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Sample records for aav inverted terminal

  1. Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.

    PubMed

    Ping, Han; Liu, Xiaomei; Zhu, Dongqin; Li, Taiming; Zhang, Chun

    2015-04-01

    The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.

  2. Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles.

    PubMed Central

    Chiorini, J A; Yang, L; Liu, Y; Safer, B; Kotin, R M

    1997-01-01

    We have cloned and characterized the full-length genome of adeno-associated virus type 4 (AAV4). The genome of AAV4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to AAV2 and AAV3. Within the inverted terminal repeat (ITR), several base changes were identified with respect to AAV2. However, these changes did not affect the ability of this region to fold into a hairpin structure. Within the ITR, the terminal resolution site and Rep binding sites were conserved; however, the Rep binding site was expanded from three GAGC repeats to four. The Rep gene product of AAV4 shows greater than 90% homology to the Rep products of serotypes 2 and 3, with none of the changes occurring in regions which had previously been shown to affect the known functions of Rep68 or Rep78. Most of the differences in the capsid proteins lie in regions which are thought to be on the exterior surface of the viral capsid. It is these unique regions which are most likely to be responsible for the lack of cross-reacting antibodies and the altered tissue tropism compared to AAV2. The results of our studies, performed with a recombinant version of AAV4 carrying a lacZ reporter gene, suggest that AAV4 can transduce human, monkey, and rat cells. Furthermore, comparison of transduction efficiencies in a number of cell lines, competition cotransduction experiments, and the effect of trypsin on transduction efficiency all suggest that the cellular receptor for AAV4 is distinct from that of AAV2. PMID:9261407

  3. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  4. Four new cases of inverted terminal duplication: A modified hypothesis of mechanism of origin

    SciTech Connect

    Hoo, J.J.; Chao, M.; Szego, K.

    1995-09-25

    We present 4 recently diagnosed cases of inverted tandem duplication with involvement of the respective terminal band. Based on these 4 cases and review of the literature, the term {open_quotes}inverted terminal duplication{close_quotes} is proposed to designate specifically the type of inverted tandem duplication which involves the terminal band. A modification of the previous hypothesis of mechanism of origin is advanced. It is speculated further that a telomeric deletion of a meiotic chromosome followed by a U-type reunion of the chromatids, considered to be the first steps of the proposed mechanism of origin, may not be a rare gonadal event. 12 refs., 5 figs.

  5. HPV E1 up-regulates replication-related biochemistries of AAV Rep78.

    PubMed

    Bandyopadhyay, Sarmistha; Cao, Maohua; Liu, Yong; Hermonat, Paul L

    2010-06-20

    Human papillomavirus type 16 (HPV) E1 protein provides helper function for the adeno-associated virus type 2 (AAV) life cycle. E1 is the replication protein of HPV, analogous to AAV Rep78, but without the endonuclease/covalent attachment activity of Rep78. Previously we have shown that E1 and Rep78 interact in vitro. Here we investigated E1's effects on Rep78 interaction with AAV's inverted terminal repeat (ITR) DNA in vitro, using purified Rep78 and E1 proteins from bacteria. E1 enhanced Rep78-ITR binding, ATPase activity, Rep78-ITR-covalent linkage and Rep78-ITR-endonuclease activity (central to AAV replication). These enhancements occurred in a dose-dependent manner whenever assayed. However, overall Rep78-plus-E1 helicase activity was lower than Rep78's helicase activity. These data suggest that E1's broad-based helper function for the AAV life cycle (AAV DNA, mRNA, and protein levels are up-regulated by E1) is likely through its ability to enhance Rep78's critical replication-required biochemistries on ITR DNA.

  6. 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.

  7. Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences.

    PubMed

    François, V; Louarn, J; Patte, J; Rebollo, J E; Louarn, J M

    1990-04-01

    Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.

  8. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.

    PubMed

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L; Polishchuk, Roman S; Auricchio, Alberto

    2015-12-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.

  9. Complete nucleotide sequences of two adjacent early vaccinia virus genes located within the inverted terminal repetition.

    PubMed

    Venkatesan, S; Gershowitz, A; Moss, B

    1982-11-01

    The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine . thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3' end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine . thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

  10. Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells. PMID:26986720

  11. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

    PubMed

    D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  12. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    PubMed Central

    D’Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field. PMID:27069952

  13. Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

    PubMed Central

    Vanrell, Lucia; Di Scala, Marianna; Blanco, Laura; Otano, Itziar; Gil-Farina, Irene; Baldim, Victor; Paneda, Astrid; Berraondo, Pedro; Beattie, Stuart G; Chtarto, Abdelwahed; Tenenbaum, Lilianne; Prieto, Jesús; Gonzalez-Aseguinolaza, Gloria

    2011-01-01

    Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (TetbidirAlb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (TetbidirCMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the TetbidirAlb was significantly higher than that of TetbidirCMV, whereas leakage of TetbidirAlb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tetbidir-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tetbidir-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer. PMID:21364542

  14. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-10-11

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.

  15. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  16. An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model.

    PubMed

    Amaro, I Alexandra; Henderson, Lee A

    2016-01-01

    Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease. PMID:27595037

  17. An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

    PubMed Central

    2016-01-01

    Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease.

  18. An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

    PubMed Central

    2016-01-01

    Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease. PMID:27595037

  19. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

    PubMed Central

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V.; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L.; Polishchuk, Roman S.; Auricchio, Alberto

    2015-01-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. PMID:26420842

  20. Terminal Bacteroid Differentiation Is Associated With Variable Morphological Changes in Legume Species Belonging to the Inverted Repeat-Lacking Clade.

    PubMed

    Montiel, Jesús; Szűcs, Attila; Boboescu, Iulian Z; Gherman, Vasile D; Kondorosi, Éva; Kereszt, Attila

    2016-03-01

    Medicago and closely related legume species from the inverted repeat-lacking clade (IRLC) impose terminal differentiation onto their bacterial endosymbionts, manifested in genome endoreduplication, cell enlargement, and loss of cell-division capacity. Nodule-specific cysteine-rich (NCR) secreted host peptides are plant effectors of this process. As bacteroids in other IRLC legumes, such as Cicer arietinum and Glycyrrhiza lepidota, were reported not to display features of terminal differentiation, we investigated the fate of bacteroids in species from these genera as well as in four other species representing distinct genera of the phylogenetic tree for this clade. Bacteroids in all tested legumes proved to be larger in size and DNA content than cultured cells; however, the degree of cell elongation was rather variable in the different species. In addition, the reproductive ability of the bacteroids isolated from these legumes was remarkably reduced. In all IRLC species with available sequence data, the existence of NCR genes was found. These results indicate that IRLC legumes provoke terminal differentiation of their endosymbionts with different morphotypes, probably with the help of NCR peptides.

  1. A coalescence of two syndromes in a girl with terminal deletion and inverted duplication of chromosome 5

    PubMed Central

    2014-01-01

    Background Rearrangements involving chromosome 5p often result in two syndromes, Cri-du-chat (CdC) and Trisomy 5p, caused by a deletion and duplication, respectively. The 5p15.2 has been defined as a critical region for CdC syndrome; however, genotype-phenotype studies allowed isolation of particular characteristics such as speech delay, cat-like cry and mental retardation, caused by distinct deletions of 5p. A varied clinical outcome was also observed in patients with Trisomy 5p. Duplications of 5p10-5p13.1 manifest themselves in a more severe phenotype, while trisomy of regions distal to 5p13 mainly causes mild and indistinct features. Combinations of a terminal deletion and inverted duplication of 5p are infrequent in literature. Consequences of these chromosomal rearrangements differ, depending on size of deletion and duplication in particular cases, although authors mainly describe the deletion as the cause of the observed clinical picture. Case presentation Here we present a 5-month-old Slovenian girl, with de novo terminal deletion and inverted duplication of chromosome 5p. Our patient presents features of both CdC and Trisomy 5. The most prominent features observed in our patient are a cat-like cry and severe malformations of the right ear. Conclusion The cat-like cry, characteristic of CdC syndrome, is noted in our patient despite the fact that the deletion is not fully consistent with previously defined cat-like cry critical region in this syndrome. Features like dolichocephaly, macrocephaly and ear malformations, associated with duplication of the critical region of Trisomy 5p, are also present, although this region has not been rearranged in our case. Therefore, the true meaning of the described chromosomal rearrangements is discussed. PMID:24517234

  2. Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

  3. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

    PubMed

    Namadurai, Sivakumar; Jain, Deepti; Kulkarni, Dhananjay S; Tabib, Chaitanya R; Friedhoff, Peter; Rao, Desirazu N; Nair, Deepak T

    2010-01-01

    The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage. PMID:21060849

  4. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    PubMed

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.

  5. Reengineered AAV vectors: old dog, new tricks.

    PubMed

    Asokan, Aravind

    2010-05-01

    Adeno-associated viral (AAV) vectors have emerged in recent years as powerful tools for therapeutic gene transfer. Successes in clinical trials and the discovery of several hundreds of naturally occurring AAV isolates have triggered efforts to understand and manipulate this deceptively simple parvovirus for a myriad of gene therapy applications. Exciting breakthroughs based on directed evolution of novel tissue-specific variants from combinatorial AAV libraries have been reported. Recent approaches driven by the availability of structural information have yielded a new generation of reengineered AAV vectors. PMID:20515607

  6. Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

    PubMed

    Wang, Jianye; Huang, Yu; Zhou, Mingxu; Zhu, Guoqiang

    2016-09-01

    Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure. PMID:27344160

  7. Independent post-zygotic breaks of a dicentric chromosome result in mosaicism for an inverted duplication deletion 9p and terminal deletion 9p.

    PubMed

    Schlade-Bartusiak, Kamilla; Tucker, Tracy; Safavi, Holly; Livingston, Janet; van Allen, Margot I; Eydoux, Patrice; Armstrong, Linlea

    2013-05-01

    Mosaicism with two cell lines having different rearrangements of the same chromosome is rare. Only a few cases of mosaicism have been described in association with chromosomal inverted duplication deletion (inv dup del) rearrangements. A well-established mechanism of formation of inv dup del rearrangements involves a dicentric intermediate, which undergoes breakage during cell division, generating cells with either an inv dup del or a simple deletion. A patient with developmental delay and dysmorphic features was found to carry two cell lines with rearrangements of 9p: an inv dup del 9p and a terminal deletion 9p. Microarray and FISH analysis showed that these cell lines do not constitute the reciprocal products of a single dicentric breakage event. We propose that independent post-zygotic breaks of a dicentric chromosome as a likely mechanism leading to the generation of the observed cell lines. The post-zygotic origin of the inv dup del rearrangements and the associated mosaicism can be a more frequent phenomenon than currently appreciated. Therefore, genotype-phenotype correlations in the inv dup del rearrangements need to take into account the possible presence of other abnormal cell lines during early development.

  8. Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

    PubMed

    Wang, Jianye; Huang, Yu; Zhou, Mingxu; Zhu, Guoqiang

    2016-09-01

    Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure.

  9. Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the Inverted Repeat-Lacking Clade legume Glycyrrhiza uralensis.

    PubMed

    Crespo-Rivas, Juan C; Guefrachi, Ibtissem; Mok, Kenny C; Villaécija-Aguilar, José A; Acosta-Jurado, Sebastián; Pierre, Olivier; Ruiz-Sainz, José E; Taga, Michiko E; Mergaert, Peter; Vinardell, José M

    2016-09-01

    In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation. PMID:26521863

  10. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

    PubMed

    Fuchs, Sebastian P; Martinez-Navio, José M; Gao, Guangping; Desrosiers, Ronald C

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  11. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    PubMed Central

    Fuchs, Sebastian P.; Martinez-Navio, José M.; Gao, Guangping; Desrosiers, Ronald C.

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  12. Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

    PubMed Central

    Balagúe, C; Kalla, M; Zhang, W W

    1997-01-01

    Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78. PMID:9060699

  13. Comparative Study of Liver Gene Transfer With AAV Vectors Based on Natural and Engineered AAV Capsids.

    PubMed

    Wang, Lili; Bell, Peter; Somanathan, Suryanarayan; Wang, Qiang; He, Zhenning; Yu, Hongwei; McMenamin, Deirdre; Goode, Tamara; Calcedo, Roberto; Wilson, James M

    2015-12-01

    Vectors based on the clade E family member adeno-associated virus (AAV) serotype 8 have shown promise in patients with hemophilia B and have emerged as best in class for human liver gene therapies. We conducted a thorough evaluation of liver-directed gene therapy using vectors based on several natural and engineered capsids including the clade E AAVrh10 and the largely uncharacterized and phylogenically distinct AAV3B. Included in this study was a putatively superior hepatotropic capsid, AAVLK03, which is very similar to AAV3B. Vectors based on these capsids were benchmarked against AAV8 and AAV2 in a number of in vitro and in vivo model systems including C57BL/6 mice, immune-deficient mice that are partially repopulated with human hepatocytes, and nonhuman primates. Our studies in nonhuman primates and human hepatocytes demonstrated high level transduction of the clade E-derived vectors and equally high transduction with vectors based on AAV3B. In contrast to previous reports, AAVLK03 vectors are not superior to either AAV3B or AAV8. Vectors based on AAV3B should be considered for liver-directed gene therapy when administered following, or before, treatment with the serologically distinct clade E vectors.

  14. The ANCA Vasculitis Questionnaire (AAV-PRO©)

    ClinicalTrials.gov

    2016-05-10

    Eosinophilic Granulomatosis With Polyangiitis (Churg-Strauss) (EGPA); Churg-Strauss Syndrome (CSS); Granulomatosis With Polyangiitis (Wegener's) (GPA); Wegener Granulomatosis (WG); Microscopic Polyangiitis (MPA); ANCA-Associated Vasculitis (AAV); Vasculitis

  15. Dosage Thresholds for AAV2 and AAV8 Photoreceptor Gene Therapy in Monkey

    PubMed Central

    Vandenberghe, Luk H.; Bell, Peter; Maguire, Albert M.; Cearley, Cassia N.; Xiao, Ru; Calcedo, Roberto; Wang, Lili; Castle, Michael J.; Maguire, Alexandra C.; Grant, Rebecca; Wolfe, John H.; Wilson, James M.; Bennett, Jean

    2016-01-01

    Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors. PMID:21697530

  16. rAAV human trial experience.

    PubMed

    High, Katherine A; Aubourg, Patrick

    2011-01-01

    Recombinant AAV vectors have been used in clinical trials since the mid-1990s, with over 300 subjects enrolled in studies. Although there are not yet licensed AAV products, there are several clear examples of clinical efficacy, and recombinant AAV vectors have a strong safety record after administration both locally and systemically. This chapter provides a review of two types of studies that have shown efficacy, including studies for Leber's congenital amaurosis, a hereditary retinal degenerative disorder in which subretinal administration of AAV has shown efficacy in terms of improvement in multiple measures of visual/retinal function; and of Parkinson's disease which has also shown improvement in clinical and imaging studies after gene transfer to the CNS. The chapter also provides a detailed review of the results of studies of gene therapy for hemophilia, in which short-term efficacy was achieved, but expression of the donated gene failed to persist, likely due to an immune response to the vector. Safety issues relating to AAV-mediated gene transfer are discussed, including a detailed review of the single death to have occurred in an AAV gene therapy trial (likely unrelated to the AAV vector), and of issues related to integration and insertional mutagenesis, risk of germline transmission, and risks related to immune responses to either vector or transgene product. Finally, protocols for determining the presence of vector DNA in body fluids using real-time quantitative PCR, and for isolating, cryopreserving, and testing peripheral blood mononuclear cells for interferon-γ (IFN-γ) responses to capsid are described in detail. PMID:22034041

  17. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2.

    PubMed

    Grimm, D; Kern, A; Pawlita, M; Ferrari, F; Samulski, R; Kleinschmidt, J

    1999-07-01

    We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.

  18. Comparative antiatherogenic effects of intravenous AAV8- and AAV2-mediated ApoA-IMilano gene transfer in hypercholesterolemic mice.

