacological blockade to improve the therapeutic index of the vector.Further optimization of preferential liver targeting by HDAd through balloon catheter delivery has the potential of providing a clinically attractive method of vector delivery.Further assessment of Ad PEGylation and modulation of the liver fenestrations may provide attractive strategies to increase the therapeutic index of the vector.Capsid modification to increase the affinity of Ad for hepatocytes has the potential to improve

  16. E1a promotes c-Myc-dependent replicative stress

    PubMed Central

    Valero, María Llanos; Cimas, Francisco Jose; Arias, Laura; Melgar-Rojas, Pedro; García, Elena; Callejas-Valera, Juan Luis; García-Cano, Jesús; Serrano-Oviedo, Leticia; Ángel de la Cruz-Morcillo, Miguel; Sánchez-Pérez, Isabel; Sánchez-Prieto, Ricardo

    2014-01-01

    The E1a gene from adenovirus is known to be a potent inducer of chemo/radiosensitivity in a wide range of tumors. However, the molecular bases of its radiosensitizer properties are still poorly understood. In an attempt to study this effect, U87MG cells, derived from a radio-resistant tumor as glioblastoma, where infected with lentivirus carrying E1a gene developing an acute sensitivity to ionizing radiation. The induction of radiosensitivity correlated with a marked G2/M phase accumulation and a potent apoptotic response. Our findings demonstrate that c-Myc plays a pivotal role in E1a-associated radiosensitivity through the induction of a replicative stress situation, as our data support by genetic approaches, based in interference and overexpression in U87MG cells. In fact, we present evidence showing that Chk1 is a novel transcriptional target of E1a gene through the effect exerted by this adenoviral protein onto c-Myc. Moreover, c-Myc upregulation also explains the marked phosphorylation of H2AX associated to E1a expression in the absence of DNA damage. Indeed, all these observations were applicable to other experimental models, such as T98G, LN-405 and A172, rendering the same pattern in terms of radiosensitivity, cell cycle distribution, upregulation of Chk1, c-Myc, and phosphorylation pattern of H2AX. In summary, our data propose a novel mechanism to explain how E1a mediates radiosensitivity through the signaling axis E1a→c-Myc→ replicative stress situation. This novel mechanism of E1a-mediated radiosensitivity could be the key to open new possibilities in the current therapy of glioblastoma. PMID:24196438

  17. Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

    PubMed Central

    Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

    2015-01-01

    SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

  18. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    PubMed Central

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of > 95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. PMID:25173815

  19. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  20. In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV.

    PubMed

    Montenegro-Miranda, P S; Pichard, V; Aubert, D; Ten Bloemendaal, L; Duijst, S; de Waart, D R; Ferry, N; Bosma, P J

    2014-02-01

    Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

  1. Tropism-modification strategies for targeted gene delivery using adenoviral vectors.

    PubMed

    Coughlan, Lynda; Alba, Raul; Parker, Alan L; Bradshaw, Angela C; McNeish, Iain A; Nicklin, Stuart A; Baker, Andrew H

    2010-10-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and "bridging" interactions. "Bridging" interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of "bridging interactions" such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated "stealth" vectors

  2. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    PubMed

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

  3. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases. PMID:27245510

  4. Efficient gene transfer into normal human B lymphocytes with the chimeric adenoviral vector Ad5/F35.

    PubMed

    Jung, Daniel; Néron, Sonia; Drouin, Mathieu; Jacques, Annie

    2005-09-01

    The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naïve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.

  5. Tumorigenicity and adenovirus-transformed cells: Collagen interaction and cell surface laminin are controlled by the serotype origin of the E1A and E1B genes

    SciTech Connect

    Bober, F.J.; Birk, D.E.; Raska, K. Jr. ); Shenk, T. )

    1988-02-01

    A library of cells transformed with recombinant adenoviruses was used to study tumorigenicity and interaction with extracellular matrix. Cells expressing the complete E1 region of highly oncogenic adenovirus type 12 (Ad12) are tumorigenic, adhere preferentially to type IV collagen, and express cell surface laminin. Weakly tumorigenic cells, which express the E1A oncogene of Ad12 and the E1B genes of Ad5, also attach preferentially to type IV collagen but do not contain laminin on their surface. Cells which express the E1A oncogene of Ad5 and the E1B genes of Ad12 are nontumorigenic and do not preferentially attach to type IV versus type I collagen but have laminin on their surface. There is no significant difference in the amounts of laminin secreted into the culture medium among cells expressing the E1B genes of Ad5 or Ad12. In vitro assays show that cells which express the E1B genes of Ad12, irrespective of the origin of the E1A genes, can bind three times more exogenously added {sup 125}I-laminin than cells expressing the E1B genes of nononcogenic Ad5. The interaction of adenovirus-transformed cells with collagen is controlled by the serotype origin of the E1A oncogene, whereas cell surface laminin is controlled by the serotype origin of the E1B genes.

  6. Novel recombinant adenoviral vector that targets the interleukin-13 receptor alpha2 chain permits effective gene transfer to malignant glioma.

    PubMed

    Ulasov, Ilya V; Tyler, Matthew A; Han, Yu; Glasgow, Joel N; Lesniak, Maciej S

    2007-02-01

    Transduction of malignant glioma with adenovirus serotype 5 (Ad5) vectors is limited by the low levels of coxsackievirus and adenovirus receptor (CAR) on tumor cells. However, malignant brain tumors have been found to overexpress a glioma-associated receptor, interleukin-13 receptor alpha2 chain (IL-13Ralpha2), a marker of both glial transformation and tumor grade. To selectively target Ad5 to IL-13Ralpha2, we constructed a replication-deficient adenoviral vector that possesses an IL-13 ligand presented by a T4 phage fibritin shaft, and designated the new virus LU-13. Western blot and sequence analyses confirmed proper trimerization and ligand presentation by the T4 fibritin shaft. Confocal microscopy analysis of primary glioma suspensions incubated with viral recombinants showed that LU-13 colocalized with IL-13Ralpha2. Luciferase transduction assays conducted in both primary and passaged glioma cell cultures exhibited at least 10-fold enhanced gene transduction. Moreover, the virus preferentially bound to glioma cells, as documented by increased adenoviral E4 DNA copy number. In vitro competition assays performed with anti-human IL-13 monoclonal antibody confirmed significant attenuation of LU-13 transduction. These results were further confirmed in vivo, where LU-13 showed a 300-fold increase in transgene expression. In summary, we describe here the development of a novel and targeted adenoviral vector that binds IL-13Ralpha2. Our findings confirm the ability of LU-13 to bind IL-13Ralpha2 and increase transgene expression, making it an attractive gene therapy vector for the treatment of malignant glioma in a clinical setting.

  7. Enhanced Prostacyclin Synthesis by Adenoviral Gene Transfer Reduced Glial Activation and Ameliorated Dopaminergic Dysfunction in Hemiparkinsonian Rats

    PubMed Central

    Tsai, May-Jywan; Weng, Ching-Feng; Yu, Nien-Chu; Liou, Dann-Ying; Kuo, Fu-San; Huang, Ming-Chao; Huang, Wen-Cheng; Tam, Kabik; Shyue, Song-Kun; Cheng, Henrich

    2013-01-01

    Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway. PMID:23691265

  8. Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling.

    PubMed

    Cittadini, A; Monti, M G; Iaccarino, G; Di Rella, F; Tsichlis, P N; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

    2006-01-01

    The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the beta-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca(2+)) handling, oxygen consumption, and beta-adrenergic pathway. To this aim, Sprague-Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca(2+) handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca(2+)-force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6+/-0.2 versus 2.7+/-0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca(2+) available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca(2+) responsiveness, documented by an increased maximal Ca(2+)-activated pressure (+19% versus controls) and a shift to the left of the Ca(2+)-force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca(2+) ATPase protein levels were significantly increased in Ad.Akt rats. beta-Adrenergic receptor density, affinity, kinase-1

  9. Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

    PubMed Central

    Yoshida, K; Narita, M; Fujinaga, K

    1989-01-01

    Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor. Images PMID:2532319

  10. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein.

    PubMed Central

    Hateboer, G; Timmers, H T; Rustgi, A K; Billaud, M; van 't Veer, L J; Bernards, R

    1993-01-01

    Using a protein binding assay, we show that the amino-terminal 204 amino acids of the c-Myc protein interact directly with a key component of the basal transcription factor TFIID, the TATA box-binding protein (TBP). Essentially the same region of the c-Myc protein also binds the product of the retinoblastoma gene, the RB protein. c-Myc protein coimmunoprecipitates with TBP in lysates of mammalian cells, demonstrating that the proteins are also complexed in vivo. A short peptide that spans the RB binding site of the E7 protein of human papilloma virus type 16 interferes with the binding of c-Myc to TBP. The same peptide also blocks binding of adenovirus E1A protein to TBP, suggesting that c-Myc and E1A bind to RB and TBP through overlapping epitopes. Furthermore, we show that binding of RB to E1A prevents association of E1A with TBP. Our data suggest that one of the functions of RB and RB-like proteins is to prevent interaction of viral and cellular oncoproteins, such as c-Myc and E1A, with TBP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7690963

  11. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  12. Combined use of adenoviral vector Ad5/F35-mediated APE1 siRNA enhances the therapeutic efficacy of adenoviral-mediated p53 gene transfer in hepatoma cells in vitro and in vivo.

    PubMed

    Cun, Yanping; Zhang, Qinhong; Xiong, Chengjie; Li, Mengxia; Dai, Nan; Zhang, Shiheng; Wang, Dong

    2013-06-01

    Gene therapy has emerged as a novel therapeutic approach for the treatment of cancer. In order to establish a more effective therapeutic strategy against unresectable hepatocellular carcinoma (HCC), we evaluated, in the present study, the effects of combined treatment with adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1) and adenoviral-mediated p53 gene transfer (Ad-p53) in hepatoma cells in vitro and in vivo. Infection of SMMC-7721 cells with Ad5/F35-siAPE1 resulted in a time- and dose-dependent decrease of APE1 protein, while Ad-p53 treatment led to a time- and dose-dependent increase of p53 protein expression. Ad5/F35-siAPE1 significantly enhanced the cytotoxic effect of SMMC-7721 cells to Ad-p53 in cell survival assays, associated with increased cell apoptosis. Moreover, administration of Ad5/F35-siAPE1 and Ad-p53 into nude mice resulted in tumor growth inhibition and apoptosis induction in SMMC-7721 xenografts compared to administration of either agent alone. These results suggest that combination of Ad5/F35-siAPE1 and Ad-p53 could be a promising gene therapeutic approach against human HCC.

  13. Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease.

    PubMed

    Ziegler, Robin J; Li, Chester; Cherry, Maribeth; Zhu, Yunxiang; Hempel, Donna; van Rooijen, Nico; Ioannou, Yiannis A; Desnick, Robert J; Goldberg, Mark A; Yew, Nelson S; Cheng, Seng H

    2002-05-20

    Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the

  14. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

    PubMed Central

    Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

    2016-01-01

    Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

  15. Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system.

    PubMed

    Zhang, Feng; Yao, Yongchang; Su, Kai; Fang, Yu; Citra, Fudiman; Wang, Dong-An

    2015-09-01

    Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

  16. MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

    SciTech Connect

    Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

    2013-05-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC

  17. Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

    PubMed

    Li, Rongying; Oka, Kazuhiro; Yechoor, Vijay

    2012-01-01

    Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice. PMID:23093064

  18. Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

    PubMed Central

    Stoff, Alexander; Rivera, Angel A.; Banerjee, N. S.; Mathis, J. Michael; Espinosa-de-los-Monteros, Antonio; Le, Long P.; De la Torre, Jorge I.; Vasconez, Luis O.; Broker, Thomas R.; Richter, Dirk F.; Stoff-Khalili, Mariam A.; Curiel, David T.

    2007-01-01

    Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, αυ integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of αυ integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy. PMID:17014674

  19. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses. PMID:14654922

  20. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

  1. Delivery of adenoviral DNA to mouse liver.

    PubMed

    Connelly, Sheila; Mech, Christine

    2004-01-01

    The liver represents a major target organ for gene delivery owing to its high biosynthetic capacity and access to the bloodstream. Adenoviral vectors are highly efficient gene-transfer vehicles, making them among the most promising systems for in vivo gene transfer to the liver. Following intravenous administration of adenoviral vectors to a variety of mammalian models, including mice, dogs, and monkeys, hepatocytes are efficiently transduced. Several delivery methods to the liver have been described, including portal vein (2-4), hepatic artery (3,5), and peripheral vein infusions (6). This chapter describes the simple, nonsurgical method of intravenous (iv) administration of adenoviral vectors in mice, and an immunohistochemical method to qualitatively evaluate liver transduction efficiency following delivery of an adenoviral vector encoding a bgalactosidase (beta-gal) marker gene. Additionally, several alternative methods to verify efficient liver transduction are introduced.

  2. Chimeric Adenoviral Vectors Incorporating a Fiber of Human Adenovirus 3 Efficiently Mediate Gene Transfer into Prostate Cancer Cells

    PubMed Central

    Murakami, Miho; Ugai, Hideyo; Belousova, Natalya; Pereboev, Alexander; Dent, Paul; Fisher, Paul B.; Everts, Maaike; Curiel, David T.

    2010-01-01

    BACKGROUND We have developed a range of adenoviral (Ad) vectors based on human adenovirus serotype 5 (HAdV-5) displaying the fiber shaft and knob domains of species B viruses (HAdV-3, HAdV-11, or HAdV-35). These species B Ads utilize different cellular receptors than HAdV-5 for infection. We evaluated whether Ad vectors displaying species B fiber shaft and knob domains (Ad5F3Luc1, Ad5F11Luc1, and Ad5F35Luc1) would efficiently infect cancer cells of distinct origins, including prostate cancer. METHODS The fiber chimeric Ad vectors were genetically generated and compared with the original Ad vector (Ad5Luc1) for transductional efficiency in a variety of cancer cell lines, including prostate cancer cells and primary prostate epithelial cells (PrEC), using luciferase as a reporter gene. RESULTS Prostate cancer cell lines infected with Ad5F3Luc1 expressed higher levels of luciferase than Ad5Luc1, as well as the other chimeric Ad vectors. We also analyzed the transductional efficiency via monitoring of luciferase activity in prostate cancer cells when expressed as a fraction of the gene transfer in PrEC cells. In the PC-3 and DU145 cell lines, the gene transfer ratio of cancer cells versus PrEC was once again highest for Ad5F3Luc1. CONCLUSION Of the investigated chimeric HAdV-5/species B vectors, Ad5F3Luc1 was judged to be the most suitable for targeting prostate cancer cells as it showed the highest transductional efficiency in these cells. It is foreseeable that an Ad vector incorporating the HAdV-3 fiber could potentially be used for prostate cancer gene therapy. PMID:19902467

  3. Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

    PubMed Central

    Cela, Racel G.; Suzuki, Masataka; Lee, Brendan; Lipshutz, Gerald S.

    2011-01-01

    Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential “cure.” Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained. PMID:21245323

  4. MKP1 mediates chemosensitizer effects of E1a in response to cisplatin in non-small cell lung carcinoma cells

    PubMed Central

    Pascual-Serra, Raquel; García-Cano, Jesus; Garcia-Gil, Elena; De la Cruz-Morcillo, Miguel; Ortega-Muelas, Marta; Serrano-Oviedo, Leticia; Gutkind, J. Silvio; Sánchez-Prieto, Ricardo

    2015-01-01

    The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells. In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene. PMID:26689986

  5. INSM1 promoter-driven adenoviral herpes simplex virus thymidine kinase cancer gene therapy for the treatment of primitive neuroectodermal tumors.

    PubMed

    Wang, Hong-Wei; Breslin, Mary B; Chen, Chiachen; Akerstrom, Victoria; Zhong, Qiu; Lan, Michael S

    2009-11-01

    The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs. PMID:19604042

  6. Construction of an adenoviral expression vector carrying FLAG and hrGFP-1 genes and its expression in bone marrow mesenchymal stem cells.

    PubMed

    Wang, G X; Hu, L; Zhang, Z; Liu, D P

    2014-02-20

    The aim of this study was to construct an adenoviral expression vector for vascular endothelium growth factor 121 (VEGF121)-FLAG and humanized Renilla reniformis green fluorescent protein (hrGFP-1) genes, and to observe their expressions in bone marrow mesenchymal stem cells. Using pTG19T-VEGF121 as a template, polymerase chain reaction technology was adopted to mutate the VEGF121 gene by removing the stop codon and inserting NotI and XhoI restriction sites both before and after the gene sequences. The resultant gene was then subcloned into a pMD19-T plasmid, the pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids were double-digested, and small and large fragments were linked after gel recovery to complete the construction of recombinant adenovirus vectors. After titer determination, the recombinant adenovirus vectors were used to affect rabbit bone marrow mesenchymal stem cells, and fluorescence intensity was observed under fluorescence microscopy. Enzyme digestion identification and sequencing confirmed that the recombinant plasmids were successfully constructed, and observations under fluorescence microscopy showed significant expression of green fluorescent protein in recombinant adenovirus-infected bone marrow mesenchymal stem cells. The constructed adenoviral gene expression vectors carrying VEGF121-FLAG and hrGFP-1 can be expressed in eukaryotic cells, which may be used for gene therapy of ischemic disorders.

  7. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

  8. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  9. Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein.

    PubMed

    Lears, K A; Parry, J J; Andrews, R; Nguyen, K; Wadas, T J; Rogers, B E

    2015-03-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy owing to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that both the SSTR2 and yCD were functional in binding assays, conversion assays and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  10. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  11. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  12. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

    PubMed Central

    Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

    1999-01-01

    The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

  13. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma.

    PubMed

    Wang, Yi-Gang; Huang, Pan-Pan; Zhang, Rong; Ma, Bu-Yun; Zhou, Xiu-Mei; Sun, Yan-Fang

    2016-01-01

    Human hepatocellular carcinoma (HCC) heavily endangers human heath worldwide. HCC is one of most frequent cancers in China because patients with liver disease, such as chronic hepatitis, have the highest cancer susceptibility. Traditional therapeutic approaches have limited efficacy in advanced liver cancer, and novel strategies are urgently needed to improve the limited treatment options for HCC. This review summarizes the basic knowledge, current advances, and future challenges and prospects of adeno-associated virus (AAV) and adenoviruses as vectors for gene therapy of HCC. This paper also reviews the clinical trials of gene therapy using adenovirus vectors, immunotherapy, toxicity and immunological barriers for AAV and adenoviruses, and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors. PMID:26755879

  14. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma

    PubMed Central

    Wang, Yi-Gang; Huang, Pan-Pan; Zhang, Rong; Ma, Bu-Yun; Zhou, Xiu-Mei; Sun, Yan-Fang

    2016-01-01

    Human hepatocellular carcinoma (HCC) heavily endangers human heath worldwide. HCC is one of most frequent cancers in China because patients with liver disease, such as chronic hepatitis, have the highest cancer susceptibility. Traditional therapeutic approaches have limited efficacy in advanced liver cancer, and novel strategies are urgently needed to improve the limited treatment options for HCC. This review summarizes the basic knowledge, current advances, and future challenges and prospects of adeno-associated virus (AAV) and adenoviruses as vectors for gene therapy of HCC. This paper also reviews the clinical trials of gene therapy using adenovirus vectors, immunotherapy, toxicity and immunological barriers for AAV and adenoviruses, and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors. PMID:26755879

  15. Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

    PubMed

    Carr, David J; Wallace, Jacqueline M; Aitken, Raymond P; Milne, John S; Martin, John F; Zachary, Ian C; Peebles, Donald M; David, Anna L

    2016-06-01

    Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period. PMID:27103444

  16. Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

    PubMed

    Carr, David J; Wallace, Jacqueline M; Aitken, Raymond P; Milne, John S; Martin, John F; Zachary, Ian C; Peebles, Donald M; David, Anna L

    2016-06-01

    Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period.

  17. Efficacy of recombinant adenoviral human p53 gene in the treatment of lung cancer-mediated pleural effusion

    PubMed Central

    LI, KUN-LIN; KANG, JUN; ZHANG, PENG; LI, LI; WANG, YU-BO; CHEN, HENG-YI; HE, YONG

    2015-01-01

    Pleural effusion induced by lung cancer exerts a negative impact on quality of life and prognosis. The aim of the present study was to evaluate the value of the recombinant adenoviral human p53 gene (rAd-p53) in the local treatment of lung cancer and its synergistic effect with chemotherapy. The present study retrospectively recruited 210 patients with lung cancer-mediated pleural effusion who had adopted a treatment strategy of platinum chemotherapy. Pleurodesis was performed via the injection of cisplatin or rAd-p53. Long-term follow-up was conducted to investigate the therapeutic effects of cisplatin and rAd-p53 administration on pleural effusion and other relevant clinical indicators. The short-term effect of pleurodesis was as follows: The efficacy rate of rAd-p53 therapy was significantly higher compared with cisplatin therapy (71.26 vs. 54.47%), and the efficacy of treatment with ≥2×1012 viral particles of rAd-p53 for pleurodesis was significantly greater than treatment with 40 mg cisplatin (P<0.05). Furthermore, efficacy analysis performed 6 and 12 months after pleurodesis indicated that the efficacy rate of rAd-p53 was significantly greater than that of cisplatin (P<0.05). A comparison of median progression-free survival (PFS) time identified a significant difference (P<0.05) between rAd-p53 and cisplatin therapy (3.3 vs. 2.7 months); however, a comparison of median overall survival time identified no significant difference (P>0.05) between rAd-p53 and cisplatin therapy (9.6 vs. 8.7 months). In addition, Cox regression analysis indicated that PFS was not affected by clinical indicators such as age, gender, prognostic staging and smoking status; however, PFS was affected by pathological subtype (adenocarcinoma or squamous carcinoma) in the rAd-p53 group. rAd-p53 administration for pleurodesis exerts long-term therapeutic effects on the local treatment of lung cancer. Thus, a combination of rAd-p53 and chemotherapy may exert a synergistic effect and

  18. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  19. Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.

    PubMed

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-06-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  20. Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates.

    PubMed

    Smith, Theodore A G; Idamakanti, Neeraja; Marshall-Neff, Jennifer; Rollence, Michele L; Wright, Patrick; Kaloss, Michele; King, Laura; Mech, Christine; Dinges, Lisa; Iverson, William O; Sherer, Alfred D; Markovits, Judit E; Lyons, Russette M; Kaleko, Michael; Stevenson, Susan C

    2003-11-20

    Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.

  1. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  2. Good manufacturing practice production of adenoviral vectors for clinical trials.

    PubMed

    Lusky, Monika

    2005-03-01

    The increasing importance of recombinant adenoviral vectors for gene therapy, cancer therapy, and the development of prophylactic and therapeutic vaccines has led to worldwide efforts toward scalable process development suitable for commercial manufacturing of replication-deficient adenoviral vectors. This review focuses on the manufacturing of adenovirus for clinical trials in the context of good manufacturing practice conditions and regulations. PMID:15812223

  3. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

    SciTech Connect

    Peng, Cheng-Fei; Han, Ya-Ling; Jie-Deng,; Yan, Cheng-Hui; Jian-Kang,; Bo-Luan,; Jie-Li

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study

  4. Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

    PubMed Central

    Deng, Gang; Huang, Xiang-Jun; Luo, Hong-Wu; Huang, Fei-Zhou; Liu, Xun-Yang; Wang, Yong-Heng

    2013-01-01

    AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 1011 vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups

  5. Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

    PubMed Central

    2010-01-01

    Background Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time. Methods Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis. Results We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed. Conclusions Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals. PMID:21176193

  6. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    PubMed

    Yang, Xuechao; van der Kraan, Peter M; van den Dolder, Juliette; Walboomers, X Frank; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2007-11-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium. PMID:17824831

  7. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  8. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  9. Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.

    PubMed

    Ishihara, Akikazu; Shields, Kathleen M; Litsky, Alan S; Mattoon, John S; Weisbrode, Steven E; Bartlett, Jeffrey S; Bertone, Alicia L

    2008-06-01

    This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.

  10. Induction of E1A-responsive negative factors for transcription of the fibronectin gene in adenovirus E1-transformed rat cells.

    PubMed Central

    Nakamura, T; Nakajima, T; Tsunoda, S; Nakada, S; Oda, K; Tsurui, H; Wada, A

    1992-01-01

    The level of fibronectin (FN) gene expression is very high in resting rat 3Y1 cells but greatly decreased in adenovirus E1-transformed cells. To study the mechanism of this down-regulation, nuclear factors binding to the 5'-flanking region of the FN gene were analyzed by gel retardation assay and DNase I footprinting. Nuclear factors that were present in the transformed cells but nearly absent in resting 3Y1 cells interacted with multiple sites of the promoter region. Oligonucleotide competition with the FN promoter-chloramphenicol acetyltransferase (CAT) reporter constructs (pFCAT) for these factors in the transformed cells indicated that all of them had a negative effect on FN gene expression. Of them, a factor(s) (G10BP) binding to the G10 stretch from positions -239 to -230 and to two GC boxes consisting of the G10 stretch with one internal C residue insertion from positions -105 to -95 and -54 to -44 had the strongest repressive activity. Introduction of substitutive mutations into these G-rich sequences resulted in the increase in CAT activity of pFCAT in the transformed cells. The recognition sequences of G10BP and Sp1 overlap in two GC boxes. G10BP has stronger affinity for heparin and GC boxes than does Sp1, suggesting that G10BP may repress FN gene transcription by displacing Sp1. Images PMID:1404598

  11. Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.

    PubMed

    Bertone, A L; Pittman, D D; Bouxsein, M L; Li, J; Clancy, B; Seeherman, H J

    2004-11-01

    This study evaluated healing of rabbit bilateral ulnar osteotomies 6 and 8 weeks after surgery in response to percutaneous injection of transgenic adenoviral (Ad) bone morphogenetic protein-6 (BMP-6) vector or green fluorescent protein vector control (Ad-GFP) administered 7 days after surgery compared to untreated osteotomy controls. The amount, composition and biomechanical properties of the healing bone repair tissue were compared among groups and to historical data for intact rabbit ulnae obtained from similar studies at the same institution. Quantitative computed tomography was used to determine area, density and mineral content of the mineralized callus in the harvested ulnae. Maximum torque, torsional stiffness, and energy absorbed to failure were determined at 1.5 degrees /s. Calcified sections of excised ulnae (5 microm) were stained with Goldner's Trichrome and Von Kossa, and evaluated for callus composition, maturity, cortical continuity, and osteotomy bridging. Radiographic assessment of bone formation indicated greater mineralized callus in the ulnae injected with Ad-hBMP-6 as early as 1 week after treatment (2 weeks after surgery) compared to untreated osteotomy ulnae (p < 0.006) and Ad-GFP treated osteotomy ulnae (p < 0.002). Quantitative computed tomography confirmed greater bone area and bone mineral content at the osteotomy at 6 weeks in Ad-BMP-6 treated osteotomy as compared to untreated osteotomy ulnae (p < 0.001) and Ad-GFP treated osteotomy ulnae (p < 0.01). Ad-BMP-6 treated osteotomy ulnae were stronger (p < 0.001 and 0.003) and stiffer (p < 0.004 and 0.003) in torsion at 6 weeks than untreated osteotomy ulnae or Ad-GFP treated osteotomy ulnae, respectively. Maximum torque, torsional stiffness, and energy absorbed to failure were greater in Ad-BMP-6 treated osteotomy ulnae compared to their respective untreated contralateral osteotomy ulnae at 8 weeks [p < 0.03]. Maximum torque and torsional stiffness in the Ad-BMP-6 treated osteotomy ulnae

  12. Adeno-associated virus protects the retinoblastoma family of proteins from adenoviral-induced functional inactivation.