    PubMed

    Tian, Fang; Wang, Lai; Arias, Ana; Yang, Mingjie; Sharifi, Behrooz G; Shah, Prediman K

    2015-01-01

    Apolipoprotein A-IMilano (ApoA-IM), a naturally occurring Arg173 to Cys mutant of ApoA-I, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown superior atheroprotective effects of ApoA-IM gene compared with wild-type ApoA-I gene using transplantation of retrovirally transduced bone marrow in ApoA-I/ApoE null mice. In this study, we compared the antiatherogenic efficacy of ApoA-IM gene transfer using Recombinant adeno-associated virus (rAAV) 2 or rAAV8 as vectors in ApoA-I/ApoE null mice. Mice received a single intravenous injection of 1.2 × 10(12) vector genomes of AAV2 or AAV8 vectors expressing ApoA-IM or control empty vectors (12 mice/group). Circulating levels of ApoA-IM were higher in recipients of AAV8 compared with AAV2 at 4, 12, and 20 weeks postinjection. Qualitative polymerase chain reaction analysis of RNA collected from different tissues showed that the AAV8-mediated gene transfer resulted in a more efficient transgene expression in the heart, brain, liver, lung, spleen, and kidney of the recipient mice compared with AAV2. Intravenous AAV8-ApoA-IM injection reduced atherosclerosis in the whole aorta (P < .01), aortic sinuses (P < .05), and brachiocephalic arteries (P < .05) compared with the vector control, whereas there was no statistically significant reduction in atherosclerosis in mice receiving intravenous AAV2-ApoA-IM. The ApoA-IM gene was expressed in the aortic tissue of mice receiving AAV8 ApoA-IM but not in those receiving AAV2 ApoA-IM. Immunostaining showed that compared with the vector control, there was reduced macrophage content in the brachiocephalic (P < .05) and aortic sinus plaques (P < .05) of AAV8 ApoA-IM recipients but not in the recipients of AAV2 ApoA-IM. Thus, intravenous injection of AAV8 is more effective than intravenous injection of AAV2 in the expression of ApoA-IM gene. These data provide support for the potential feasibility of this approach for atheroprotection in

  19. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  20. Power inverters

    SciTech Connect

    Miller, David H.; Korich, Mark D.; Smith, Gregory S.

    2011-11-15

    Power inverters include a frame and a power module. The frame has a sidewall including an opening and defining a fluid passageway. The power module is coupled to the frame over the opening and includes a substrate, die, and an encasement. The substrate includes a first side, a second side, a center, an outer periphery, and an outer edge, and the first side of the substrate comprises a first outer layer including a metal material. The die are positioned in the substrate center and are coupled to the substrate first side. The encasement is molded over the outer periphery on the substrate first side, the substrate second side, and the substrate outer edge and around the die. The encasement, coupled to the substrate, forms a seal with the metal material. The second side of the substrate is positioned to directly contact a fluid flowing through the fluid passageway.

  1. Viral and Cellular Components of AAV2 Replication Compartments

    PubMed Central

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  2. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  3. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    PubMed Central

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  4. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    PubMed

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  5. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

    PubMed

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O'Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  6. Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors.

    PubMed

    Hirsch, Matthew L; Wolf, Sonya J; Samulski, R J

    2016-01-01

    Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber's congenital amaurosis. In addition to rAAV's high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package "large" genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6-8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6-8, 2010; Duan et al., J Virol 73(1):161-169, 1999; Duan et al., J Virol 72(11):8568-8577, 1998; Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002). This method involves "splitting" the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002; Ghosh et al., Mol Ther 16(1):124-130, 2008; Ghosh et al., Mol Ther 15(4):750-755, 2007). The other major

  7. AAV vector integration sites in mouse hepatocellular carcinoma.

    PubMed

    Donsante, Anthony; Miller, Daniel G; Li, Yi; Vogler, Carole; Brunt, Elizabeth M; Russell, David W; Sands, Mark S

    2007-07-27

    Adeno-associated viruses (AAV) are promising gene therapy vectors that have little or no acute toxicity. We show that normal mice and mice with mucopolysaccharidosis VII (MPS VII) develop hepatocellular carcinoma (HCC) after neonatal injection of an AAV vector expressing b-glucuronidase. AAV proviruses were isolated from four tumors and were all located within a 6-kilobase region of chromosome 12. This locus encodes several imprinted transcripts, small nucleolar RNAs (snoRNAs), and microRNAs. Transcripts from adjacent genes encoding snoRNAs and microRNAs were overexpressed in tumors. Our findings implicate this locus in the development of HCC and raise concerns over the clinical use of AAV vectors. PMID:17656716

  8. Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy.

    PubMed

    Hagen, Sven; Baumann, Tobias; Wagner, Hanna J; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M; Müller, Kristian M

    2014-01-01

    The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

  9. Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs

    PubMed Central

    2014-01-01

    Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting. PMID:25183392

  10. Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice

    PubMed Central

    Chapdelaine, P; Gérard, C; Sanchez, N; Cherif, K; Rousseau, J; Ouellet, D L; Jauvin, D; Tremblay, J P

    2016-01-01

    Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases. PMID:27082765

  11. Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy

    PubMed Central

    Bisset, Darren R.; Stepniak-Konieczna, Ewa A.; Zavaljevski, Maja; Wei, Jessica; Carter, Gregory T.; Weiss, Michael D.; Chamberlain, Joel R.

    2015-01-01

    RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3′ UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUGexp) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUGexp mRNA in the human α-skeletal muscle actin long-repeat (HSALR) mouse model of DM1. RNAi expression cassettes were delivered to HSALR mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSALR mice, including a reduction in the CUGexp mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUGexp mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSALR mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies. PMID:26082468

  12. Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

    PubMed

    Ye, Guo-jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Miller, Paul E; McPherson, Leslie; Ver Hoeve, James N; Smith, Leia M; Arndt, Tara; Mandapati, Savitri; Robinson, Paulette M; Calcedo, Roberto; Knop, David R; Hauswirth, William W; Chulay, Jeffrey D

    2016-03-01

    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. PMID:27003752

  13. Engineering the AAV capsid to optimize vector-host-interactions.

    PubMed

    Büning, Hildegard; Huber, Anke; Zhang, Liang; Meumann, Nadja; Hacker, Ulrich

    2015-10-01

    Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of pre-existing neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAV's tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAV's host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously non-permissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes. These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine.

  14. AAV Gene Therapy for MPS1-associated Corneal Blindness

    PubMed Central

    Vance, Melisa; Llanga, Telmo; Bennett, Will; Woodard, Kenton; Murlidharan, Giridhar; Chungfat, Neil; Asokan, Aravind; Gilger, Brian; Kurtzberg, Joanne; Samulski, R. Jude; Hirsch, Matthew L.

    2016-01-01

    Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness. PMID:26899286

  15. A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

    PubMed Central

    Stutika, Catrin; Gogol-Döring, Andreas; Botschen, Laura; Mietzsch, Mario; Weger, Stefan; Feldkamp, Mirjam; Chen, Wei

    2015-01-01

    ABSTRACT Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. IMPORTANCE Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable

  16. Mapping the AAV Capsid Host Antibody Response toward the Development of Second Generation Gene Delivery Vectors

    PubMed Central

    Tseng, Yu-Shan; Agbandje-McKenna, Mavis

    2013-01-01

    The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed. PMID:24523720

  17. Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

    PubMed

    Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

    2016-06-01

    Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

  18. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  19. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington’s disease

    PubMed Central

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O’Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington’s disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  20. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies.

    PubMed

    Tseng, Yu-Shan; Vliet, Kim Van; Rao, Lavanya; McKenna, Robert; Byrne, Barry J; Asokan, Aravind; Agbandje-McKenna, Mavis

    2016-10-01

    Adeno-associated viruses (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of six new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors being developed for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism. PMID:27424005

  1. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

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

    2016-01-01

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

  2. Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1.

    PubMed

    Díaz-Lezama, Nundehui; Wu, Zhijian; Adán-Castro, Elva; Arnold, Edith; Vázquez-Membrillo, Miguel; Arredondo-Zamarripa, David; Ledesma-Colunga, Maria G; Moreno-Carranza, Bibiana; Martinez de la Escalera, Gonzalo; Colosi, Peter; Clapp, Carmen

    2016-03-01

    Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvβ5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated

  3. Exosome-associated AAV vector as a robust and convenient neuroscience tool

    PubMed Central

    Hudry, Eloise; Martin, Courtney; Gandhi, Sheetal; György, Bence; Scheffer, Deborah I.; Mu, Dakai; Merkel, Steven F.; Mingozzi, Federico; Fitzpatrick, Zachary; Dimant, Hemi; Masek, Marissa; Ragan, Tim; Tan, Sisareuth; Brisson, Alain R.; Ramirez, Servio H.; Hyman, Bradley T.; Maguire, Casey A.

    2016-01-01

    Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill, and time intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared to standard AAV. Here we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation, results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing 3-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex-vivo serial 2-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant. PMID:26836117

  4. The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences.

    PubMed Central

    Smith, R H; Spano, A J; Kotin, R M

    1997-01-01

    The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences. PMID:9151837

  5. IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression.

    PubMed

    Gil-Fariña, Irene; Di Scala, Marianna; Vanrell, Lucia; Olagüe, Cristina; Vales, Africa; High, Katherine A; Prieto, Jesus; Mingozzi, Federico; Gonzalez-Aseguinolaza, Gloria

    2013-01-01

    Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents. PMID:23844082

  6. IL12-Mediated Liver Inflammation Reduces the Formation of AAV Transcriptionally Active Forms but Has No Effect over Preexisting AAV Transgene Expression

    PubMed Central

    Gil-Fariña, Irene; Di Scala, Marianna; Vanrell, Lucia; Olagüe, Cristina; Vales, Africa; High, Katherine A.; Prieto, Jesus; Mingozzi, Federico; Gonzalez-Aseguinolaza, Gloria

    2013-01-01

    Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents. PMID:23844082

  7. Microvesicle-associated AAV Vector as a Novel Gene Delivery System

    PubMed Central

    Maguire, Casey A; Balaj, Leonora; Sivaraman, Sarada; Crommentuijn, Matheus HW; Ericsson, Maria; Mincheva-Nilsson, Lucia; Baranov, Vladimir; Gianni, Davide; Tannous, Bakhos A; Sena-Esteves, Miguel; Breakefield, Xandra O; Skog, Johan

    2012-01-01

    Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery. PMID:22314290

  8. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    SciTech Connect

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-04-15

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

  9. AAV's Anatomy: Roadmap for Optimizing Vectors for Translational Success

    PubMed Central

    Samulski, R. Jude

    2014-01-01

    Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success. PMID:20712583

  10. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

    PubMed Central

    Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

    2009-01-01

    Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

  11. Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

    PubMed Central

    Hüser, Daniela; Gogol-Döring, Andreas; Lutter, Timo; Weger, Stefan; Winter, Kerstin; Hammer, Eva-Maria; Cathomen, Toni; Reinert, Knut; Heilbronn, Regine

    2010-01-01

    Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome. PMID:20628575

  12. Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

    PubMed Central

    Tseng, Yu-Shan; Gurda, Brittney L.; Chipman, Paul; McKenna, Robert; Afione, Sandra; Chiorini, John A.; Muzyczka, Nicholas; Olson, Norman H.; Baker, Timothy S.; Kleinschmidt, Jürgen

    2014-01-01

    ABSTRACT The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCE The adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that

  13. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

    PubMed Central

    Deverman, Benjamin E.; Pravdo, Piers L.; Simpson, Bryan P.; Kumar, Sripriya Ravindra; Chan, Ken Y.; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P.; Gradinaru, Viviana

    2015-01-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1-6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7-13. Here we describe a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV914-17, and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  14. Intranasal Vaccination with AAV5 and 9 Vectors Against Human Papillomavirus Type 16 in Rhesus Macaques

    PubMed Central

    Nieto, Karen; Stahl-Hennig, Christiane; Leuchs, Barbara; Müller, Martin; Gissmann, Lutz

    2012-01-01

    Abstract Cervical cancer is the second most common cancer in women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as the causative event for the development of this type of cancer. Recombinant adeno-associated viruses (rAAVs) are currently being developed and evaluated as vaccine vector. In previous work, we demonstrated that rAAVs administered intranasally in mice induced high titers and long-lasting neutralizing antibodies against HPV type 16 (HPV16). To extend this approach to a more human-related species, we immunized rhesus macaques (Macaca mulatta) with AAVs expressing an HPV16 L1 protein using rAAV5 and 9 vectors in an intranasal prophylactic setting. An rAAV5-L1 vector followed by a boost with rAAV9-L1 induced higher titers of L1-specific serum antibodies than a single rAAV5-L1 immunization. L1-specific antibodies elicited by AAV9 vector neutralized HPV16 pseudovirions and persisted for at least 7 months post immunization. Interestingly, nasal application of rAAV9 was immunogenic even in the presence of high AAV9 antibody titers, allowing reimmunization with the same serotype without prevention of the transgene expression. Two of six animals did not respond to AAV-mediated intranasal vaccination, although they were not tolerant, as both developed antibodies after intramuscular vaccination with HPV16 virus-like particles. These data clearly show the efficacy of an intranasal immunization using rAAV9-L1 vectors without the need of an adjuvant. We conclude from our results that rAAV9 vector is a promising candidate for a noninvasive nasal vaccination strategy. PMID:22401308

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

    SciTech Connect

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-11-25

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

  16. Intravenous delivery of AAV9 vector mediates effective gene expression in ischemic stroke lesion and brain angiogenic foci

    PubMed Central

    Shen, Fanxia; Kuo, Robert; Milon-Camus, Marine; Han, Zhenying; Jiang, Lidan; Young, William L.; Su, Hua

    2012-01-01

    Background and Purpose Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary, but not single-stranded, AAV9 vector (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. Methods Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO), and focal angiogenesis, by injecting an AAV vector expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have CMV promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid, and injected into mice through the jugular vein one hour after MCAO or four weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs five days post LacZ vector-injection. Results LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice, and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not AAV9-H9LacZ-injected mice. Conclusion ssAAV9 vector could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci. PMID:23250995

  17. Multilevel DC link inverter

    DOEpatents

    Su, Gui-Jia

    2003-06-10

    A multilevel DC link inverter and method for improving torque response and current regulation in permanent magnet motors and switched reluctance motors having a low inductance includes a plurality of voltage controlled cells connected in series for applying a resulting dc voltage comprised of one or more incremental dc voltages. The cells are provided with switches for increasing the resulting applied dc voltage as speed and back EMF increase, while limiting the voltage that is applied to the commutation switches to perform PWM or dc voltage stepping functions, so as to limit current ripple in the stator windings below an acceptable level, typically 5%. Several embodiments are disclosed including inverters using IGBT's, inverters using thyristors. All of the inverters are operable in both motoring and regenerating modes.

  18. AAV-mediated gene targeting methods for human cells

    PubMed Central

    Khan, Iram F; Hirata, Roli K; Russell, David W

    2013-01-01

    Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks. PMID:21455185

  19. Retinal gene delivery by rAAV and DNA electroporation

    PubMed Central

    Venkatesh, Aditya; Ma, Shan; Langellotto, Fernanda; Gao, Guangping; Punzo, Claudio

    2013-01-01

    Ocular gene therapy is a fast growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged, easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye specific genes tend to be more common. To date, over 40 eye specific genes have been identified which harbor mutations that lead to blindness. Gene therapy with recombinant Adeno Associated Virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, as well as subsequent tissue processing methods are described. PMID:23408132

  20. Measuring Immune Responses to recombinant AAV Gene Transfer

    PubMed Central

    Martino, Ashley T.; Herzog, Roland W.; Anegon, Ignacio; Adjali, Oumeya

    2013-01-01

    Following AAV-based gene transfer, the occurrence of adaptive immune responses specific to the vector or the transgene product is a major roadblock to successful clinical translation. These responses include antibodies against the AAV capsid, which can be neutralizing and therefore prevent the ability to repeatedly administer the vector, and CD8+ cytotoxic T lymphocytes, which can eliminate transduced cells. In addition, humans may have both humoral and cellular pre-existing immunity, as a result from natural infection with parent virus or related serotypes. The need for assays to detect and measure these anti-capsid immune responses in humans and in experimental animals is profound. Here, ELISPOT, immunocapture (ELISA), and neutralization assays are explained and provided in detail. Furthermore, such techniques can readily be adapted to monitor and quantify immune responses against therapeutic transgene products encoded by the vector genome. PMID:22034034

  1. Inhibition of pathological brain angiogenesis through systemic delivery of AAV vector expressing soluble FLT1

    PubMed Central

    Shen, Fanxia; Mao, Lei; Zhu, Wan; Lawton, Michael T.; Pechan, Peter; Colosi, Peter; Wu, Zhijian; Scaria, Abraham; Su, Hua

    2015-01-01

    The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, e.g., age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region. AAV vectors were packaged in AAV serotypes 1 and 2 (stereotactic injection) and 9 (IV-injection). Brain angiogenesis was induced in adult mice through stereotactic injection of AAV1-VEGF. AAV2-sFLT02 containing sFLT1 VEGF-binding domain (domain 2) was injected into the brain angiogenic region, and AAV9-sFLT1 was injected into the jugular vein at the time of or 4 weeks after AAV1-VEGF injection. We showed that AAV2-sFLT02 inhibited brain angiogenesis at both time points. Intravenous injection of AAV9-sFLT1 inhibited angiogenesis only when the vector was injected 4 weeks after angiogenic induction. Neither lymphocyte infiltration nor neuron loss was observed in AAV9-sFLT1-treated mice. Our data show that systemically delivered AAV9-sFLT1 inhibits angiogenesis in the mouse brain, which could be utilized to treat brain angiogenic diseases such as brain arteriovenous malformation. PMID:26090874

  2. Reprogramming Immune Response With Capsid-Optimized AAV6 Vectors for Immunotherapy of Cancer.

    PubMed

    Pandya, Munjal; Britt, Kellee; Hoffman, Brad; Ling, Chen; Aslanidi, George V

    2015-09-01

    In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors.

  3. AAV-mediated photoreceptor transduction of the pig cone-enriched retina

    PubMed Central

    Mussolino, C; della Corte, M; Rossi, S; Viola, F; Di Vicino, U; Marrocco, E; Neglia, S; Doria, M; Testa, F; Giovannoni, R; Crasta, M; Giunti, M; Villani, E; Lavitrano, M; Bacci, M L; Ratiglia, R; Simonelli, F; Auricchio, A; Surace, E M

    2011-01-01

    Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases. PMID:21412286

  4. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    PubMed Central

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  5. Resonant snubber inverter

    DOEpatents

    Lai, Jih-Sheng; Young, Sr., Robert W.; Chen, Daoshen; Scudiere, Matthew B.; Ott, Jr., George W.; White, Clifford P.; McKeever, John W.

    1997-01-01

    A resonant, snubber-based, soft switching, inverter circuit achieves lossless switching during dc-to-ac power conversion and power conditioning with minimum component count and size. Current is supplied to the resonant snubber branches solely by the main inverter switches. Component count and size are reduced by use of a single semiconductor switch in the resonant snubber branches. Component count is also reduced by maximizing the use of stray capacitances of the main switches as parallel resonant capacitors. Resonance charging and discharging of the parallel capacitances allows lossless, zero voltage switching. In one embodiment, circuit component size and count are minimized while achieving lossless, zero voltage switching within a three-phase inverter.

  6. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  7. High density recombinant AAV particles are competent vectors for in vivo transduction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  8. Prevalence of AAV1 neutralizing antibodies and consequences for a clinical trial of gene transfer for advanced heart failure.

    PubMed

    Greenberg, B; Butler, J; Felker, G M; Ponikowski, P; Voors, A A; Pogoda, J M; Provost, R; Guerrero, J; Hajjar, R J; Zsebo, K M

    2016-03-01

    Adeno-associated virus serotype 1 (AAV1) has many advantages as a gene therapy vector, but the presence of pre-existing neutralizing antibodies (NAbs) is an important limitation. This study was designed to determine: (1) characteristics of AAV NAbs in human subjects, (2) prevalence of AAV1 NAbs in heart failure patients and (3) utility of aggressive immunosuppressive therapy in reducing NAb seroconversion in an animal model. NAb titers were assessed in a cohort of heart failure patients and in patients screened for a clinical trial of gene therapy with AAV1 carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a). AAV1 NAbs were found in 59.5% of 1552 heart failure patients. NAb prevalence increased with age (P=0.001) and varied geographically. The pattern of NAb titers suggested that exposure is against AAV2, with AAV1 NAb seropositivity due to crossreactivity. The effects of immunosuppression on NAb formation were tested in mini-pigs treated with immunosuppressant therapy before, during and after a single AAV1/SERCA2a infusion. Aggressive immunosuppression did not prevent formation of AAV1 NAbs. We conclude that immunosuppression is unlikely to be a viable solution for repeat AAV1 dosing. Strategies to reduce NAbs in heart failure patients are needed to increase eligibility for gene transfer using AAV vectors.

  9. 75 FR 55808 - Prospective Grant of Exclusive License: Development of AAV5 Based Therapeutics To Treat Human...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-14

    ..., 517, entitled ``AAV5 and Uses Thereof,'' U.S. Patent 7, 479, 554, entitled ``AAV5 Nucleic Acids'' and... of delivering nucleic acids to a cell by using the AAV5 vectors and particles. More specifically, the ] technology provides the methods of delivering nucleic acids to cells of specific regions, tissues and...

  10. Analyzing Cellular Immunity to AAV in a Canine Model Using ELISpot Assay

    PubMed Central

    Wang, Zejing; Storb, Rainer; Tapscott, Stephen J; Riddell, Stanley

    2015-01-01

    Adeno-associated viral vector (AAV) mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine specific ELISpot assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T cell responses in peripheral blood in dogs following intra-muscular injection of AAV. This high-throughput method allows the identification of T cell epitopes without the need for large cell numbers and the need for MHC matched cell lines. PMID:21956501

  11. Inverting the Achievement Pyramid

    ERIC Educational Resources Information Center

    White-Hood, Marian; Shindel, Melissa

    2006-01-01

    Attempting to invert the pyramid to improve student achievement and increase all students' chances for success is not a new endeavor. For decades, educators have strategized, formed think tanks, and developed school improvement teams to find better ways to improve the achievement of all students. Currently, the No Child Left Behind Act (NCLB) is…

  12. Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo.

    PubMed

    Li, Baozheng; Ma, Wenqin; Ling, Chen; Van Vliet, Kim; Huang, Lin-Ya; Agbandje-McKenna, Mavis; Srivastava, Arun; Aslanidi, George V

    2015-12-01

    The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant adeno-associated virus 2 (AAV2) vectors, which negatively impacts the transduction efficiency of these vectors. Because ubiquitination occurs on lysine (K) residues, we performed site-directed mutagenesis where we replaced each of 10 surface-exposed K residues (K258, K490, K507, K527, K532, K544, K549, K556, K665, and K706) with glutamic acid (E) because of similarity of size and lack of recognition by modifying enzymes. The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors increased in HeLa cells in vitro up to 5-fold compared with wild-type (WT) AAV2 vectors, with the K556E mutant being the most efficient. Intravenous delivery of WT and K-mutant ssAAV2 vectors further corroborated these results in murine hepatocytes in vivo. Because AAV8 vectors transduce murine hepatocytes exceedingly well, and because some of the surface-exposed K residues are conserved between these serotypes, we generated and tested two single mutants (K547E and K569E), and one double-mutant (K547 + 569E) AAV8 vector. However, no significant increase in the transduction efficiency of any of these mutant AAV8 vectors was observed in murine hepatocytes in vivo. These studies suggest that although targeting the surface-exposed K residues is yet another strategy to improve the transduction efficiency of AAV vectors, phenotypic outcome is serotype specific.

  13. Program Predicts Nonlinear Inverter Performance

    NASA Technical Reports Server (NTRS)

    Al-Ayoubi, R. R.; Oepomo, T. S.

    1985-01-01

    Program developed for ac power distribution system on Shuttle orbiter predicts total load on inverters and node voltages at each of line replaceable units (LRU's). Mathematical model simulates inverter performance at each change of state in power distribution system.

  14. Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

    PubMed Central

    Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

    2016-01-01

    Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

  15. Development of novel AAV serotype 6 based vectors with selective tropism for human cancer cells.

    PubMed

    Sayroo, R; Nolasco, D; Yin, Z; Colon-Cortes, Y; Pandya, M; Ling, C; Aslanidi, G

    2016-01-01

    Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified to be particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to approximately 5-fold, by the mutagenesis of surface-exposed tyrosine and threonine residues involved in the intracellular trafficking of AAV vectors. Therefore, in our study, the AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD peptide, tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency, approximately 8-fold in vitro. In addition, we mutated lysine (K531E) to impair the affinity of AAV6 vectors to heparan sulfate proteoglycan. Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V+K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells.

  16. Milliwatt dc/dc Inverter

    NASA Technical Reports Server (NTRS)

    Mclyman, C. W.

    1983-01-01

    Compact dc/dc inverter uses single integrated-circuit package containing six inverter gates that generate and amplify 100-kHz square-wave switching signal. Square-wave switching inverts 10-volt local power to isolated voltage at another desired level. Relatively high operating frequency reduces size of filter capacitors required, resulting in small package unit.

  17. Inverting the Linear Algebra Classroom

    ERIC Educational Resources Information Center

    Talbert, Robert

    2014-01-01

    The inverted classroom is a course design model in which students' initial contact with new information takes place outside of class meetings, and students spend class time on high-level sense-making activities. The inverted classroom model is so called because it inverts or "flips" the usual classroom design where typically class…

  18. Inverted organic photovoltaic cells.

    PubMed

    Wang, Kai; Liu, Chang; Meng, Tianyu; Yi, Chao; Gong, Xiong

    2016-05-21

    The advance in lifestyle, modern industrialization and future technological revolution are always at high expense of energy consumption. Unfortunately, there exist serious issues such as limited storage, high cost and toxic contamination in conventional fossil fuel energy sources. Instead, solar energy represents a renewable, economic and green alternative in the future energy market. Among the photovoltaic technologies, organic photovoltaics (OPVs) demonstrate a cheap, flexible, clean and easy-processing way to convert solar energy into electricity. However, OPVs with a conventional device structure are still far away from industrialization mainly because of their short lifetime and the energy-intensive deposition of top metal electrode. To address the stability and cost issue simultaneously, an inverted device structure has been introduced into OPVs, bridging laboratory research with practical application. In this review, recent progress in device structures, working mechanisms, functions and advances of each component layer as well their correlations with the efficiency and stability of inverted OPVs are reviewed and illustrated.

  19. Inverted organic photovoltaic cells.

    PubMed

    Wang, Kai; Liu, Chang; Meng, Tianyu; Yi, Chao; Gong, Xiong

    2016-05-21

    The advance in lifestyle, modern industrialization and future technological revolution are always at high expense of energy consumption. Unfortunately, there exist serious issues such as limited storage, high cost and toxic contamination in conventional fossil fuel energy sources. Instead, solar energy represents a renewable, economic and green alternative in the future energy market. Among the photovoltaic technologies, organic photovoltaics (OPVs) demonstrate a cheap, flexible, clean and easy-processing way to convert solar energy into electricity. However, OPVs with a conventional device structure are still far away from industrialization mainly because of their short lifetime and the energy-intensive deposition of top metal electrode. To address the stability and cost issue simultaneously, an inverted device structure has been introduced into OPVs, bridging laboratory research with practical application. In this review, recent progress in device structures, working mechanisms, functions and advances of each component layer as well their correlations with the efficiency and stability of inverted OPVs are reviewed and illustrated. PMID:27087582

  20. Inverted organic photosensitive device

    SciTech Connect

    Forrest, Stephen R.; Tong, Xiaoran; Lee, Jun Yeob; Cho, Yong Joo

    2015-09-08

    There is disclosed a method for preparing the surface of a metal substrate. The present disclosure also relates to an organic photovoltaic device including a metal substrate made by such method. Also disclosed herein is an inverted photosensitive device including a stainless steel foil reflective electrode, an organic donor-acceptor heterojunction over the reflective electrode, and a transparent electrode over the donor-acceptor heterojunction.

  1. Effective delivery of large genes to the retina by dual AAV vectors

    PubMed Central

    Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

  2. No tumour-initiating risk associated with scAAV transduction in newborn rat liver.

    PubMed

    Gauttier, V; Pichard, V; Aubert, D; Kaeppel, C; Schmidt, M; Ferry, N; Conchon, S

    2013-07-01

    Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.

  3. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

    PubMed

    Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1999-06-01

    Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

  4. Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

    PubMed Central

    Lipinski, Daniel M.; Singh, Mandeep S.; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R.; Hankins, Mark W.; During, Matthew J.; MacLaren, Robert E.

    2013-01-01

    Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4−/− mouse which is a model for Stargardt disease and in the Pde6brd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments. PMID:23593201

  5. Gene Therapy for Mucopolysaccharidosis Type VI Is Effective in Cats Without Pre-Existing Immunity to AAV8

    PubMed Central

    Ferla, Rita; O'Malley, Thomas; Calcedo, Roberto; O'Donnell, Patricia; Wang, Ping; Cotugno, Gabriella; Claudiani, Pamela; Wilson, James M.; Haskins, Mark

    2013-01-01

    Abstract Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy. PMID:23194248

  6. Inverted glass harp

    NASA Astrophysics Data System (ADS)

    Quinn, Daniel B.; Rosenberg, Brian J.

    2015-08-01

    We present an analytical treatment of the acoustics of liquid-filled wine glasses, or "glass harps." The solution is generalized such that under certain assumptions it reduces to previous glass harp models, but also leads to a proposed musical instrument, the "inverted glass harp," in which an empty glass is submerged in a liquid-filled basin. The versatility of the solution demonstrates that all glass harps are governed by a family of solutions to Laplace's equation around a vibrating disk. Tonal analyses of recordings for a sample glass are offered as confirmation of the scaling predictions.

  7. Inverted glass harp.

    PubMed

    Quinn, Daniel B; Rosenberg, Brian J

    2015-08-01

    We present an analytical treatment of the acoustics of liquid-filled wine glasses, or "glass harps." The solution is generalized such that under certain assumptions it reduces to previous glass harp models, but also leads to a proposed musical instrument, the "inverted glass harp," in which an empty glass is submerged in a liquid-filled basin. The versatility of the solution demonstrates that all glass harps are governed by a family of solutions to Laplace's equation around a vibrating disk. Tonal analyses of recordings for a sample glass are offered as confirmation of the scaling predictions. PMID:26382336

  8. A multifunctional AAV-CRISPR-Cas9 and its host response.

    PubMed

    Chew, Wei Leong; Tabebordbar, Mohammadsharif; Cheng, Jason K W; Mali, Prashant; Wu, Elizabeth Y; Ng, Alex H M; Zhu, Kexian; Wagers, Amy J; Church, George M

    2016-10-01

    CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

  9. Gas cooled traction drive inverter

    SciTech Connect

    Chinthavali, Madhu Sudhan

    2013-10-08

    The present invention provides a modular circuit card configuration for distributing heat among a plurality of circuit cards. Each circuit card includes a housing adapted to dissipate heat in response to gas flow over the housing. In one aspect, a gas-cooled inverter includes a plurality of inverter circuit cards, and a plurality of circuit card housings, each of which encloses one of the plurality of inverter cards.

  10. Applications of cascade multilevel inverters.

    PubMed

    Peng, Fang-zen; Qian, Zhao-ming

    2003-01-01

    Cascade multilevel inverters have been developed for electric utility applications. A cascade M-level inverter consists of (M-1)/2 H-bridges in which each bridge's dc voltage is supported by its own dc capacitor. The new inverter can: (1) generate almost sinusoidal waveform voltage while only switching one time per fundamental cycle; (2) dispense with multi-pulse inverters' transformers used in conventional utility interfaces and static var compensators; (3) enables direct parallel or series transformer-less connection to medium- and high-voltage power systems. In short, the cascade inverter is much more efficient and suitable for utility applications than traditional multi-pulse and pulse width modulation (PWM) inverters. The authors have experimentally demonstrated the superiority of the new inverter for power supply, (hybrid) electric vehicle (EV) motor drive, reactive power (var) and harmonic compensation. This paper summarizes the features, feasibility, and control schemes of the cascade inverter for utility applications including utility interface of renewable energy, voltage regulation, var compensation, and harmonic filtering in power systems. Analytical, simulated, and experimental results demonstrated the superiority of the new inverters. PMID:14566981

  11. Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles.