    PubMed

    Batchu, Ramesh B; Shammas, Masood A; Wang, Jing Yi; Freeman, John; Rosen, Nancy; Munshi, Nikhil C

    2002-05-15

    Adeno-associated virus type 2 (AAV) is known to inhibit virally mediated oncogenic transformation. One of the early events of adenovirus (Ad) infection is the functional inactivation of cell cycle regulatory retinoblastoma (RB) family of proteins, which consists of retinoblastoma protein (pRB), p107, and p130. In an effort to understand the molecular basis of anti-oncogenic properties of AAV, we studied the effects of AAV expression on these proteins in cells infected with Ad. Western blot analysis showed that AAV interferes with the adenoviral-induced degradation and hyperphosphorylation of the pRB family of proteins in normal human fibroblasts as well as in HeLa and 293 cell lines. RNase protection assay showed enhanced expression of pocket protein gene by AAV expression. We also demonstrate that Rep proteins, the major AAV regulatory proteins, bind to E1A, the immediate early gene of Ad responsible for hyperphosphorylation and dissociation of pRB-E2F complex. This binding of AAV Rep proteins to E1A leads to decreased association between E1A and pRB leading to protection of pocket proteins from degradation, decreased expression of S phase genes and inhibition of cell cycle progression. These results suggest that the antiproliferative activity of AAV against Ad is mediated, at least in part, by effects of AAV Rep proteins on the Rb family of proteins.

  13. Gene therapy for rhesus monkeys heterozygous for LDL receptor deficiency by balloon-catheter hepatic delivery of helper-dependent adenoviral vector

    PubMed Central

    Oka, Kazuhiro; Mullins, Charles E.; Kushwaha, Rampratap S.; Leen, Ann M; Chan, Lawrence

    2014-01-01

    Autosomal dominant familial hypercholesterolemia (FH) is a monogenic life-threatening disease. We tested the efficacy of low-density lipoprotein receptor (LDLR) gene therapy using helper-dependent adenoviral vector (HDAd) in a nonhuman primate model of FH, comparing intravenous injection versus intrahepatic arterial injection in the presence of balloon catheter-based hepatic venous occlusion. Rhesus monkeys heterozygous for mutant LDLR gene (LDLR+/−) developed hypercholesterolemia while on a high cholesterol diet. We treated them with HDAd-LDLR either by intravenous delivery, or by catheter-based intra-hepatic artery injection. Intravenous injection of ≤1.1×1012 viral particles (vp)/kg failed to have any effect on plasma cholesterol. Increasing the dose to 5×1012 vp/kg led to a 59% lowering of the plasma cholesterol that lasted for 30 days before it returned to pretreatment levels by day 40. A further increase in dose to 8.4×1012 vp/kg resulted in severe lethal toxicity. In contrast, direct hepatic artery injection following catheter-based hepatic venous occlusion enabled the use of a reduced HDAd-LDLR dose of 1×1012 vp/kg that lowered plasma cholesterol within a week, and reached a nadir of 59% pretreatment level on days 20 to 48 after injection. Serum alanine aminotransaminase (ALT) remained normal until day 48 when it went up slightly and stayed mildly elevated on day 72 before it returned to normal on day 90. In this monkey, the HDAd-LDLR-induced trough of hypocholesterolemia started trending upwards on day 72 and returned to pretreatment levels on day 120. We measured the LDL apolipoprotein B turnover rate at 10 days before, and again 79 days after, HDAd-LDLR treatment in two monkeys that exhibited a cholesterol lowering response. HDAd-LDLR therapy increased the LDL fractional catabolic rate by 78% and 50%, respectively, in the two monkeys, coincident with an increase in hepatic LDLR mRNA expression. In conclusion, HDAd-mediated LDLR gene delivery to

  14. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    SciTech Connect

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.; Freimuth, P.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  15. 24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer.

    PubMed

    Wiechert, S; El-Armouche, A; Rau, T; Zimmermann, W-H; Eschenhagen, T

    2003-08-01

    The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to approximately 165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 108 active virus particles yielded homogeneous transfection of approximately 80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.

  16. Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit.

    PubMed Central

    Hearing, P; Shenk, T

    1985-01-01

    The adenovirus E1A gene is known to be autoregulated at the level of transcription. Autoregulation was found to be mediated by products of the E1A 13S mRNA, which induced a fivefold increase in E1A transcription rate. Deletion analysis suggested that the autoregulation did not require any specific sequence in the E1A transcriptional control region. This conclusion was reinforced by the demonstration that a cellular alpha-globin gene substituted for the E1A gene on the adenovirus chromosome was also positively regulated by E1A gene products. Images PMID:2943984

  17. Adenoviral p16/CDKN2 gene transfer to malignant glioma: role of p16 in growth, invasion, and senescence.

    PubMed

    Kim, Seung-Ki; Wang, Kyu-Chang; Cho, Byung-Kyu; Lim, Su-Young; Kim, Young-Yim; Oh, Chang-Wan; Chung, You-Nam; Kim, Chae-Yong; Lee, Choon-Taek; Kim, Hyun Jib

    2003-01-01

    In gliomas, a high frequency of homozygous p16 gene deletions have been demonstrated, which are believed to be linked with malignant progression. The aim of this study was to assess the role of p16 in growth, invasion, and senescence. The human glioma cell lines U87 MG and U373 MG were transduced with Ad-p16, and cell viability was assessed by trypan blue staining. To examine the mechanism of cell growth inhibition, cell cycle analyses and annexin assays were performed. The invasive potential of Ad-p16 transduced cells was evaluated using a Matrigel invasion assay, and trimolecular complex (MMP-2/MT1-MMP/TIMP-2) synthesis was proven by zymography and Western blotting. To establish the link between p16 and cell senescence, we stained for Senescence-Associated beta-galactosidase activity. A cell proliferation assay demonstrated that Ad-p16 treatment significantly inhibits cell growth. Moreover, this cell growth inhibition was induced by cell cycle arrest, not by apoptosis. In vitro treatment of malignant glioma cells with Ad-p16 significantly decreased their invasive potential by Matrigel invasion assay. However, we were unable to demonstrate any differences in the constitutive productions and secretions of MMP-2, MT1-MMP, and TIMP-2, among the mock-treated, Ad-lacZ-transduced, and Ad-p16-transduced cells. p16 expression caused an enlargement of all cells, and these were morphologically similar to senescent cells. Staining for Senescence-Associated beta-galactosidase activity showed that the enlarged cells stained positively. Taken together these data strongly suggest that the anti-cancer effect of p16 is modulated by p16-mediated cell cycle arrest and by the induction of senescence.

  18. High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

    PubMed

    Gaillet, Bruno; Gilbert, Rénald; Amziani, Rachid; Guilbault, Claire; Gadoury, Christine; Caron, Antoine W; Mullick, Alaka; Garnier, Alain; Massie, Bernard

    2007-01-01

    To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

  19. Enhanced anti-tumor effects of combined MDR1 RNA interference and human sodium/iodide symporter (NIS) radioiodine gene therapy using an adenoviral system in a colon cancer model

    PubMed Central

    Ahn, S J; Jeon, Y H; Lee, Y J; Lee, Y L; Lee, S-W; Ahn, B-C; Ha, J-H; Lee, J

    2010-01-01

    Using an adenoviral system as a delivery mediator of therapeutic gene, we investigated the therapeutic effects of the use of combined MDR1 shRNA and human NIS (hNIS) radioiodine gene therapy in a mouse colon xenograft model. In vitro uptake of Tc-99m sestamibi was increased approximately two-fold in cells infected with an adenovirus vector that expressed MDR1 shRNA (Ad-shMDR1) and I-125 uptake was 25-fold higher in cells infected with an adenovirus vector that expressed human NIS (Ad-hNIS) as compared with control cells. As compared with doxorubicin or I-131 treatment alone, the combination of doxorubicin and I-131 resulted in enhanced cytotoxicity for both Ad-shMDR1- and Ad-hNIS-infected cells, but not for control cells. In vivo uptake of Tc-99m sestamibi and Tc-99m pertechnetate was twofold and 10-fold higher for Ad-shMDR1 and Ad-hNIS-infected tumors as compared with tumors infected with a control adenovirus construct that expressed β-galactrosidase (Ad-LacZ), respectively. In mice treated with either doxorubicin or I-131 alone, there was a slight delay in tumor growth as compared to mice treated with Ad-LacZ. However, combination therapy with doxorubicin and I-131 induced further significant inhibition of tumor growth as compared with mice treated with Ad-LacZ. We have shown successful therapeutic efficacy of combined MDR shRNA and hNIS radioiodine gene therapy using an adenoviral vector system in a mouse colon cancer model. Adenovirus-mediated cancer gene therapy using MDR1 shRNA and hNIS would be a useful tool for the treatment of cancer cells expressing multi-drug resistant genes. PMID:20186172

  20. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

    PubMed

    Campos, Samuel K; Barry, Michael A

    2004-11-01

    There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

  1. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  2. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  3. Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

    PubMed Central

    Hooley, R P; Paterson, M; Brown, P; Kerr, K; Saunders, P T K

    2009-01-01

    Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. This paper describes a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems. PMID:18955374

  4. Rb function is required for E1A-induced S-phase checkpoint activation.

    PubMed

    Nemajerova, A; Talos, F; Moll, U M; Petrenko, O

    2008-09-01

    It is widely accepted that adenoviral E1A exerts its influence on recipient cells through binding to the retinoblastoma (Rb) family proteins, followed by a global release of E2F factors from pocket-protein control. Our study challenges this simple paradigm by demonstrating previously unappreciated complexity. We show that E1A-expressing primary and transformed cells are characterized by the persistence of Rb-E2F1 complexes. We provide evidence that E1A causes Rb stabilization by interfering with its proteasomal degradation. Functional experiments supported by biochemical data reveal not only a dramatic increase in Rb and E2F1 protein levels in E1A-expressing cells but also demonstrate their activation throughout the cell cycle. We further show that E1A activates an Rb- and E2F1-dependent S-phase checkpoint that attenuates the growth of cells that became hyperploid through errors in mitosis and supports the fidelity DNA replication even in the absence of E2F complexes with other Rb family proteins, thereby functionally substituting for the loss of p53. Our results support the essential role of Rb and E2F1 in the regulation of genomic stability and DNA damage checkpoints.

  5. pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

    PubMed

    Seifried, L A; Talluri, S; Cecchini, M; Julian, L M; Mymryk, J S; Dick, F A

    2008-05-01

    Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

  6. Photochemical control of the infectivity of adenoviral vectors using a novel photocleavable biotinylation reagent.

    PubMed

    Pandori, Mark W; Hobson, David A; Olejnik, Jerzy; Krzymanska-Olejnik, Edyta; Rothschild, Kenneth J; Palmer, Abraham A; Phillips, Tamara J; Sano, Takeshi

    2002-05-01

    We have explored a novel strategy for controlling the infectivity of adenoviral vectors. This strategy involves a method whereby the infectivity of adenoviral vectors is neutralized by treatment of viral particles with a water-soluble, photocleavable biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to 365 nm light induces a reversal of the neutralizing, chemical modification, resulting in restoration of infectivity to the viral vectors. The light-directed transduction of target cells by photoactivatable adenoviral vectors was demonstrated successfully both in vitro and in vivo. This photochemical infectivity trigger possesses great potential, both as a research tool and as a novel tactic for the delivery of gene-transfer agents, since the infectivity of adenoviral vectors can be controlled externally in a versatile manner. PMID:12031663

  7. Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

    PubMed Central

    Fiory, Francesca; Nigro, Cecilia; Ulianich, Luca; Castanò, Ilenia; D’Esposito, Vittoria; Terracciano, Daniela; Pastore, Lucio; Formisano, Pietro; Beguinot, Francesco; Miele, Claudia

    2013-01-01

    Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance. PMID:23585839

  8. Adenoviral-mediated glial cell line-derived neurotrophic factor gene transfer has a protective effect on sciatic nerve following constriction-induced spinal cord injury.

    PubMed

    Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren

    2014-01-01

    Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good

  9. The evolution of adenoviral vectors through genetic and chemical surface modifications.

    PubMed

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-02-17

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

  10. Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells

    PubMed Central

    Radko, Sandi; Jung, Richard; Olanubi, Oladunni; Pelka, Peter

    2015-01-01

    Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts. PMID:26448631

  11. Adenoviral vector-based strategies against infectious disease and cancer

    PubMed Central

    Zhang, Chao; Zhou, Dongming

    2016-01-01

    ABSTRACT Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vector-based strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. PMID:27105067

  12. Identification of a cellular repressor of transcription of the adenoviral late IVa(2) gene that is unaltered in activity in infected cells.

    PubMed

    Lin, H J; Flint, S J

    2000-11-25

    The gene encoding the adenovirus type 2 IVa(2) protein, a sequence-specific activator of transcription from the viral major late promoter, is itself transcribed only during the late phase of infection. We previously identified a cellular protein (IVa(2)-RF) that binds specifically to an intragenic sequence of the IVa(2) transcription unit. We now report that precise substitutions within the IVa(2)-RF-binding site that decreased binding affinity increased the efficiency of IVa(2) transcription in in vitro reactions containing IVa(2)-RF. Consistent with the conclusion that this cellular protein represses IVa(2) transcription, mutations that led to more efficient transcription in the presence of IVa(2)-RF were without effect in reactions lacking this cellular protein. No change in the concentration or activity of IVa(2)-RF could be detected in adenovirus-infected cells during the period in which the IVa(2) gene is transcribed. We therefore propose that restriction of IVa(2) transcription to the late phase is the result of titration of this cellular repressor as the number of copies of the IVa(2) promoter increases upon replication of the viral genome.

  13. E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

    PubMed

    Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

    1999-05-01

    Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

  14. Polyethyleneimine-coating enhances adenoviral transduction of mesenchymal stem cells.

    PubMed

    Yao, Xinglei; Zhou, Na; Wan, Li; Su, Xiaodong; Sun, Zhao; Mizuguchi, Hiroyuki; Yoshioka, Yasuo; Nakagawa, Shinsaku; Zhao, Robert Chunhua; Gao, Jian-Qing

    2014-05-01

    Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs. PMID:24727452

  15. Immunocompromised Children with Severe Adenoviral Respiratory Infection

    PubMed Central

    Tylka, Joanna C.; McCrory, Michael C.; Gertz, Shira J.; Custer, Jason W.; Spaeder, Michael C.

    2016-01-01

    Purpose. To investigate the impact of severe respiratory adenoviral infection on morbidity and case fatality in immunocompromised children. Methods. Combined retrospective-prospective cohort study of patients admitted to the intensive care unit (ICU) in four children's hospitals with severe adenoviral respiratory infection and an immunocompromised state between August 2009 and October 2013. We performed a secondary case control analysis, matching our cohort 1 : 1 by age and severity of illness score with immunocompetent patients also with severe respiratory adenoviral infection. Results. Nineteen immunocompromised patients were included in our analysis. Eleven patients (58%) did not survive to hospital discharge. Case fatality was associated with cause of immunocompromised state (p = 0.015), multiple organ dysfunction syndrome (p = 0.001), requirement of renal replacement therapy (p = 0.01), ICU admission severity of illness score (p = 0.011), and treatment with cidofovir (p = 0.005). Immunocompromised patients were more likely than matched controls to have multiple organ dysfunction syndrome (p = 0.01), require renal replacement therapy (p = 0.02), and not survive to hospital discharge (p = 0.004). One year after infection, 43% of immunocompromised survivors required chronic mechanical ventilator support. Conclusions. There is substantial case fatality as well as short- and long-term morbidity associated with severe adenoviral respiratory infection in immunocompromised children. PMID:27242924

  16. Production of human epidermal growth factor using adenoviral based system

    PubMed Central

    Negahdari, Babak; Shahosseini, Zahra; Baniasadi, Vahid

    2016-01-01

    Epidermal growth factor (EGF), a growth factor involved in cell growth and differentiation, is a small polypeptide with molecular weight of approximately 6 kDa known to be present in a number of different mammalian species. Experimental studies in animals and humans have demonstrated that the topical application of EGF accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Due to its commercial applications, Human EGF (hEGF) has been cloned in several forms. In the present study, adenoviral based expression system was used to produce biologically active recombinant hEGF. The presence of secreted recombinant hEGF was confirmed by a dot blot and its expression level was determined by enzyme-linked immuno-sorbent assay. Moreover, biological activity of secreted hEGF was evaluated by a proliferation assay performed on A549 cells. For production of hEGF in a secretory form, a chimeric gene coding for the hEGF fused to the signal peptide was expressed using adenoviral based method. This method enables the production of hEGF at the site of interest and moreover it could be used for cell proliferation and differentiation assays in tissue engineering research experiments instead of using commercially available EGF. PMID:27051431

  17. The E1A transcriptional control region is efficiently activated in proliferating tissues of transgenic mice.

    PubMed

    Dieckmann, A; Krippl, B

    1994-08-01

    To study the in vivo regulation of the adenovirus E1A transcriptional regulatory region in transgenic mice, we have constructed two hybrid genes in which the viral control element regulates the expression of the CAT and the lacZ reporter gene. The fusion constructs were introduced into the mouse germline. The expression of the transgenes were monitored during embryogenesis and during postnatal development as well as in adult organs. We show that the E1A regulatory region is recognized and activated in undifferentiated cells during early embryonic cleavage, in the morula, in the inner cell mass and in the trophectoderm of the blastocyst. Transcription initiation at the E1A promoter leads to higher marker gene expression in proliferative centers in postimplantation embryos at the beginning of the neural tube closure. Analysing marker gene expression during postnatal development, a correlation of transcriptional activity of the E1A regulatory region and cell proliferation could be demonstrated. The expression profile of the transgene in different adult organs parallels with DNA synthesis. Marker gene expression was high in cells of organs known to have a high mitotic rate, such as the intestine, the stomach, the skin and the bone marrow, whereas little activity of the E1A control region was observed in the post-proliferative brain. These results are consistent with the finding that activation of the viral cis-regulating elements dramatically increased in the kidney after mitotic stimulation by folic acid. These observations strongly suggests a cell cycle regulated expression from the E1A enhancer/promoter in the absence of the E1A autoregulatory proteins in the living animal. PMID:8036008

  18. Generation of helper-dependent adenoviral vectors by homologous recombination.

    PubMed

    Toietta, Gabriele; Pastore, Lucio; Cerullo, Vincenzo; Finegold, Milton; Beaudet, Arthur L; Lee, Brendan

    2002-02-01

    Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo. The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb). The ligation to produce the viral plasmid is generally very inefficient. Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183. Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb. To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter. The viruses were rescued and tested in vitro and for in vivo expression in mice. The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E. coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector. PMID:11829528

  19. Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

    PubMed

    Lopez-Gordo, Estrella; Podgorski, Iva I; Downes, Nicholas; Alemany, Ramon

    2014-04-01

    Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.

  20. Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

    PubMed Central

    Cook, J L; May, D L; Wilson, B A; Walker, T A

    1989-01-01

    The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization. Images PMID:2526229

  1. Adenoviral vector-based strategies for cancer therapy

    PubMed Central

    Sharma, Anurag; Tandon, Manish; Bangari, Dinesh S.; Mittal, Suresh K.

    2009-01-01

    Definitive treatment of cancer has eluded scientists for decades. Current therapeutic modalities like surgery, chemotherapy, radiotherapy and receptor-targeted antibodies have varied degree of success and generally have moderate to severe side effects. Gene therapy is one of the novel and promising approaches for therapeutic intervention of cancer. Viral vectors in general and adenoviral (Ad) vectors in particular are efficient natural gene delivery systems and are one of the obvious choices for cancer gene therapy. Clinical and preclinical findings with a wide variety of approaches like tumor suppressor and suicide gene therapy, oncolysis, immunotherapy, anti-angiogenesis and RNA interference using Ad vectors have been quite promising, but there are still many hurdles to overcome. Shortcomings like increased immunogenicity, prevalence of preexisting anti-Ad immunity in human population and lack of specific targeting limit the clinical usefulness of Ad vectors. In recent years, extensive research efforts have been made to overcome these limitations through a variety of approaches including the use of conditionally-replicating Ad and specific targeting of tumor cells. In this review, we discuss the potential strengths and limitations of Ad vectors for cancer therapy. PMID:20160875

  2. A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

    PubMed Central

    Buo, Atum M; Williams, Mark S; Kerr, Jaclyn P; Stains, Joseph P

    2016-01-01

    We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research. PMID:27547486

  3. Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells.

    PubMed

    Gueret, Vincent; Negrete-Virgen, Juan A; Lyddiatt, Andrew; Al-Rubeai, Mohamed

    2002-01-01

    There is a constant and growing interest in exploitingadenoviruses as vectors for gene therapy when transientexpression of a therapeutic protein is necessary. Therequirement for an increased viral titre has prompted asearch for techniques by which this virus may be assayedwith greater speed and simplicity. Conventional plaqueassay for quantification of adenoviral vectors titre incurrent use is laborious and time-consuming (up to 14days). We report herein a method for the monitoring ofadenovirus expressing green fluorescent protein thatincorporates rapid and easy sample handling by means offlow cytometric analysis. Cells (HEK293) were infectedwith adenovirus at various multiplicity of infection(MOI), harvested 17 to 20 h post infection and analysedby flow cytometry. Assumptions were made that onefluorescent cell was infected by a single infectiousparticle at a relatively low MOI. The adenoviral titrewas subsequently estimated from cell analysis in arelatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with thatobtained using a non-complementing cell line (A549). APoisson distribution successfully modelled the profile ofinfection as a function of MOI. This provided a betterunderstanding of adenoviral infection at the earlieststage possible. Monitoring of GFP fluorescence and viruspropagation in a batch culture of infected cells wassubsequently used as a practical application of thevalidated method.

  4. Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex.

    PubMed Central

    Geisberg, J V; Chen, J L; Ricciardi, R P

    1995-01-01

    Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex. PMID:7565781

  5. Current Strategies and Future Directions for Eluding Adenoviral Vector Immunity

    PubMed Central

    Bangari, Dinesh S.; Mittal, Suresh K.

    2006-01-01

    Adenoviral (Ad) vectors can efficiently transduce a broad range of cell types and have been used extensively in preclinical and clinical studies for gene delivery applications. The presence of preexisting Ad immunity in the majority of human population and a rapid development of immune response against the Ad vector backbone following the first inoculation with the vector have impeded clinical use of these vectors. In addition, a number of animal inoculation studies have demonstrated that high systemic doses of Ad vectors invariably lead to initiation of acute inflammatory responses. This is mainly due to activation of innate immunity by vector particles. In general, vector and innate immune responses drastically limit the vector transduction efficiency and the duration of transgene expression. In order to have a predictable response with Ad vectors for gene therapy applications, the above limitations must be overcome. Strategies that are being examined to circumvent these drawbacks of Ad vectors include immunosuppression, immunomodulation, serotype switching, use of targeted Ad vectors, microencapsulation of Ad vectors, use of helper-dependent (HD) Ad vectors, and development of nonhuman Ad vectors. Here we review the current understanding of immune responses to Ad vectors, and recent advances in the strategies for immune evasion to improve the vector transduction efficiency and the duration of transgene expression. Development of novel strategies for targeting specific cell types would further boost the utility of Ad vectors by enhancing the safety, efficacy and duration of transgene expression. PMID:16611043

  6. The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC

    PubMed Central

    Vijayalingam, S.; Subramanian, T.; Zhao, Ling-jun

    2015-01-01

    . Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb. PMID:26559831

  7. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

  8. A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

    PubMed

    Zhang, Peng; Du, Enqi; Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 ± 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

  9. Reduced inflammation and improved airway expression using helper-dependent adenoviral vectors with a K18 promoter.

    PubMed

    Toietta, Gabriele; Koehler, David R; Finegold, Milton J; Lee, Brendan; Hu, Jim; Beaudet, Arthur L

    2003-05-01

    Efforts have been made to deliver transgenes to the airway epithelia of laboratory animals and humans to develop gene therapy for cystic fibrosis. These investigations have been disappointing due to combinations of transient and low-level gene expression, acute toxicity, and inflammation. We have developed new helper-dependent adenoviral vectors to deliver an epithelial cell-specific keratin 18 expression cassette driving the beta-galactosidase (beta-gal) or human alpha-fetoprotein (AFP) reporter genes. Following intranasal administration to mice, we found that the reporter genes were widely expressed in airway epithelial and submucosal cells, and secreted human AFP was also detectable in serum. In contrast to a first-generation adenoviral vector, inflammation was negligible at doses providing efficient transduction, and expression lasted longer than typically reported-up to 28 days with beta-gal and up to 15 weeks with human AFP. These results suggest that delivery to the airway of helper-dependent adenoviral vectors utilizing a tissue-specific promoter could be a significant advance in the development of gene therapy for cystic fibrosis. PMID:12718908

  10. Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis.

    PubMed Central

    Marcellus, R C; Teodoro, J G; Wu, T; Brough, D E; Ketner, G; Shore, G C; Branton, P E

    1996-01-01

    In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis. PMID:8709247

  11. Aptamer modification improves the adenoviral transduction of malignant glioma cells.

    PubMed

    Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

    2013-12-01

    Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.

  12. A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.

    PubMed Central

    Lamb, B T; Satyamoorthy, K; Solter, D; Basu, A; Xu, M Q; Weinmann, R; Howe, C C

    1992-01-01

    The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element. Images PMID:1406664

  13. Rare serotype adenoviral vectors for HIV vaccine development.

    PubMed

    Michael, Nelson L

    2012-01-01

    Human adenoviral vectors are being developed for use in candidate vaccines for HIV-1 and other pathogens. However, this approach suffered a setback when an HIV-1 vaccine using an adenovirus type 5 (Ad5) vector failed to reduce, and might even have increased, the rate of HIV infection in men who were uncircumcised and who had preexisting antibodies specific for Ad5. This increased interest in the evaluation of serologically distinct adenoviral vectors. In this issue of the JCI, Frahm and coworkers report evidence that preexisting cellular immune responses directed toward Ad5 reduce the immunogenicity of antigens expressed in Ad5-vectored vaccines and have cross-reacting potential with non-Ad5 adenoviral vectors. The implications of this observation need to be carefully evaluated in future clinical trials of all serotypes of adenovirus-vectored vaccines.

  14. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.

    PubMed Central

    Zerler, B; Moran, B; Maruyama, K; Moomaw, J; Grodzicker, T; Ruley, H E

    1986-01-01

    Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243-amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that E1A functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, E1A transforming functions are not associated with activities of the 289-amino acid E1A proteins required for efficient transcriptional activation of viral early region promoters. Images PMID:3022137

  15. The adenovirus E1A protein targets the SAGA but not the ADA transcriptional regulatory complex through multiple independent domains.

    PubMed

    Shuen, Michael; Avvakumov, Nikita; Walfish, Paul G; Brandl, Chris J; Mymryk, Joe S

    2002-08-23

    Expression of the adenovirus E1A protein in the simple eukaryote Saccharomyces cerevisiae inhibits growth. We tested four regions of E1A that alter growth and transcription in mammalian cells for their effects in yeast when expressed as fusions to the Gal4p DNA binding domain. Expression of the N-terminal/conserved region (CR) 1 or CR3, but not of the CR2 or the C-terminal portion of E1A, inhibited yeast growth. Growth inhibition was relieved by deletion of the genes encoding the yGcn5p, Ngg1p, or Spt7p components of the SAGA transcriptional regulatory complex, but not the Ahc1p component of the related ADA complex, indicating that the N-terminal/CR1 and CR3 regions of E1A target the SAGA complex independently. Expression of the pCAF acetyltransferase, a mammalian homologue of yGcn5p, also suppressed growth inhibition by either portion of E1A. Furthermore, the N-terminal 29 residues and the CR3 portion of E1A interacted independently with yGcn5p and pCAF in vitro. Thus, two separate regions of E1A target the yGcn5p component of the SAGA transcriptional activation complex. A subregion of the N-terminal/CR1 fragment spanning residues 30-69 within CR1 also inhibited yeast growth in a SAGA-dependent fashion. However, this region did not interact with yGcn5p or pCAF, suggesting that it makes a third contact with another SAGA component. Our results provide a new model system to elucidate mechanisms by which E1A and the SAGA complex regulate transcription and growth. PMID:12070146

  16. A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes.

    PubMed Central

    Richter, J D; Young, P; Jones, N C; Krippl, B; Rosenberg, M; Ferguson, B

    1985-01-01

    The purified Escherichia coli-expressed human subgroup C adenovirus E1A 13S mRNA product induces expression from the adenovirus type 5 E3 promoter when injected into Xenopus oocytes. In the present communication, the E. coli-expressed E1A 13S and 12S mRNA products are shown to undergo a posttranslational modification in microinjected Xenopus oocytes, which causes a 2- to 4-kDa increase in apparent molecular size, identical to that occurring in HeLa cells expressing the E1A gene. The E. coli-expressed E1A proteins are similarly modified in vitro in a soluble rabbit reticulocyte lysate. The modified form of the E1A proteins preferentially localizes to the oocyte nucleus following cytoplasmic microinjection. The use of various deleted forms of E1A protein synthesized in E. coli shows that a first exon-encoded domain of E1A, residing between amino acid residues 23 and 120, is sufficient for the posttranslational modification and nuclear localization of E1A and also for the trans-activation of the E3 promoter by E1A in Xenopus oocytes. These results suggest that the posttranslational modification of E1A protein may be important for its function. Images PMID:2934733

  17. Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in beta-cell lines.

    PubMed

    Langlois, Allan; Bietiger, William; Sencier, Marie-Christine; Maillard, Elisa; Pinget, Michel; Kessler, Laurence; Sigrist, Severine

    2009-07-01

    Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in beta-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). beta-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 micromol/L of DFO (103.95 +/- 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected beta-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation. PMID:19527112

  18. DprE1, a new taxonomic marker in mycobacteria.

    PubMed

    Incandela, Maria Loreto; Perrin, Elena; Fondi, Marco; de Jesus Lopes Ribeiro, Ana Luisa; Mori, Giorgia; Moiana, Alessia; Gramegna, Maurizio; Fani, Renato; Riccardi, Giovanna; Pasca, Maria Rosalia

    2013-11-01

    Among the species of the Mycobacterium genus, more than 50 have been recognized as human pathogens. In spite of the different diseases caused by mycobacteria, the interspecies genetic similarity ranges from 94% to 100%, and for some species, this value is higher than in other bacteria. Consequently, it is important to understand the relationships existing among mycobacterial species. In this context, the possibility to use Mycobacterium tuberculosis dprE1 gene as new phylogenetic/taxonomic marker has been explored. The dprE1 gene codes for the target of benzothiazinones, belonging to a very promising class of antitubercular drugs. Mutations in cysteine 387 of DprE1 are responsible for benzothiazinone resistance. The DprE1 tree, obtained with 73 amino acid sequences of mycobacterial species, revealed that concerning the benzothiazinone sensitivity/resistance, it is possible to discriminate two clusters. To validate it, a concatamer obtained from the amino acid sequences of nine mycobacterial housekeeping genes was performed. The concatamer revealed that there is no separation between the benzothiazinone-susceptible and benzothiazinone-resistant species; consequently, this parameter is not linked to the phylogeny. DprE1 tree might represent a good taxonomic marker for the assignment of a mycobacterial isolate to a species. Moreover, the concatamer represents a good reference phylogeny for the Mycobacterium genus. PMID:24024613

  19. Development of an adenoviral vector with robust expression driven by p53

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.

  20. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  1. Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

    PubMed Central

    Chatterjee, P K; Vayda, M E; Flint, S J

    1986-01-01

    The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes. Images Fig. 1. Fig. 3. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3743550

  2. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  3. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  4. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  5. Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

    PubMed Central

    Krippl, B; Andrisani, O; Jones, N; Westphal, H; Rosenberg, M; Ferguson, B

    1986-01-01

    We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein. Images PMID:2942704

  6. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

    PubMed Central

    Campos, Samuel K.; Barry, Michael A.

    2008-01-01

    Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting. PMID:17584037

  7. Hepatic Delivery of Artificial Micro RNAs Using Helper-Dependent Adenoviral Vectors.

    PubMed

    Crowther, Carol; Mowa, Betty; Arbuthnot, Patrick

    2016-01-01

    The potential of RNA interference (RNAi)-based gene therapy has been demonstrated in many studies. However, clinical application of this technology has been hampered by a paucity of efficient and safe methods of delivering the RNAi activators. Prolonged transgene expression and improved safety of helper-dependent adenoviral vectors (HD AdVs) makes them well suited to delivery of engineered artificial intermediates of the RNAi pathway. Also, AdVs' natural hepatotropism makes them potentially useful for liver-targeted gene delivery. HD AdVs may be used for efficient delivery of cassettes encoding short hairpin RNAs and artificial primary microRNAs to the mouse liver. Methods for the characterization of HD AdV-mediated delivery of hepatitis B virus-targeting RNAi activators are described here.

  8. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    SciTech Connect

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R.; Yousef, Ahmed F.; Zhang, Zhiying; Mymryk, Joe S.

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  9. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 8 2014-04-01 2014-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  10. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 8 2012-04-01 2012-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  11. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 8 2011-04-01 2011-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  12. Adenoviral-mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma.

    PubMed

    Fan, Rui; Li, Xiaohua; Du, Wenqi; Zou, Xue; Du, Rui; Zhao, Lina; Luo, Guanhong; Mo, Ping; Xia, Lin; Pan, Yanglin; Shi, Yongquan; Lian, Zhaorui; Feitelson, Mark A; Nie, Yongzhan; Liu, Jie; Fan, Daiming

    2011-06-15

    Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5-year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1-S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl-2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl-2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad-URG11-siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad-URG11-siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.

  13. Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells

    PubMed Central

    Mitsuishi, Michiko; Masuda, Seiko; Kudo, Ichiro; Murakami, Makoto

    2005-01-01

    sPLA2 (secretory phospholipase A2) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA2s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA2s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA2s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA2s. Thus hydrolysis of the plasma membrane by these sPLA2s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA2s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA2 is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA2s may play a role in innate immunity against adenoviral infection in the respiratory tract. PMID:16146426

  14. Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1

    SciTech Connect

    Wierstra, Inken Alves, Juergen

    2008-03-28

    FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27.

  15. Dilated cardiomyopathy alters the expression patterns of CAR and other adenoviral receptors in human heart.

    PubMed

    Toivonen, Raine; Mäyränpää, Mikko I; Kovanen, Petri T; Savontaus, Mikko

    2010-03-01

    Gene therapy trials for heart failure have demonstrated the key role of efficient gene transfer in achieving therapeutic efficacy. An attractive approach to improve adenoviral gene transfer is to use alternative virus serotypes with modified tropism. We performed a detailed analysis of cardiac expression of receptors for several adenovirus serotypes with a focus on differential expression of CAR and CD46, as adenoviruses targeting these receptors have been used in various applications. Explanted hearts from patients with DCM and healthy donors were analyzed using Q-RT-PCR, western blot and immunohistochemistry. Q-RT-PCR and Western analyses revealed robust expression of all receptors except CD80 in normal hearts with lower expression levels in DCM. Immunohistochemical analyses demonstrated that CD46 expression was somewhat higher than CAR both in normal and DCM hearts with highest levels of expression in intramyocardial coronary vessels. Total CAR expression was upregulated in DCM. Triple staining on these vessels demonstrated that both CAR and CD46 were confined to the subendothelial layer in normal hearts. The situation was clearly different in DCM, where both CAR and CD46 were expressed by endothelial cells. The induction of expression of CAR and CD46 by endothelial cells in DCM suggests that viruses targeting these receptors could more easily gain entry to heart cells after intravascular administration. This finding thus has potential implications for the development of targeted gene therapy for heart failure.

  16. Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

    SciTech Connect

    Kanagawa, Naoko; Koretomo, Ryosuke; Murakami, Sayaka |; Sakurai, Fuminori; Mizuguchi, Hiroyuki |; Nakagawa, Shinsaku; Fujita, Takuya |; Yamamoto, Akira; Okada, Naoki |

    2008-05-10

    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35{delta}RGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The {alpha}{sub v}-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-{alpha}, and interferon-{alpha} in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of {alpha}{sub v}-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.

  17. Chromatography purification of canine adenoviral vectors.

    PubMed

    Segura, María Mercedes; Puig, Meritxell; Monfar, Mercè; Chillón, Miguel

    2012-06-01

    Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks. PMID:22799886

  18. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  19. The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

    PubMed Central

    Schmitz, M L; Indorf, A; Limbourg, F P; Städtler, H; Traenckner, E B; Baeuerle, P A

    1996-01-01

    The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB. PMID:8754803

  20. The C-terminal region of E1A: a molecular tool for cellular cartography.

    PubMed

    Yousef, Ahmed F; Fonseca, Gregory J; Cohen, Michael J; Mymryk, Joe S

    2012-04-01

    The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.

  1. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes.

    PubMed

    Ruan, Merry Zc; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan Hl

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  2. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    PubMed Central

    Ruan, Merry ZC; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan HL

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  3. Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus

    SciTech Connect

    Qi, Yanxin; Guo, Huanhuan; Hu, Ningning; He, Dongyun; Zhang, Shi; Chu, Yunjie; Huang, Yubin; Li, Xiao; Sun, LiLi; Jin, Ningyi

    2014-10-15

    Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose > 5 × 10{sup 10} VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25 × 10{sup 10} VP/kg) and beagles (2.5 × 10{sup 9} VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35 × 10{sup 10} VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67 × 10{sup 8} VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. - Highlights: • We use the rodents and non-rodents animal models to evaluation Ad-hTERT-E1a-Apoptin. • Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. • Demonstrate the safety and feasibility dose of injected Ad-hTERT-E

  4. Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

    PubMed

    Barbeau, D; Marcellus, R C; Bacchetti, S; Bayley, S T; Branton, P E

    1992-01-01

    Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably. PMID:1297336

  5. An Adenoviral Vector Based Vaccine for Rhodococcus equi.

    PubMed

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  6. An Adenoviral Vector Based Vaccine for Rhodococcus equi

    PubMed Central

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D.; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  7. Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.

    PubMed

    Garcia-Sanchez, F; Pizzorno, G; Fu, S Q; Nanakorn, T; Krause, D S; Liang, J; Adams, E; Leffert, J J; Yin, L H; Cooperberg, M R; Hanania, E; Wang, W L; Won, J H; Peng, X Y; Cote, R; Brown, R; Burtness, B; Giles, R; Crystal, R; Deisseroth, A B

    1998-07-15

    Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for

  8. Pancreatic Transduction by Helper-Dependent Adenoviral Vectors via Intraductal Delivery

    PubMed Central

    Morró, Meritxell; Teichenne, Joan; Jimenez, Veronica; Kratzer, Ramona; Marletta, Serena; Maggioni, Luca; Mallol, Cristina; Ruberte, Jesus; Kochanek, Stefan; Bosch, Fatima

    2014-01-01

    Abstract Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue. PMID:25046147

  9. PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile.

    PubMed

    Croyle, M A; Le, H T; Linse, K D; Cerullo, V; Toietta, G; Beaudet, A; Pastore, L

    2005-04-01

    Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic. PMID:15647765

  10. [Antiapoptotic oncogene bcl-2 induces a program of senescence in E1A + c-Ha-ras-transformants treated with adriamycin].

    PubMed

    Neliudova, A M; Zubova, S G; Aksenov, N D; Pospelov, V A; Pospelova, T V

    2005-01-01

    Introduction of bcl-2 gene in EIA + c-Ha-ras-transformed rat embryo fibroblasts, which are unable to be arrested after damaging influences and possess high proapoptotic sensitivity, results not only in suppression of cell death but also in re-establishment of cell cycle block following DNA damage and serum starvation. Flow cytometry showed that E1A + c-Ha-ras + bcl-2-transformants treated with DNA-intercalator adriamycin are capable of being arrested at G1/S boundary for a long time (for less than 5 days). According to the growth curve data, the number of Bcl-2-overexpressing cells remanins constant for a week of cultivation with adriamycin. Clonogenic efficacy of E1A + c-Ha-ras + bcl-2-cells is brought to no already in 16 h after adriamycin addition. Apoptotic death, revealed by oligonucleosomic fragmentation of DNA, as well as cell death, occurring due to mitotic catastrophe, after adriamycin treatment are almost absent in Bcl-2-overexpressing transformants, as compared with parental E1A + c-Ha-ras-transformants. Bcl-2 introduction in E1A + c-Ha-ras-transformants is accompanied by a rise of SA beta-Gal (Senescence Associated beta-Galactosidase) activity, which is commonly considered to be a marker of cell senescence. Adriamycin treatment of E1A + c-Ha-ras + bcl-2-transformants results in a much higher rise in SA beta-Gal activity, as compared with untreated cells. Co-immunoprecipitation experiments demonstrated the introduction of Bcl-2 to result in formation of Bcl-2 complexes with early region E1A oncoproducts, which are thought to be responsible for proapoptotic susceptibility of E1A-expressing transformants. The data obtained lead to suggestion that bcl-2 transfer to E1A + c-Ha-ras-transformants may induce a switch from the cell death program on the program of senescence after DNA damage, due, presumably, to Bcl-2 interaction with the apoptosis activator the viral oncoprotein E1A. PMID:16711390

  11. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture.

  12. Adenoviral vector DNA for accurate genome editing with engineered nucleases.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Henriques, Sara F D; Janssen, Josephine M; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-10-01

    Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

  13. Isolation and Characterization of Anti-Adenoviral Secondary Metabolites from Marine Actinobacteria

    PubMed Central

    Strand, Mårten; Carlsson, Marcus; Uvell, Hanna; Islam, Koushikul; Edlund, Karin; Cullman, Inger; Altermark, Björn; Mei, Ya-Fang; Elofsson, Mikael; Willassen, Nils-Peder; Wadell, Göran; Almqvist, Fredrik

    2014-01-01

    Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure. PMID:24477283

  14. Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

    SciTech Connect

    Ugai, Hideyo; Dobbins, George C.; Wang, Minghui; Le, Long P.; Matthews, David A.; Curiel, David T.

    2012-10-25

    Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

  15. Recruitment of CBP/p300, TATA-binding protein, and S8 to distinct regions at the N terminus of adenovirus E1A.

    PubMed

    Rasti, Mozhgan; Grand, Roger J A; Mymryk, Joe S; Gallimore, Phillip H; Turnell, Andrew S

    2005-05-01

    The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for

  16. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation

    PubMed Central

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122

  17. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    PubMed

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122

  18. Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

    PubMed

    Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development.

  19. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  20. De-repression of RaRF-mediated RAR repression by adenovirus E1A in the nucleolus.

    PubMed

    Um, Soo-Jong; Youn, Hye Sook; Kim, Eun-Joo

    2014-02-21

    Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation.

  1. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  2. trans-dominant mutants of E1A provide genetic evidence that the zinc finger of the trans-activating domain binds a transcription factor.

    PubMed Central

    Webster, L C; Ricciardi, R P

    1991-01-01

    The 289R E1A protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by E1A is mediated through cellular transcription factors. In particular, the ability of the trans-dominant E1A point mutant hr5 (Ser-185 to Asn) to inhibit wild-type E1A trans-activation was proposed to result from the sequestration of a cellular factor. Using site-directed mutagenesis, we individually replaced every residue within and flanking the trans-activating domain with a conservative amino acid, revealing 16 critical residues. Six of the individual substitutions lying in a contiguous stretch C terminal to the zinc finger (carboxyl region183-188) imparted a trans-dominant phenotype. trans-Dominance was even produced by deletion of the entire carboxyl region183-188. Conversely, an intact finger region147-177 was absolutely required for trans-dominance, since second-site substitution of every critical residue in this region abrogated the trans-dominant phenotype of the hr5 protein. These data indicate that the finger region147-177 bind a limiting cellular transcription factor and that the carboxyl region183-188 provides a separate and essential function. In addition, we show that four negatively charged residues within the trans-activating domain do not comprise a distinct acidic activating region. We present a model in which the trans-activating domain of E1A binds to two different cellular protein targets through the finger and carboxyl regions. Images PMID:1831535

  3. Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

    PubMed Central

    Whalen, S G; Marcellus, R C; Whalen, A; Ahn, N G; Ricciardi, R P; Branton, P E

    1997-01-01

    A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter. PMID:9094626

  4. Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

    PubMed Central

    Lucher, L A; Brackmann, K H; Symington, J S; Green, M

    1986-01-01

    The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the

  5. [Transfection efficiency of adenoviral vector AD5/F35 to malignant hematopoietic cells of different origins].

    PubMed

    Wabg, Kai; Peng, Jian-Qinag; Yuan, Zhen-Hua; Wu, Xiao-Bin

    2006-06-01

    This study was aimed to investigate the transfection efficiency of adenoviral vector AD5/F35 to hematopoietic malignant cells lines of various origins and AD5/F35 cytotoxicity. The hematologic malignant cell lines of various origins were transfected by AD5/F35-EGFP at different multiple of infection (MOI) and AD5-EGFP was used as control; the proportion of fluorescence positive cells was detected by flow cytometry; the killing effect of virus on infective target cells was assayed by MTT and observed by fluorescence microscopy. The results showed that the transfection efficiency of AD5/F35 vector to cell line of myeloid origin was > 99% at MOI = 30, the transfective efficiency of AD5 vector was 26.4% at MOI = 1,000; the transfection efficiency of AD5/F35 vector and AD5 vector to cell line of B cell origin were 11.7% and 5.7%, respectively, at MOI = 1,000. AD5/F35 and AD5 vectors could not effectively transfect cells of T cell origin, no fluorescence positive cells were detected at MOI = 1,000; no significant killing effect of AD5/F35 vector on infective target cells was observed at MOI = 1,000. It is concluded that AD5/F35 vector infection has definite selectivity to hematologic malignant cells of various origin, the infection ability of AD5/F35 vector to cells of myeloid origin is stronger than that to cells of B cell origin, the cytotoxicity of AD5/F35 vector to infective target cells is small. The AD5/F35 vector is preferable to AD5 vector in respect of infection ability and offers good prospects of application in gene therapy for myeloid leukemia cells as target cells.

  6. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    SciTech Connect

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  7. Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

    PubMed Central

    Chatterjee, P K; Flint, S J

    1986-01-01

    The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells. Images PMID:3023654

  8. Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells

    PubMed Central

    Connell, Claire M.; Shibata, Atsushi; Tookman, Laura A.; Archibald, Kyra M.; Flak, Magdalena B.; Pirlo, Katrina J.; Lockley, Michelle; Wheatley, Sally P.; McNeish, Iain A.

    2011-01-01

    Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer. PMID:21383502

  9. The prevalence of adenoviral conjunctivitis at the Clinical Hospital of the State University of Campinas, Brazil

    PubMed Central

    Pinto, Roberto Damian Pacheco; Lira, Rodrigo Pessoa Cavalcanti; Arieta, Carlos Eduardo Leite; de Castro, Rosane Silvestre; Bonon, Sandra Helena Alves

    2015-01-01

    OBJECTIVES: Viral conjunctivitis is a common, highly contagious disease that is often caused by an adenovirus. The aim of this study was to evaluate the prevalence of adenoviral conjunctivitis by analyzing data from a prospective clinical study of 122 consecutively enrolled patients who were treated at the Clinical Hospital of the State University of Campinas (UNICAMP) after a clinical diagnosis of infectious conjunctivitis between November 2011 and June 2012. METHODS: Polymerase chain reaction was used to evaluate all cases of clinically diagnosed infectious conjunctivitis and based on the laboratory findings, the prevalence of adenoviral infections was determined. The incidence of subepithelial corneal infiltrates was also investigated. RESULTS: Of the 122 patients with acute infectious conjunctivitis included, 72 had positive polymerase chain reaction results for adenoviruses and 17 patients developed subepithelial corneal infiltrates (13.93%). CONCLUSIONS: The polymerase chain reaction revealed that the prevalence of adenoviral conjunctivitis was 59% in all patients who presented with a clinical diagnosis of infectious conjunctivitis from November 2011 to June 2012. The prevalence of adenoviral conjunctivitis in the study population was similar to its prevalence in other regions of the world. PMID:26602522

  10. Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

    PubMed Central

    Richter, J D; Slavicek, J M; Schneider, J F; Jones, N C

    1988-01-01

    The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels. Images PMID:2835499

  11. Arg-Gly-Asp (RGD)-Modified E1A/E1B Double Mutant Adenovirus Enhances Antitumor Activity in Prostate Cancer Cells In Vitro and in Mice.

    PubMed

    Shen, Yue-Hong; Yang, Fei; Wang, Hua; Cai, Zhi-Jian; Xu, Yi-Peng; Zhao, An; Su, Ying; Zhang, Gu; Zhu, Shao-Xing

    2016-01-01

    CAR is a transmembrane protein that is expressed in various epithelial and endothelial cells. CAR mediates adenoviral infection, as well as adenovirus-mediated oncolysis of AxdAdB-3, an E1A/E1B double-restricted oncolytic adenovirus, in prostate cancer cells. This study further assessed the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, in prostate cancer. Susceptibility of prostate cancer cells LNCaP, PC3, and DU145 to adenovirus infection was associated with CAR expression. All of the prostate cancer cell lines expressed integrin αvβ3 and αvβ5. AxdAdB-3 was more cytopathic in CAR-positive prostate cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused potent oncolysis in both CAR-positive and CAR-negative prostate cancer cells. In contrast, AxdAdB3-F/RGD was not cytopathic against normal prostate epithelial cells, RWPE-1. Intratumoral injection of AxdAdB3-F/RGD into CAR-negative prostate cancer cell xenografts in nude mice inhibited tumor growth. The current study demonstrates that E1A/E1B double-restricted oncolytic adenovirus with an RGD-fiber modification enhances infection efficiency and anti-tumor activity in CAR-deficient prostate cancer cells, while sparing normal cells. Future studies will evaluate the therapeutic potential of AxdAdB3-F/RGD in prostate cancer. PMID:26799485

  12. Interleukin-encoding adenoviral vectors as genetic adjuvant for vaccination against retroviral infection.

    PubMed

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4(+) T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4(+) T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4(+) T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity.

  13. Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration.

    PubMed

    Pei, Zheng; Kondo, Saki; Kanegae, Yumi; Saito, Izumu

    2012-01-20

    Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.

  14. Short-term Correction of Arginase Deficiency in a Neonatal Murine Model With a Helper-dependent Adenoviral Vector

    PubMed Central

    Gau, Chia-Ling; Rosenblatt, Robin A; Cerullo, Vincenzo; Lay, Fides D; Dow, Adrienne C; Livesay, Justin; Brunetti-Pierri, Nicola; Lee, Brendan; Cederbaum, Stephen D; Grody, Wayne W; Lipshutz, Gerald S

    2009-01-01

    Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 × 109 viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg−/− mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg−/− livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg−/− mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV. PMID:19367256

  15. First and second line imatinib treatment in chronic myelogenous leukemia patients expressing rare e1a2 or e19a2 BCR-ABL transcripts.

    PubMed

    Andrikovics, Hajnalka; Nahajevszky, Sarolta; Szilvási, Anikó; Bors, András; Adám, Emma; Kozma, András; Kajtár, Béla; Barta, Anikó; Poros, Anna; Tordai, Attila

    2007-09-01

    During the formation of the Philadelphia (Ph) chromosome, in the majority of chronic myelogenous leukemia (CML) patients, the chromosome 22 breakpoint is located in the major breakpoint cluster region of the BCR gene (M-bcr). Minor and micro breakpoint cluster regions (m-bcr with e1a2 transcript and micro-bcr with e19a2 transcript) are rarely affected and have been suggested to be associated with peculiar CML phenotypes. Despite the different clinical characteristics, it is currently not established, whether different therapeutic options are preferably recommended for the treatment of e1a2 or e19a2 CML. Here we report two patients with e1a2 and one patient with e19a2 translocations, treated with different approaches including imatinib. First and second line imatinib treatments induced haematologic response in all of the three patients, and major cytogenetic response in one patient with e1a2, as well as in the patient with e19a2 CML. However, relapse occurred in the patient with e19a2 CML, possibly caused by the presence of additional chromosomal abnormalities such as an extra Ph chromosome, and loss of chromosome Y. Stem cell transplantation (SCT) therapy caused complete haematologic response with molecular remission; however, the patient died of infectious complication. We conclude that in patients with rare BCR-ABL variants, the effectiveness of imatininb treatment may be influenced by the CML stage besides the actual molecular type of the rare transcript. However, this conclusion cannot be generalized to larger patient groups with rare BCR-ABL variants for which further studies may be needed.

  16. 11 CFR 300.61 - Federal elections (2 U.S.C. 441i(e)(1)(A)).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... elections (2 U.S.C. 441i(e)(1)(A)). No person described in 11 CFR 300.60 shall solicit, receive, direct... any Federal election activity as defined in 11 CFR 100.24, unless the amounts consist of Federal funds... 11 Federal Elections 1 2010-01-01 2010-01-01 false Federal elections (2 U.S.C. 441i(e)(1)(A))....

  17. Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

    PubMed

    Kindy, M S; King, A R; Yu, J; Gerardot, C; Whitley, J; de Beer, F C

    1998-06-15

    Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro

  18. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

    PubMed

    Griffin, Bryan D; Nagy, Eva

    2011-06-01

    Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

  19. Treatment for retinopathy of prematurity in an infant with adenoviral conjunctivitis.

    PubMed

    Gunay, Murat; Celik, Gokhan; Con, Rahim

    2015-01-01

    Retinopathy of prematurity (ROP) has been a major problematic disorder during childhood. Laser photocoagulation (LPC) has been proven to be effective in most of the ROP cases. Adenoviral conjunctivitis (AVC) is responsible for epidemics among adult and pediatric population. It has also been reported to be a cause of outbreaks in neonatal intensive care units (NICU) several times. We herein demonstrate a case with AVC who underwent LPC for ROP. And we discuss the treatment methodology in such cases.

  20. Inhibition of apoptosis reduces immunogeneic potential of adenoviral-treated syngeneic liver grafts.

    PubMed

    Puellmann, Kerstin; Beham, Alexander; Kienle, Klaus; Vogel, Mandy; Schlitt, Hans Juergen; Jauch, Karl Walter; Rentsch, Markus

    2006-11-27

    Effects of adenoviral therapy and reduced apoptosis on immune response were investigated in a rat liver transplantation model after prolonged ischemia-reperfusion. Liver donors were treated i.v. either with an adenoviral construct, expressing bcl-2, green-fluorescent-protein, or doxycyclin. Intrahepatic apoptosis was assessed by terminal transferase dUTP nick end labeling assay. The intrahepatic presence of CD4, CD8a, CD163, immunoglobulin (Ig)beta, tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO) was quantified by realtime polymerase chain reaction at 24 hours and seven days after transplantation. Bcl-2 expression abrogated the TNF-alpha elevation and reduced apoptosis of hepatocytes and sinusoidal endothelial cells as compared to advCMV green fluorescent protein. No effects on CD4, CD8a, CD163 and MPO expression were noticed in bcl-2 pretreated livers, whereas Igbeta was slightly enhanced compared to controls. Adenoviral infected liver grafts trigger an immune response but reduced apoptosis resulted in down-regulation of TNF-alpha. Thus, bcl-2 transfer might simultaneously reduce graft ischemia reperfusion injury and immunogenicity. PMID:17130789

  1. The Role of Endosomal Escape and Mitogen-Activated Protein Kinases in Adenoviral Activation of the Innate Immune Response

    PubMed Central

    Smith, Jeffrey S.; Xu, Zhili; Tian, Jie; Palmer, Donna J.; Ng, Philip; Byrnes, Andrew P.

    2011-01-01

    Adenoviral vectors (AdV) activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs). In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK) and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases. PMID:22046344

  2. The spread of adenoviral vectors to central nervous system through pathway of cochlea in mimetic aging and young rats.