    PubMed

    Rohr, Ulrich-Peter; Heyd, Florian; Neukirchen, Judith; Wulf, Marc-Andre; Queitsch, Iris; Kroener-Lux, Gabriele; Steidl, Ulrich; Fenk, Roland; Haas, Rainer; Kronenwett, Ralf

    2005-07-01

    In this report, we present a fast, reliable and easy to perform method to quantify infectious titers of recombinant AAV-2 (rAAV-2) particles using the LightCycler technology, which is independent from the therapeutic transgene and without the presence of a marker gene. The method is based on the life cycle of AAV-2: after infection of the host cell, the single stranded (ss) AAV-2 genome is converted into a double stranded (ds) form. Following infection with rAAV-2, HeLa cells were lysed and ssDNA of transcriptionally inactive particles were efficiently removed by ssDNA-specific S1 nuclease digestion. The remaining viral dsDNA can be quantified by quantitative real-time PCR (qPCR). For validation of the new method, rAAV-2 preparations were analyzed by two other standard methods for titration of infectious particles in parallel, i.e. the infectious center assay (ICA) as well as flow cytometry using GFP as a marker. Comparing the infectious titers of 40 different AAV-2 fractions assessed by qPCR with the titers determined by FACS analysis a significant correlation (r=0.87, p<0.001) with a mean ratio of the titers assessed by qPCR and FACS of 1.92 (S.D.+/-1.59) was found. Further, the titers of seven rAAV-2 fractions using qPCR and ICA covering 5 log ranges were compared and a significant correlation was found between the results (r=0.80, p<0.001) with a mean ratio of 3.38 (S.D.+/-1.79), respectively.

  12. Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

    PubMed Central

    Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A.; Song, Juan

    2016-01-01

    Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design. PMID:27699236

  13. Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

    PubMed Central

    Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A.; Song, Juan

    2016-01-01

    Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.

  14. Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors.

    PubMed

    Li, Shaoyong; Ling, Chen; Zhong, Li; Li, Mengxin; Su, Qin; He, Ran; Tang, Qiushi; Greiner, Dale L; Shultz, Leonard D; Brehm, Michael A; Flotte, Terence R; Mueller, Christian; Srivastava, Arun; Gao, Guangping

    2015-12-01

    Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

  15. Microglia-specific targeting by novel capsid-modified AAV6 vectors

    PubMed Central

    Rosario, Awilda M; Cruz, Pedro E; Ceballos-Diaz, Carolina; Strickland, Michael R; Siemienski, Zoe; Pardo, Meghan; Schob, Keri-Lyn; Li, Andrew; Aslanidi, George V; Srivastava, Arun; Golde, Todd E; Chakrabarty, Paramita

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies. PMID:27308302

  16. High cerebrospinal fluid levels of interleukin-10 attained by AAV in dogs.

    PubMed

    Pleticha, J; Malkmus, S A; Heilmann, L F; Veesart, S L; Rezek, R; Xu, Q; Yaksh, T L; Beutler, A S

    2015-02-01

    Intrathecal (IT) gene transfer using adeno-associated virus (AAV) may be clinically promising as a treatment for chronic pain if it can produce sufficiently high levels of a transgene product in the cerebrospinal fluid (CSF). Although this strategy was developed in rodents, no studies investigating CSF levels of an analgesic or antiallodynic protein delivered by IT AAV have been performed in large animals. Interleukin-10 (IL-10) is an antiallodynic cytokine for which target therapeutic levels have been established in rats. The present study tested IT AAV8 encoding either human IL-10 (hIL-10) or enhanced green fluorescent protein (EGFP) in a dog model of IT drug delivery. AAV8/hIL-10 at a dose of 3.5 × 10(12) genome copies induced high hIL-10 levels in the CSF, exceeding the target concentration previously found to be antiallodynic in rodents by >1000-fold. AAV8/EGFP targeted the primary sensory and motor neurons and the meninges. hIL-10, a xenogeneic protein in dogs, induced anti-hIL-10 antibodies detectable in the CSF and serum of dogs. The high hIL-10 levels demonstrate the efficacy of AAV for delivery of secreted transgenes into the IT space of large animals, suggesting a strong case for further development toward clinical testing.

  17. High cerebrospinal fluid levels of interleukin-10 attained by AAV in dogs

    PubMed Central

    Pleticha, Josef; Malkmus, Shelle A.; Heilmann, Lukas F.; Veesart, Samantha L.; Rezek, Rahaf; Xu, Qinghao; Yaksh, Tony L.; Beutler, Andreas S.

    2016-01-01

    Intrathecal (IT) gene transfer using adeno-associated virus (AAV) may be clinically promising as a treatment for chronic pain if it can produce sufficiently high levels of a transgene product in the cerebrospinal fluid (CSF). While this strategy was developed in rodents, no studies investigating CSF levels of an analgesic or anti-allodynic protein delivered by IT AAV have been performed in large animals. Interleukin-10 (IL-10) is an anti-allodynic cytokine, for which target therapeutic levels have been established in rats. The present study tested IT AAV8 encoding either human IL-10 (hIL-10) or enhanced green fluorescent protein (EGFP) in a dog model of IT drug delivery. AAV8/hIL-10 at a dose of 3.5×1012 genome copies induced high hIL-10 levels in the CSF, exceeding the target concentration previously found to be anti-allodynic in rodents by >1000-fold. AAV8/EGFP targeted the primary sensory and motor neurons and the meninges. hIL-10, a xenogeneic protein in dogs, induced anti-hIL-10 antibodies detectable in the dogs’ CSF and serum. The high hIL-10 levels demonstrate the efficacy of AAV for delivery of secreted transgenes into the IT space of large animals suggesting a strong case for further development towards clinical testing. PMID:25354684

  18. CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

    PubMed Central

    Steines, Benjamin; Dickey, David D.; Bergen, Jamie; Excoffon, Katherine J.D.A.; Weinstein, John R.; Li, Xiaopeng; Yan, Ziying; Alaiwa, Mahmoud H. Abou; Shah, Viral S.; Bouzek, Drake C.; Powers, Linda S.; Gansemer, Nicholas D.; Ostedgaard, Lynda S.; Engelhardt, John F.; Stoltz, David A.; Welsh, Michael J.; Sinn, Patrick L.; Schaffer, David V.

    2016-01-01

    The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl– transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways. PMID:27699238

  19. CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

    PubMed Central

    Steines, Benjamin; Dickey, David D.; Bergen, Jamie; Excoffon, Katherine J.D.A.; Weinstein, John R.; Li, Xiaopeng; Yan, Ziying; Alaiwa, Mahmoud H. Abou; Shah, Viral S.; Bouzek, Drake C.; Powers, Linda S.; Gansemer, Nicholas D.; Ostedgaard, Lynda S.; Engelhardt, John F.; Stoltz, David A.; Welsh, Michael J.; Sinn, Patrick L.; Schaffer, David V.

    2016-01-01

    The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl– transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

  20. The anti-tumor effect and increased tregs infiltration mediated by rAAV-SLC vector.

    PubMed

    Li, Rilun; Hu, Heng; Ma, Huiying; Chen, Long; Zhou, Binbin; Liu, Yinkun; Liang, Chunmin

    2013-10-01

    To explore the anti-tumor effect and immune mechanism mediated by a new recombinant adeno-associated virus (rAAV) encoding secondary lymphoid tissue chemokine (SLC) mature peptide gene. AAV Helper-Free system was used for rAAV-SLC package. The anti-tumor effect of SLC was detected by bearing tumor established from Hepal-6 cells both in C57BL/6J and nude mice. Flow cytometry analysis and IHC for Tumor-infiltrating T cells and CD11c+DCs were also investigated to explore the immunological mechanism. rAAV-SLC was successfully packaged in AAV293 cells and transfected Hepal-6 tumor cells at high efficiency. The anti-tumor effect was demonstrated by less tumor weight and longer survival outcome. Coincident with the anti-tumor response, local elaboration of SLC within the tumor bed elicited a heavy infiltration of CD4+, CD8+T cells and CD11c+ dendritic cells into the tumor sites. More importantly, there was higher infiltration of Foxp3+ regulatory T cells (Tregs). Local elaboration of SLC mediated by rAAV-SLC has strong T cell mediated anti-tumor effect. The study also suggested that Tregs in the tumor microenvironment tampered the anti-tumor effect.

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

    PubMed Central

    Chou, JY; Mansfield, BC

    2011-01-01

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

  2. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10

    PubMed Central

    Gilkes, J A; Bloom, M D; Heldermon, C D

    2016-01-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB. PMID:26674264

  3. Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis.

    PubMed

    Uhrig, S; Coutelle, O; Wiehe, T; Perabo, L; Hallek, M; Büning, H

    2012-02-01

    Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

  4. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector.

    PubMed

    Murlidharan, Giridhar; Sakamoto, Kensuke; Rao, Lavanya; Corriher, Travis; Wang, Dan; Gao, Guangping; Sullivan, Patrick; Asokan, Aravind

    2016-01-01

    Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities. PMID:27434683

  5. In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy.

    PubMed

    Choudhury, Sourav R; Fitzpatrick, Zachary; Harris, Anne F; Maitland, Stacy A; Ferreira, Jennifer S; Zhang, Yuanfan; Ma, Shan; Sharma, Rohit B; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Alonso, Laura C; Punzo, Claudio; Wagner, Kathryn R; Maguire, Casey A; Kotin, Robert M; Martin, Douglas R; Sena-Esteves, Miguel

    2016-08-01

    Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy. PMID:27117222

  6. Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

    PubMed

    Choudhury, Sourav R; Harris, Anne F; Cabral, Damien J; Keeler, Allison M; Sapp, Ellen; Ferreira, Jennifer S; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Su, Qin; Stoica, Lorelei; DiFiglia, Marian; Aronin, Neil; Martin, Douglas R; Gao, Guangping; Sena-Esteves, Miguel

    2016-04-01

    Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

  7. Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

    PubMed Central

    Wagner, Bettina; Calcedo, Roberto; Wilson, James; Schaefer, Deanna; Nixon, Alan

    2015-01-01

    Abstract Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×1011 vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA. PMID:25705927

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

    PubMed Central

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

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  9. AAV9-mediated central nervous system–targeted gene delivery via cisterna magna route in mice

    PubMed Central

    Lukashchuk, Vera; Lewis, Katherine E; Coldicott, Ian; Grierson, Andrew J; Azzouz, Mimoun

    2016-01-01

    Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off-target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken-β-actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. We demonstrate that synapsin 1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10-fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9-GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin 1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor-related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intracranial or intracerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications. PMID:26942208

  10. Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.

    PubMed

    Melo, Sandra P; Lisowski, Leszek; Bashkirova, Elizaveta; Zhen, Hanson H; Chu, Kirk; Keene, Douglas R; Marinkovich, M Peter; Kay, Mark A; Oro, Anthony E

    2014-04-01

    Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder. PMID:24390279

  11. AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

    PubMed

    Santiago-Ortiz, J; Ojala, D S; Westesson, O; Weinstein, J R; Wong, S Y; Steinsapir, A; Kumar, S; Holmes, I; Schaffer, D V

    2015-12-01

    Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

  12. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

    PubMed

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-04-30

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished.

  13. Intranasal Delivery of Recombinant NT4-NAP/AAV Exerts Potential Antidepressant Effect.

    PubMed

    Ma, Xian-Cang; Chu, Zheng; Zhang, Xiao-Ling; Jiang, Wen-Hui; Jia, Min; Dang, Yong-Hui; Gao, Cheng-Ge

    2016-06-01

    The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment. PMID:26846142

  14. Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy

    PubMed Central

    Okada, Takashi; Takeda, Shin'ichi

    2013-01-01

    Various characteristics of adeno-associated virus (AAV)-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD). Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response. PMID:24276316

  15. Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

    PubMed Central

    Ahn, Misol; Bajsarowicz, Krystyna; Oehler, Abby; Lemus, Azucena; Bankiewicz, Krystof; DeArmond, Stephen J.

    2014-01-01

    Prion disease is caused by a single pathogenic protein (PrPSc), an abnormal conformer of the normal cellular prion protein PrPC. Depletion of PrPC in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrPC expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrPC levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrPSc formation in the thalamic infusion site by ∼75%, it did not suppress PrPSc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrPC in the brain is required for successful therapy of prion diseases. PMID:24866748

  16. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids

    PubMed Central

    Castle, Michael J.; Turunen, Heikki T.; Vandenberghe, Luk H.; Wolfe, John H.

    2016-01-01

    More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina. PMID:26611584

  17. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

    PubMed

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-04-30

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished. PMID:24583077

  18. AAV Vectors for Cardiac Gene Transfer: Experimental Tools and Clinical Opportunities

    PubMed Central

    Pacak, Christina A; Byrne, Barry J

    2011-01-01

    Since the first demonstration of in vivo gene transfer into myocardium there have been a series of advancements that have driven the evolution of cardiac gene delivery from an experimental tool into a therapy currently at the threshold of becoming a viable clinical option. Innovative methods have been established to address practical challenges related to tissue-type specificity, choice of delivery vehicle, potency of the delivered material, and delivery route. Most importantly for therapeutic purposes, these strategies are being thoroughly tested to ensure safety of the delivery system and the delivered genetic material. This review focuses on the development of recombinant adeno-associated virus (rAAV) as one of the most valuable cardiac gene transfer agents available today. Various forms of rAAV have been used to deliver “pre-event” cardiac protection and to temper the severity of hypertrophy, cardiac ischemia, or infarct size. Adeno-associated virus (AAV) vectors have also been functional delivery tools for cardiac gene expression knockdown studies and successfully improving the cardiac aspects of several metabolic and neuromuscular diseases. Viral capsid manipulations along with the development of tissue-specific and regulated promoters have greatly increased the utility of rAAV-mediated gene transfer. Important clinical studies are currently underway to evaluate AAV-based cardiac gene delivery in humans. PMID:21792180

  19. Nanowire NMOS Logic Inverter Characterization.

    PubMed

    Hashim, Yasir

    2016-06-01

    This study is the first to demonstrate characteristics optimization of nanowire N-Channel Metal Oxide Semiconductor (NW-MOS) logic inverter. Noise margins and inflection voltage of transfer characteristics are used as limiting factors in this optimization. A computer-based model used to produce static characteristics of NW-NMOS logic inverter. In this research two circuit configuration of NW-NMOS inverter was studied, in first NW-NMOS circuit, the noise margin for (low input-high output) condition was very low. For second NMOS circuit gives excellent noise margins, and results indicate that optimization depends on applied voltage to the inverter. Increasing gate to source voltage with (2/1) nanowires ratio results better noise margins. Increasing of applied DC load transistor voltage tends to increasing in decreasing noise margins; decreasing this voltage will improve noise margins significantly. PMID:27427653

  20. Inverter Ground Fault Overvoltage Testing

    SciTech Connect

    Hoke, Andy; Nelson, Austin; Chakraborty, Sudipta; Chebahtah, Justin; Wang, Trudie; McCarty, Michael

    2015-08-12

    This report describes testing conducted at NREL to determine the duration and magnitude of transient overvoltages created by several commercial PV inverters during ground fault conditions. For this work, a test plan developed by the Forum on Inverter Grid Integration Issues (FIGII) has been implemented in a custom test setup at NREL. Load rejection overvoltage test results were reported previously in a separate technical report.

  1. Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element

    PubMed Central

    Wang, Lizheng; Wang, Zixuan; Zhang, Fangfang; Zhu, Rui; Bi, Jinpeng; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-01-01

    Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors. PMID:27076785

  2. Serotype-specific Binding Properties and Nanoparticle Characteristics Contribute to the Immunogenicity of rAAV1 Vectors.