    PubMed

    Chen, X; Zhao, X; Hu, Y; Lan, F; Sun, H; Fan, G; Sun, Y; Wu, J; Kong, W; Kong, W

    2015-11-01

    There is no definitive conclusion concerning the spread of viral vectors to the brain after a cochlear inoculation. In addition, some studies have reported different distribution profiles of viral vectors in the central auditory system after a cochlear inoculation. Thus, rats were grouped into either a mimetic aging group or a young group and transfected with adenoviral vectors (AdVs) by round window membrane injection. The distribution of AdV in central nervous system (CNS) was demonstrated in the two groups with transmission electron microscopy and immunofluorescence. We found that the AdV could disseminate into the CNS and that the neuronal damage and stress-induced GRP78 expression were reduced after transfection with PGC-1α, as compared with the control vectors, especially in the mimetic aging group. We also found that the host immune response was degraded in CNS in the mimetic aging group after transduction through the cochlea, as compared with the young group. These results demonstrate that viral vectors can disseminate into the CNS through the cochlea. Moreover, mimetic aging induced by D-galactose could facilitate the spread of viral vectors into the CNS from the cochlea. These findings may indicate a new potential approach for gene therapy against age-related diseases in the CNS.

  3. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation.

    PubMed

    Knippertz, Ilka; Deinzer, Andrea; Dörrie, Jan; Schaft, Niels; Nettelbeck, Dirk M; Steinkasserer, Alexander

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  4. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

    PubMed Central

    Deinzer, Andrea

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B′ core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8+ T cells. Second, we generated the two-vector-based “modular promoter” system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B′ core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  5. Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.

    PubMed Central

    Kraus, V B; Inostroza, J A; Yeung, K; Reinberg, D; Nevins, J R

    1994-01-01

    Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex. Images PMID:8022773

  6. Adenovirus 12 E1A gene detection by polymerase chain reaction in both the normal and coeliac duodenum.

    PubMed Central

    Lawler, M; Humphries, P; O'Farrelly, C; Hoey, H; Sheils, O; Jeffers, M; O'Briain, D S; Kelleher, D

    1994-01-01

    A 12 amino acid sequence from the adenovirus 12 E1B protein is homologous at the protein level with a similar 12-mer derived from the wheat protein A-gliadin. It has been suggested that exposure to Ad 12 could sensitise individuals to gliadins with resultant gluten sensitive enteropathy. In this study, the polymerase chain reaction (PCR) was used to analyse duodenal biopsy tissue from patients with coeliac disease for the presence of Ad 12. The sensitivity of the assay system was at least 1 in 10(5) cells and specificity was confirmed both by probing with an internal oligonucleotide and by direct sequencing. Ad 12 sequences were detected in three of 17 patients with adult coeliac disease and in five of 16 adult controls with normal duodenal biopsies. Since exposure to the virus would be predicted to occur in infancy we also studied patients with childhood coeliac disease diagnosed at less than 1 year of age. Ad 12 was positive in three of 10 childhood coeliac patients and one of seven controls. In addition, we studied a cohort of patients who presented with a diarrhoeal illness and associated anti alpha gliadin antibodies in 1983. These patients had duodenal biopsies performed at this time. One of three patients with abnormal histology had detectable Ad 12 while two of 14 with normal findings were positive for Ad 12. Finally, the potential oncogenic nature of Ad 12 prompted examination of a group of patients with intestinal tumours. Ad 12 DNA was, however, in only two of 19 tumour samples tested. These data indicate that Ad 12 can be successfully detected using PCR on paraffin embedded tissue. Furthermore, Ad 12 was detected at a relatively high level in normal duodenum. The results do not, however, support the hypothesis that prior exposure to Ad 12 is implicated in the pathogenesis of coeliac disease. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:7959228

  7. The role of chromatin in adenoviral vector function.

    PubMed

    Wong, Carmen M; McFall, Emily R; Burns, Joseph K; Parks, Robin J

    2013-06-01

    Vectors based on adenovirus (Ad) are one of the most commonly utilized platforms for gene delivery to cells in molecular biology studies and in gene therapy applications. Ad is also the most popular vector system in human clinical gene therapy trials, largely due to its advantageous characteristics such as high cloning capacity (up to 36 kb), ability to infect a wide variety of cell types and tissues, and relative safety due to it remaining episomal in transduced cells. The latest generation of Ad vectors, helper-dependent Ad (hdAd), which are devoid of all viral protein coding sequences, can mediate high-level expression of a transgene for years in a variety of species ranging from rodents to non-human primates. Given the importance of histones and chromatin in modulating gene expression within the host cell, it is not surprising that Ad, a nuclear virus, also utilizes these proteins to protect the genome and modulate virus- or vector-encoded genes. In this review, we will discuss our current understanding of the contribution of chromatin to Ad vector function. PMID:23771241

  8. Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system.

    PubMed

    Rubinchik, S; Ding, R; Qiu, A J; Zhang, F; Dong, J

    2000-05-01

    Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege. PMID:10845726

  9. Construction and evaluation of an adenoviral vector for the liver-specific expression of the serine/arginine-rich splicing factor, SRSF3.

    PubMed

    Suchanek, Amanda L; Salati, Lisa M

    2015-11-01

    Serine/arginine-rich splicing factor-3 (SRSF3), alternatively known as SRp20, is a member of the highly-conserved SR protein family of mRNA splicing factors. SRSF3 generally functions as an enhancer of mRNA splicing by binding to transcripts in a sequence-specific manner to both recruit and stabilize the binding of spliceosomal components to the mRNA. In liver, expression of SRSF3 is relatively low and its activity is increased in response to insulin and feeding a high carbohydrate diet. We sought to over-express SRSF3 in primary rat hepatocytes to identify regulatory targets. A standard adenoviral shuttle vector system containing an epitope-tagged SRSF3 under the transcriptional control of the CMV promoter could not be used to produce infectious adenoviral particles. SRSF3 over-expression in the packaging cell line prevented the production of infectious adenovirus particles by interfering with the viral splicing program. To circumvent this issue, SRSF3 expression from the shuttle vector was blocked by placing its expression under the control of the liver-specific albumin promoter. In this system, the FLAG-SRSF3 transgene is only expressed in the target cells (hepatocytes) but not in the packaging cell line. An additional benefit of the albumin promoter is that expression of the transgene does not require the addition of hormones or antibiotics to drive SRSF3 expression in the hepatocytes. Robust expression of FLAG-SRSF3 protein is detected in both HepG2 cells and primary rat hepatocytes infected with adenovirus prepared from this new shuttle vector. Furthermore, abundances of several known and suspected mRNA targets of SRSF3 action are increased in response to over-expression using this virus. This report details the construction of the albumin promoter-driven adenoviral shuttle vector, termed pmAlbAd5-FLAG.SRSF3, that can be used to generate functional adenovirus to express FLAG-SRSF3 specifically in liver. This vector would be suitable for over-expression of

  10. Correction of Hyperbilirubinemia in Gunn Rats Using Clinically Relevant Low Doses of Helper-Dependent Adenoviral Vectors

    PubMed Central

    Dimmock, David; Brunetti-Pierri, Nicola; Palmer, Donna J.; Beaudet, Arthur L.

    2011-01-01

    Abstract Crigler–Najjar syndrome type I is a severe inborn error of bilirubin metabolism caused by a complete deficiency of uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) and results in life-threatening unconjugated hyperbilirubinemia. Lifelong correction of hyperbilirubinemia by liver-directed gene therapy using a helper-dependent adenoviral (HDAd) vector has been previously reported in the Gunn rat, a model of Crigler–Najjar syndrome, but was only achieved using high doses (≥3 × 1012 viral particles [vp]/kg), which are likely to elicit a severe toxic response in humans. Therefore, in this study, we investigate strategies to achieve correction of hyperbilirubinemia in the Gunn rat using clinically relevant low HDAd doses. We have found that correction of hyperbilirubinemia in the Gunn rat can be achieved with a low dose of 5 × 1011 vp/kg by using an HDAd vector bearing a more potent UGT1A1 expression cassette. Furthermore, by using hydrodynamic injection of the improved HDAd vector, correction of hyperbilirubinemia in the Gunn rat can be achieved using an even lower dose of 5 × 1010 vp/kg. Although hydrodynamic injection as performed in rats is not acceptable in humans, clinically attractive, minimally invasive methods have been successfully developed to mimic hydrodynamic injection of HDAd vector in non-human primates. Therefore, using an improved expression cassette combined with a more efficient method of vector delivery permits correction of hyperbilirubinemia in the Gunn rat using clinically relevant low HDAd doses and may thus pave the way to clinical application of HDAd vectors for Crigler–Najjar syndrome gene therapy. PMID:20973621

  11. Gene therapy and nasopharyngeal carcinoma.

    PubMed

    Hughes, J; Alusi, G; Wang, Y

    2012-06-01

    In 2003, a non-replicating adenoviral gene therapy product received the world`s first government licence for the treatment of head and neck cancer. Two years later approval was granted to a replication-selective adenovirus for the treatment of nasopharyngeal carcinoma in combination with chemotherapy. This review introduces the reader to gene therapy as an emerging treatment modality, and outlines its application to the management of nasopharyngeal carcinoma by examining recent pre-clinical and clinical research.

  12. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

    PubMed Central

    Moayeri, Mahtab; Tremblay, Jacqueline M.; Debatis, Michelle; Dmitriev, Igor P.; Kashentseva, Elena A.; Yeh, Anthony J.; Cheung, Gordon Y. C.; Curiel, David T.; Leppla, Stephen

    2016-01-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  13. Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

    PubMed

    Campos, Samuel K; Parrott, M Brandon; Barry, Michael A

    2004-06-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

  14. Two domains within the adenovirus type 12 E1A unique spacer have disparate effects on the interaction of E1A with P105-Rb and the transformation of primary mouse cells.

    PubMed

    Rumpf, H; Esche, H; Kirch, H C

    1999-04-25

    Transformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the unique spacer region of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of the spacer binds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a second spacer mutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1 diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awt or, least efficient, E1ASpm2. These properties of the spacer mutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1 (plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2 or Ad12 E1AdelCR2 (lacking the entire CR2) died within 6-10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes approximately 120 h. Moreover, in the established cell lines, the affinity of E1ASpm1 to p105-Rb was higher than with that of E1Awt. Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-unique spacer, whereas the hydrophobic stretch of six alanines in the spacer is required for stable transformation. PMID:10208919

  15. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

  16. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker. PMID:26723876

  17. Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector

    PubMed Central

    Toietta, Gabriele; Mane, Viraj P.; Norona, Wilma S.; Finegold, Milton J.; Ng, Philip; McDonagh, Antony F.; Beaudet, Arthur L.; Lee, Brendan

    2005-01-01

    Crigler–Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to «1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 × 1012 viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIα and XIIIα in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 × 1011 viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler–Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity. PMID:15753292

  18. Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector.

    PubMed

    Toietta, Gabriele; Mane, Viraj P; Norona, Wilma S; Finegold, Milton J; Ng, Philip; McDonagh, Antony F; Beaudet, Arthur L; Lee, Brendan

    2005-03-15

    Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity. PMID:15753292

  19. Vector systems for prenatal gene therapy: principles of adenovirus design and production.

    PubMed

    Alba, Raul; Baker, Andrew H; Nicklin, Stuart A

    2012-01-01

    Adenoviruses have many attributes, which have made them one of the most widely investigated vectors for gene therapy applications. These include ease of genetic manipulation to produce replication-deficient vectors, ability to readily generate high titer stocks, efficiency of gene delivery into many cell types, and ability to encode large genetic inserts. Recent advances in adenoviral vector engineering have included the ability to genetically manipulate the tropism of the vector by engineering of the major capsid proteins, particularly fiber and hexon. Furthermore, simple replication-deficient adenoviral vectors deleted for expression of a single gene have been complemented by the development of systems in which the majority of adenoviral genes are deleted, generating sophisticated Ad vectors which can mediate sustained transgene expression following a single delivery. This chapter outlines methods for developing simple transgene over expressing Ad vectors and detailed strategies to engineer mutations into the major capsid proteins.

  20. Altered hyaluronic acid content in tear fluid of patients with adenoviral conjunctivitis.

    PubMed

    Dreyfuss, Juliana L; Regatieri, Caio V; Coelho, Bruno; Barbosa, José B; De Freitas, Denise; Nader, Helena B; Martins, João R

    2015-03-01

    The adenoviral conjunctivitis is one of the biggest causes of conjunctival infection in the world. Conjunctivitis causes relatively nonspecific symptoms, as hyperaemia and chemosis. Even after biomicroscopy, complex laboratory tests, such as viral culture, are necessary to identify the pathogen or its etiology. To contribute to the better understanding of the pathobiology of the adenoviral conjunctivitis, the tear fluids of patients with unilateral acute adenovirus conjunctivitis (UAAC), normal donors (control) and patients with allergic conjunctivitis were analyzed. Tear samples were collected with Schirmer strips from control, allergic conjunctivitis and UAAC patients, diagnosed by clinical signs. UAAC tears were tested positive in viral cultures. After the elution, HA was quantified using an ELISA-like fluorometric assay and the protein profile was determined by SDS-PAGE. A profound increase in the HA tear content in UAAC patients was found when compared to control and ALC. This HA increase in UAAC tears remarkably was not observed in tears from contralateral eyes without clinical signs, nor in allergic conjunctivitis. In addition a distinct profile of UAAC tear proteins was observed in patients with UAAC. The quantification of HA in the tear fluid is a rapid, sensitive and specific test. This molecule might be a biomarker candidate for acute conjunctivitis.

  1. Disseminated adenoviral infection masquerading as lower urinary tract voiding dysfunction in a kidney transplant recipient.

    PubMed

    Aboumohamed, Ahmed; Flechner, Stuart M; Chiesa-Vottero, Andres; Srinivas, Titte R; Mossad, Sherif B

    2014-11-01

    Viral infections continue to cause significant morbidity in immunosuppressed kidney transplant patients. Although cytomegalovirus, Epstein-Barr virus and polyoma "BK" virus are more frequently encountered, the Adenovirus can cause multi-organ system infections, and may be difficult to diagnose because it is not often considered in the initial work up in kidney transplant recipients. We present an unusual case of a kidney recipient 1 year post-transplant with disseminated adenoviral infection, who had an initial presentation of lower urinary tract voiding dysfunction with hematuria and sterile pyuria. This progressed to a severe tubulointerstitial nephritis and acute kidney injury that improved with reduction of immunosuppression. Serial blood viral loads are useful for monitoring the course of infection. Urinary adenoviral infection should be considered in the differential diagnosis whenever a kidney transplant recipient presents with unexplained lower tract voiding dysfunction, hematuria, and sterile pyuria. The allograft kidney and bladder can be targets of viral proliferation. Early diagnosis with reduction of immunosuppressive therapy is essential to clear the virus and maintain allograft function. PMID:23816478

  2. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-07-19

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

  3. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose. PMID:27434682

  4. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis

    PubMed Central

    Dolzhikova, Inna V.; Shcherbinin, Dmitry N.; Zubkova, Olga V.; Ivanova, Tatiana I.; Tukhvatulin, Amir I.; Shmarov, Maxim M.; Logunov, Denis Y.; Naroditsky, Boris S.; Gintsburg, Aleksandr L.

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  5. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    PubMed

    Burmistrova, Daria A; Tillib, Sergey V; Shcheblyakov, Dmitry V; Dolzhikova, Inna V; Shcherbinin, Dmitry N; Zubkova, Olga V; Ivanova, Tatiana I; Tukhvatulin, Amir I; Shmarov, Maxim M; Logunov, Denis Y; Naroditsky, Boris S; Gintsburg, Aleksandr L

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  6. Functional similarity between E6 proteins of cutaneous human papillomaviruses and the adenovirus E1A tumor-restraining module.

    PubMed

    Kuppuswamy, Mohan; Subramanian, T; Kostas-Polston, Elizabeth; Vijayalingam, S; Zhao, Ling-jun; Varvares, Mark; Chinnadurai, G

    2013-07-01

    The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins. PMID:23637414

  7. 26 CFR 1.404(e)-1A - Contributions on behalf of a self-employed individual to or under a qualified pension, annuity...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... individual to or under a qualified pension, annuity, or profit-sharing plan. 1.404(e)-1A Section 1.404(e)-1A...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1A Contributions on behalf of a self-employed individual to or under a qualified pension, annuity, or profit-sharing plan....

  8. 26 CFR 1.404(e)-1A - Contributions on behalf of a self-employed individual to or under a qualified pension, annuity...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... individual to or under a qualified pension, annuity, or profit-sharing plan. 1.404(e)-1A Section 1.404(e)-1A...) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1A Contributions on behalf of a self-employed individual to or under a qualified pension, annuity, or profit-sharing plan. (a)...

  9. An enhancer element is located 340 base pairs upstream from the adenovirus-2 E1A capsite.

    PubMed Central

    Hen, R; Borrelli, E; Sassone-Corsi, P; Chambon, P

    1983-01-01

    A chimeric recombinant, containing the 270 bp left-terminal fragment of Adenovirus-2 (Ad2) inserted upstream from the -34 to +33 Ad2 major late promoter (Ad2MLP) element, has been used to characterize the transcription stimulatory element which is located at least 231 bp upstream from the E1A capsite in the left-end of Ad2 (Ref. 1). We demonstrate that this element, which acts in cis, possesses several properties characteristic of transcriptional enhancers. Firstly, it potentiates initiation of transcription from the capsite of the heterologous Ad2MLP and from "cryptic" sites often preceded by TATA box-like sequences. Secondly, although there is no critical distance requirement between the enhancer element and the Ad2MLP, the extent of stimulation decreases as the distance between the two element increases. However, in contrast to the other known viral or cellular enhancers which are bidirectional, the Ad2 enhancer is unidirectional, i.e. it potentiates the Ad2MLP element only when it is inserted in its "natural" orientation with respect to the direction of transcription. Using two convergent series of deletions, we have localized the Ad2 enhancer element within a 24 bp segment located at approximately 160 bp from the Ad2 left-end, i.e. 340 bp upstream from the E1A capsite. This 24 bp segment contains a sequence which exhibits a striking homology with the consensus sequence of several viral and cellular enhancers. Images PMID:6324099

  10. Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications.

    PubMed

    Montesinos, Mónica S; Satterfield, Rachel; Young, Samuel M

    2016-01-01

    Neuroscience research has been revolutionized by the use of recombinant viral vector technology from the basic, preclinical and clinical levels. Currently, multiple recombinant viral vector types are employed with each having its strengths and weaknesses depending on the proposed application. Helper-dependent adenoviral vectors (HdAd) are emerging as ideal viral vectors that solve a major need in the neuroscience field: (1) expression of transgenes that are too large to be packaged by other viral vectors and (2) rapid onset of transgene expression in the absence of cytotoxicity. Here, we describe the methods for large-scale production of HdAd viral vectors for in vivo use with neurospecific transgene expression. PMID:27515075

  11. Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

    PubMed

    Biasiotto, Roberta; Akusjärvi, Göran

    2015-01-28

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

  12. Modulation of TNFalpha, a determinant of acute toxicity associated with systemic delivery of first-generation and helper-dependent adenoviral vectors.

    PubMed

    Mane, V P; Toietta, G; McCormack, W M; Conde, I; Clarke, C; Palmer, D; Finegold, M J; Pastore, L; Ng, P; Lopez, J; Lee, B

    2006-09-01

    Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemically administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing beta-galactosidase expression cassettes. A preliminary screen for modulation of vector-induced thrombocytopenia revealed no role for interferon-gamma, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFalpha)-deficient mice. Moreover, we also demonstrated that TNFalpha blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (>40% increase in platelet count) and IL-6 expression (>80% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduction in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFalpha immunomodulation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials. PMID:16708078

  13. p202, an interferon-inducible negative regulator of cell growth, is a target of the adenovirus E1A protein.

    PubMed

    Xin, H; D'Souza, S; Fang, L; Lengyel, P; Choubey, D

    2001-10-18

    Studies have revealed that human adenovirus-encoded E1A protein promotes cell proliferation through the targeted interaction with cellular proteins that act as key negative regulators of cell growth. The targets of E1A protein include the retinoblastoma tumor suppressor protein (pRb). Because p202, an interferon (IFN)-inducible murine protein (52-kDa), negatively regulates cell growth in part through the pRb/E2F pathway, we tested whether the p202 is a target of the adenovirus-encoded E1A protein for functional inactivation. Here we report that the expression of E1A protein overcame p202-mediated inhibition of cell growth and this correlated with an alleviation of p202-mediated inhibition of the transcriptional activity of E2F. Furthermore, E1A protein relieved p202-mediated inhibition of the specific DNA-binding activity of E2F complexes, including those containing the pocket proteins. Additionally, the E1A protein bound to p202 both in vitro and in vivo and a deletion of four amino acids in the conserved region 2 (CR2) of E1A protein significantly reduced the binding of E1A to p202. Interestingly, ectopic expression of p202 under reduced serum conditions significantly reduced E1A-mediated apoptosis. Taken together, our observations provide support to the idea that the p202 and adenovirus E1A protein functionally counteract each other and E1A protein targets p202 to promote cell proliferation. PMID:11687962

  14. Dissection of the C-terminal region of E1A redefines the roles of CtBP and other cellular targets in oncogenic transformation.

    PubMed

    Cohen, M J; Yousef, A F; Massimi, P; Fonseca, G J; Todorovic, B; Pelka, P; Turnell, A S; Banks, L; Mymryk, J S

    2013-09-01

    Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A. PMID:23864635

  15. Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers.

    PubMed

    Grafl, Beatrice; Prokofieva, Irina; Wernsdorf, Patricia; Steinborn, Ralf; Hess, Michael

    2014-08-01

    Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

  16. Viral and nonviral delivery systems for gene delivery.

    PubMed

    Nayerossadat, Nouri; Maedeh, Talebi; Ali, Palizban Abas

    2012-01-01

    Gene therapy is the process of introducing foreign genomic materials into host cells to elicit a therapeutic benefit. Although initially the main focus of gene therapy was on special genetic disorders, now diverse diseases with different patterns of inheritance and acquired diseases are targets of gene therapy. There are 2 major categories of gene therapy, including germline gene therapy and somatic gene therapy. Although germline gene therapy may have great potential, because it is currently ethically forbidden, it cannot be used; however, to date human gene therapy has been limited to somatic cells. Although numerous viral and nonviral gene delivery systems have been developed in the last 3 decades, no delivery system has been designed that can be applied in gene therapy of all kinds of cell types in vitro and in vivo with no limitation and side effects. In this review we explain about the history of gene therapy, all types of gene delivery systems for germline (nuclei, egg cells, embryonic stem cells, pronuclear, microinjection, sperm cells) and somatic cells by viral [retroviral, adenoviral, adeno association, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex, pox virus, lentivirus, Epstein-Barr virus)] and nonviral systems (physical: Naked DNA, DNA bombardant, electroporation, hydrodynamic, ultrasound, magnetofection) and (chemical: Cationic lipids, different cationic polymers, lipid polymers). In addition to the above-mentioned, advantages, disadvantages, and practical use of each system are discussed. PMID:23210086

  17. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  18. Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1.

    PubMed

    Alevizopoulos, K; Sanchez, B; Amati, B

    2000-04-13

    Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context. PMID:10803468

  19. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  20. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

    SciTech Connect

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.

  1. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    PubMed

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies. PMID:16482201

  2. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    PubMed

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

  3. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection.

    PubMed

    Subramanian, T; Zhao, Ling-Jun; Chinnadurai, G

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. PMID:23747199

  4. Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements.

    PubMed

    Gálvez, J; Lecina, M; Solà, C; Cairó, J J; Gòdia, F

    2012-01-01

    The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10×10(5)cell/mL was infected, producing 1.3×10(9) infectious viral particles/mL (IVP/mL). To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78×10(5)cell/mL with the consequent production of 44×10(9)IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.

  5. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

    PubMed Central

    Neumann, Alexander J.; Gardner, Oliver F. W.; Williams, Rebecca; Alini, Mauro; Archer, Charles W.; Stoddart, Martin J.

    2015-01-01

    Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a

  6. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-09-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. PMID:2143540

  7. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  8. Melphalan, prednisone, and thalidomide vs melphalan, prednisone, and lenalidomide (ECOG E1A06) in untreated multiple myeloma.

    PubMed

    Stewart, A Keith; Jacobus, Susanna; Fonseca, Rafael; Weiss, Matthias; Callander, Natalie S; Chanan-Khan, Asher A; Rajkumar, S Vincent

    2015-09-10

    This phase 3 trial (Eastern Cooperative Oncology Group [ECOG] E1A06) compared melphalan, prednisone, and thalidomide (MPT-T) with melphalan, prednisone, and lenalidomide (mPR-R) in patients with untreated multiple myeloma (MM). A noninferiority design was used, and inferiority was defined as a progression-free survival (PFS) hazard ratio (HR) of MPT-T/mPR-R ≤0.82. A total of 306 patients enrolled, with a median age of 75.7 years. Median follow-up was 40.7 months. Median time on therapy was 12.1 months and 23.1 months for the 46.6% of treated patients who received maintenance, with no differences by arm. Median PFS was 21 months on MPT-T and 18.7 months on mPR-R (HR, 0.84; 95% confidence interval, 0.64-1.09). Overall survival was 52.6 months (MPT-T) vs 47.7 months (mPR-R) (P = .476). Per-protocol response rates were 63.6% (MPT-T) and 59.9% (mPR-R) (P = .557). Grade ≥3 nonhematologic toxicity was 59.5% for MPT-T vs 40.0% for mPR-R (P = .001). Second malignancies were observed in 18 MPT-T patients vs 14 mPR-R patients. Quality-of-life analysis favored mPR-R by induction end (P = .007). Use of MPT-T or mPR-R in elderly patients with untreated MM demonstrates no statistical or clinically relevant differences in response rates, PFS, and OS; however, quality of life at end of induction was improved and lower toxicity reported with mPR-R. This trial was registered at www.clinicaltrials.gov as #NCT00602641.

  9. Transformation by E1A Oncoprotein Involves Ubiquitin-Mediated Proteolysis of the Neuronal and Tumor Repressor REST in the Nucleus

    PubMed Central

    Guan, Hancheng

    2012-01-01

    The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCFβ-TrCP, since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST. PMID:22419809

  10. Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors.

    PubMed Central

    Koyama, K; Shimabukuro, M; Chen, G; Wang, M Y; Lee, Y; Kalra, P S; Dube, M G; Kalra, S P; Newgard, C B; Unger, R H

    1998-01-01

    Leptin regulates appetite and body weight via hypothalamic targets, but it can act directly on cultured pancreatic islets to regulate their fat metabolism. To obtain in vivo evidence that leptin may act peripherally as well as centrally, we compared the effect of adenovirally induced hyperleptinemia on food intake, body weight, and islet fat content in ventromedial hypothalamic-lesioned (VMHL) rats, sham-lesioned (SL) controls, and Zucker Diabetic Fatty (ZDF) rats in which the leptin receptor is mutated. Infusion with recombinant adenovirus containing the rat leptin cDNA increased plasma leptin by approximately 20 ng/ml in VMHL and ZDF rats but had no effect on their food intake, body weight, or fat tissue weight. Caloric matching of hyperphagic VMHL rats to SL controls did not reduce their resistance to hyperleptinemia. Whereas prediabetic ZDF rats had a fourfold elevation in islet fat, in VMHL rats islet fat was normal and none of them became diabetic. Isolated islets from ZDF rats were completely resistant to the lipopenic action of leptin, while VMHL islets exhibited 50% of the normal response; caloric matching of VMHL rats to SL controls increased leptin responsiveness of their islets to 92% of controls. We conclude that leptin regulation of adipocyte fat requires an intact VMH but that islet fat content is regulated independently of the VMH. PMID:9710441

  11. Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.

    PubMed

    Lesch, Hanna P; Heikkilä, Kati M; Lipponen, Eevi M; Valonen, Piia; Müller, Achim; Räsänen, Eva; Tuunanen, Tarja; Hassinen, Minna M; Parker, Nigel; Karhinen, Minna; Shaw, Robert; Ylä-Herttuala, Seppo

    2015-08-01

    Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors. PMID:26176404

  12. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy

    PubMed Central

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-01-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer. PMID:27229159

  13. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy.

    PubMed

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-07-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer.

  14. Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

    PubMed

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E; Ehrhardt, Anja

    2015-02-01

    High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy.