    PubMed

    Ferrand, Maxime; Da Rocha, Sylvie; Corre, Guillaume; Galy, Anne; Boisgerault, Florence

    2015-06-01

    The immunogenic properties of recombinant adeno-associated virus (rAAV) gene transfer vectors remain incompletely characterized in spite of their usage as gene therapy vectors or as vaccines. Molecular interactions between rAAV and various types of antigen-presenting cells (APCs), as well as the impact of these interactions on transgene or capsid-specific immunization remain unclear. We herein show that binding motifs recognized by the capsid and which determine the vector tissue tropism are also critical for key immune activation processes. Using rAAV capsid serotype 1 (rAAV1) vectors which primary receptors on target cells are α2,3 and α2,6 N-linked sialic acids, we show that sialic acid-dependent binding of rAAV1 on APCs is essential to trigger CD4(+) T-cell responses by increasing rAAV1 uptake and contributing to antigenic presentation of both the capsid and transgene product although this involves different APCs. In addition, the nanoparticulate structure of the vector in itself appears to be sufficient to trigger mobilization and activation of some APCs. Therefore, combinations of structural and of serotype-specific cell-targeting properties of rAAV1 determine its complex immunogenicity. These findings may be useful to guide a selection of rAAV variants depending on the intended level of immunogenicity for either gene therapy or vaccination applications.

  3. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

  4. Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors.

    PubMed

    Hoyng, S A; De Winter, F; Gnavi, S; van Egmond, L; Attwell, C L; Tannemaat, M R; Verhaagen, J; Malessy, M J A

    2015-10-01

    Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this article, we compared adeno-associated viral (AAV) vectors based on serotypes 1-9 for their capability to transduce cultured primary rat and human SCs and nerve segments. AAV1 is the best serotype to transduce rat SCs, whereas AAV2 and AAV6 performed equally well in human SCs. Transduction of monolayers of cultured rat and human SCs did not accurately predict the transduction efficiency in nerve segments. Rat nerve segments could be genetically modified equally well by a set of four AAV vectors (AAV1, AAV5, AAV7, AAV9), whereas AAV2 was superior in human nerve segments. The current experiments were undertaken as a first step towards future clinical implementation of ex vivo AAV-based gene therapy in surgical nerve repair. The transduction of rat and human SCs and nerve segments by entirely different AAV serotypes, as documented here, highlights one of the challenges of translating gene therapy from experimental animals to human patients.

  5. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation.

    PubMed

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  6. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    PubMed Central

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  7. 21 CFR 184.1859 - Invert sugar.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Invert sugar. 184.1859 Section 184.1859 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD....1859 Invert sugar. (a) Invert sugar (CAS Reg. No. 8013-17-0) is an aqueous solution of inverted...

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

    PubMed Central

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

    2011-01-01

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

  9. Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference

    PubMed Central

    Borel, Florie; Kay, Mark A; Mueller, Christian

    2014-01-01

    RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. PMID:24352214

  10. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    2012-02-05

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  11. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  12. Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII.

    PubMed

    Marcos-Contreras, Oscar A; Smith, Shannon M; Bellinger, Dwight A; Raymer, Robin A; Merricks, Elizabeth; Faella, Armida; Pavani, Giulia; Zhou, Shangzhen; Nichols, Timothy C; High, Katherine A; Margaritis, Paris

    2016-02-01

    Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency. PMID:26702064

  13. Termination Documentation

    ERIC Educational Resources Information Center

    Duncan, Mike; Hill, Jillian

    2014-01-01

    In this study, we examined 11 workplaces to determine how they handle termination documentation, an empirically unexplored area in technical communication and rhetoric. We found that the use of termination documentation is context dependent while following a basic pattern of infraction, investigation, intervention, and termination. Furthermore,…

  14. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    PubMed

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  15. Accelerating development of advanced inverters :

    SciTech Connect

    Neely, Jason C.; Gonzalez, Sigifredo; Ropp, Michael; Schutz, Dustin

    2013-11-01

    The high penetration of utility interconnected photovoltaic (PV) systems is causing heightened concern over the effect that variable renewable generation will have on the electrical power system (EPS). These concerns have initiated the need to amend the utility interconnection standard to allow advanced inverter control functionalities that provide: (1) reactive power control for voltage support, (2) real power control for frequency support and (3) better tolerance of grid disturbances. These capabilities are aimed at minimizing the negative impact distributed PV systems may have on EPS voltage and frequency. Unfortunately, these advanced control functions may interfere with island detection schemes, and further development of advanced inverter functions requires a study of the effect of advanced functions on the efficacy of antiislanding schemes employed in industry. This report summarizes the analytical, simulation and experimental work to study interactions between advanced inverter functions and anti-islanding schemes being employed in distributed PV systems.

  16. Comparative impact of AAV and enzyme replacement therapy on respiratory and cardiac function in adult Pompe mice

    PubMed Central

    Falk, Darin J; Soustek, Meghan S; Todd, Adrian Gary; Mah, Cathryn S; Cloutier, Denise A; Kelley, Jeffry S; Clement, Nathalie; Fuller, David D; Byrne, Barry J

    2015-01-01

    Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa–/– mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa–/– animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease. PMID:26029718

  17. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

    PubMed Central

    Lecomte, Emilie; Tournaire, Benoît; Cogné, Benjamin; Dupont, Jean-Baptiste; Lindenbaum, Pierre; Martin-Fontaine, Mélanie; Broucque, Frédéric; Robin, Cécile; Hebben, Matthias; Merten, Otto-Wilhelm; Blouin, Véronique; François, Achille; Redon, Richard; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles. PMID:26506038

  18. Systemically administered AAV9-sTRAIL combats invasive glioblastoma in a patient-derived orthotopic xenograft model

    PubMed Central

    Crommentuijn, Matheus HW; Kantar, Rami; Noske, David P; Vandertop, W Peter; Badr, Christian E; Würdinger, Thomas; Maguire, Casey A; Tannous, Bakhos A

    2016-01-01

    Adeno-associated virus (AAV) vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise, however, invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA) promoter and the neuron-specific enolase (NSE) promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns, although the NSE promoter yielded 100-fold lower expression in the abdomen (liver), with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes, neurons and endothelial cells, while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival, with the CBA promoter having higher efficacy. To our knowledge, this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors. PMID:27382645

  19. Selective In Vivo Targeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

    PubMed Central

    Wang, Yuan; Zhang, Yuanhui; Ejjigani, Anila; Yin, Zifei; Lu, Yuan; Wang, Lina; Wang, Meng; Li, Jun; Hu, Zhongbo; Aslanidi, George V.; Zhong, Li; Gao, Guangping

    2014-01-01

    Abstract Current challenges for recombinant adeno-associated virus (rAAV) vector–based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer. PMID:25296041

  20. Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model.

    PubMed

    Ling, Chen; Wang, Yuan; Zhang, Yuanhui; Ejjigani, Anila; Yin, Zifei; Lu, Yuan; Wang, Lina; Wang, Meng; Li, Jun; Hu, Zhongbo; Aslanidi, George V; Zhong, Li; Gao, Guangping; Srivastava, Arun; Ling, Changquan

    2014-12-01

    Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

  1. More than chemotaxis: a new anti-tumor DC vaccine modified by rAAV2-SLC.

    PubMed

    Liang, Chun-min; Ye, Sheng-long; Zhong, Cui-ping; Zheng, Ning; Bian, Wei; Sun, Rui-xia; Chen, Jun; Li, Ri-lun; Zhou, Shuang; Liu, Yin-kun

    2007-07-01

    Secondary lymphoid tissue chemokine (SLC) is strongly expressed in secondary lymphoid organs. Its ability to facilitate chemotaxis of both dendritic cells (DC) and T cells makes it a promising candidate for cancer therapy. In this study, we modified a BMDC vaccine by incorporating the SLC mature peptide gene. The efficacy of this vaccine was evaluated using a mouse hepatocellular carcinoma (HCC) model, with rAAV2 as the gene delivery vector. The rAAV2 encoding SLC (rAAV2-SLC) transfected immature BMDCs at high efficiency and the anti-tumor effects of SLC gene modified BMDCs (rAAV2-SLC/BMDC) were evaluated. In addition, rAAV2-SLC/BMDC vaccine injected directly into tumors attracted more CD4(+) and CD8(+) T lymphocytes into tumors and showed stronger anti-tumor effects than footpad delivery. Moreover, we found that the phenotypic expression of MHC II, the secretion of IL-12 and IFN-gamma, and T cell stimulation were increased in vitro following treatment with rAAV2-SLC/BMDC vaccine and these responses were inhibited by PTX. In vivo, PTX also inhibited the anti-tumor effects of the vaccine. The results suggest that the expression of SLC by rAAV2-SLC/BMDC plays more than a chemotactic role in anti-tumor responses, thus these studies further demonstrate that SLC has potential to be valuable in cancer therapy.

  2. Successful transgene expression with serial doses of aerosolized rAAV2 vectors in rhesus macaques.

    PubMed

    Fischer, Anne C; Beck, Suzanne E; Smith, Carolina I; Laube, Beth L; Askin, Frederic B; Guggino, Sandra E; Adams, Robert J; Flotte, Terence R; Guggino, William B

    2003-12-01

    Bronchoscopic microspraying of recombinant adeno-associated viral (rAAV) vectors targets high doses of vector directly to pulmonary epithelium. Single-dose endobronchial gene therapy trials have been accomplished in cystic fibrosis patients; however, repeated dosing strategies are likely essential for lifetime correction. These studies address whether serial redosing with rAAV2 vectors results in an antiserotypic response and, furthermore, whether it triggers an inflammatory response prohibitive to transgene expression. Serial redosing of 9 x 10(11) infectious units of aerosolized rAAV2 vectors to rhesus macaques resulted in successful gene transfer by quantitative PCR (1.43 x 10(9) copies/g tissue) and transgene expression. Additionally, confocal microscopy and immunohistochemical analysis demonstrated in situ expression localized to the pulmonary epithelium. Although serial redosing did induce a heightened anti-neutralizing antibody response in sera, gene transfer prevailed with resultant expression. This study is the first to demonstrate successful gene transfer subsequent to repeated aerosolized doses of rAAV2 in immunocompetent nonhuman primates without associated inflammatory responses prohibitive to transgene expression. PMID:14664794

  3. AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models

    PubMed Central

    Sondergaard, Patricia C; Griffin, Danielle A; Pozsgai, Eric R; Johnson, Ryan W; Grose, William E; Heller, Kristin N; Shontz, Kim M; Montgomery, Chrystal L; Liu, Joseph; Clark, Kelly Reed; Sahenk, Zarife; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-01-01

    Objective Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment. Methods Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety. Results Treated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns. Interpretation Treated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients. PMID:25815352

  4. Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

    PubMed

    Thorne, Barbara A; Quigley, Paulene; Nichols, Gina; Moore, Christine; Pastor, Eric; Price, David; Ament, Jon W; Takeya, Ryan K; Peluso, Richard W

    2008-01-01

    Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

  5. Responses of One First Grade Class to the Representation of AAVE in Picture Books

    ERIC Educational Resources Information Center

    McCreight, Jennifer

    2011-01-01

    The following article will address the need for classrooms to promote the use of children's literature whose characters speak in a dialect other than Standard English (specifically African American Vernacular English, or AAVE). It will begin by drawing attention to the lack of authentic representation of African Americans in picture books…

  6. AAV-mediated gene therapy in mouse models of recessive retinal degeneration

    PubMed Central

    Pang, Ji-jing; Lei, Lei; Dai, Xufeng; Shi, Wei; Liu, Xuan; Dinculescu, Astra; McDowell, J. Hugh

    2013-01-01

    In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adeno-associated viral vector (AAV). Following delivery to the immuno-priviledged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases. PMID:22300136

  7. Microgrid and Inverter Control and Simulator Software

    SciTech Connect

    2012-09-13

    A collection of software that can simulate the operation of an inverter on a microgrid or control a real inverter. In addition, it can simulate the control of multiple nodes on a microgrid." Application: Simulation of inverters and microgrids; control of inverters on microgrids." The MMI submodule is designed to control custom inverter hardware, and to simulate that hardware. The INVERTER submodule is only the simulator code, and is of an earlier generation than the simulator in MMI. The MICROGRID submodule is an agent-based simulator of multiple nodes on a microgrid which presents a web interface. The WIND submodule produces movies of wind data with a web interface.

  8. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    PubMed

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  9. Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia

    PubMed Central

    Shanmugam, Prakash Srinivasan Timiri; Dayton, Robert D.; Palaniyandi, Senthilnathan; Abreo, Fleurette; Caldito, Gloria; Klein, Ronald L.

    2013-01-01

    Abstract Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (1011 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection

  10. Advanced Modular Inverter Technology Development

    SciTech Connect

    Adam Szczepanek

    2006-02-04

    Electric and hybrid-electric vehicle systems require an inverter to convert the direct current (DC) output of the energy generation/storage system (engine, fuel cells, or batteries) to the alternating current (AC) that vehicle propulsion motors use. Vehicle support systems, such as lights and air conditioning, also use the inverter AC output. Distributed energy systems require an inverter to provide the high quality AC output that energy system customers demand. Today's inverters are expensive due to the cost of the power electronics components, and system designers must also tailor the inverter for individual applications. Thus, the benefits of mass production are not available, resulting in high initial procurement costs as well as high inverter maintenance and repair costs. Electricore, Inc. (www.electricore.org) a public good 501 (c) (3) not-for-profit advanced technology development consortium assembled a highly qualified team consisting of AeroVironment Inc. (www.aerovironment.com) and Delphi Automotive Systems LLC (Delphi), (www.delphi.com), as equal tiered technical leads, to develop an advanced, modular construction, inverter packaging technology that will offer a 30% cost reduction over conventional designs adding to the development of energy conversion technologies for crosscutting applications in the building, industry, transportation, and utility sectors. The proposed inverter allows for a reduction of weight and size of power electronics in the above-mentioned sectors and is scalable over the range of 15 to 500kW. The main objective of this program was to optimize existing AeroVironment inverter technology to improve power density, reliability and producibility as well as develop new topology to reduce line filter size. The newly developed inverter design will be used in automotive and distribution generation applications. In the first part of this program the high-density power stages were redesigned, optimized and fabricated. One of the main tasks

  11. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

    PubMed Central

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis

    2016-01-01

    ABSTRACT We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to

  12. Inverting an Introductory Statistics Classroom

    ERIC Educational Resources Information Center

    Kraut, Gertrud L.

    2015-01-01

    The inverted classroom allows more in-class time for inquiry-based learning and for working through more advanced problem-solving activities than does the traditional lecture class. The skills acquired in this learning environment offer benefits far beyond the statistics classroom. This paper discusses four ways that can make the inverted…

  13. Enhancement of flap survival and changes in angiogenic gene expression after AAV2-mediated VEGF gene transfer to rat ischemic flaps.

    PubMed

    Wang, Xiao Tian; Avanessian, Bella; Ma, Qiangzhong; Durfee, Heather; Tang, Yu Qing; Liu, Paul Y

    2011-01-01

    Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno-associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2-GFP group, and one saline group. AAV2-VEGF or AAV2-GFP were injected intradermally into the rat dorsum in the AAV2-VEGF or AAV2-GFP group. The saline group received saline injection. A 3 × 10 cm flap was raised in each rat two weeks post-injection. One week after surgery, flap viability was evaluated. Angiogenesis real-time PCR array was performed to analyze the expression of angiogenesis-associated genes. The AAV2-VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2-VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis-associated genes in AAV2-VEGF treated flaps. Particularly, EGF, PDGF-A and VEGF-B genes were up-regulated in these flaps. In contrast, FGF2 gene expression was down-regulated. In conclusion, AAV2-VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival. PMID:21649787

  14. 21 CFR 184.1859 - Invert sugar.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Invert sugar. 184.1859 Section 184.1859 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1859 Invert sugar. (a) Invert sugar (CAS Reg. No. 8013-17-0) is an...

  15. 21 CFR 184.1859 - Invert sugar.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Invert sugar. 184.1859 Section 184.1859 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1859 Invert sugar. (a) Invert sugar (CAS Reg. No. 8013-17-0) is an...

  16. 21 CFR 184.1859 - Invert sugar.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Invert sugar. 184.1859 Section 184.1859 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1859 Invert sugar. (a) Invert sugar (CAS Reg. No. 8013-17-0) is an...

  17. 21 CFR 184.1859 - Invert sugar.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Invert sugar. 184.1859 Section 184.1859 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1859 Invert sugar. (a) Invert sugar (CAS Reg. No. 8013-17-0) is an...