  15. Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

    PubMed

    Widjojoatmodjo, Myra N; Bogaert, Lies; Meek, Bob; Zahn, Roland; Vellinga, Jort; Custers, Jerome; Serroyen, Jan; Radošević, Katarina; Schuitemaker, Hanneke

    2015-10-01

    RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

  16. Label-free biochemical analytic method for the early detection of adenoviral conjunctivitis using human tear biofluids.

    PubMed

    Choi, Samjin; Moon, Sung Woon; Shin, Jae-Ho; Park, Hun-Kuk; Jin, Kyung-Hyun

    2014-11-18

    Cell culture and polymerase chain reaction are currently regarded as the gold standard for adenoviral conjunctivitis diagnosis. They maximize sensitivity and specificity but require several days to 3 weeks to get the results. The aim of this study is to determine the potential of Raman spectroscopy as a stand-alone analytical tool for clinical diagnosis of adenoviral conjunctivitis using human tear fluids. A drop-coating deposition surface enhanced Raman scattering (DCD-SERS) method was identified as the most effective method of proteomic analysis in tear biofluids. The proposed DCD-SERS method (using a 2-μL sample) led to Raman spectra with high reproducibility, noise-independence, and uniformity. Additionally, the spectra were independent of the volume of biofluids used and detection zones, including the ring, middle, and central zone, with the exception of the outer layer of the ring zone. Assessments with an intensity ratio of 1242-1342 cm(-1) achieved 100% sensitivity and 100% specificity in the central zone. Principal component analysis assessments achieved 0.9453 in the area under the receiver operating characteristic curve (AUC) as well as 93.3% sensitivity and 94.5% specificity in the central zone. Multi-Gaussian peak assessments showed that the differences between these two groups resulted from the reduction of the amide III α-helix structures of the proteins. The presence of adenovirus in tear fluids could be detected more accurately in the center of the sample than in the periphery. The DCD-SERS technique allowed for high chemical structure sensitivity without additional tagging or chemical modification, making it a good alternative for early clinical diagnosis of adenoviral conjunctivitis. Therefore, we are hopeful that the DCD-SERS method will be approved for use in ophthalmological clinics in the near future.

  17. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  18. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

    PubMed Central

    Raza, M. Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M. Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S.; Drayna, Dennis

    2015-01-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  19. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.

  20. Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase.

    PubMed

    Smethells, John R; Swalve, Natashia; Brimijoin, Stephen; Gao, Yang; Parks, Robin J; Greer, Adam; Carroll, Marilyn E

    2016-05-01

    A promising approach in treating cocaine abuse is to metabolize cocaine in the blood using a mutated butyrylcholinesterase (BChE) that functions as a cocaine hydrolase (CocH). In rats, a helper-dependent adenoviral (hdAD) vector-mediated delivery of CocH abolished ongoing cocaine use and cocaine-primed reinstatement of drug-seeking for several months. This enzyme also metabolizes ghrelin, an effect that may be beneficial in maintaining healthy weights. The effect of a single hdAD-CocH vector injection was examined in rats on measures of anxiety, body weight, cocaine self-administration, and cocaine-induced locomotor activity. To examine anxiety, periadolescent rats were tested in an elevated-plus maze. Weight gain was then examined under four rodent diets. Ten months after CocH-injection, adult rats were trained to self-administer cocaine intravenously and, subsequently, cocaine-induced locomotion was tested. Viral gene transfer produced sustained plasma levels of CocH for over 13 months of testing. CocH-treated rats did not differ from controls in measures of anxiety, and only showed a transient reduction in weight gain during the first 3 weeks postinjection. However, CocH-treated rats were insensitive to cocaine. At 10 months postinjection, none of the CocH-treated rats initiated cocaine self-administration, unlike 90% of the control rats. At 13 months postinjection, CocH-treated rats showed no cocaine-induced locomotion, whereas control rats showed a dose-dependent enhancement of locomotion. CocH vector produced a long-term blockade of the rewarding and behavioral effects of cocaine in rats, emphasizing its role as a promising therapeutic intervention in cocaine abuse.

  1. Long-term reduction of cocaine self-administration in rats treated with adenoviral vector-delivered cocaine hydrolase: evidence for enzymatic activity.

    PubMed

    Zlebnik, Natalie E; Brimijoin, Stephen; Gao, Yang; Saykao, Amy T; Parks, Robin J; Carroll, Marilyn E

    2014-05-01

    A new pharmacokinetic approach treating cocaine addiction involves rapidly metabolizing cocaine before it reaches brain reward centers using mutated human butyrylcholinesterase (BChE) or cocaine hydrolase (CocH). Recent work has shown that helper-dependent adenoviral (hdAD) vector-mediated plasma CocH reduced the locomotor-activating effects of cocaine and prevented reinstatement of cocaine-seeking behavior up to 6 months in rats. The present study investigated whether hdAD-CocH could decrease ongoing intravenous cocaine (0.4 mg/kg) self-administration. The hdAD-CocH vector was injected into self-administering rats, and after accumulation of plasma CocH, there was a dramatic reduction in cocaine infusions earned under a fixed ratio 1 schedule of reinforcement that lasted for the length of the study (>2 months). Pretreatment with the selective BChE and CocH inhibitor iso-OMPA (1.5 mg/kg) restored cocaine intake; therefore, the decline in self-administration was likely due to rapid CocH-mediated cocaine metabolism. Direct measurements of cocaine levels in plasma and brain samples taken after the conclusion of behavioral studies provided strong support for this conclusion. Further, rats injected with hdAD-CocH did not experience a deficit in operant responding for drug reinforcement and self-administered methamphetamine (0.05 mg/kg) at control levels. Overall, these outcomes suggest that viral gene transfer can yield plasma CocH levels that effectively diminish long-term cocaine intake and may have potential treatment implications for cocaine-dependent individuals seeking to become and remain abstinent. PMID:24407266

  2. Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-beta1 inhibits fibrogenic activities of hepatic stellate cells.

    PubMed

    Arias, Monica; Lahme, Birgit; Van de Leur, Eddy; Gressner, Axel M; Weiskirchen, Ralf

    2002-06-01

    Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.

  3. Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase.

    PubMed

    Smethells, John R; Swalve, Natashia; Brimijoin, Stephen; Gao, Yang; Parks, Robin J; Greer, Adam; Carroll, Marilyn E

    2016-05-01

    A promising approach in treating cocaine abuse is to metabolize cocaine in the blood using a mutated butyrylcholinesterase (BChE) that functions as a cocaine hydrolase (CocH). In rats, a helper-dependent adenoviral (hdAD) vector-mediated delivery of CocH abolished ongoing cocaine use and cocaine-primed reinstatement of drug-seeking for several months. This enzyme also metabolizes ghrelin, an effect that may be beneficial in maintaining healthy weights. The effect of a single hdAD-CocH vector injection was examined in rats on measures of anxiety, body weight, cocaine self-administration, and cocaine-induced locomotor activity. To examine anxiety, periadolescent rats were tested in an elevated-plus maze. Weight gain was then examined under four rodent diets. Ten months after CocH-injection, adult rats were trained to self-administer cocaine intravenously and, subsequently, cocaine-induced locomotion was tested. Viral gene transfer produced sustained plasma levels of CocH for over 13 months of testing. CocH-treated rats did not differ from controls in measures of anxiety, and only showed a transient reduction in weight gain during the first 3 weeks postinjection. However, CocH-treated rats were insensitive to cocaine. At 10 months postinjection, none of the CocH-treated rats initiated cocaine self-administration, unlike 90% of the control rats. At 13 months postinjection, CocH-treated rats showed no cocaine-induced locomotion, whereas control rats showed a dose-dependent enhancement of locomotion. CocH vector produced a long-term blockade of the rewarding and behavioral effects of cocaine in rats, emphasizing its role as a promising therapeutic intervention in cocaine abuse. PMID:26968195

  4. Adenovirus E1A-induced apoptosis elicits a steep decrease in the topoisomerase II alpha level during the latent phase.

    PubMed

    Nakajima, T; Ohi, N; Arai, T; Nozaki, N; Kikuchi, A; Oda, K

    1995-02-16

    The human KB derivative cell line MA1, established by introduction of the adenovirus E1A 12S cDNA linked to the hormone-inducible promoter, elicits apoptosis upon treatment with dexamethasone. The cell lines partially refractory to apoptosis were established by introducing the expression plasmid for the adenovirus E1B 19k protein to MA1 cells. After induction of E1A in MA1 cells by dexamethasone, the level of p53 increased to about 10-fold within 24 h, and morphological changes characteristics of apoptosis began to be observed within 48 h. Most of cells were killed at 72 h releasing apoptotic bodies. The level of topoisomerase II alpha began to decrease steeply within 36 h, preceding the onset of DNA degradation while its mRNA level unchanged throughout the apoptotic process. E1B 19k protected the decrease in topoisomerase II alpha as well as DNA fragmentation depending on its expression levels. Topoisomerase II alpha is induced specifically at G2/M, and computer search revealed the presence of cyclin B type destruction box in topoisomerase II alpha. These results strongly suggest that E1A or E1A stabilized p53 induces apoptosis by targeting topoisomerase II alpha to the ubiquitination pathway and E1B 19k alleviates its action. PMID:7862442

  5. Transgene Expression up to 7 Years in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors

    PubMed Central

    Brunetti-Pierri, Nicola; Ng, Thomas; Iannitti, David; Cioffi, William; Stapleton, Gary; Law, Mark; Breinholt, John; Palmer, Donna; Grove, Nathan; Rice, Karen; Bauer, Cassondra; Finegold, Milton; Beaudet, Arthur; Mullins, Charles

    2013-01-01

    Abstract Helper-dependent adenoviral vectors (HDAd) have been shown to mediate a considerably longer duration of transgene expression than first-generation adenoviral vectors. We have previously shown that transgene expression from HDAd-transduced hepatocytes can persist at high levels for up to 2.6 years in nonhuman primates following a single-vector administration. Because duration of transgene expression and long-term toxicity are critical for risk:benefit assessment, we have continued to monitor these animals. We report here that transgene expression has persisted for the entire observation period of up to 7 years for all animals without long-term adverse effects. However, in all cases, transgene expression level slowly declined over time to less than 10% of peak values by the end of the observation period but remained 2.3–111-fold above baseline values. These results will provide important information for a more informed risk:benefit assessment before clinical application of HDAd. PMID:23902403

  6. Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector

    PubMed Central

    Palmer, Donna J; Grove, Nathan C; Ng, Philip

    2016-01-01

    Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells, especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report, we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3’ untranslated region of the expression cassette in the HDAd. Thus during vector production, when the producer cells are coinfected with both the helper virus (HV) and the HDAd, the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified, transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. PMID:27331077

  7. Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli.

    PubMed

    Lucher, L A; Kimelman, D; Symington, J S; Brackmann, K H; Cartas, M A; Thornton, H; Green, M

    1984-10-01

    A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.

  8. Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli.

    PubMed Central

    Lucher, L A; Kimelman, D; Symington, J S; Brackmann, K H; Cartas, M A; Thornton, H; Green, M

    1984-01-01

    A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4. Images PMID:6384554

  9. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  10. STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Standardization and Validation of Adenoviral Transduction of an Androgen Receptor Positive Cell Line with an MMTV-Luc Reporter for Endocrine Screening P. Hartig, K . Bobseine,
    M. Cardon, C. Lambright and L. E. Gray, Jr. USEPA, Reproductive Toxicology Division, NHEERL, RTP, NC...

  11. Quantification of High-Capacity Helper-Dependent Adenoviral Vector Genomes In Vitro and In Vivo, Using Quantitative TaqMan Real-Time Polymerase Chain Reaction

    PubMed Central

    PUNTEL, M.; CURTIN, J.F.; ZIRGER, J.M.; MUHAMMAD, A.K.M.; XIONG, W.; LIU, C.; HU, J.; KROEGER, K.M.; CZER, P.; SCIASCIA, S.; MONDKAR, S.; LOWENSTEIN, P.R.; CASTRO, M.G.

    2006-01-01

    First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host’s immune system. Another advantage is their large cloning capacity (up to ~35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials. PMID:16716110

  12. Ultrasound-assisted nonviral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion.

    PubMed

    Wang, Z; Zourelias, L; Wu, C; Edwards, P C; Trombetta, M; Passineau, M J

    2015-09-01

    Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial using Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system used in this pioneering trial preclude its advancement to a Phase II trial, and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted nonviral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Swine irradiated unilaterally with a 10-Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 h and 1 week post treatment (1.62±0.48 ml and 1.87±0.45 ml) to preinjury levels (1.34±0.14 ml) in a manner comparable to Adenoviral delivery (2.32±0.6 ml and 1.33±0.97 ml). UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model, and it is a candidate for advancement to a Phase I human clinical trial.

  13. Ultrasound-assisted non-viral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion

    PubMed Central

    Wang, Z; Zourelias, L; Wu, C; Edwards, PC; Trombetta, M; Passineau, MJ

    2015-01-01

    Rationale Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial utilizing Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system utilized in this pioneering trial preclude its advancement to a Phase II trial and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted non-viral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Findings Swine irradiated unilaterally with a 10Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 hours and one week post-treatment (1.62+/−0.48ml, 1.87+/−0.45ml) to pre-injury levels (1.34+/−0.14ml) in a manner comparable to Adenoviral delivery (2.32+/−0.6ml, 1.33+/−0.97ml). Conclusions UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model and is a candidate for advancement to a Phase I human clinical trial. PMID:25871828

  14. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  15. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  16. A novel adenoviral vector-mediated mouse model of Charcot-Marie-Tooth type 2D (CMT2D).

    PubMed

    Seo, Ah Jung; Shin, Youn Ho; Lee, Seo Jin; Kim, Doyeun; Park, Byung Sun; Kim, Sunghoon; Choi, Kyu Ha; Jeong, Na Young; Park, Chan; Jang, Ji-Yeon; Huh, Youngbuhm; Jung, Junyang

    2014-04-01

    Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.

  17. Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment.

    PubMed

    Alyahya, S Anisah; Nolan, Scott T; Smith, Cara M R; Bishai, William R; Sadoff, Jerald; Lamichhane, Gyanu

    2015-01-01

    To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies. PMID:25996375

  18. Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment

    PubMed Central

    Alyahya, S. Anisah; Nolan, Scott T.; Smith, Cara M. R.; Bishai, William R.; Sadoff, Jerald; Lamichhane, Gyanu

    2015-01-01

    To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies. PMID:25996375

  19. Transfection of fetal rat intestinal epithelial cells by viral oncogenes: establishment and characterization of the E1A-immortalized SLC-11 cell line.

    PubMed Central

    Emami, S; Mir, L; Gespach, C; Rosselin, G

    1989-01-01

    Intestinal epithelial cells from 19-day-old rat fetuses underwent electropermeabilization and were successfully transfected by three recombinant plasmids containing the cloned oncogenes from the human adenovirus type 2 early region E1A (SLC-11 cells) and polyoma virus and simian virus 40 large T tumor antigens (SLC-21 and SLC-41 cells). SLC-11 cells were propagated for 21 months in culture (current passage, 76; doubling time, 17 hr) and were immortalized by E1A, as shown by RNA transfer blot (Northern blot) analysis and indirect immunofluorescence of the nuclear oncoproteins. These cells were not tumorigenic in either athymic nude mice or syngeneic Wistar rats and showed a nearly normal karyotype with minimal chromosomal changes. The immortalized epithelial cell line SLC-11 retained several of the phenotypes observed in the parent cells of the intestinal mucosa, including cytoplasmic villin, cytokeratins, enkephalinase, and cell surface receptors sensitive to vasoactive intestinal peptide. It is concluded that immortal SLC-11 cells are a suitable model for studying the proliferation and differentiation of epithelial intestinal cells and analyzing cancer progression in the gastrointestinal tract. Images PMID:2470094

  20. PEA-15 is inhibited by adenovirus E1A and plays a role in ERK nuclear export and Ras-induced senescence.

    PubMed

    Gaumont-Leclerc, Marie-France; Mukhopadhyay, Uptal Kumar; Goumard, Stéphane; Ferbeyre, Gerardo

    2004-11-01

    Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.

  1. Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

    PubMed Central

    Vasconcelos, José Ronnie; Bruña–Romero, Oscar; Araújo, Adriano F.; Dominguez, Mariana R.; Ersching, Jonatan; de Alencar, Bruna C. G.; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bortoluci, Karina R.; Amarante-Mendes, Gustavo P.; Lopes, Marcela F.; Rodrigues, Mauricio M.

    2012-01-01

    MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination. PMID:22615561

  2. Ex vivo adenovirus-mediated gene transfer and immunomodulatory protein production in human cornea.

    PubMed

    Oral, H B; Larkin, D F; Fehervari, Z; Byrnes, A P; Rankin, A M; Haskard, D O; Wood, M J; Dallman, M J; George, A J

    1997-07-01

    One attractive strategy to prevent or control allograft rejection is to genetically modify the donor tissue before transplantation. In this study, we have examined the feasibility of gene transfer to human corneal endothelium, using a number of recombinant adenovirus constructs. Ex vivo infection of human corneas with adenoviral vectors containing lacZ, under transcriptional control of either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters, provided high-level gene expression, which was largely restricted to endothelium. Expression of the reporter gene persisted at relatively high levels for up to 7 days, followed by a decline to indetectable levels by 28 days. RT-PCR analysis of lacZ transcription showed a similar picture with a short period (3-7 days) of RNA transcription after infection. In contrast, adenoviral DNA persisted for at least 56 days. Subsequently, we examined the expression of a potential therapeutic gene, CTLA-4 Ig fusion protein. Following infection of human corneas with adenoviral vectors encoding CTLA-4 Ig protein, high levels of the fusion protein were detected in corneal culture supernatants for up to 28 days. This protein was functionally active, as determined by binding to B7.1 (CD80)-expressing transfectants. This study suggests that genetic alteration of donor cornea before transplantation is a feasible approach for preventing or controlling allograft rejection. Similar gene-based strategies might also be feasible to prevent rejection of other transplanted tissues or organs. PMID:9282165

  3. Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

    PubMed

    Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2016-08-01

    Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

  4. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    PubMed Central

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  5. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  6. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients.

    PubMed

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as "off-the-shelf" therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  7. Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds.

    PubMed

    Stoff, Alexander; Rivera, Angel A; Mathis, J Michael; Moore, Steven T; Banerjee, N S; Everts, Maaike; Espinosa-de-los-Monteros, Antonio; Novak, Zdenek; Vasconez, Luis O; Broker, Thomas R; Richter, Dirk F; Feldman, Dale; Siegal, Gene P; Stoff-Khalili, Mariam A; Curiel, David T

    2007-05-01

    Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-beta(1) and TGF-beta(2) precursor proteins, but an increase in expression of TGF-beta(3) precursor protein and TGF-beta type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

  8. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    PubMed Central

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients.

  9. Comparison of Replication-Competent, First Generation, and Helper-Dependent Adenoviral Vaccines

    PubMed Central

    Weaver, Eric A.; Nehete, Pramod N.; Buchl, Stephanie S.; Senac, Julien S.; Palmer, Donna; Ng, Philip; Sastry, K. Jagannadha; Barry, Michael A.

    2009-01-01

    All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines. PMID:19333387

  10. Adenoviral transduction of naive CD4 T cells to study Treg differentiation.

    PubMed

    Warth, Sebastian C; Heissmeyer, Vigo

    2013-08-13

    Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44(low), CD62L(high)) and resting (CD25(-), CD69(-)) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

  11. Gene therapy prospects--intranasal delivery of therapeutic genes.

    PubMed

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  12. PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers

    PubMed Central

    Klanrit, P; Taebunpakul, P; Flinterman, MB; Odell, EW; Riaz, MA; Melino, G; Salomoni, P; Mymryk, JS; Gäken, J; Farzaneh, F; Tavassoli, M

    2010-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic ΔNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73β, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73β-induced EGFR suppression and apoptosis. Expression of TAp73β efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression. PMID:19597475

  13. Adenoviral vector-mediated overexpression of osteoprotegerin accelerates osteointegration of titanium implants in ovariectomized rats.

    PubMed

    Yin, G; Chen, J; Wei, S; Wang, H; Chen, Q; Lin, Y; Hu, J; Luo, E

    2015-08-01

    This study investigated the efficacy of human osteoprotegerin (hOPG) transgene to accelerate osteointegration of titanium implant in ovariectomized (OVX) rats. Bone marrow stromal cells transduced with Ad-hOPG-EGFP could sustainedly express hOPG. Osteoclast precursor RAW264.7 cells treated by the hOPG were examined by tartrate-resistant acid phosphatase (TRAP) staining and bone slice resorption assay. The results showed differentiation and function of osteoclasts were significantly suppressed by hOPG in vitro. Ad-hOPG-EGFP was locally administered to the bone defect prior to implant placement in OVX and sham rats. After 3, 7, 28 days of implantation, the femurs were harvested for molecular and histological analyses. Successful transgene expression was confirmed by western blot and cryosectioning. A significant reduction in TRAP+ numbers was detected in Ad-hOPG-EGFP group. Real-time reverse transcriptase-PCR examination revealed that hOPG transgene markedly diminished the expression of cathepsin K and receptor activator for nuclear factor-κ B ligand in vivo. The transgene hOPG modification revealed a marked increasing osteointegration and restored implant stability in OVX rats (P<0.01), compared with the control groups (Ad-EGFP or sterilized phosphate-buffered saline) 28 days after implantation. In conclusion, hOPG via direct adenovirus-mediated gene transfer could accelerate osteointegration of titanium implants in OVX rats. Osteoprotegerin gene therapy may be an effective strategy to osteointegration of implants under osteoporotic conditions.

  14. Adenoviral E4orf3 and E4orf6 Proteins, But Not E1B55K, Increase Killing of Cancer Cells by Radiotherapy in vivo

    SciTech Connect

    Liikanen, Ilkka; Dias, Joao D.; Nokisalmi, Petri; Sloniecka, Marta; Kangasniemi, Lotta; Rajecki, Mari; Dobner, Thomas; Tenhunen, Mikko; Kanerva, Anna; Pesonen, Sari; Ahtiainen, Laura Ph.D.; Hemminki, Akseli

    2010-11-15

    Purpose: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. Methods and Materials: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. Results: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. Conclusions: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

  15. Potent antitumor immunity generated by a CD40-targeted adenoviral vaccine.

    PubMed

    Hangalapura, Basav N; Oosterhoff, Dinja; de Groot, Jan; Boon, Louis; Tüting, Thomas; van den Eertwegh, Alfons J; Gerritsen, Winald R; van Beusechem, Victor W; Pereboev, Alexander; Curiel, David T; Scheper, Rik J; de Gruijl, Tanja D

    2011-09-01

    In situ delivery of tumor-associated antigen (TAA) genes into dendritic cells (DC) has great potential as a generally applicable tumor vaccination approach. Although adenoviruses (Ad) are an attractive vaccine vehicle in this regard, Ad-mediated transduction of DCs is hampered by the lack of expression of the Ad receptor CAR on the DC surface. DC activation also requires interaction of CD40 with its ligand CD40L to generate protective T-cell-mediated tumor immunity. Therefore, to create a strategy to target Ads to DCs in vivo, we constructed a bispecific adaptor molecule with the CAR ectodomain linked to the CD40L extracellular domain via a trimerization motif (CFm40L). By targeting Ad to CD40 with the use of CFm40L, we enhanced both transduction and maturation of cultured bone marrow-derived DCs. Moreover, we improved transduction efficiency of DCs in lymph node and splenic cell suspensions in vitro and in skin and vaccination site-draining lymph nodes in vivo. Furthermore, CD40 targeting improved the induction of specific CD8(+) T cells along with therapeutic efficacy in a mouse model of melanoma. Taken together, our findings support the use of CD40-targeted Ad vectors encoding full-length TAA for in vivo targeting of DCs and high-efficacy induction of antitumor immunity.

  16. Adenoviral Transduction of Human Acid Sphingomyelinase into Neo-Angiogenic Endothelium Radiosensitizes Tumor Cure

    PubMed Central

    Fuller, John D.; Rotolo, Jimmy A.; García-Barros, Mónica; Feldman, Regina; Rao, Shyam; Weichselbaum, Ralph R.; Harats, Dror; Haimovitz-Friedman, Adriana; Fuks, Zvi; Sadelain, Michel; Kolesnick, Richard

    2013-01-01

    These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors. PMID:23936314

  17. Biophysical characterization of the DNA binding and condensing properties of adenoviral core peptide mu.

    PubMed

    Keller, Michael; Tagawa, Toshiaki; Preuss, Monika; Miller, Andrew D

    2002-01-15

    Cationic peptides containing Lys and Arg residues interact with DNA via charge-charge interactions and are known to play an important role in DNA charge neutralization and condensation processes. In this paper, we describe investigations of the interaction of the cationic adenovirus core complex peptide mu with a dodecameric ODN (12 bp) and pDNA (7528 bp) using a combination of fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and photon correlation spectroscopy. Comparisons are made with protamine, a cationic peptide well-known for DNA charge neutralization and condensation. Equilibrium dissociation constants are derived independently by both CD and ITC methods for the interaction between protamine or mu with pDNA (K(d) = 0.6-1 microM). Thermodynamic data are also obtained by ITC, indicating strong charge-charge interactions. The interaction of protamine with pDNA takes place with decreasing entropy (-28.7 cal mol(-1) K(-1)); unusually, the interaction of mu with pDNA takes place with increasing entropy (Delta S degrees (bind) = 11.3 cal mol(-1) K(-1)). Although protamine and mu appear to destabilize pDNA double helix character to similar extents, according to CD thermal titration analyses, PCS studies show that interactions between mu and pDNA result in the formation of significantly more size-stable condensed particles than protamine. The enhanced flexibility and size stability of mu-DNA (MD) particles (80-110 nm) compared to protamine counterparts suggest that MD particles are ideal for use as a part of new nonviral gene delivery vectors.

  18. Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors.

    PubMed

    Schiedner, Gudrun; Hertel, Sabine; Johnston, Marion; Dries, Volker; van Rooijen, Nico; Kochanek, Stefan

    2003-01-01

    Tissue macrophages, in particular hepatic Kupffer cells (KCs), contribute to early inflammatory responses following adenoviral vector administration. This study evaluates the effect of selective and transient (3 days) depletion of KCs by a single injection of clodronate liposomes on the in vivo performance of high-capacity adenoviral (HC-Ad) vectors. In KC-depleted C57BL/6 and C3H mice increased and stabilized hAAT levels were observed following intravenous injection of HC-Ad vectors expressing human alpha-1 anti-trypsin (hAAT) either from the hAAT promoter or from the human cytomegalovirus promoter. Comparable increases in hAAT levels were obtained in mice preinjected with a transcriptionally silent HC-Ad vector. Interestingly, in the majority of animals of both strains depletion of KCs was sufficient to prevent the generation of anti-hAAT antibodies, resulting in prolonged transgene expression. Thus, short-term and selective depletion of hepatic macrophages at the same time significantly increased hepatic transgene expression and reduced the humoral immune response to the transgenic protein.

  19. Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

    PubMed Central

    Kosinska, Anna; Schumann, Alexandra; Brovko, Olena; Walker, Andreas; Lu, Mengji; Johrden, Lena; Mayer, Anja; Wildner, Oliver; Roggendorf, Michael

    2013-01-01

    Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8+ and CD4+ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven. PMID:23637419

  20. Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

    PubMed

    Günther, Patrick S; Peper, Janet K; Faist, Benjamin; Kayser, Simone; Hartl, Lena; Feuchtinger, Tobias; Jahn, Gerhard; Neuenhahn, Michael; Busch, Dirk H; Stevanović, Stefan; Dennehy, Kevin M

    2015-09-01

    Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

  1. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  2. Adenoviral-E2F-1 radiosensitizes p53{sup wild-type} and p53{sup null} human prostate cancer cells

    SciTech Connect

    Nguyen, Khanh H.; Hachem, Paul; Khor, L.-Y.; Salem, Naji; Hunt, Kelly K.; Calkins, Peter R.; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2005-09-01

    Purpose: E2F-1 is a transcription factor that enhances the radiosensitivity of various cell lines by inducing apoptosis. However, there are conflicting data concerning whether this enhancement is mediated via p53 dependent pathways. Additionally, the role of E2F-1 in the response of human prostate cancer to radiation has not been well characterized. In this study, we investigated the effect of Adenoviral-E2F-1 (Ad-E2F-1) on the radiosensitivity of p53{sup wild-type} (LNCaP) and p53{sup null} (PC3) prostate cancer cell lines. Methods and Materials: LNCaP and PC3 cells were transduced with Ad-E2F-1, Adenoviral-Luciferase (Ad-Luc) control vector, or Adenoviral-p53 (Ad-p53). Expression of E2F-1 and p53 was examined by Western blot analysis. Annexin V and caspase 3 + 7 assays were performed to estimate the levels of apoptosis. Clonogenic survival assays were used to determine overall cell death. Statistical significance was determined by analysis of variance, using the Bonferroni method to correct for multiple comparisons. Results: Western blot analysis confirmed the efficacy of transductions with Ad-E2F-1 and Ad-p53. Ad-E2F-1 transduction significantly enhanced apoptosis and decreased clonogenic survival in both cell lines. These effects were compounded by the addition of RT. Although E2F-1-mediated radiosensitization was independent of p53 status, this effect was more pronounced in p53{sup wild-type} LNCaP cells. When PC3 cells were treated with Ad-p53 in combination with RT and Ad-E2F-1, there was at least an additive reduction in clonogenic survival. Conclusions: Our results suggest that Ad-E2F-1 significantly enhances the response of p53{sup wild-type} and p53{sup null} prostate cancer cells to radiation therapy, although radiosensitization is more pronounced in the presence of p53. Ad-E2F-1 may be a useful adjunct to radiation therapy in the treatment of prostate cancer.

  3. The effectiveness of the oncolytic activity induced by Ad5/F35 adenoviral vector is dependent on the cumulative cellular conditions of survival and autophagy.

    PubMed

    Kim, So Y; Kang, Sujin; Song, Jae J; Kim, Joo-Hang

    2013-04-01

    To overcome the poor tumor transduction efficiency of adenovirus serotype 5 (Ad5) observed in several types of cancer, the fiber region of Ad5, apart from its tail, was replaced by adenovirus serotype 35 (Ad35). The chimeric Ad5/F35 adenoviral vector did not exhibit any significant enhancement of transduction efficiency. CD46, a receptor for Ad35, was expressed in relatively small amounts in most of the cancer cells examined. Therefore, we investigated the pivotal factor(s) that render cancer cells susceptible to transduction. We discovered that the tumor transduction efficiency of Ad5/F35 was enhanced in the presence of rapamycin, an autophagy inducer, in some cancer cells. Analysis of survival potential and cell proliferation rates revealed that Ad5/F35 exerted a more pronounced oncolytic effect in cancer cells with higher survival potential in the presence of rapamycin.

  4. [Experimental gene therapy using p21/WAF1 gene in esophageal squamous cell carcinoma--adenovirus infection and gene gun technology].

    PubMed

    Fujii, T; Tanaka, Y; Tanaka, T; Matono, S; Sueyoshi, S; Fujita, H; Shirouzu, K; Kato, S; Yamana, H

    2001-10-01

    p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

  5. A Human Vaccine Strategy Based On Chimpanzee Adenoviral and MVA Vectors That Primes, Boosts and Sustains Functional HCV Specific T-Cell Memory*

    PubMed Central

    Swadling, Leo; Capone, Stefania; Antrobus, Richard D.; Brown, Anthony; Richardson, Rachel; Newell, Evan W.; Halliday, John; Kelly, Christabel; Bowen, Dan; Fergusson, Joannah; Kurioka, Ayako; Ammendola, Virginia; Sorbo, Mariarosaria Del; Grazioli, Fabiana; Esposito, Maria Luisa; Siani, Loredana; Traboni, Cinzia; Hill, Adrian; Colloca, Stefano; Davis, Mark; Nicosia, Alfredo; Cortese, Riccardo; Folgori, Antonella; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies and assessment of host immunity during acute infection highlight the critical role that effective T-cell immunity plays in viral control. In this first-in-man study we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A and NS5B proteins of HCV genotype-1b. Analysis employed single cell mass cytometry (CyTOF), and HLA class-I peptide tetramer technology in healthy human volunteers. We show that HCV specific T-cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV specific T-cells targeting multiple HCV antigens. Sustained memory and effector T-cell populations are generated and T-cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) following heterologous MVA boost. We have developed a HCV vaccine strategy, with durable, broad, sustained and balanced T-cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine. PMID:25378645

  6. Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

    PubMed

    Deng, Guangcun; Li, Wu; Wu, Xiaoling; Bao, Shaowen; Zeng, Jin; Zhao, Ning; Luo, Meihui; Liu, Xiaoming; Wang, Yujiong

    2015-12-01

    The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation.

  7. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

    PubMed Central

    Cristiano, R J; Smith, L C; Kay, M A; Brinkley, B R; Woo, S L

    1993-01-01

    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies. Images Fig. 2 Fig. 4 PMID:8265587

  8. Potential of Gene Therapy for the Treatment of Pituitary Tumors

    PubMed Central

    Goya, R G.; Sarkar, D.K.; Brown, O.A.; Hereñú, C.B.

    2010-01-01

    Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy—the transfer of genetic material for therapeutic purposes—has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary

  9. Development of hybrid viral vectors for gene therapy.

    PubMed

    Huang, Shuohao; Kamihira, Masamichi

    2013-01-01

    Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.

  10. Acetylation at a lysine residue adjacent to the CtBP binding motif within adenovirus 12 E1A causes structural disruption and limited reduction of CtBP binding

    SciTech Connect

    Molloy, David; Mapp, Katie L.; Webster, Rachel; Gallimore, Phillip H.; Grand, Roger J.A. . E-mail: R.J.A.Grand@bham.ac.uk

    2006-11-25

    C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disrupts the binding to CtBP1 and CtBP2 and alters the K {sub i} of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of {beta}-turns over the region Val{sup 254}-Arg{sup 262}. Within the acetylated isoform, the {beta}-turn conformation is less extensive, Val{sup 26}-Arg{sup 262} adopting a random confirmation. We conclude that secondary structure ({beta}-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the {beta}-turn conformation at the C-terminus of AdE1A contributes to binding to {alpha} importin and nuclear import. Acetylation of lysine {sup 261} could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.

  11. Gene Therapy in Cardiac Surgery: Clinical Trials, Challenges, and Perspectives

    PubMed Central

    Katz, Michael G.; Fargnoli, Anthony S.; Kendle, Andrew P.; Hajjar, Roger J.; Bridges, Charles R.

    2016-01-01

    The concept of gene therapy was introduced in the 1970s after the development of recombinant DNA technology. Despite the initial great expectations, this field experienced early setbacks. Recent years have seen a revival of clinical programs of gene therapy in different fields of medicine. There are many promising targets for genetic therapy as an adjunct to cardiac surgery. The first positive long-term results were published for adenoviral administration of vascular endothelial growth factor with coronary artery bypass grafting. In this review we analyze the past, present, and future of gene therapy in cardiac surgery. The articles discussed were collected through PubMed and from author experience. The clinical trials referenced were found through the Wiley clinical trial database (http://www.wiley.com/legacy/wileychi/genmed/clinical/) as well as the National Institutes of Health clinical trial database (Clinicaltrials.gov). PMID:26801060

  12. Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

    PubMed Central

    Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

    2014-01-01

    Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

  13. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via “Antigen Capsid-Incorporation” strategy

    PubMed Central

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L.; Derdeyn, Cynthia A.; Matthews, Qiana L.

    2016-01-01

    Adenoviral (Ad) vectors in combination with the “Antigen Capsid-Incorporation” strategy have been applied in developing HIV-1 vaccines, due to the vectors’ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the “Antigen Capsid-Incorporation” strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. PMID:26499044

  14. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

    PubMed

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

    2016-01-01

    Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2.

  15. Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

    PubMed

    Souza, Sandra C; Muliro, Kizito V; Liscum, Laura; Lien, Ping; Yamamoto, Mia T; Schaffer, Jean E; Dallal, Gerard E; Wang, Xinzhong; Kraemer, Fredric B; Obin, Martin; Greenberg, Andrew S

    2002-03-01

    Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells. PMID:11751901

  16. Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.

    PubMed

    Gordon, Ashley; Kozin, Elliott D; Keswani, Sundeep G; Vaikunth, Sachin S; Katz, Anna B; Zoltick, Philip W; Favata, Michele; Radu, Antoneta P; Soslowsky, Louis J; Herlyn, Meenhard; Crombleholme, Timothy M

    2008-01-01

    Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1-90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment. PMID:18086289

  17. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  18. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    SciTech Connect

    Fu, Yuanhui; He, Jinsheng; Zheng, Xianxian; Wu, Qiang; Zhang, Mei; Wang, Xiaobo; Wang, Yan; Xie, Can; Tang, Qian; Wei, Wei; Wang, Min; Song, Jingdong; Qu, Jianguo; Zhang, Ying; Wang, Xin; Hong, Tao

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  19. Off-the-shelf adenoviral-mediated immunotherapy via bicistronic expression of tumor antigen and iMyD88/CD40 adjuvant.

    PubMed

    Kemnade, Jan Ole; Seethammagari, Mamatha; Narayanan, Priya; Levitt, Jonathan M; McCormick, Alison A; Spencer, David M

    2012-07-01

    Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.

  20. Enhancement of Protective Efficacy through Adenoviral Vectored Vaccine Priming and Protein Boosting Strategy Encoding Triosephosphate Isomerase (SjTPI) against Schistosoma japonicum in Mice

    PubMed Central

    Dai, Yang; Wang, Xiaoting; Tang, Jianxia; Zhao, Song; Xing, Yuntian; Dai, Jianrong; Jin, Xiaolin; Zhu, Yinchang

    2015-01-01

    Background Schistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice. Methodology/Principal Findings Adenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice. Conclusions/Significance The rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China. PMID:25793406

  1. Computer-assisted hydrodynamic gene delivery.

    PubMed

    Suda, Takeshi; Suda, Kieko; Liu, Dexi

    2008-06-01

    The recently developed hydrodynamic delivery method makes it possible to deliver DNA and RNA into parenchyma cells by intravascular injection of nucleic acid-containing solution. While this procedure is effective in rodents, it is difficult to perform in large animals, because manual control while delivering the injection cannot be sufficiently reliable for achieving a just-right hydrodynamic pressure in targeted tissue. In order to overcome this problem, we have developed a computer-controlled injection device that uses real-time intravascular pressure as a regulator. Using the new injection device, and mouse liver as the model organ, we demonstrated continuous injection at a single pressure and different pressures, and also serial (repeated) injections at intervals of 250 ms, by programming the computer according to the need. When assessed by reporter plasmids, the computer-controlled injection device exhibits gene delivery efficiency similar to that of conventional hydrodynamic injection. The device is also effective in gene delivery to kidney and muscle cells in rats, with plasmids or adenoviral vectors as gene carriers. Successful gene delivery to liver and kidney was also demonstrated in pigs, with the computer-controlled injection being combined with image-guided catheterization. These results represent a significant advance in in vivo gene delivery research, with potential for use in gene therapy in humans.

  2. Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5–based Constructs

    PubMed Central

    Alonso-Padilla, Julio; Papp, Tibor; Kaján, Győző L; Benkő, Mária; Havenga, Menzo; Lemckert, Angelique; Harrach, Balázs; Baker, Andrew H

    2016-01-01

    Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes. PMID:26478249

  3. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    SciTech Connect

    Luo, Chunxia; Yi, Bin; Bai, Li; Xia, Yongzhi; Wang, Guansong; Qian, Guisheng; Feng, Hua

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  4. Adenoviral-mediated gene transfer of vascular endothelial growth factor in critical limb ischemia: safety results from a phase I trial.

    PubMed

    Mohler, Emile R; Rajagopalan, Sanjay; Olin, Jeffrey W; Trachtenberg, Jeffrey D; Rasmussen, Henrik; Pak, Raphael; Crystal, Ronald G

    2003-01-01

    Critical limb ischemia (CLI) is typified by rest pain and/or tissue necrosis secondary to advanced peripheral arterial disease (PAD) and is characterized by diminution in limb perfusion at rest. We tested the safety of an angiogenic strategy with CI-1023 (Ad(GV)VEGF121.10), a replication-deficient adenovirus encoding human vascular endothelial growth factor isoform 121 in patients with CLI as part of a phase I trial. Fifteen subjects >35 years of age with CLI and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 x 10(8) to 4 x 10(10) particle units, n = 13) or placebo (n = 2). All of the patients tolerated the injection well and there were no serious complications related to the procedure. Transient edema was noted in one patient. A total of 79 adverse events were reported over the course of one year. One death (day 136) and one malignancy (day 332) occurred in the CI-1023 group. CI-1023 appears to be well tolerated and safe for single-dose administration in patients with critical limb ischemia due to PAD. Further studies are needed to determine the efficacy of this form of therapeutic angiogenesis. PMID:12866606

  5. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

  6. A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

    PubMed Central

    Pérez de Val, Bernat; Vidal, Enric; Villarreal-Ramos, Bernardo; Gilbert, Sarah C.; Andaluz, Anna; Moll, Xavier; Martín, Maite; Nofrarías, Miquel; McShane, Helen; Vordermeier, H. Martin; Domingo, Mariano

    2013-01-01

    The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. PMID:24278420

  7. Chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA enhances sensitivity of human colorectal cancer cells to radiotherapy in vitro and in vivo.

    PubMed

    Xiang, D-B; Chen, Z-T; Wang, D; Li, M-X; Xie, J-Y; Zhang, Y-S; Qing, Y; Li, Z-P; Xie, J

    2008-10-01

    Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with radioresistance. Here we investigate whether targeted inhibition of APE1 can sensitize tumor cells to irradiation in vitro and in vivo. We first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA). The infectivity of chimeric Ad5/F35 to LOVO colon cancer cells was greater than that of Ad5. APE1 was strongly expressed and nuclear factor kappaB (NF-kappaB), a downstream molecule of APE1, known as a radioresistance factor, was constitutively active in LOVO cells. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of LOVO cells to irradiation in clonogenic survival assays, associated with increased cell apoptosis. The APE1 expression in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of DNA-binding activity of NF-kappaB and Ad5/F35-APE1 siRNA effectively inhibited constitutive and irradiation-induced APE1 expression and NF-kappaB activation. In a subcutaneous nude mouse colon cancer model, Ad5/F35-APE1 siRNA (5 x 10(8) IU, intratumoral injection) inhibited the expression of APE1 protein in LOVO xenografts, and significantly enhanced inhibition of tumor growth by irradiation. In conclusion, APE1 may be involved as one of the radioresistance factors, and targeted inhibition of APE1 shows an effective means of enhancing tumor sensitivity to radiotherapy.

  8. Adenovirus Specific Pre-Immunity Induced by Natural Route of Infection Does Not Impair Transduction by Adenoviral Vaccine Vectors in Mice

    PubMed Central

    de Andrade Pereira, Bruna; E. Maduro Bouillet, Leoneide; Dorigo, Natalia A.; Fraefel, Cornel; Bruna-Romero, Oscar

    2015-01-01

    Recombinant human adenovirus serotype 5 (HAd5V) vectors are gold standards of T-cell immunogenicity as they efficiently induce also humoral responses to exogenous antigens, in particular when used in prime-boost protocols. Some investigators have shown that pre-existing immunity to adenoviruses interferes with transduction by adenoviral vectors, but the actual extent of this interference is not known since it has been mostly studied in mice using unnatural routes of infection and virus doses. Here we studied the effects of HAd5V-specific immune responses induced by intranasal infection on the transduction efficiency of recombinant adenovirus vectors. Of interest, when HAd5V immunity was induced in mice by the natural respiratory route, the pre-existing immunity against HAd5V did not significantly interfere with the B and T-cell immune responses against the transgene products induced after a prime/boost inoculation protocol with a recombinant HAd5V-vector, as measured by ELISA and in vivo cytotoxic T-cell assays, respectively. We also correlated the levels of HAd5V-specific neutralizing antibodies (Ad5NAbs) induced in mice with the levels of Ad5NAb titers found in humans. The data indicate that approximately 60% of the human serum samples tested displayed Ad5NAb levels that could be overcome with a prime-boost vaccination protocol. These results suggest that recombinant HAd5V vectors are potentially useful for prime-boost vaccination strategies, at least when pre-existing immunity against HAd5V is at low or medium levels. PMID:26679149

  9. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons.

    PubMed

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T; Kasperski, Jeffrey; Gierasch, William W; Edvinsson, Lars; Heistad, Donald D; Faraci, Frank M; Russo, Andrew F

    2004-01-30

    We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT(1B/D) agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT(1B/D) receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis. PMID:14715155

  10. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  11. Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

    PubMed Central

    DeHart, Caroline J.; Perlman, David H.

    2015-01-01

    ABSTRACT Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1–17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076–1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3

  12. Tuning Surface Charge and PEGylation of Biocompatible Polymers for Efficient Delivery of Nucleic Acid or Adenoviral Vector.

    PubMed

    Choi, Joung-Woo; Kim, Jaesung; Bui, Quang Nam; Li, Yi; Yun, Chae-Ok; Lee, Doo Sung; Kim, Sung Wan

    2015-08-19

    As an effective and safe strategy to overcome the limits of therapeutic nucleic acid or adenovirus (Ad) vectors for in vivo application, various technologies to modify the surface of vectors with nonimmunogenic/biocompatible polymers have been emerging in the field of gene therapy. However, the transfection efficacy of the polymer to transfer genetic materials is still relatively weak. To develop more advanced and effective polymers to deliver not only Ad vectors, but also nucleic acids, 6 biocompatible polymers were newly designed and synthesized to different sizes (2k, 3.4k, or 5k) of poly(ethylene) glycol (PEG) and different numbers of amine groups (2 or 5) based on methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-l-glutamate (PNLG). We characterized size distribution and surface charge of 6 PNLGs after complexation with either nucleic acid or Ad. Among all 6 PNLGs, the 5 amine group PNLG showed the strongest efficacy in delivering nucleic acid as well as Ad vectors. Interestingly, cellular uptake results showed higher uptake ability in Ad complexed with 2 amine group PNLG than Ad/5 amine group PNLG, suggesting that the size of Ad/PNLGs is more essential than the surface charge for cellular uptake in polymers with charges greater than 30 mV. Moreover, the endosome escape ability of Ad/PNLGs increased depending on the number of amine groups, but decreased by PEG size. Cancer cell killing efficacy and immune response studies of oncolytic Ad/PNLGs showed 5 amine group PNLG to be a more effective and safe carrier for delivering Ad. Overall, these studies provide new insights into the functional mechanism of polymer-based approaches to either nucleic acid or Ad/nanocomplex. Furthermore, the identified ideal biocompatible PNLG polymer formulation (5 amine/2k PEG for nucleic acid, 5 amine/5k PEG for Ad) demonstrated high transduction efficiency as well as therapeutic value (efficacy and safety) and thus has strong potential for in vivo therapeutic

  13. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  14. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

    Nikitenko, N. A.; Speiseder, T.; Lam, E.; Rubtsov, P. M.; Tonaeva, Kh. D.; Borzenok, S. A.; Dobner, T.; Prassolov, V. S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

  15. The pyruvate dehydrogenase E1 alpha gene is testosterone and prolactin regulated in prostate epithelial cells.

    PubMed

    Costello, L C; Liu, Y; Zou, J; Franklin, R B

    2000-02-01

    The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells. PMID:10711720

  16. Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies

    PubMed Central

    Uusi-Kerttula, Hanni; Legut, Mateusz; Davies, James; Jones, Rachel; Hudson, Emma; Hanna, Louise; Stanton, Richard J.; Chester, John D.

    2015-01-01

    Abstract Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCARlow/EGFRhigh cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of

  17. A complementation method for functional analysis of mammalian genes

    PubMed Central

    Gonzalez-Santos, Juana Maria; Cao, Huibi; Wang, Anan; Koehler, David R.; Martin, Bernard; Navab, Roya; Hu, Jim

    2005-01-01

    Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies. PMID:15944448

  18. Targeted gene therapy for the treatment of heart failure.

    PubMed

    Rapti, Kleopatra; Chaanine, Antoine H; Hajjar, Roger J

    2011-01-01

    Chronic heart failure is one of the leading causes of morbidity and mortality in Western countries and is a major financial burden to the health care system. Pharmacologic treatment and implanting devices are the predominant therapeutic approaches. They improve survival and have offered significant improvement in patient quality of life, but they fall short of producing an authentic remedy. Cardiac gene therapy, the introduction of genetic material to the heart, offers great promise in filling this void. In-depth knowledge of the underlying mechanisms of heart failure is, obviously, a prerequisite to achieve this aim. Extensive research in the past decades, supported by numerous methodological breakthroughs, such as transgenic animal model development, has led to a better understanding of the cardiovascular diseases and, inadvertently, to the identification of several candidate genes. Of the genes that can be targeted for gene transfer, calcium cycling proteins are prominent, as abnormalities in calcium handling are key determinants of heart failure. A major impediment, however, has been the development of a safe, yet efficient, delivery system. Nonviral vectors have been used extensively in clinical trials, but they fail to produce significant gene expression. Viral vectors, especially adenoviral, on the other hand, can produce high levels of expression, at the expense of safety. Adeno-associated viral vectors have emerged in recent years as promising myocardial gene delivery vehicles. They can sustain gene expression at a therapeutic level and maintain it over extended periods of time, even for years, and, most important, without a safety risk.

  19. Vector platforms for gene therapy of inherited retinopathies

    PubMed Central

    Trapani, Ivana; Puppo, Agostina; Auricchio, Alberto

    2014-01-01

    Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina’s compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs’ limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations. PMID:25124745

  20. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  1. Hepatocellular carcinoma in a gene therapy research subject with ornithine transcarbamylase deficiency

    PubMed Central

    Wilson, James M.; Shchelochkov, Oleg A.; Gallagher, Renata C.; Batshaw, Mark L.

    2012-01-01

    A 66 year old woman who is a manifesting heterozygote for ornithine transcarbamylase deficiency (OTCD) presented with hepatocellular carcinoma (HCC). Fourteen years prior to this presentation she participated in a phase I gene therapy study which used an adenoviral vector, thought to be non-oncogenic, to deliver a normal OTC gene to hepatocytes [1]. A recent review of data collected through a national longitudinal study of individuals with urea cycle defects [2, 3] suggests that early urea cycle disorders (UCDs) are associated with hepatocellular damage and liver dysfunction in many cases. This may predispose an affected individual to a substantially increased risk of developing HCC, as has been observed in certain other inborn errors of metabolism. We speculate that the underlying urea cycle defect may be the cause of HCC in this individual. PMID:22129577

  2. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    PubMed

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  3. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  4. Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation

    PubMed Central

    Quinn, Kylie M.; Costa, Andreia Da; Yamamoto, Ayako; Berry, Dana; Lindsay, Ross W.B.; Darrah, Patricia A.; Wang, Lingshu; Cheng, Cheng; Kong, Wing-Pui; Gall, Jason G.D.; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gomez, Carmen E.; Esteban, Mariano; Wyatt, Linda S.; Moss, Bernard; Morgan, Cecilia; Roederer, Mario; Bailer, Robert T.; Nabel, Gary J.; Koup, Richard A.; Seder, Robert A.

    2013-01-01

    Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively. PMID:23390298

  5. Inhibition of Experimental Liver Cirrhosis in Mice by Telomerase Gene Delivery

    NASA Astrophysics Data System (ADS)

    Rudolph, Karl Lenhard; Chang, Sandy; Millard, Melissa; Schreiber-Agus, Nicole; DePinho, Ronald A.

    2000-02-01

    Accelerated telomere loss has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis. To test this hypothesis directly, telomerase-deficient mice, null for the essential telomerase RNA (mTR) gene, were subjected to genetic, surgical, and chemical ablation of the liver. Telomere dysfunction was associated with defects in liver regeneration and accelerated the development of liver cirrhosis in response to chronic liver injury. Adenoviral delivery of mTR into the livers of mTR-/- mice with short dysfunctional telomeres restored telomerase activity and telomere function, alleviated cirrhotic pathology, and improved liver function. These studies indicate that telomere dysfunction contributes to chronic diseases of continual cellular loss-replacement and encourage the evaluation of ``telomerase therapy'' for such diseases.

  6. Osmotically-induced genes are controlled by the transcription factor TonEBP in cultured cardiomyocytes.

    PubMed

    Navarro, Paola; Chiong, Mario; Volkwein, Karen; Moraga, Francisco; Ocaranza, María Paz; Jalil, Jorge E; Lim, Sun Woo; Kim, Jeong-Ah; Kwon, H Moo; Lavandero, Sergio

    2008-07-25

    Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes. PMID:18502201

  7. Osmotically- induced genes are controlled by the transcription factor TonEBP in cultured cardiomyocytes

    PubMed Central

    Navarro, Paola; Chiong, Mario; Volkwein, Karen; Moraga, Francisco; Ocaranza, María Paz; Jalil, Jorge E.; Lim, Sun Woo; Kim, Jeong-Ah; Kwon, H. Moo; Lavandero, Sergio

    2008-01-01

    Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes. PMID:18502201

  8. Gene Targeting to the Uteroplacental Circulation of Pregnant Guinea Pigs.

    PubMed

    Mehta, Vedanta; Ofir, Keren; Swanson, Anna; Kloczko, Ewa; Boyd, Michael; Barker, Hannah; Avdic-Belltheus, Adnan; Martin, John; Zachary, Ian; Peebles, Donald; David, Anna L

    2016-08-01

    Our study aimed to target adenoviral gene therapy to the uteroplacental circulation of pregnant guinea pigs in order to develop a novel therapy for fetal growth restriction. Four methods of delivery of an adenovirus encoding β-galactosidase (Ad.LacZ) were evaluated: intravascular injection using phosphate-buffered saline (PBS) into (1) uterine artery (UtA) or (2) internal iliac artery or external administration in (3) PBS or (4) pluronic F-127 gel (Sigma Aldrich). Postmortem examination was performed 4 to 7 days after gene transfer. Tissue transduction was assessed by X-gal histochemistry and enzyme-linked immunosorbent assay. External vascular application of the adenovirus vector in combination with pluronic gel had 91.7% success rate in terms of administration (85% maternal survival) and gave the best results for maternal/fetal survival and local transduction efficiency without any spread to maternal or fetal tissues. This study suggests an optimal method of gene delivery to the UtAs of a small rodent for preclinical studies.

  9. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

    PubMed

    Régulier, E; Paul, S; Marigliano, M; Kintz, J; Poitevin, Y; Ledoux, C; Roecklin, D; Cauet, G; Calenda, V; Homann, H E

    2001-01-01

    Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors. PMID:11219493

  10. Tumor Restrictive Suicide Gene Therapy for Glioma Controlled by the FOS Promoter

    PubMed Central

    Hu, Jiliang; Song, Weijian; Luo, Jie; Jiang, Shan; Yan, Fei; Zhai, Baojin

    2015-01-01

    Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog) promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373). Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk) driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement. PMID:26571389

  11. Cis and trans activation of adenovirus IVa2 gene transcription.

    PubMed Central

    Natarajan, V; Salzman, N P

    1985-01-01

    The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter in front of a gene coding for bacterial chloramphenicol acetyl transferase (CATase) and estimating levels of CATase and IVa2 promoter specific RNA synthesized after transfection. To produce detectable amounts of CATase with the IVa2 promoter, an enhancer has to be present in cis. In the absence of enhancer sequences, the adenovirus E1A gene can not stimulate CATase synthesis. When cells were transfected with plasmids containing enhancer sequences and various IVa2 mutant promoters upstream of the CAT gene, we observed that CATase activity was not reduced significantly even after deletion of all sequences upstream of the RNA initiation site. Synthesis of IVa2 specific RNA was dependent on plasmids containing an enhancer (SV40 72 bp repeat) that was present in cis. In the absence of enhancer sequences, co-transfection to provide the adenovirus E1A gene in trans also stimulated IVa2 RNA synthesis. When HeLa cells were transfected with various deletion mutants with an enhancer in cis it was seen that sequences -38 to -64 base pairs upstream of the RNA initiation site are necessary for efficient transcription. The E1A gene in trans and an enhancer in cis have an additive effect on RNA synthesis from both IVa2 and major late promoters. The basis for the conflicting results between transcription and CATase synthesis is discussed. Images PMID:2989786

  12. Glial fibrillary acidic protein promoters direct adenovirus early 1A gene and human telomerase reverse transcriptase promoters direct sodium iodide symporter expression for malignant glioma radioiodine therapy.