  18. Security of Invertible Media Authentication Schemes Revisited

    NASA Astrophysics Data System (ADS)

    Dönigus, Daniel; Endler, Stefan; Fischlin, Marc; Hülsing, Andreas; Jäger, Patrick; Lehmann, Anja; Podrazhansky, Sergey; Schipp, Sebastian; Tews, Erik; Vowe, Sven; Walthart, Matthias; Weidemann, Frederik

    Dittmann, Katzenbeisser, Schallhart and Veith (SEC 2005) introduced the notion of invertible media authentication schemes, embedding authentication data in media objects via invertible watermarks. These invertible watermarks allow to recover the original media object (given a secret encryption key), as required for example in some medical applications where the distortion must be removable.

  19. Improved Performance via the Inverted Classroom

    ERIC Educational Resources Information Center

    Weinstein, Randy D.

    2015-01-01

    This study examined student performance in an inverted thermodynamics course (lectures provided by video outside of class) compared to a traditional lecture class. Students in the inverted class performed better on their exams. Students in the bottom third of the inverted course showed the greatest improvement. These bottom third students had a C…

  20. Single-electron differential-amplifier/inverter/non-inverter

    NASA Astrophysics Data System (ADS)

    Hung, K.-M.; Chen, C.-S.; Lin, T.-W.

    2006-07-01

    This work presents a single-electron differential amplifier (SEDA), inverter, and non-inverter based on the triple single-dopant quantum-dot (TSDQD) configuration, with new structures. The competition between the field-induced and confinement-related shifts in the wavefunction of the quantum dots yields a field-controllable spatial-displacement single-electron transistor. Deeper impurity levels in quantum dots promise a higher operating temperature and higher on/off current ratios. The I- V characteristics of the device, studied using the transfer Hamiltonian approach (THA), show that the ratio of on/off currents is >80 000 and the voltage gain is >4 eV/Ry, where V is the applied voltage.

  1. Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

    PubMed Central

    Singer, Josef; Manzano-Szalai, Krisztina; Fazekas, Judit; Thell, Kathrin; Bentley-Lukschal, Anna; Stremnitzer, Caroline; Roth-Walter, Franziska; Weghofer, Margit; Ritter, Mirko; Pino Tossi, Kerstin; Hörer, Markus; Michaelis, Uwe; Jensen-Jarolim, Erika

    2016-01-01

    ABSTRACT Background: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients. PMID:27622022

  2. Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

    PubMed Central

    Singer, Josef; Manzano-Szalai, Krisztina; Fazekas, Judit; Thell, Kathrin; Bentley-Lukschal, Anna; Stremnitzer, Caroline; Roth-Walter, Franziska; Weghofer, Margit; Ritter, Mirko; Pino Tossi, Kerstin; Hörer, Markus; Michaelis, Uwe; Jensen-Jarolim, Erika

    2016-01-01

    ABSTRACT Background: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients.

  3. [Novel qPCR strategy for quantification of recombinant adeno-associated virus serotype 2 vector genome-titer].

    PubMed

    Meng, Qinglin; Zhang, Binbin; Zhang, Chun

    2013-02-01

    Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs' vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.

  4. Thermal Study of Inverter Components: Preprint

    SciTech Connect

    Sorensen, N. R.; Thomas, E. V.; Quintana, M. A.; Barkaszi, S.; Rosenthal, A.; Zhang, Z.; Kurtz, S.

    2012-06-01

    Thermal histories of inverter components were collected from operating inverters from several manufacturers and three locations. The data were analyzed to determine thermal profiles, the dependence on local conditions, and to assess the effect on inverter reliability. Inverter temperatures were shown to increase with the power dissipation of the inverters, follow diurnal and annual cycles, and have a dependence on wind speed. An accumulated damage model was applied to the temperature profiles and an example of using these data to predict reliability was explored.

  5. Fault-tolerant three-level inverter

    DOEpatents

    Edwards, John; Xu, Longya; Bhargava, Brij B.

    2006-12-05

    A method for driving a neutral point clamped three-level inverter is provided. In one exemplary embodiment, DC current is received at a neutral point-clamped three-level inverter. The inverter has a plurality of nodes including first, second and third output nodes. The inverter also has a plurality of switches. Faults are checked for in the inverter and predetermined switches are automatically activated responsive to a detected fault such that three-phase electrical power is provided at the output nodes.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells

    PubMed Central

    Mizutani, Takeomi; Haga, Hisashi; Kawabata, Kazushige

    2016-01-01

    This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1) locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]). To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article “Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85” [1]. PMID:26937449

  10. Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats

    PubMed Central

    Bucher, T; Dubreil, L; Colle, M-A; Maquigneau, M; Deniaud, J; Ledevin, M; Moullier, P; Joussemet, B

    2014-01-01

    Systemic and intracerebrospinal fluid delivery of adeno-associated virus serotype 9 (AAV9) has been shown to achieve widespread gene delivery to the central nervous system (CNS). However, after systemic injection, the neurotropism of the vector has been reported to vary according to age at injection, with greater neuronal transduction in newborns and preferential glial cell tropism in adults. This difference has not yet been reported after cerebrospinal fluid (CSF) delivery. The present study analyzed both neuronal and glial cell transduction in the CNS of cats according to age of AAV9 CSF injection. In both newborns and young cats, administration of AAV9-GFP in the cisterna magna resulted in high levels of motor neurons (MNs) transduction from the cervical (84±5%) to the lumbar (99±1%) spinal cord, demonstrating that the remarkable tropism of AAV9 for MNs is not affected by age at CSF delivery. Surprisingly, numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates, indicating that (i) age of CSF delivery influences the tropism of AAV9 for glial cells and (ii) AAV9 intracisternal delivery could be relevant for both the treatment of MN and demyelinating disorders. PMID:24572783

  11. The Inverted Snow Globe Shadow

    NASA Astrophysics Data System (ADS)

    Ribeiro, Jair Lúcio Prados

    2015-01-01

    Our high school optics course finishes with an assignment that students usually appreciate. They must take pictures of everyday situations representing optical phenomena such as reflection, refraction, or dispersion, and post them on Instagram.1 When the photos were presented to the class, one student revealed an intriguing photo, similar to Fig. 1, showing a snow globe exposed to sunlight and its inverted shadow. This paper offers an explanation of the problem, which occurs due to light refraction from the globe.

  12. Effective switching frequency multiplier inverter

    DOEpatents

    Su, Gui-Jia; Peng, Fang Z.

    2007-08-07

    A switching frequency multiplier inverter for low inductance machines that uses parallel connection of switches and each switch is independently controlled according to a pulse width modulation scheme. The effective switching frequency is multiplied by the number of switches connected in parallel while each individual switch operates within its limit of switching frequency. This technique can also be used for other power converters such as DC/DC, AC/DC converters.

  13. Gripper deploying and inverting linkage

    DOEpatents

    Minichan, Richard L.; Killian, Mark A.

    1993-01-01

    An end effector deploying and inverting linkage. The linkage comprises an air cylinder mounted in a frame or tube, a sliding bracket next to the air cylinder, a stopping bracket depending from the frame and three, pivotally-attached links that are attached to the end effector and to each other in such a way as to be capable of inverting the end effector and translating it laterally. The first of the three links is a straight element that is moved up and down by the shaft of the air cylinder. The second link is attached at one end to the stopping bracket and to the side of the end effector at the other end. The first link is attached near the middle of the second, sharply angled link so that, as the shaft of the air cylinder moves up and down, the second link rotates about an axis perpendicular to the frame and inverts and translates the end effector. The rotation of the second link is stopped at both ends when the link engages stops on the stopping bracket. The third link, slightly angled, is attached to the sliding bracket at one end and to the end of the end effector at the other. The third helps to control the end effector in its motion.

  14. Gripper deploying and inverting linkage

    DOEpatents

    Minichan, R.L.; Killian, M.A.

    1993-03-02

    An end effector deploying and inverting linkage. The linkage comprises an air cylinder mounted in a frame or tube, a sliding bracket next to the air cylinder, a stopping bracket depending from the frame and three, pivotally-attached links that are attached to the end effector and to each other in such a way as to be capable of inverting the end effector and translating it laterally. The first of the three links is a straight element that is moved up and down by the shaft of the air cylinder. The second link is attached at one end to the stopping bracket and to the side of the end effector at the other end. The first link is attached near the middle of the second, sharply angled link so that, as the shaft of the air cylinder moves up and down, the second link rotates about an axis perpendicular to the frame and inverts and translates the end effector. The rotation of the second link is stopped at both ends when the link engages stops on the stopping bracket. The third link, slightly angled, is attached to the sliding bracket at one end and to the end of the end effector at the other. The third helps to control the end effector in its motion.

  15. Inverted annular flow experimental study

    SciTech Connect

    De Jarlais, G.; Ishii, M.

    1985-04-01

    Steady-state inverted annular flow of Freon 113 in up flow was established in a transparent test section. Using a special inlet configuration consisting of long aspect-ratio liquid nozzles coaxially centered within a heated quartz tube, idealized inverted annular flow initial geometry (cylindrical liquid core surrounded by coaxial annulus of gas) could be established. Inlet liquid and gas flowrates, liquid subcooling, and gas density (using various gas species) were measured and varied systematically. The hydrodynamic behavior of the liquid core, and the subsequent downstream break-up of this core into slugs, ligaments and/or droplets of various sizes, was observed. In general, for low inlet liquid velocities it was observed that after the initial formation of roll waves on the liquid core surface, an agitated region of high surface area, with attendant high momentum and energy transfers, occurs. This agitated region appears to propagate downsteam in a quasi-periodic pattern. Increased inlet liquid flow rates, and high gas annulus flow rates tend to diminish the significance of this agitated region. Observed inverted annular flow (and subsequent downstream flow pattern) hydrodynamic behavior is reported, and comparisons are drawn to data generated by previous experimenters studying post-CHF flow.

  16. AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington's disease

    SciTech Connect

    Zuleta, Amparo; Vidal, Rene L.; Armentano, Donna; Parsons, Geoffrey; Hetz, Claudio

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer The contribution of ER stress to HD has not been directly addressed. Black-Right-Pointing-Pointer Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. Black-Right-Pointing-Pointer We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptation to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588{sup Q95}-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588{sup Q95}-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.

  17. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

    PubMed Central

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Cappellari, Gianluca Gortan; Baldini, Giovanna; Zweyer, Marina; Barazzoni, Rocco; Graziani, Andrea; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy. PMID:26066847

  18. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  19. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N P; Riedmayr, Lisa M; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  20. Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice

    PubMed Central

    Lai, N. Chin; Gao, Mei Hua; Giamouridis, Dimosthenis; Suarez, Jorge; Miyanohara, Atsushi; Parikh, Jay; Hightower, Stephen; Guo, Tracy; Dillmann, Wolfgang; Kim, Young-Chul; Diaz-Juarez, Julieta

    2015-01-01

    Abstract Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, is costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. We previously showed that a single intravenous (IV) injection of AAV8.UCn2 increases plasma UCn2 and left ventricular (LV) systolic and diastolic function for at least 7 months in normal mice. Here we test the hypothesis that IV delivery of AAV8.UCn2 increases function of the failing heart. Myocardial infarction (MI, by coronary ligation) was used to induce heart failure, which was assessed by echocardiography 3 weeks after MI. Mice with LV ejection fraction (EF) <25% received IV delivery of AAV8.UCn2 (5×1011 gc) or saline, and 5 weeks later echocardiography showed increased LV EF in mice that received UCn2 gene transfer (p=0.01). In vivo physiological studies showed a 2-fold increase in peak rate of LV pressure development (LV +dP/dt; p<0.0001) and a 1.6-fold increase in peak rate of LV pressure decay (LV −dP/dt; p=0.0007), indicating increased LV systolic and diastolic function in treated mice. UCn2 gene transfer was associated with increased peak systolic Ca2+ transient amplitude and rate of Ca2+ decline and increased SERCA2a expression. In addition, UCn2 gene transfer reduced Thr286 phosphorylation of Cam kinase II, and increased expression of cardiac myosin light chain kinase, findings that would be anticipated to increase function of the failing heart. We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart. The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy. PMID:25760560

  1. AAV-IL-22 modifies liver chemokine activity and ameliorates portal inflammation in murine autoimmune cholangitis.

    PubMed

    Hsueh, Yu-Hsin; Chang, Yun-Ning; Loh, Chia-En; Gershwin, M Eric; Chuang, Ya-Hui

    2016-01-01

    There remain significant obstacles in developing biologics to treat primary biliary cholangitis (PBC). Although a number of agents have been studied both in murine models and human patients, the results have been relatively disappointing. IL-22 is a member of the IL-10 family and has multiple theoretical reasons for predicting successful usage in PBC. We have taken advantage of an IL-22 expressing adeno-associated virus (AAV-IL-22) to address the potential role of IL-22 in not only protecting mice from autoimmune cholangitis, but also in treating animals with established portal inflammation. Using our established mouse model of 2-OA-OVA immunization, including α-galactosylceramide (α-GalCer) stimulation, we treated mice both before and after the onset of clinical disease with AAV-IL-22. Firstly, AAV-IL-22 treatment given prior to 2-OA-OVA and α-GalCer exposure, i.e. before the onset of disease, significantly reduces the portal inflammatory response, production of Th1 cytokines and appearance of liver fibrosis. It also reduced the liver lymphotropic chemokines CCL5, CCL19, CXCL9, and CXCL10. Secondly, and more importantly, therapeutic use of AAV-IL-22, administered after the onset of disease, achieved a greater hurdle and significantly improved portal pathology. Further the improvements in inflammation were negatively correlated with levels of CCL5 and CXCL10 and positively correlated with levels of IL-22. In conclusion, we submit that the clinical use of IL-22 has a potential role in modulating the inflammatory portal process in patients with PBC.

  2. Long-term Amelioration of Feline Mucopolysaccharidosis VI After AAV-mediated Liver Gene Transfer

    PubMed Central

    Cotugno, Gabriella; Annunziata, Patrizia; Tessitore, Alessandra; O'Malley, Thomas; Capalbo, Anita; Faella, Armida; Bartolomeo, Rosa; O'Donnell, Patricia; Wang, Ping; Russo, Fabio; Sleeper, Meg M; Knox, Van W; Fernandez, Steven; Levanduski, Leah; Hopwood, John; De Leonibus, Elvira; Haskins, Mark; Auricchio, Alberto

    2011-01-01

    Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 1013 genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 1012 gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients. PMID:21119624

  3. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    PubMed

    Senís, Elena; Fatouros, Chronis; Große, Stefanie; Wiedtke, Ellen; Niopek, Dominik; Mueller, Ann-Kristin; Börner, Kathleen; Grimm, Dirk

    2014-11-01

    Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. PMID:25186301

  4. AAV1.NT-3 Gene Therapy for Charcot–Marie–Tooth Neuropathy

    PubMed Central

    Sahenk, Zarife; Galloway, Gloria; Clark, Kelly Reed; Malik, Vinod; Rodino-Klapac, Louise R; Kaspar, Brian K.; Chen, Lei; Braganza, Cilwyn; Montgomery, Chrystal; Mendell, Jerry R

    2014-01-01

    Charcot–Marie–Tooth (CMT) neuropathies represent a heterogeneous group of peripheral nerve disorders affecting 1 in 2,500 persons. One variant, CMT1A, is a primary Schwann cell (SC) disorder, and represents the single most common variant. In previous studies, we showed that neurotrophin-3 (NT-3) improved the tremblerJ (TrJ) mouse and also showed efficacy in CMT1A patients. Long-term treatment with NT-3 was not possible related to its short half-life and lack of availability. This led to considerations of NT-3 gene therapy via adenoassociated virus (AAV) delivery to muscle, acting as secretory organ for widespread distribution of this neurotrophic agent. In the TrJ model of demyelinating CMT, rAAV1.NT-3 therapy resulted in measurable NT-3 secretion levels in blood sufficient to provide improvement in motor function, histopathology, and electrophysiology of peripheral nerves. Furthermore, we showed that the compound muscle action potential amplitude can be used as surrogate for functional improvement and established the therapeutic dose and a preferential muscle-specific promoter to achieve sustained NT-3 levels. These studies of intramuscular (i.m.) delivery of rAAV1.NT-3 serve as a template for future CMT1A clinical trials with a potential to extend treatment to other nerve diseases with impaired nerve regeneration. PMID:24162799

  5. Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease

    PubMed Central

    Bradbury, Allison M; Gray-Edwards, Heather L; Shirley, Jamie L; McCurdy, Victoria J; Colaco, Alexandria N; Randle, Ashley N; Christopherson, Pete W; Bird, Allison C; Johnson, Aime K; Wilson, Diane U; Hudson, Judith A; De Pompa, Nicholas L; Sorjonen, Donald C; Brunson, Brandon L; Jeyakumar, Mylvaganam; Platt, Frances M; Baker, Henry J; Cox, Nancy R; Sena-Esteves, Miguel; Martin, Douglas R

    2014-01-01

    The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme β-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated GM2 patients and those in future clinical trials. PMID:25284324

  6. Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease.