    PubMed

    Li, Wei; Tan, Jian; Wang, Peng; Li, Ning; Li, Chengxia

    2015-01-01

    Malignant glioma can be treated with radioiodine following transfection with human sodium iodide symporter (hNIS) gene. Ad-Tp-E1A-Gp-NIS is engineered with human telomerase reverse transcriptase (hTERT) and glial fibrillary acidic protein (GFAP) promoters to express early region 1A (E1A) and hNIS genes, which may be useful in targeted gene therapy. The Ad-Tp-E1A-Gp-NIS was constructed and purified using the E1A and hNIS genes regulated by the hTERT and GFAP promoters, respectively. Glioma cells were infected by Ad-Tp-E1A-Gp-NIS. Selective replication ability of Ad-Tp-E1A-Gp-NIS was then evaluated by plaque forming assay, transgene expression by Western blot, (125)I-iodide uptake and efflux, clonogenicity following (131)I-iodide treatment in the tumor cells, and radioiodine therapy using nude mouse model. The Ad-Tp-E1A-Gp-NIS could selectively replicate; the hNIS gene was successfully expressed under the GFAP promoter. Western blot analyses using E1A- and hNIS-specific antibodies revealed two bands of approximately 40 and 70 kDa. In addition, the cells showed about 93.4 and 107.1 times higher (125)I uptake in U251 and U87 cells than in the control cells, respectively. Clonogenic assay indicated that >90% of cells transfected with Ad-Tp-E1A-Gp-NIS were killed. The Ad-Tp-E1A-Gp-NIS-transfected and 2 mCi (131)I-injected U87 xenograft nude mice survived the longest among the three groups. Ad-Tp-E1A-Gp-NIS has a good ability of selective replication and strong antitumor selectivity. An effective therapy of (131)I was achieved activity in malignant glioma cells after induction of tumor-specific iodide uptake activity by GFAP promoter-directed hNIS gene expression in vitro and in vivo.

  13. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

    PubMed

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.

  14. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter–Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth

    PubMed Central

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W. K.; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor–promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter–driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers. PMID:27054343

  15. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

    PubMed

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers. PMID:27054343

  16. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  17. Gene-based vaccines and immunotherapeutics.

    PubMed

    Liu, Margaret; Acres, Bruce; Balloul, Jean-Marc; Bizouarne, Nadine; Paul, Stephane; Slos, Philippe; Squiban, Patrick

    2004-10-01

    DNA vaccines, comprised of plasmid DNA encoding proteins from pathogens, allergens, and tumors, are being evaluated as prophylactic vaccines and therapeutic treatments for infectious diseases, allergies, and cancer; plasmids encoding normal human proteins are likewise being tested as vaccines and treatments for autoimmune diseases. Examples of in vivo prophylaxis and immunotherapy, based on different types of immune responses (humoral and cellular), in a variety of disease models and under evaluation in early phase human clinical trials are presented. Viral vectors continue to show better levels of expression than those achieved by DNA plasmid vectors. We have focused our clinical efforts, at this time, on the use of recombinant viral vectors for both vaccine as well as cytokine gene transfer studies. We currently have four clinical programs in cancer immunotherapy. Two nonspecific immunotherapy programs are underway that apply adenoviral vectors for the transfer of cytokine genes into tumors in situ. An adenovirus-IFN gamma construct (TG1042) is currently being tested in phase II clinical trials in cutaneous lymphoma. A similar construct, adenovirus-IL2 (TG1024), also injected directly into solid tumors, is currently being tested in patients with solid tumors (about one-half of which are melanoma). Encouraging results are seen in both programs. Two cancer vaccine immunotherapy programs focus on two cancer-associated antigens: human papilloma virus E6 and E7 proteins and the epithelial cancer-associated antigen MUC1. Both are encoded by a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] and both are coexpressed with IL-2. Encouraging results seen in both of these programs are described. PMID:15333750

  18. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    PubMed

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-01-01

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism. PMID:27404720

  19. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    PubMed

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-07-12

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

  20. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  1. Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

    PubMed Central

    Chick, Helen E.; Nowrouzi, Ali; Fronza, Raffaele; McDonald, Robert A.; Kane, Nicole M.; Alba, Raul; Delles, Christian; Sessa, William C.; Schmidt, Manfred; Thrasher, Adrian J.

    2012-01-01

    Abstract We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer. PMID:22931362

  2. Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

    PubMed

    Lee, Seoghyun; Sohn, Kyung-Cheol; Choi, Dae-Kyoung; Won, Minho; Park, Kyeong Ah; Ju, Sung-Kyu; Kang, Kidong; Bae, Young-Ki; Hur, Gang Min; Ro, Hyunju

    2016-01-01

    Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion. PMID:27673563

  3. Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues

    PubMed Central

    Lee, Seoghyun; Sohn, Kyung-Cheol; Choi, Dae-Kyoung; Won, Minho; Park, Kyeong Ah; Ju, Sung-Kyu; Kang, Kidong; Bae, Young-Ki; Hur, Gang Min; Ro, Hyunju

    2016-01-01

    Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion. PMID:27673563

  4. Micro-PET/CT Monitoring of Herpes Thymidine Kinase Suicide Gene Therapy in a Prostate Cancer Xenograft: The Advantage of a Cell-specific Transcriptional Targeting Approach

    PubMed Central

    Johnson, Mai; Sato, Makoto; Burton, Jeremy; Gambhir, Sanjiv S.; Carey, Michael; Wu, Lily

    2010-01-01

    Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols. PMID:16285908

  5. Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model.

    PubMed

    Majewski, M; Betz, O; Ochsner, P E; Liu, F; Porter, R M; Evans, C H

    2008-08-01

    The aim of our study was to evaluate the histological and biomechanical effects of BMP-12 gene transfer on the healing of rat Achilles tendons using a new approach employing a genetically modified muscle flap. Biopsies of autologous skeletal muscle were transduced with a type-five, first-generation adenovirus carrying the human BMP-12 cDNA (Ad.BMP-12) and surgically implanted around experimentally transected Achilles tendons in a rat model. The effect of gene transfer on healing was evaluated by mechanical and histological testing after 1, 2, 4 and 8 weeks. One week after surgery, the maximum failure load of the healing tendons was significantly increased in the BMP-12 group, compared with the controls, and the tendon stiffness was significantly higher at 1, 2 and 4 weeks. Moreover, the size of the rupture callus was increased in the presence of BMP-12 and there was evidence of accelerated remodeling of the lesion in response to BMP-12. Histological examination showed a much more organized and homogeneous pattern of collagen fibers at all time points in lesions treated with the BMP-12 cDNA muscle graft. Both single fibrils and the collagen fibers had a greater diameter, with a higher degree of collagen crimp than the collagen of the control groups. This was confirmed by sirius red staining in conjunction with polarized light microscopy, which showed a higher shift of small yellow-green fibers to strong yellow-orange fibers after 2, 4 and 8 weeks in the presence of BMP-12 cDNA. There was also an earlier shift from fibroblasts to fibrocytes within the healing tendon, with less fat cells present in the tendons of the BMP-12 group compared with the controls. Treatment with BMP-12 cDNA-transduced muscle grafts thus produced a promising acceleration and improvement of tendon healing, particularly influencing early tissue regeneration, leading to quicker recovery and improved biomechanical properties of the Achilles tendon. Further development of this approach could have

  6. Ferret and Pig Models of Cystic Fibrosis: Prospects and Promise for Gene Therapy

    PubMed Central

    Yan, Ziying; Stewart, Zoe A.; Sinn, Patrick L.; Olsen, John C.; Hu, Jim; McCray, Paul B.

    2015-01-01

    Abstract Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies. PMID:25675143

  7. Combination Gene Therapy for Liver Metastasis of Colon Carcinoma in vivo

    NASA Astrophysics Data System (ADS)

    Chen, Shu-Hsai; Chen, X. H. Li; Wang, Yibin; Kosai, Ken-Ichiro; Finegold, Milton J.; Rich, Susan S.

    1995-03-01

    The efficacy of combination therapy with a "suicide gene" and a cytokine gene to treat metastatic colon carcinoma in the liver was investigated. Tumor in the liver was generated by intrahepatic injection of a colon carcinoma cell line (MCA-26) in syngeneic BALB/c mice. Recombinant adenoviral vectors containing various control and therapeutic genes were injected directly into the solid tumors, followed by treatment with ganciclovir. While the tumors continued to grow in all animals treated with a control vector or a mouse interleukin 2 vector, those treated with a herpes simplex virus thymidine kinase vector, with or without the coadministration of the mouse interleukin 2 vector, exhibited dramatic necrosis and regression. However, only animals treated with both vectors developed an effective systemic antitumoral immunity against challenges of tumorigenic doses of parental tumor cells inoculated at distant sites. The antitumoral immunity was associated with the presence of MCA-26 tumor-specific cytolytic CD8^+ T lymphocytes. The results suggest that combination suicide and cytokine gene therapy in vivo can be a powerful approach for treatment of metastatic colon carcinoma in the liver.

  8. Adenoviral delivery of truncated MMP-8 fused with the hepatocyte growth factor mutant 1K1 ameliorates liver cirrhosis and promotes hepatocyte proliferation

    PubMed Central

    Liu, Jinghua; Li, Jianbo; Fu, Weiwei; Tang, Jiacheng; Feng, Xu; Chen, Jiang; Liang, Yuelong; Jin, Ren’an; Xie, Anyong; Cai, Xiujun

    2015-01-01

    Liver cirrhosis is a chronic liver disease caused by chronic liver injury, which activates hepatic stellate cells (HSCs) and the secretion of extracellular matrix (ECM). Cirrhosis accounts for an extensive level of morbidity and mortality worldwide, largely due to lack of effective treatment options. In this study, we have constructed a fusion protein containing matrix metal-loproteinase 8 (MMP-8) and the human growth factor mutant 1K1 (designated cMMP8-1K1) and delivered it into hepatocytes and in vivo and in cell culture via intravenous injection of fusion protein-harboring adenovirus. In doing so, we found that the cMMP8-1K1 fusion protein promotes the proliferation of hepatocytes, likely resulting from the combined inhibition of type I collagen secretion and the degradation of the ECM in the HSCs. This fusion protein was also observed to ameliorate liver cirrhosis in our mouse model. These changes appear to be linked to changes in downstream gene expression. Taken together, these results suggest a possible strategy for the treatment of liver cirrhosis and additional work is warranted. PMID:26527860

  9. Adenoviral delivery of truncated MMP-8 fused with the hepatocyte growth factor mutant 1K1 ameliorates liver cirrhosis and promotes hepatocyte proliferation.

    PubMed

    Liu, Jinghua; Li, Jianbo; Fu, Weiwei; Tang, Jiacheng; Feng, Xu; Chen, Jiang; Liang, Yuelong; Jin, Ren'an; Xie, Anyong; Cai, Xiujun

    2015-01-01

    Liver cirrhosis is a chronic liver disease caused by chronic liver injury, which activates hepatic stellate cells (HSCs) and the secretion of extracellular matrix (ECM). Cirrhosis accounts for an extensive level of morbidity and mortality worldwide, largely due to lack of effective treatment options. In this study, we have constructed a fusion protein containing matrix metal-loproteinase 8 (MMP-8) and the human growth factor mutant 1K1 (designated cMMP8-1K1) and delivered it into hepatocytes and in vivo and in cell culture via intravenous injection of fusion protein-harboring adenovirus. In doing so, we found that the cMMP8-1K1 fusion protein promotes the proliferation of hepatocytes, likely resulting from the combined inhibition of type I collagen secretion and the degradation of the ECM in the HSCs. This fusion protein was also observed to ameliorate liver cirrhosis in our mouse model. These changes appear to be linked to changes in downstream gene expression. Taken together, these results suggest a possible strategy for the treatment of liver cirrhosis and additional work is warranted.

  10. Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus.

    PubMed

    Singh, Neetu; Pandey, Aseem; Jayashankar, Lakshmi; Mittal, Suresh K

    2008-05-01

    Because of the high prevalence of adenovirus (Ad) infections in humans, it is believed that pre-existing Ad-neutralizing antibodies (vector immunity) may negatively impact the immune response to vaccine antigens when delivered by human Ad (HAd) vectors. In order to evaluate whether bovine Ad subtype 3 (BAd3), a non-HAd vector, can effectively elude high levels of pre-existing vector immunity, naïve and HAd serotype 5 (HAd)-primed mice were immunized with BAd-H5HA [BAd3 vector expressing the hemagglutinin (HA) gene from H5N1 influenza virus]. Even in the presence of very high levels of HAd-specific neutralizing antibody, no significant reductions in HA-specific humoral and cell-mediated immune (CMI) responses were observed in HAd-primed mice immunized with BAd-H5HA. In naïve mice immunized with HAd-H5HA (HAd5 vector expressing H5N1 HA) and boosted with BAd-H5HA, the humoral responses elicited were significantly higher (P < 0.01) than with either HAd-H5HA or BAd-H5HA alone, while the CMI responses were comparable in the groups. This finding underlines the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of naïve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness.

  11. Neurogenin 3 Recruits CBP Co-activator to Facilitate Histone H3/H4 Acetylation in the Target Gene INSM1

    PubMed Central

    Breslin, Mary B.; Wang, Hong-Wei; Pierce, Amy; Aucoin, Rebecca; Lan, Michael S.

    2007-01-01

    INSM1 is a downstream target gene of ngn3. A promoter construct containing the −426/+40bp region transiently co-transfected into NIH-3T3 cells with a ngn3 expression plasmid resulted in a 12 fold increase in promoter activity. The ngn3/E47 heterodimer selectively binds and activates the E-box3 of the INSM1 promoter. The endogenous ngn3 and CBP co-activator occupy the INSM1 promoter, resulting in hyper-acetylation of histone H3/H4 chromatin in a human neuroblastoma cell line, IMR-32. Additionally, adenoviral ngn3 can induce endogenous INSM-1 expression in PANC-1 cells through the recruitment of CBP to the INSM1 promoter and increase the acetylation of the INSM1 promoter region. PMID:17300785

  12. Identification of Four Mouse Diabetes Candidate Genes Altering β-Cell Proliferation

    PubMed Central

    Kamitz, Anne; Jähnert, Markus; Vogel, Heike; Scherneck, Stephan; Schulze, Matthias; Staiger, Harald; Machicao, Fausto; Häring, Hans-Ulrich; Joost, Hans-Georg; Schürmann, Annette

    2015-01-01

    Beta-cell apoptosis and failure to induce beta-cell regeneration are hallmarks of type 2-like diabetes in mouse models. Here we show that islets from obese, diabetes-susceptible New Zealand Obese (NZO) mice, in contrast to diabetes-resistant C57BL/6J (B6)-ob/ob mice, do not proliferate in response to an in-vivo glucose challenge but lose their beta-cells. Genome-wide RNAseq based transcriptomics indicated an induction of 22 cell cycle-associated genes in B6-ob/ob islets that did not respond in NZO islets. Of all genes differentially expressed in islets of the two strains, seven mapped to the diabesity QTL Nob3, and were hypomorphic in either NZO (Lefty1, Apoa2, Pcp4l1, Mndal, Slamf7, Pydc3) or B6 (Ifi202b). Adenoviral overexpression of Lefty1, Apoa2, and Pcp4l1 in primary islet cells increased proliferation, whereas overexpression of Ifi202b suppressed it. We conclude that the identified genes in synergy with obesity and insulin resistance participate in adaptive islet hyperplasia and prevention from severe diabetes in B6-ob/ob mice. PMID:26348837

  13. In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer

    PubMed Central

    White, SJ; Papadakis, ED; Rogers, CA; Johnson, JL; Biessen, EAL; Newby, AC

    2010-01-01

    Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer. PMID:17989704

  14. In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer.

    PubMed

    White, S J; Papadakis, E D; Rogers, C A; Johnson, J L; Biessen, E A L; Newby, A C

    2008-03-01

    Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer. PMID:17989704

  15. Identification of Four Mouse Diabetes Candidate Genes Altering β-Cell Proliferation.

    PubMed

    Kluth, Oliver; Matzke, Daniela; Kamitz, Anne; Jähnert, Markus; Vogel, Heike; Scherneck, Stephan; Schulze, Matthias; Staiger, Harald; Machicao, Fausto; Häring, Hans-Ulrich; Joost, Hans-Georg; Schürmann, Annette

    2015-09-01

    Beta-cell apoptosis and failure to induce beta-cell regeneration are hallmarks of type 2-like diabetes in mouse models. Here we show that islets from obese, diabetes-susceptible New Zealand Obese (NZO) mice, in contrast to diabetes-resistant C57BL/6J (B6)-ob/ob mice, do not proliferate in response to an in-vivo glucose challenge but lose their beta-cells. Genome-wide RNAseq based transcriptomics indicated an induction of 22 cell cycle-associated genes in B6-ob/ob islets that did not respond in NZO islets. Of all genes differentially expressed in islets of the two strains, seven mapped to the diabesity QTL Nob3, and were hypomorphic in either NZO (Lefty1, Apoa2, Pcp4l1, Mndal, Slamf7, Pydc3) or B6 (Ifi202b). Adenoviral overexpression of Lefty1, Apoa2, and Pcp4l1 in primary islet cells increased proliferation, whereas overexpression of Ifi202b suppressed it. We conclude that the identified genes in synergy with obesity and insulin resistance participate in adaptive islet hyperplasia and prevention from severe diabetes in B6-ob/ob mice.

  16. Emerging strategies for cell and gene therapy of the muscular dystrophies

    PubMed Central

    Muir, Lindsey A.; Chamberlain, Jeffrey S.

    2016-01-01

    The muscular dystrophies are a heterogeneous group of over 40 disorders that are characterised by muscle weakness and wasting. The most common are Duchenne muscular dystrophy and Becker muscular dystrophy, which result from mutations within the gene encoding dystrophin; myotonic dystrophy type 1, which results from an expanded trinucleotide repeat in the myotonic dystrophy protein kinase gene; and facioscapulohumeral dystrophy, which is associated with contractions in the subtelomeric region of human chromosome 1. Currently the only treatments involve clinical management of symptoms, although several promising experimental strategies are emerging. These include gene therapy using adeno-associated viral, lentiviral and adenoviral vectors and nonviral vectors, such as plasmid DNA. Exon-skipping and cell-based therapies have also shown promise in the effective treatment and regeneration of dystrophic muscle. The availability of numerous animal models for Duchenne muscular dystrophy has enabled extensive testing of a wide range of therapeutic approaches for this type of disorder. Consequently, we focus here on the therapeutic developments for Duchenne muscular dystrophy as a model of the types of approaches being considered for various types of dystrophy. We discuss the advantages and limitations of each therapeutic strategy, as well as prospects and recent successes in the context of future clinical applications. PMID:19555515

  17. Evidence for an indirect transcriptional regulation of glucose-6-phosphatase gene expression by liver X receptors

    SciTech Connect

    Grempler, Rolf . E-mail: rolfgrempler@yahoo.de; Guenther, Susanne; Steffensen, Knut R.; Nilsson, Maria; Barthel, Andreas; Schmoll, Dieter

    2005-12-16

    Liver X receptor (LXR) paralogues {alpha} and {beta} (LXR{alpha} and LXR{beta}) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXR{alpha} or LXR{beta} suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors.

  18. Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

    PubMed

    Nalbantoglu, J; Pari, G; Karpati, G; Holland, P C

    1999-04-10

    Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

  19. The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression

    PubMed Central

    Kelloniemi, Annina; Szabo, Zoltan; Serpi, Raisa; Näpänkangas, Juha; Ohukainen, Pauli; Tenhunen, Olli; Kaikkonen, Leena; Koivisto, Elina; Bagyura, Zsolt; Kerkelä, Risto; Leosdottir, Margret; Hedner, Thomas; Melander, Olle

    2015-01-01

    The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio. PMID:26098115

  20. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    NASA Astrophysics Data System (ADS)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  1. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    PubMed Central

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  2. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy.

    PubMed

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  3. Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis

    PubMed Central

    Villarreal-Ramos, Bernardo; Xing, Zhou; Singh, Mahavir; Vordermeier, H. Martin

    2012-01-01

    Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy. PMID:23300440

  4. Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

    PubMed Central

    Chamorro, Cristina; Mencía, Angeles; Almarza, David; Duarte, Blanca; Büning, Hildegard; Sallach, Jessica; Hausser, Ingrid; Del Río, Marcela; Larcher, Fernando; Murillas, Rodolfo

    2016-01-01

    Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction. PMID:27045209

  5. Global gene transcriptome analysis in vaccinated cattle revealed a dominant role of IL-22 for protection against bovine tuberculosis.

    PubMed

    Bhuju, Sabin; Aranday-Cortes, Elihu; Villarreal-Ramos, Bernardo; Xing, Zhou; Singh, Mahavir; Vordermeier, H Martin

    2012-12-01

    Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.

  6. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

    PubMed Central

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M.; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D.; Townes, Tim M.

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  7. Efficient human growth hormone gene expression in the milk of non-transgenic goats.

    PubMed

    Han, Z; Wu, S; Li, Q; Li, J; Gao, D; Li, K; Liu, Z W; Zhao, H

    2009-01-01

    Heterogenous expression of recombinant proteins in milk of livestock at a large scale is very labour-intensive to be achieved with current transgenic animals, and usually seen as time-consuming, expensive and technically most challenging. Here we describe a convenient system for transient production of recombinant human growth hormone and its extensive use in recombinant protein production for therapeutic purposes. In this study, an adenoviral vector containing the GFP gene and hGH gene was constructed for direct infusion into the epithelium of mammary glands of goats via the teat canal during the period of natural lactation. Western-blot analysis of milk samples obtained from all of the viral-treated founders indicated that the recombinant hGH (rhGH) was secreted into the milk of the goats. The concentrations of rhGH in milk ranged from 0.6 to 2.4 mg/ml and lasted for more than 10 days during lactation. These data suggest that it is possible to produce larger amounts of recombinant human growth hormone in the milk of livestock animals by using replication-defective adenoviruses.

  8. Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab

    PubMed Central

    Xing, Man; Wang, Xiang; Chi, Yudan; Zhou, Dongming

    2016-01-01

    Cetuximab is a chimeric monoclonal antibody, approved to treat patients with metastatic colorectal cancer (mCRC), head and neck squamous cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC) for years. It functions by blocking the epidermal growth factor receptor (EGFR) from receiving signals or interacting with other proteins. Although the demand for cetuximab for the treatment of cancer patients in clinics is increasing, the complicated techniques involved and its high cost limit its wide applications. Here, a new, cheaper form of cetuximab was generated for cancer gene therapy. This was achieved by cloning the full-length cetuximab antibody into two serotypes of adenoviral vectors, termed as AdC68-CTB and Hu5-CTB. In vivo studies showed that a single dose of AdC68-CTB or Hu5-CTB induced sustained cetuximab expression and dramatically suppressed tumor growth in NCI-H508– or DiFi-inoculated nude mice. In conclusion, gene therapy using adenovirus expressing full-length cetuximab could be a novel alternative method for the effective treatment of colorectal cancer. PMID:27058423

  9. A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma.

    PubMed

    Sangro, B; Mazzolini, G; Ruiz, M; Ruiz, J; Quiroga, J; Herrero, I; Qian, C; Benito, A; Larrache, J; Olagüe, C; Boan, J; Peñuelas, I; Sádaba, B; Prieto, J

    2010-12-01

    The aim of this phase I clinical trial was to assess the feasibility and safety of intratumoral administration of a first-generation adenoviral vector encoding herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.TK) followed by systemic ganciclovir to patients with advanced hepatocellular carcinoma (HCC). Secondarily, we have analyzed its antitumor effect. Ten patients were enrolled in five dose-level cohorts that received from 10¹⁰ to 2 × 10¹² viral particles (vp). Ad.TK was injected intratumorally and patients received up to three doses at 30-day intervals. Positron emission tomography was used to monitor TK gene expression. Ad.TK injection was feasible in 100% of cases. Treatment was well tolerated and dose-limiting toxicity was not achieved. Cumulative toxicity was not observed. Hepatic toxicity was absent even in cirrhotic patients. Fever, flu-like syndrome, pain at the injection site and pancytopenia were the most common side effects. No partial responses were observed and 60% of patients showed tumor stabilization of the injected lesion. Importantly, two patients who received the highest dose showed signs of intratumoral necrosis by imaging procedures. One of them achieved a sustained stabilization and survived for 26 months. In conclusion, Ad.TK can be safely administered by intratumoral injection to patients with HCC up to 2 × 10¹² vp per patient. PMID:20689572

  10. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  11. Gene doping.

    PubMed

    Harridge, Stephen D R; Velloso, Cristiana P

    2008-01-01

    Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities.

  12. Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

    PubMed

    Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-01-01

    Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors. PMID:26530405

  13. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  14. A New Technique of ex vivo Gene Delivery of VEGF to Wounds Using Genetically Modified Skin Particles Promotes Wound Angiogenesis

    PubMed Central

    Koyama, Taro; Hackl, Florian; Aflaki, Pejman; Bergmann, Juri; Zuhaili, Baraa; Waisbren, Emily; Govindarajulu, Usha; Yao, Feng; Eriksson, Elof

    2011-01-01

    Background Transplantation of genetically modified keratinocytes has been shown to accelerate wound healing. However, this method is labor-intensive and time-consuming. We have developed a new technique of intraoperative gene-delivery to wounds that involves transplantation of transfected minced skin particles (MSPs) derived from harvested partial-thickness skin. Study Design MSPs measuring 0.8×0. ×0.35 mm were created from a split-thickness skin graft of a pig. In vitro transfection was carried out with adenoviral-LacZ (Ad-LacZ) for qualitative and adenoviral-VEGF (Ad-VEGF) for quantitative analysis. Transfected MSPs were transplanted to each of 2.5×2.5 cm full-thickness wounds on the dorsum of the pig. Non-transfected MSPs served as controls. Wound chambers were applied and injected with saline to create a wet environment. Results LacZ expression was detected in migrating cells originating from MSPs both in vitro and in vivo. VEGF expression in the wound fluid of Ad-VEGF-MSPs transplanted wounds on each of days 2–4 (mean±SEM (ng/ml): 6.74±1.89: day 2, 9.88±2.27: day 3, 9.87±1.28: day 4) was significantly higher (p<0.0001) compared to wounds transplanted with either untransfected MSPs, Ad-LacZ-MSPs or untransplanted controls. In vitro VEGF expression was significantly higher (p<0.0001) in Ad-VEGF 1×1010 transfected-MSPs compared to either Ad-VEGF 1×109 transfected MSPs or untransfected MSPs. Wounds transplanted with Ad-VEGF-MSPs showed significantly higher (p<0.0001) numbers of newly formed blood vessels (12.6±0.9 vessels/HPF) compared to wounds transplanted either Ad-LacZ-MSPs (4.4±0.5) or untransfected MSPs (5.2±0.7). All MSP transplanted wounds (Ad-VEGF-MSPs, Untransfected-MSPs, Ad-LacZ-MSPs) showed significantly higher reepithelialization compared to untransplanted wounds on days 10 and 14 (p<0.0001). Conclusions We demonstrated successful transfection of MSPs that can be transplanted to wounds as a source of gene expressing cells. This technique

  15. Alternative promoter usage and differential expression of multiple transcripts of mouse Prkar1a gene.

    PubMed

    Banday, Abdul Rouf; Azim, Shafquat; Tabish, Mohammad

    2011-11-01

    Prkar1a gene encodes regulatory type 1 alpha subunit (RIα) of cAMP-dependent protein kinase (PKA) in mouse. The role of this gene has been implicated in Carney complex and many cancer types that suggest its involvement in physiological processes like cell cycle regulation, growth and/or proliferation. We have identified and sequenced partial cDNA clones encoding four alternatively spliced transcripts of mouse Prkar1a gene. These transcripts have alternate 5' UTR structure which results from splicing of three exons (designated as E1a, E1b, and E1c) to canonical exon 2. The designated transcripts T1, T2, T3, and T4 contain 5' UTR exons as E1c, E1a + E1b, E1a, and E1b, respectively. The transcript T1 corresponded to earlier reported transcript in GenBank. In silico study of genomic DNA sequence revealed three distinct promoter regions namely, P1, P2, and P3 upstream of the exons E1a, E1b, and E1c, respectively. P1 is non-CpG-related promoter but P2 and P3 are CpG-related promoters; however, all three are TATA less. RT-PCR analysis demonstrated the expression of all four transcripts in late postnatal stages; however, these were differentially regulated in early postnatal stages of 0.5 day, 3 day, and 15 day mice in different tissue types. Variations in expression of Prkar1a gene transcripts suggest their regulation from multiple promoters that respond to a variety of signals arising in or out of the cell in tissue and developmental stage-specific manner. PMID:21638026

  16. Trichoderma genes

    SciTech Connect

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  17. [Gene therapy].

    PubMed

    Rodríguez-Fragoso, L

    1997-01-01

    In the last years there has been much progress in our understanding of molecular mechanisms in the pathogenesis of disease. In this review we provide an overview of gene therapy, its most actualized techniques for gene delivery, and we give specific examples of laboratory and clinical achievements to date. The development of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. As a result, concepts and methods that would have been considered purely science fiction 50 years ago are now used in the treatment of diseases. The widespread application of gene therapy technology to many diseases is already breaking down the traditional boundaries of modern medicine. However, despite its progress, several key technical drawbacks need to be overcome before gene therapy can be used safely and effectively in clinical settings. Technological developments, particularly in the areas of gene delivery and cell transplantation, will be critical for the successful practice of gene therapy.

  18. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  19. Control of Gene Expression by the Retinoic Acid-Related Orphan Receptor Alpha in HepG2 Human Hepatoma Cells

    PubMed Central

    Chauvet, Caroline; Vanhoutteghem, Amandine; Duhem, Christian; Saint-Auret, Gaëlle; Bois-Joyeux, Brigitte; Djian, Philippe; Staels, Bart; Danan, Jean-Louis

    2011-01-01

    Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin. PMID:21818335

  20. S100A1 gene therapy preserves in vivo cardiac function after myocardial infarction.

    PubMed

    Pleger, Sven T; Remppis, Andrew; Heidt, Beatrix; Völkers, Mirko; Chuprun, J Kurt; Kuhn, Matthew; Zhou, Rui-Hai; Gao, Erhe; Szabo, Gabor; Weichenhan, Dieter; Müller, Oliver J; Eckhart, Andrea D; Katus, Hugo A; Koch, Walter J; Most, Patrick

    2005-12-01

    Myocardial infarction (MI) represents an enormous clinical challenge as loss of myocardium due to ischemic injury is associated with compromised left ventricular (LV) function often leading to acute cardiac decompensation or chronic heart failure. S100A1 was recently identified as a positive inotropic regulator of myocardial contractility in vitro and in vivo. Here, we explore the strategy of myocardial S100A1 gene therapy either at the time of, or 2 h after, MI to preserve global heart function. Rats underwent cryothermia-induced MI and in vivo intracoronary delivery of adenoviral transgenes (4 x 10(10) pfu). Animals received saline (MI), the S100A1 adenovirus (MI/AdS100A1), a control adenovirus (MI/AdGFP), or a sham operation. S100A1 gene delivery preserved global in vivo LV function 1 week after MI. Preservation of LV function was due mainly to S100A1-mediated gain of contractility of the remaining, viable myocardium since contractile parameters and Ca(2+) transients of isolated MI/AdS100A1 myocytes were significantly enhanced compared to myocytes isolated from both MI/AdGFP and sham groups. Moreover, S100A1 gene therapy preserved the cardiac beta-adrenergic inotropic reserve, which was associated with the attenuation of GRK2 up-regulation. Also, S100A1 overexpression reduced cardiac hypertrophy 1 week post-MI. Overall, our data indicate that S100A1 gene therapy provides a potential novel treatment strategy to maintain contractile performance of the post-MI heart.

  1. Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function.

    PubMed

    Lin, Huei-Min; Lee, Ji-Hyeon; Yadav, Hariom; Kamaraju, Anil K; Liu, Eric; Zhigang, Duan; Vieira, Anthony; Kim, Seong-Jin; Collins, Heather; Matschinsky, Franz; Harlan, David M; Roberts, Anita B; Rane, Sushil G

    2009-05-01

    Pancreatic islet beta-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate beta-cell function is of immense clinical relevance. Transforming growth factor (TGF)-beta signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-beta pathway in regulation of insulin gene transcription and beta-cell function. We identify insulin as a TGF-beta target gene and show that the TGF-beta signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-beta signaling regulates expression of genes involved in beta-cell function. Together, these studies emphasize TGF-beta/Smad3 signaling as an important regulator of insulin gene transcription and beta-cell function and suggest that components of the TGF-beta signaling pathway may be dysregulated in diabetes.

  2. Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming

    PubMed Central

    Marthaler, Adele G.; Adachi, Kenjiro; Tiemann, Ulf; Wu, Guangming; Sabour, Davood; Velychko, Sergiy; Kleiter, Ingo; Schöler, Hans R.; Tapia, Natalia

    2016-01-01

    Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming. PMID:26762895

  3. Histone Deacetylase Inhibitors Interact with Melanoma Differentiation Associated-7/Interleukin-24 to Kill Primary Human Glioblastoma Cells

    PubMed Central

    Hamed, Hossein A.; Yacoub, Adly; Park, Margaret A.; Archer, Kellie; Das, Swadesh K.; Sarkar, Devanand; Grant, Steven; Fisher, Paul B.

    2013-01-01

    We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca2+. Quenching of reactive oxygen species and Ca2+ blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic. PMID:23661648

  4. Efficient Clinical Scale Gene Modification via Zinc Finger Nuclease–Targeted Disruption of the HIV Co-receptor CCR5

    PubMed Central

    Maier, Dawn A.; Brennan, Andrea L.; Jiang, Shuguang; Binder-Scholl, Gwendolyn K.; Lee, Gary; Plesa, Gabriela; Zheng, Zhaohui; Cotte, Julio; Carpenito, Carmine; Wood, Travis; Spratt, S. Kaye; Ando, Dale; Gregory, Philip; Holmes, Michael C.; Perez, Elena. E.; Riley, James L.; Carroll, Richard G.; June, Carl H.

    2013-01-01

    Abstract Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4+ T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >1010 CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection. PMID:23360514

  5. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    SciTech Connect

    Mu, Haixi; Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu; Li, Lili; Ren, Guosheng; Xu, Yongzhu; Zhou, Xiangyang; Xiang, Tingxiu

    2015-03-15

    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

  6. Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5.

    PubMed

    Maier, Dawn A; Brennan, Andrea L; Jiang, Shuguang; Binder-Scholl, Gwendolyn K; Lee, Gary; Plesa, Gabriela; Zheng, Zhaohui; Cotte, Julio; Carpenito, Carmine; Wood, Travis; Spratt, S Kaye; Ando, Dale; Gregory, Philip; Holmes, Michael C; Perez, Elena E; Riley, James L; Carroll, Richard G; June, Carl H; Levine, Bruce L

    2013-03-01

    Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

  7. Gene therapy for human colorectal cancer cell lines with recombinant adenovirus 5 based on loss of the insulin-like growth factor 2 imprinting.

    PubMed

    Sun, Huiling; Pan, Yuqin; He, Bangshun; Deng, Qiwen; Li, Rui; Xu, Yeqiong; Chen, Jie; Gao, Tianyi; Ying, Houqun; Wang, Feng; Liu, Xian; Wang, Shukui

    2015-04-01

    The recombinant oncolytic adenovirus is a novel anticancer agent to replicate selectively in colon cancer cell lines. Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon. We utilized the IGF2 LOI in gene therapy for the malignant tumor cell lines. We investigated the tumoricidal effects of IGF2 LOI on four cell lines by oncolytic adenovirus, and constructed novel adenovirus vectors Ad312-E1A and Ad312-EGFP. The expression of E1A was monitored by real-time PCR and western blot analysis. The viability and apoptosis of colorectal cells infected with Ad312-E1A were tested by CCK-8 and flow cytometry. In addition, we established a colorectal cancer model in nude mice. The results showed that HCT-8 and HT-29 with IGF2 LOI were infected with Ad312-EGFP and then produced the EGFP. Nevertheless, SW480 and GES-1, which were IGF2 MOI, did not produce the EGFP. The Ad312-E1A obviously reduced the cell viability and induced apoptosis in HCT-8 and HT-29 in vitro, and successfully suppressed tumor growth in HT-29 xenografts in nude mice. In summary, the conditionally replicative adenovirus with loss of IGF2 imprinting system has a positive effect on gene therapy.

  8. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

    PubMed

    Wise, Andrew K; Tu, Tian; Atkinson, Patrick J; Flynn, Brianna O; Sgro, Beatrice E; Hume, Cliff; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2011-08-01

    A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the

  9. Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity.

    PubMed

    Lymperopoulos, Anastasios; Rengo, Giuseppe; Zincarelli, Carmela; Soltys, Stephen; Koch, Walter J

    2008-02-01

    We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

  10. Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a Myc-Max binding motif.

    PubMed

    Nishino, K; Osaki, T; Kumagai, T; Kijima, T; Tachibana, I; Goto, H; Arai, T; Kimura, H; Funakoshi, T; Takeda, Y; Tanio, Y; Hayashi, S

    2001-03-15

    Recent clinical trials of gene therapy for patients with thoracic cancers have shown that these treatments were well tolerated with minimal side effects and that we need to further enhance specificity as well as efficiency of gene transfer to target cancer cells. We previously reported that myc-overexpressing SCLC cell lines became selectively sensitive to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the Myc-Max response elements (a core nucleotide sequence, CACGTG) and that this construct (MycTK) could be utilized to develop a novel treatment against chemo-radio-resistant SCLC. We report here in vivo antitumor effects and safety of a replication-deficient adenoviral vector containing the Myc-Max binding motif (AdMycTK) on SCLC cells. In vitro infection with AdMycTK selectively rendered myc-overexpressing SCLC cell lines 63- to 307-fold more sensitive to GCV. In vivo injections with AdMycTK followed by GCV administration markedly suppressed the growth of myc-overexpressing tumors established in the subcutis or in the peritoneal cavity of athymic mice. On the other hand, infection with AdMycTK did not significantly affect either in vitro GCV sensitivity of the cells expressing very low levels of the myc genes or the growth of their subcutaneous tumors. Moreover, we observed no apparent side effects of this treatment including body weight loss or biochemical abnormalities in contrast to the treatment with AdCATK that conferred strong but nonspecific expression of the HSV-TK gene. These results suggested that AdMycTK/GCV therapy is effective on SCLC patients whose tumors overexpress myc family oncogenes.

  11. Generation of Insulin-Producing Cells from the Mouse Liver Using β Cell-Related Gene Transfer Including Mafa and Mafb

    PubMed Central

    Oishi, Hisashi; Tai, Pei-Han; Sekiguchi, Yukari; Koshida, Ryusuke; Jung, Yunshin; Kudo, Takashi; Takahashi, Satoru

    2014-01-01

    Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb. PMID:25397325

  12. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  13. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  14. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  15. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    PubMed

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-06-01

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy. PMID:27195521

  16. Using a Commercial Ultrasound Contrast Agent for Viral-Mediated Gene Transfer In Vitro and In Vivo

    NASA Astrophysics Data System (ADS)

    Howard, Candace M.; Forsberg, Flemming; Liu, Ji-Bin; Merton, Daniel A.; Minimo, Corrado; Claudio, Pier P.

    2007-05-01

    This study evaluated the feasibility of site-specific gene delivery mediated by diagnostic ultrasound using genes encapsulated in commercially available ultrasound contrast agents in vitro and in vivo. Five different commercially available contrast agents were tested in vitro for their ability to enclose an adenoviral vector carrying GFP. Prostate cancer cells (DU 145) or non small cell lung cancer cells (H23) were plated in 80 culture wells and insonified at 207 or 535 kPa peak negative pressure for 1 min after administration of 0.1 ml of bubbles reconstituted with the viral vector. Experiments were repeated with the delivery vehicle incubated with complement to inactivate unenclosed Adeno-GFP and with controls. After 24 hours transduction efficiency was demonstrated by fluorescent microscopy. In vivo 15 nude mice with 21 melanoma tumors (DB-1) implanted received 0.1 ml injections of contrast. Mice were split into 3 control and 4 active groups and ultrasound was performed for 4 min at 4 MHz using an Aplio scanner (Toshiba America Medical Systems, Tustin, CA). Tumors, heart, lungs and liver were harvested 48 hours later. Specimens underwent regular and fluorescent microscopy and were stained using an antibody against GFP. In vitro all contrast agents produced more fluorescence at 207 kPa than at 535 kPa. However, only Imagent (IMCOR Pharmaceuticals, San Diego, CA) was able to induce marked gene transduction with the inactivating agent. In vivo systemic delivery of Adeno-GFP carrying microbubbles following pre-treatment with the inactivating agent resulted in specific transduction of the tumor cells only with no uptake in heart, lungs or liver (unlike the controls). In conclusion, specific viral gene transduction has been obtained in vitro and in vivo through the use of ultrasound and Imagent microbubbles as delivery vehicles.

  17. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    PubMed

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-06-01

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.

  18. Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma

    PubMed Central

    Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

    2016-01-01

    Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy. PMID:26909111

  19. Hair cell regeneration after ATOH1 gene therapy in the cochlea of profoundly deaf adult guinea pigs.

    PubMed

    Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Richardson, Rachael T

    2014-01-01

    The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness.

  20. Efficient, Long-term Hepatic Gene Transfer Using Clinically Relevant HDAd Doses by Balloon Occlusion Catheter Delivery in Nonhuman Primates

    PubMed Central

    Brunetti-Pierri, Nicola; Stapleton, Gary E; Law, Mark; Breinholt, John; Palmer, Donna J; Zuo, Yu; Grove, Nathan C; Finegold, Milton J; Rice, Karen; Beaudet, Arthur L; Mullins, Charles E; Ng, Philip

    2008-01-01

    Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy. PMID:19050700

  1. Neurotrophin-3 gene modified mesenchymal stem cells promote remyelination and functional recovery in the demyelinated spinal cord of rats.

    PubMed

    Zhang, Yu-Jiao; Zhang, Wei; Lin, Cheng-Guang; Ding, Ying; Huang, Si-Fan; Wu, Jin-Lang; Li, Yan; Dong, Hongxin; Zeng, Yuan-Shan

    2012-02-15

    Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by axonal/neuronal damage that may be caused by defective remyelination. Current therapies aim to slow the rate of degeneration, however there are no treatment options that can stop or reverse the myelin sheath damage. Bone marrow mesenchymal stem cells (MSCs) are a potential candidate for the cell implantation-targeted therapeutic strategies, but the pro-remyelination effects of MSCs when directly injected into a demyelinated cord lesion have been questioned. Neurotrophin-3 (NT-3) has been shown to serve a crucial role in the proliferation, differentiation and maturation of oligodendrocyte lineages. Here, we showed that implantation of NT-3 gene-modified MSCs via a recombinant adenoviral vector (Adv) into a region of ethidium bromide (EB)-induced demyelination in the spinal cord resulted in significant improvement of locomotor function and restoration of electrophysiological properties in rats. The morphological basis of this recovery was evidenced by robust myelin basic protein (MBP) expression and the extensive remyelination. AdvNT-3-MSC implants promote the endogenous remyelinating cells to participate directly in myelination, which was confirmed under light and electron microscopy. Our study suggested that genetically modified MSCs could be a potential therapeutic avenue for improving the efficacy of stem cell treatment for neurodegenerative diseases such as MS.

  2. Hair Cell Regeneration after ATOH1 Gene Therapy in the Cochlea of Profoundly Deaf Adult Guinea Pigs

    PubMed Central

    Atkinson, Patrick J.; Wise, Andrew K.; Flynn, Brianna O.; Nayagam, Bryony A.; Richardson, Rachael T.

    2014-01-01

    The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness. PMID:25036727

  3. The acetyltransferase p300/CBP-associated factor is a p53 target gene in breast tumor cells.

    PubMed

    Watts, George S; Oshiro, Marc M; Junk, Damian J; Wozniak, Ryan J; Watterson, Summer; Domann, Frederick E; Futscher, Bernard W

    2004-01-01

    p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.

  4. Functional repair of human donor lungs by IL-10 gene therapy.

    PubMed

    Cypel, Marcelo; Liu, Mingyao; Rubacha, Matt; Yeung, Jonathan C; Hirayama, Shin; Anraku, Masaki; Sato, Masaaki; Medin, Jeffrey; Davidson, Beverly L; de Perrot, Marc; Waddell, Thomas K; Slutsky, Arthur S; Keshavjee, Shaf

    2009-10-28

    More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10-treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10-treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar-blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients. PMID:20368171

  5. Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy.

    PubMed

    Choi, Joung-Woo; Nam, Joung-Pyo; Nam, Kihoon; Lee, Young Sook; Yun, Chae-Ok; Kim, Sung Wan

    2015-07-13

    Recently, adenovirus (Ad) has been utilized as a viral vector for efficient gene delivery. However, substantial immunogenicity and toxicity have obstructed oncolytic Ad's transition into clinical studies. The goal of this study is to generate an adenoviral vector complexed with multidegradable bioreducible core-cross-linked polyethylenimine (rPEI) polymer that has low immunogenicity and toxicity while having higher transduction efficacy and stability. We have synthesized different molecular weight rPEIs and complexed with Ad at varying molar ratios to optimize delivery of the Ad/polymer complex. The size and surface charge of Ad/rPEIs were characterized. Of note, Ad/rPEIs showed significantly enhanced transduction efficiency compared to either naked Ad or Ad/25 kDa PEI in both coxsackievirus and adenovirus receptor (CAR) positive and negative cancer cells. The cellular uptake result demonstrated that the relatively small size of Ad/16 kDa rPEIs (below 200 nm) was more critical to the complex's internalization than its surface charge. Cancer cell killing effect and viral production were significantly increased when oncolytic Ad (RdB/shMet, or oAd) was complexed with 16 kDa rPEI in comparison to naked oAd-, oAd/25 kDa PEI-, or oAd/32 kDa rPEI-treated cells. This increased anticancer cytotoxicity was more readily apparent in CAR-negative MCF7 cells, implying that it can be used to treat a broad range of cancer cells. Furthermore, A549 and HT1080 cancer cells treated with oAd/16 kDa rPEI had significantly decreased Met and VEGF expression compared to either naked oAd or oAd/25 kDa PEI. Overall, these results demonstrate that shMet expressing oncolytic Ad complexed with multidegradable bioreducible core-cross-linked PEI could be used as efficient and safe cancer gene therapy. PMID:26096567

  6. Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

    PubMed Central

    Puhl, Henry L.; Ikeda, Stephen R.

    2008-01-01

    Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

  7. Vulnerability genes or plasticity genes?

    PubMed Central

    Belsky, J; Jonassaint, C; Pluess, M; Stanton, M; Brummett, B; Williams, R

    2009-01-01

    The classic diathesis–stress framework, which views some individuals as particularly vulnerable to adversity, informs virtually all psychiatric research on behavior–gene–environment (G × E) interaction. An alternative framework of ‘differential susceptibility' is proposed, one which regards those most susceptible to adversity because of their genetic make up as simultaneously most likely to benefit from supportive or enriching experiences—or even just the absence of adversity. Recent G × E findings consistent with this perspective and involving monoamine oxidase-A, 5-HTTLPR (5-hydroxytryptamine-linked polymorphic region polymorphism) and dopamine receptor D4 (DRD4) are reviewed for illustrative purposes. Results considered suggest that putative ‘vulnerability genes' or ‘risk alleles' might, at times, be more appropriately conceptualized as ‘plasticity genes', because they seem to make individuals more susceptible to environmental influences—for better and for worse. PMID:19455150

  8. Genes and Hearing Loss

    MedlinePlus

    ... Meeting Calendar Find an ENT Doctor Near You Genes and Hearing Loss Genes and Hearing Loss Patient ... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the ...

  9. Chemoprevention gene therapy (CGT): novel combinatorial approach for preventing and treating pancreatic cancer.

    PubMed

    Sarkar, S; Azab, B M; Das, S K; Quinn, B A; Shen, X; Dash, R; Emdad, L; Thomas, S; Dasgupta, S; Su, Z-Z; Wang, X-Y; Sarkar, D; Fisher, P B

    2013-08-01

    Pancreatic cancer remains one of the deadliest of all cancers despite aggressive surgical treatment combined with adjuvant radiotherapy and chemotherapy. Chemoresistance and radioresistance are the principal causes of failure of pancreatic cancer patients to respond to therapy. Conditionally replication competent adenovirus (CRCA)-based cancer gene therapy is an innovative strategy for treating cancers displaying inherent resistance to treatment. Limitations of current adenovirus (Ad)-based gene therapies for malignant tumors include lack of cancer-specificity, and effective and targeted delivery. To remedy this situation, CRCAs have been designed that express E1A, necessary for Ad replication, under the control of a cancer-specific progression elevated gene-3 promoter (PEG-Prom) with concomitant expression of an immunomodulatory cytokine, such as mda-7/IL-24 or interferon-γ (IFN-γ), under the control of a ubiquitous and strong cytomegalovirus promoter (CMV-Prom) from the E3 region. These bipartite CRCAs, when armed with a transgene, are called cancer terminator viruses (CTVs), i.e., Ad.PEG-E1A-CMV-mda-7 (CTV-M7) and Ad.PEG-E1A-CMV-IFN-γ (CTV-γ), because of their universal effectiveness in cancer treatment irrespective of p53/pRb/p16 or other genetic alterations in tumor cells. In addition to their selective oncolytic effects in tumor cells, the potent 'bystander antitumor' properties of MDA-7/IL-24 and IFN-γ embody the CTVs with expanded treatment properties for both primary and distant cancers. Pancreatic cancer cells display a "translational block" of mda-7/IL-24 mRNA, limiting production of MDA-7/IL-24 protein and cancer-specific apoptosis. Specific chemopreventive agents abrogate this "translational block" resulting in pancreatic cancer-specific killing. This novel chemoprevention gene therapy (CGT) strategy holds promise for both prevention and treatment of pancreatic cancers where all other strategies have proven ineffective.

  10. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

    PubMed Central

    Hickey, E; Brandon, S E; Smale, G; Lloyd, D; Weber, L A

    1989-01-01

    Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells. Images PMID:2527334

  11. Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

    PubMed Central

    Ghivizzani, Steven C.; Lechman, Eric R.; Kang, Richard; Tio, Caroline; Kolls, Jay; Evans, Christopher H.; Robbins, Paul D.

    1998-01-01

    Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides. PMID:9539786

  12. Roles for the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, in feedback control of inflammatory gene expression and repression by dexamethasone.

    PubMed

    Shah, Suharsh; King, Elizabeth M; Chandrasekhar, Ambika; Newton, Robert

    2014-05-01

    Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression. PMID:24692548

  13. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  14. Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis.

    PubMed

    Tatler, Amanda L; Goodwin, Amanda T; Gbolahan, Olumide; Saini, Gauri; Porte, Joanne; John, Alison E; Clifford, Rachel L; Violette, Shelia M; Weinreb, Paul H; Parfrey, Helen; Wolters, Paul J; Gauldie, Jack; Kolb, Martin; Jenkins, Gisli

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad

  15. Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis

    PubMed Central

    Tatler, Amanda L.; Goodwin, Amanda T.; Gbolahan, Olumide; Saini, Gauri; Porte, Joanne; John, Alison E.; Clifford, Rachel L.; Violette, Shelia M.; Weinreb, Paul H.; Parfrey, Helen; Wolters, Paul J.; Gauldie, Jack; Kolb, Martin; Jenkins, Gisli

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad

  16. Compare Gene Profiles

    SciTech Connect

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linux environment in serial or parallel mode.

  17. Homologous Boosting with Adenoviral Serotype 5 HIV Vaccine (rAd5) Vector Can Boost Antibody Responses despite Preexisting Vector-Specific Immunity in a Randomized Phase I Clinical Trial

    PubMed Central

    Sarwar, Uzma N.; Novik, Laura; Enama, Mary E.; Plummer, Sarah A.; Koup, Richard A.; Nason, Martha C.; Bailer, Robert T.; McDermott, Adrian B.; Roederer, Mario; Mascola, John R.; Ledgerwood, Julie E.; Graham, Barney S.

    2014-01-01

    Background Needle-free delivery improves the immunogenicity of DNA vaccines but is also associated with more local reactogenicity. Here we report the first comparison of Biojector and needle administration of a candidate rAd5 HIV vaccine. Methods Thirty-one adults, 18–55 years, 20 naive and 11 prior rAd5 vaccine recipients were randomized to receive single rAd5 vaccine via needle or Biojector IM injection at 1010 PU in a Phase I open label clinical trial. Solicited reactogenicity was collected for 5 days; clinical safety and immunogenicity follow-up was continued for 24 weeks. Results Overall, injections by either method were well tolerated. There were no serious adverse events. Frequency of any local reactogenicity was 16/16 (100%) for Biojector compared to 11/15 (73%) for needle injections. There was no difference in HIV Env-specific antibody response between Biojector and needle delivery. Env-specific antibody responses were more than 10-fold higher in subjects receiving a booster dose of rAd5 vaccine than after a single dose delivered by either method regardless of interval between prime and boost. Conclusions Biojector delivery did not improve antibody responses to the rAd5 vaccine compared to needle administration. Homologous boosting with rAd5 gene-based vectors can boost insert-specific antibody responses despite pre-existing vector-specific immunity. Trial Registration Clinicaltrials.gov NCT00709605 NCT00709605 PMID:25264782

  18. Gene gymnastics

    PubMed Central

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  19. Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge.

    PubMed

    Van Blarcom, T J; Sofer-Podesta, C; Ang, J; Boyer, J L; Crystal, R G; Georgiou, G

    2010-07-01

    Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K(D))=3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K(D) values (H8, K(D)=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdalphaV, giving rise to AdalphaV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10(9), 10(10) or 10(11) particle units (pu). After intranasal challenge with 363 LD(50) (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10(10) pu of AdalphaV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdalphaV expressing the lower-affinity 2C12.4 (P<0.04; AdalphaV versus AdalphaV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis

  20. Hepatocyte-specific Dyrk1a gene transfer rescues plasma apolipoprotein A-I levels and aortic Akt/GSK3 pathways in hyperhomocysteinemic mice.

    PubMed

    Tlili, Asma; Jacobs, Frank; de Koning, Leanne; Mohamed, Sirine; Bui, Linh-Chi; Dairou, Julien; Belin, Nicole; Ducros, Véronique; Dubois, Thierry; Paul, Jean-Louis; Delabar, Jean-Maurice; De Geest, Bart; Janel, Nathalie

    2013-06-01

    Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy. PMID:23429073

  1. Combined Cocaine Hydrolase Gene Transfer and Anti-Cocaine Vaccine Synergistically Block Cocaine-Induced Locomotion

    PubMed Central

    Carroll, Marilyn E.; Zlebnik, Natalie E.; Anker, Justin J.; Kosten, Thomas R.; Orson, Frank M.; Shen, Xiaoyun; Kinsey, Berma; Parks, Robin J.; Gao, Yang; Brimijoin, Stephen

    2012-01-01

    Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a “training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final “challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence. PMID:22912888

  2. Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

    PubMed Central

    Zheng, Fei-qun; Xu, Yin; Yang, Ren-jie; Wu, Bin; Tan, Xiao-hua; Qin, Yi-de; Zhang, Qun-wei

    2009-01-01

    Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combine