    PubMed

    Bradbury, Allison M; Gray-Edwards, Heather L; Shirley, Jamie L; McCurdy, Victoria J; Colaco, Alexandria N; Randle, Ashley N; Christopherson, Pete W; Bird, Allison C; Johnson, Aime K; Wilson, Diane U; Hudson, Judith A; De Pompa, Nicholas L; Sorjonen, Donald C; Brunson, Brandon L; Jeyakumar, Mylvaganam; Platt, Frances M; Baker, Henry J; Cox, Nancy R; Sena-Esteves, Miguel; Martin, Douglas R

    2015-01-01

    The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme β-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials. PMID:25284324

  7. Clinical Improvement of Alpha-mannosidosis Cat Following a Single Cisterna Magna Infusion of AAV1.

    PubMed

    Yoon, Sea Young; Bagel, Jessica H; O'Donnell, Patricia A; Vite, Charles H; Wolfe, John H

    2016-02-01

    Lysosomal storage diseases (LSDs) are debilitating neurometabolic disorders for most of which long-term effective therapies have not been developed. Gene therapy is a potential treatment but a critical barrier to treating the brain is the need for global correction. We tested the efficacy of cisterna magna infusion of adeno-associated virus type 1 (AAV1) expressing feline alpha-mannosidase gene in the postsymptomatic alpha-mannosidosis (AMD) cat, a homologue of the human disease. Lysosomal alpha-mannosidase (MANB) activity in the cerebrospinal fluid (CSF) and serum were increased above the control values in untreated AMD cats. Clinical neurological signs were delayed in onset and reduced in severity. The lifespan of the treated cats was significantly extended. Postmortem histopathology showed resolution of lysosomal storage lesions throughout the brain. MANB activity in brain tissue was significantly above the levels of untreated tissues. The results demonstrate that a single cisterna magna injection of AAV1 into the CSF can mediate widespread neuronal transduction of the brain and meaningful clinical improvement. Thus, cisterna magna gene delivery by AAV1 appears to be a viable strategy for treatment of the whole brain in AMD and should be applicable to many of the neurotropic LSDs as well as other neurogenetic disorders.

  8. Efficacy and Safety of rAAV2-ND4 Treatment for Leber's Hereditary Optic Neuropathy.

    PubMed

    Wan, Xing; Pei, Han; Zhao, Min-jian; Yang, Shuo; Hu, Wei-kun; He, Heng; Ma, Si-qi; Zhang, Ge; Dong, Xiao-yan; Chen, Chen; Wang, Dao-wen; Li, Bin

    2016-02-19

    Leber's hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness. A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit 4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety of a recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carrying the G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eye and then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and optical coherence tomography were performed. Physical examinations included routine blood and urine. The visual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months of follow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remained relatively stable. No other outcome measure was significantly changed. None of the nine patients had local or systemic adverse events related to the vector during the 9-month follow-up period. These findings support the feasible use of gene therapy for LHON.

  9. Efficacy and Safety of rAAV2-ND4 Treatment for Leber's Hereditary Optic Neuropathy.

    PubMed

    Wan, Xing; Pei, Han; Zhao, Min-jian; Yang, Shuo; Hu, Wei-kun; He, Heng; Ma, Si-qi; Zhang, Ge; Dong, Xiao-yan; Chen, Chen; Wang, Dao-wen; Li, Bin

    2016-01-01

    Leber's hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness. A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit 4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety of a recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carrying the G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eye and then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and optical coherence tomography were performed. Physical examinations included routine blood and urine. The visual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months of follow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remained relatively stable. No other outcome measure was significantly changed. None of the nine patients had local or systemic adverse events related to the vector during the 9-month follow-up period. These findings support the feasible use of gene therapy for LHON. PMID:26892229

  10. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    PubMed

    Senís, Elena; Fatouros, Chronis; Große, Stefanie; Wiedtke, Ellen; Niopek, Dominik; Mueller, Ann-Kristin; Börner, Kathleen; Grimm, Dirk

    2014-11-01

    Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.

  11. Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.

    PubMed

    Wang, Z; Halbert, C L; Lee, D; Butts, T; Tapscott, S J; Storb, R; Miller, A D

    2014-04-01

    Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

  12. Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV mediated gene transfer with a non-depleting CD4 antibody and cyclosporine

    PubMed Central

    McIntosh, Jenny; Cochrane, Melanie; Cobbold, Stephen; Waldmann, Herman; Davidoff, Andrew M.; Nathwani, Amit C.

    2012-01-01

    The ability of transient immunosuppression with a combination of a nondepleting anti-CD4 (NDCD4) antibody and Cyclosporine (CyA) to abrogate immune reactivity to both adeno-associated virus vector (AAV) and its transgene product was evaluated. This combination of immunosuppressants resulted in a 20-fold reduction in the resulting anti-AAV8 antibody titres, to levels in naïve mice, following intravenous administration of 2×1012 AAV8 vector particles/kg to immunocompetent mice. This allowed efficient transduction upon secondary challenge with vector pseudotyped with the same capsid. Persistent tolerance did not result, however, as an anti-AAV8 antibody response was elicited upon rechallenge with AAV8 without immunosuppression. The route of vector administration, vector dose, AAV serotype or the concomitant administration of adenoviral vector appeared to have little impact on the ability of the NDCD4 antibody and CyA combination to moderate the primary humoral response to AAV capsid proteins. The combination of NDCD4 and CyA also abrogated the humoral response to the transgene product, that otherwise invariably would occur, following intramuscular injection of AAV5, leading to stable transgene expression. These observations could significantly improve the prospects of using rAAV vectors for chronic disorders by allowing for repeated vector administration and avoiding the development of antibodies to the transgene product. PMID:21716299

  13. Bank Terminals

    NASA Technical Reports Server (NTRS)

    1978-01-01

    In the photo, employees of the UAB Bank, Knoxville, Tennessee, are using Teller Transaction Terminals manufactured by SCI Systems, Inc., Huntsville, Alabama, an electronics firm which has worked on a number of space projects under contract with NASA. The terminals are part of an advanced, computerized financial transaction system that offers high efficiency in bank operations. The key to the system's efficiency is a "multiplexing" technique developed for NASA's Space Shuttle. Multiplexing is simultaneous transmission of large amounts of data over a single transmission link at very high rates of speed. In the banking application, a small multiplex "data bus" interconnects all the terminals and a central computer which stores information on clients' accounts. The data bus replaces the maze-of wiring that would be needed to connect each terminal separately and it affords greater speed in recording transactions. The SCI system offers banks real-time data management through constant updating of the central computer. For example, a check is immediately cancelled at the teller's terminal and the computer is simultaneously advised of the transaction; under other methods, the check would be cancelled and the transaction recorded at the close of business. Teller checkout at the end of the day, conventionally a time-consuming matter of processing paper, can be accomplished in minutes by calling up a summary of the day's transactions. SCI manufactures other types of terminals for use in the system, such as an administrative terminal that provides an immediate printout of a client's account, and another for printing and recording savings account deposits and withdrawals. SCI systems have been installed in several banks in Tennessee, Arizona, and Oregon and additional installations are scheduled this year.

  14. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes

    PubMed Central

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B.; Wen, Xin; Harasta, Anne E.; Ramkumar, Roshini; Spencer, Ziggy H. T.; Housley, Gary D.; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  15. An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models

    PubMed Central

    Gérard, Catherine; Xiao, Xiao; Filali, Mohammed; Coulombe, Zoé; Arsenault, Marie; Couet, Jacques; Li, Juan; Drolet, Marie-Claude; Chapdelaine, Pierre; Chikh, Amina; Tremblay, Jacques P

    2014-01-01

    Friedreich ataxia (FRDA) is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN). This AAV was delivered by intraperitoneal (IP) injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK) or the neuron-specific enolase (NSE) promoter. In the first part of the study, different doses of virus were tested from 6 × 1011 v.p. to 6 × 109 v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 1011 v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 1011 v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA. PMID:26015982

  16. Microgrid and Inverter Control and Simulator Software

    2012-09-13

    A collection of software that can simulate the operation of an inverter on a microgrid or control a real inverter. In addition, it can simulate the control of multiple nodes on a microgrid." Application: Simulation of inverters and microgrids; control of inverters on microgrids." The MMI submodule is designed to control custom inverter hardware, and to simulate that hardware. The INVERTER submodule is only the simulator code, and is of an earlier generation than themore » simulator in MMI. The MICROGRID submodule is an agent-based simulator of multiple nodes on a microgrid which presents a web interface. The WIND submodule produces movies of wind data with a web interface.« less

  17. Multilevel Inverters for Electric Vehicle Applications

    SciTech Connect

    Habetler, T.G.; Peng, F.Z.; Tolbert, L.M.

    1998-10-22

    This paper presents multilevel inverters as an application for all-electric vehicle (EV) and hybrid-electric vehicle (HEV) motor drives. Diode-clamped inverters and cascaded H-bridge inverters, (1) can generate near-sinusoidal voltages with only fundamental frequency switching; (2) have almost no electromagnetic interference (EMI) and common-mode voltage; and (3) make an EV more accessible/safer and open wiring possible for most of an EV'S power system. This paper explores the benefits and discusses control schemes of the cascade inverter for use as an EV motor drive or a parallel HEV drive and the diode-clamped inverter as a series HEV motor drive. Analytical, simulated, and experimental results show the superiority of these multilevel inverters for this new niche.

  18. System and method for regulating resonant inverters

    DOEpatents

    Stevanovic, Ljubisa Dragoljub; Zane, Regan Andrew

    2007-08-28

    A technique is provided for direct digital phase control of resonant inverters based on sensing of one or more parameters of the resonant inverter. The resonant inverter control system includes a switching circuit for applying power signals to the resonant inverter and a sensor for sensing one or more parameters of the resonant inverter. The one or more parameters are representative of a phase angle. The resonant inverter control system also includes a comparator for comparing the one or more parameters to a reference value and a digital controller for determining timing of the one or more parameters and for regulating operation of the switching circuit based upon the timing of the one or more parameters.

  19. Inverted ductal papilloma of minor salivary glands.

    PubMed

    Hegarty, D J; Hopper, C; Speight, P M

    1994-08-01

    Inverted ductal papillomas are rare tumours of minor salivary glands. A case is reported on the lower lip of a 50-year-old man. The tumour showed a characteristic endophytic growth pattern and was composed of bulbous papillary projections of basaloid cells dilating and filling the superficial portion of the excretory duct. Histologically, inverted ductal papillomas resemble the inverted papilloma of the nose and paranasal sinuses, but are completely benign and are not associated with malignant change.

  20. Cascade Multilevel Inverters for Utility Applications

    SciTech Connect

    Peng, F.Z., McKeever, J.W., Adams, D.J.

    1997-12-31

    Cascade multilevel inverters have been developed by the authors for utility applications. A cascade M-level inverter consists of (M-1)/2 H- bridges in which each bridge has its own separate dc source. The new inverter: (1) can generate almost sinusoidal waveform voltage while only switching one time per fundamental cycle, (2) can eliminate transformers of multipulse inverters used in conventional utility interfaces and static var compensators, and (3) makes possible direct parallel or series connection to medium- and high-voltage power systems without any transformers. In other words, the cascade inverter is much more efficient and suitable for utility applications than traditional multipulse and pulse width modulation (PWM) inverters. The authors have experimentally demonstrated the superiority of the new inverter for reactive power (var) and harmonic compensation. This paper will summarize features,feasibility, and control schemes of the cascade inverter for utility applications including utility interface of renewable energy, voltage regulation, var compensation, and harmonic filtering in power systems.Analytical, simulated, and experimental results demonstrate the superiority of the new inverters.

  1. Temporal acceleration of the human papillomavirus life cycle by adeno-associated virus (AAV) type 2 superinfection in natural host tissue.

    PubMed

    Agrawal, Nalini; Mane, Michael; Chiriva-Internati, Maurizio; Roman, Juan J; Hermonat, Paul L

    2002-06-01

    Epidemiologically, certain human papillomaviruses are positively associated with cervical cancer, while adeno-associated viruses (AAV-2) are negatively associated with this same cancer. Both HPV and AAV productively replicate in differentiating keratinocytes of the skin and interact with each other. However, AAV has a relatively fast life cycle, generating infectious progeny by the third to fourth day of an organotypic epithelial raft culture. In contrast, HPV is slow, generating infectious progeny only after 10-12 days. As earlier studies indicated that these two skin-tropic virus types significantly affect each other's life cycle, we investigated if the temporal kinetics of the slow HPV life cycle was affected by the fast AAV in raft cultures. Here it is shown that the presence of AAV-2 at a variety of multiplicities of infection (m.o.i.) resulted in early onset HPV-31b DNA replication. Using plasmids which each expressed only one of the four rep proteins, an enhancement affect was seen for all four rep proteins of AAV, with Rep40 having the highest activity. Furthermore, AAV (m.o.i. of 5) also resulted in a temporally accelerated production of HPV infectious units, seen as early as Day 4, with high levels of viral progeny being produced by Day 6.5. Like earlier studies at Day 12, histological differences were seen at Day 6.5 between AAV-infected and mock-infected HPV/rafts. These data suggest that under specific conditions the AAV rep trans-factors can positively regulate HPV gene expression in addition to the usual negative regulation that has been consistently observed by the rep proteins. These data also suggest that AAV has a significant effect upon the temporal kinetics of the HPV life cycle in natural host tissue. However, it is unclear if or how this AAV-induced fast HPV life cycle mechanistically correlates with lower rates of HPV-associated cervical disease.

  2. Patterns of scAAV vector insertion associated with oncogenic events in a mouse model for genotoxicity.

    PubMed

    Rosas, Lucia E; Grieves, Jessica L; Zaraspe, Kimberly; La Perle, Krista Md; Fu, Haiyan; McCarty, Douglas M

    2012-11-01

    Recombinant adeno-associated virus (rAAV) vectors have gained an extensive record of safety and efficacy in animal models of human disease. Infrequent reports of genotoxicity have been limited to specific vectors associated with excess hepatocellular carcinomas (HCC) in mice. In order to understand potential mechanisms of genotoxicity, and identify patterns of insertion that could promote tumor formation, we compared a self-complementary AAV (scAAV) vector designed to promote insertional activation (scAAV-CBA-null) to a conventional scAAV-CMV-GFP vector. HCC-prone C3H/HeJ mice and severe combined immunodeficiency (SCID) mice were infected with vector plus secondary treatments including partial hepatectomy (HPX) and camptothecin (CPT) to determine the effects of cell cycling and DNA damage on tumor incidence. Infection with either vector led to a significant increase in HCC incidence in male C3H/HeJ mice. Partial HPX after infection reduced HCC incidence in the cytomegalovirus-green fluorescent protein (CMV-GFP)-infected mice, but not in the cognate chicken β-actin (CBA)-null infected group. Tumors from CBA-null infected, hepatectomized mice were more likely to contain significant levels of vector DNA than tumors from the corresponding CMV-GFP-infected group. Most CBA-null vector insertions recovered from tumors were associated with known proto-oncogenes or tumor suppressors. Specific patterns of insertion suggested read-through transcription, enhancer effects, and disruption of tumor suppressors as likely mechanisms for genotoxicity.

  3. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy

    PubMed Central

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  4. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy.

    PubMed

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  5. Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

    PubMed

    Mattar, Citra N; Wong, Andrew M S; Hoefer, Klemens; Alonso-Ferrero, Maria E; Buckley, Suzanne M K; Howe, Steven J; Cooper, Jonathan D; Waddington, Simon N; Chan, Jerry K Y; Rahim, Ahad A

    2015-09-01

    Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols. PMID:26062602

  6. Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis

    PubMed Central

    Schievenbusch, Stephanie; Strack, Ingo; Scheffler, Melanie; Nischt, Roswitha; Coutelle, Oliver; Hösel, Marianna; Hallek, Michael; Fries, Jochen WU; Dienes, Hans-Peter; Odenthal, Margarete; Büning, Hildegard

    2010-01-01

    In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1α1 (Col1A1), platelet-derived growth factor receptor-β (PDGFR-β), and α-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis. PMID:20424598

  7. AAV-based Neonatal Gene Therapy for Hemophilia A: Long-Term Correction and Avoidance of Immune Responses in Mice

    PubMed Central

    Hu, Chuhong; Lipshutz, Gerald S.

    2012-01-01

    Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors to the factor VIII (FVIII) protein; these ‘inhibitors’ more commonly effect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype thereby avoiding long-term consequences. Serotype rh10 AAV was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant AAV vector delivery. Coinjection of virions of each FVIII chain intravenously to mice on the second day of life was performed. Mice express sustained FVIII antigen levels of ≥5% to 22 months of life without the development of antibodies to FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development to FVIII in this disease model where AAV is administered shortly after birth. These studies support consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period. PMID:22241178

  8. Terminal structure

    DOEpatents

    Schmidt, Frank; Allais, Arnaud; Mirebeau, Pierre; Ganhungu, Francois; Lallouet, Nicolas

    2009-10-20

    A terminal structure (2) for a superconducting cable (1) is described. It consists of a conductor (2a) and an insulator (2b) that surrounds the conductor (2a), wherein the superconducting cable (1) has a core with a superconducting conductor (5) and a layer of insulation that surrounds the conductor (5), and wherein the core is arranged in such a way that it can move longitudinally in a cryostat. The conductor (2a) of the terminal structure (2) is electrically connected with the superconducting conductor (5) or with a normal conductor (6) that is connected with the superconducting conductor (5) by means of a tubular part (7) made of an electrically conductive material, wherein the superconducting conductor (5) or the normal conductor (6) can slide in the part (7) in the direction of the superconductor.

  9. Inverted File Compression through Document Identifier Reassignment.

    ERIC Educational Resources Information Center

    Shieh, Wann-Yun; Chen, Tien-Fu; Shann, Jean Jyh-Jiun; Chung, Chung-Ping

    2003-01-01

    Discusses the use of inverted files in information retrieval systems and proposes a document identifier reassignment method to reduce the average gap values in an inverted file. Highlights include the d-gap technique; document similarity; heuristic algorithms; file compression; and performance evaluation from a simulation environment. (LRW)

  10. Circuit controls transients in SCR inverters

    NASA Technical Reports Server (NTRS)

    Moore, E. T.; Wilson, T. G.

    1964-01-01

    Elimination of starting difficulties in SCR inverters is accomplished by the addition of two taps of the output winding of the inverter. On starting or under transient loads, the two additional taps deliver power through diodes without requiring quenching of SCR currents in excess of normal starting load.

  11. Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

    PubMed Central

    Leung, Alice; Katz, Anna B.; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

    2012-01-01

    Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis. PMID:22937069

  12. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  13. Termination unit

    DOEpatents

    Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

    2016-05-03

    Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

  14. Biodistribution of AAV8 Vectors Expressing Human Low-Density Lipoprotein Receptor in a Mouse Model of Homozygous Familial Hypercholesterolemia

    PubMed Central

    Chen, Shu-Jen; Sanmiguel, Julio; Lock, Martin; McMenamin, Deirdre; Draper, Christine; Limberis, Maria P.; Kassim, Sadik H.; Somanathan, Suryanarayan; Bell, Peter; Johnston, Julie C.; Rader, Daniel J.

    2013-01-01

    Abstract Recombinant adeno-associated viral vectors based on serotype 8 (AAV8) transduce liver with superior tropism following intravenous (IV) administration. Previous studies conducted by our lab demonstrated that AAV8-mediated transfer of the human low-density lipoprotein receptor (LDLR) gene driven by a strong liver-specific promoter (thyroxin-binding globulin [TBG]) leads to high level and persistent gene expression in the liver. The approach proved efficacious in reducing plasma cholesterol levels and resulted in the regression of atherosclerotic lesions in a murine model of homozygous familial hypercholesterolemia (hoFH). Prior to advancing this vector, called AAV8.TBG.hLDLR, to the clinic, we set out to investigate vector biodistribution in an hoFH mouse model following IV vector administration to assess the safety profile of this investigational agent. Although AAV genomes were present in all organs at all time points tested (up to 180 days), vector genomes were sequestered mainly in the liver, which contained levels of vector 3 logs higher than that found in other organs. In both sexes, the level of AAV genomes gradually declined and appeared to stabilize 90 days post vector administration in most organs although vector genomes remained high in liver. Vector loads in the circulating blood were high and close to those in liver at the early time point (day 3) but rapidly decreased to a level close to the limit of quantification of the assay. The results of this vector biodistribution study further support a proposed clinical trial to evaluate AAV8 gene therapy for hoFH patients. PMID:24070336

  15. Biodistribution of AAV8 vectors expressing human low-density lipoprotein receptor in a mouse model of homozygous familial hypercholesterolemia.

    PubMed

    Chen, Shu-Jen; Sanmiguel, Julio; Lock, Martin; McMenamin, Deirdre; Draper, Christine; Limberis, Maria P; Kassim, Sadik H; Somanathan, Suryanarayan; Bell, Peter; Johnston, Julie C; Rader, Daniel J; Wilson, James M

    2013-12-01

    Recombinant adeno-associated viral vectors based on serotype 8 (AAV8) transduce liver with superior tropism following intravenous (IV) administration. Previous studies conducted by our lab demonstrated that AAV8-mediated transfer of the human low-density lipoprotein receptor (LDLR) gene driven by a strong liver-specific promoter (thyroxin-binding globulin [TBG]) leads to high level and persistent gene expression in the liver. The approach proved efficacious in reducing plasma cholesterol levels and resulted in the regression of atherosclerotic lesions in a murine model of homozygous familial hypercholesterolemia (hoFH). Prior to advancing this vector, called AAV8.TBG.hLDLR, to the clinic, we set out to investigate vector biodistribution in an hoFH mouse model following IV vector administration to assess the safety profile of this investigational agent. Although AAV genomes were present in all organs at all time points tested (up to 180 days), vector genomes were sequestered mainly in the liver, which contained levels of vector 3 logs higher than that found in other organs. In both sexes, the level of AAV genomes gradually declined and appeared to stabilize 90 days post vector administration in most organs although vector genomes remained high in liver. Vector loads in the circulating blood were high and close to those in liver at the early time point (day 3) but rapidly decreased to a level close to the limit of quantification of the assay. The results of this vector biodistribution study further support a proposed clinical trial to evaluate AAV8 gene therapy for hoFH patients. PMID:24070336

  16. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization

    PubMed Central

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 108 viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

  17. An AAV vector-mediated gene delivery approach facilitates reconstitution of functional human CD8+ T cells in mice.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Wilson, James M; Tsuji, Moriya

    2014-01-01

    In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8(+) T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγ(null) (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45(+) cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8(+) T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8(+) T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting. PMID:24516613

  18. Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals

    PubMed Central

    Gompf, Heinrich S.; Budygin, Evgeny A.; Fuller, Patrick M.; Bass, Caroline E.

    2015-01-01

    Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from

  19. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

    PubMed

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

  20. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization

    PubMed Central

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 108 viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

  1. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

    PubMed

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

  2. Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.

    PubMed

    Gompf, Heinrich S; Budygin, Evgeny A; Fuller, Patrick M; Bass, Caroline E

    2015-01-01

    Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from

  3. AAV-Mediated Cone Rescue in a Naturally Occurring Mouse Model of CNGA3-Achromatopsia

    PubMed Central

    Dai, Xufeng; Lei, Bo; Everhart, Drew; Umino, Yumiko; Li, Jie; Zhang, Keqing; Mao, Song; Boye, Sanford L.; Liu, Li; Chiodo, Vince A.; Liu, Xuan; Shi, Wei; Tao, Ye; Chang, Bo; Hauswirth, William W.

    2012-01-01

    Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia. PMID:22509403

  4. AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia.

    PubMed

    Pang, Ji-jing; Deng, Wen-Tao; Dai, Xufeng; Lei, Bo; Everhart, Drew; Umino, Yumiko; Li, Jie; Zhang, Keqing; Mao, Song; Boye, Sanford L; Liu, Li; Chiodo, Vince A; Liu, Xuan; Shi, Wei; Tao, Ye; Chang, Bo; Hauswirth, William W

    2012-01-01

    Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia. PMID:22509403

  5. Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

    PubMed Central

    Sallach, Jessica; Di Pasquale, Giovanni; Larcher, Fernando; Niehoff, Nadine; Rübsam, Matthias; Huber, Anke; Chiorini, Jay; Almarza, David; Eming, Sabine A; Ulus, Hikmet; Nishimura, Stephen; Hacker, Ulrich T; Hallek, Michael; Niessen, Carien M; Büning, Hildegard

    2014-01-01

    Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy. PMID:24468915

  6. Global brain delivery of neprilysin gene by intravascular administration of AAV vector in mice

    PubMed Central

    Iwata, Nobuhisa; Sekiguchi, Misaki; Hattori, Yoshino; Takahashi, Akane; Asai, Masashi; Ji, Bin; Higuchi, Makoto; Staufenbiel, Matthias; Muramatsu, Shin-ichi; Saido, Takaomi C.

    2013-01-01

    Accumulation of amyloid-β peptide (Aβ) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aβ in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced Aβ oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding Aβ oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary. PMID:23503602

  7. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice

    PubMed Central

    Heckmann, M B; Bauer, R; Jungmann, A; Winter, L; Rapti, K; Strucksberg, K-H; Clemen, C S; Li, Z; Schröder, R; Katus, H A; Müller, O J

    2016-01-01

    Mutations of the human desmin (DES) gene cause autosomal dominant and recessive myopathies affecting skeletal and cardiac muscle tissue. Desmin knockout mice (DES-KO), which develop progressive myopathy and cardiomyopathy, mirror rare human recessive desminopathies in which mutations on both DES alleles lead to a complete ablation of desmin protein expression. Here, we investigated whether an adeno-associated virus-mediated gene transfer of wild-type desmin cDNA (AAV-DES) attenuates cardiomyopathy in these mice. Our approach leads to a partial reconstitution of desmin protein expression and the de novo formation of the extrasarcomeric desmin–syncoilin network in cardiomyocytes of treated animals. This finding was accompanied by reduced fibrosis and heart weights and improved systolic left-ventricular function when compared with control vector-treated DES-KO mice. Since the re-expression of desmin protein in cardiomyocytes of DES-KO mice restores the extrasarcomeric desmin–syncoilin cytoskeleton, attenuates the degree of cardiac hypertrophy and fibrosis, and improves contractile function, AAV-mediated desmin gene transfer may be a novel and promising therapeutic approach for patients with cardiomyopathy due to the complete lack of desmin protein expression. PMID:27101257

  8. Switching Characteristics of Ferroelectric Transistor Inverters

    NASA Technical Reports Server (NTRS)

    Laws, Crystal; Mitchell, Coey; MacLeod, Todd C.; Ho, Fat D.

    2010-01-01

    This paper presents the switching characteristics of an inverter circuit using a ferroelectric field effect transistor, FeFET. The propagation delay time characteristics, phl and plh are presented along with the output voltage rise and fall times, rise and fall. The propagation delay is the time-delay between the V50% transitions of the input and output voltages. The rise and fall times are the times required for the output voltages to transition between the voltage levels V10% and V90%. Comparisons are made between the MOSFET inverter and the ferroelectric transistor inverter.

  9. Static Characteristics of the Ferroelectric Transistor Inverter

    NASA Technical Reports Server (NTRS)

    Mitchell, Cody; Laws, crystal; MacLeond, Todd C.; Ho, Fat D.

    2010-01-01

    The inverter is one of the most fundamental building blocks of digital logic, and it can be used as the foundation for understanding more complex logic gates and circuits. This paper presents the characteristics of an inverter circuit using a ferroelectric field-effect transistor. The voltage transfer characteristics are analyzed with respect to varying parameters such as supply voltage, input voltage, and load resistance. The effects of the ferroelectric layer between the gate and semiconductor are examined, and comparisons are made between the inverters using ferroelectric transistors and those using traditional MOSFETs.

  10. Inverter Output Filter Effect on PWM Motor Drives of a Flywheel Energy Storage System

    NASA Technical Reports Server (NTRS)

    Santiago, Walter

    2004-01-01

    NASA Glenn Research Center (GRC) has been involved in the research and development of high speed flywheel systems for small satellite energy storage and attitude control applications. One research and development area has been the minimization of the switching noise produced by the pulsed width modulated (PWM) inverter that drives the flywheel permanent magnet motor/generator (PM M/G). This noise can interfere with the flywheel M/G hardware and the system avionics hampering the full speed performance of the flywheel system. One way to attenuate the inverter switching noise is by placing an AC filter at the three phase output terminals of the inverter with the filter neutral point connected to the DC link (DC bus) midpoint capacitors. The main benefit of using an AC filter in this fashion is the significant reduction of the inverter s high dv/dt switching and its harmonics components. Additionally, common mode (CM) and differential mode (DM) voltages caused by the inverter s high dv/dt switching are also reduced. Several topologies of AC filters have been implemented and compared. One AC filter topology consists of a two-stage R-L-C low pass filter. The other topology consists of the same two-stage R-L-C low pass filter with a series connected trap filter (an inductor and capacitor connected in parallel). This paper presents the analysis, design and experimental results of these AC filter topologies and the comparison between the no filter case and conventional AC filter.

  11. Expression of Human α1-Antitrypsin in Mice and Dogs Following AAV6 Vector-mediated Gene Transfer to the Lungs

    PubMed Central

    Halbert, Christine L; Madtes, David K; Vaughan, Andrew E; Wang, Zejing; Storb, Rainer; Tapscott, Stephen J; Miller, A Dusty

    2010-01-01

    We evaluated the potential of lung-directed gene therapy for α1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans. PMID:20372105

  12. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

    PubMed Central

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  13. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. PMID:26443873

  14. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy.

  15. Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    PubMed Central

    Tomono, Taro; Hirai, Yukihiko; Okada, Hironori; Adachi, Kumi; Ishii, Akiko; Shimada, Takashi; Onodera, Masafumi; Tamaoka, Akira; Okada, Takashi

    2016-01-01

    Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. PMID:26913289

  16. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.

    PubMed

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  17. The trans-inhibitory Rep78 protein of adeno-associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat.

    PubMed

    Batchu, R B; Hermonat, P L

    1995-07-01

    The large rep gene products, Rep78 and Rep68, of adeno-associated virus (AAV) are pleiotropic effector proteins which are required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, these rep products are able to inhibit the replication and gene expression of human immunodeficiency virus type 1 (HIV-1) and a number of DNA viruses. Here, it is demonstrated that Rep78, as a chimeric with the maltose binding protein, directly binds the full-length HIV-1 long terminal repeat (LTR), and to a subset of these sequences containing the trans-activation response (TAR) sequence as DNA. These interactions, an effector protein physically binding a target promoter, suggest a direct mechanism of action for Rep78 inhibition. Furthermore, competitive binding studies between the TAR region and the full-length HIV-LTR, strongly suggested that another site(s) within the LTR was also bound by Rep78. Finally, as Rep78 binding is also believed to be affected by secondary structure within the DNA, it was found that Rep78 preferentially binds with HIV-LTR sequences with promoted secondary structure generated by heat denaturation and rapid cooling.

  18. The Skeletal Muscle Environment and Its Role in Immunity and Tolerance to AAV Vector-Mediated Gene Transfer.

    PubMed

    Boisgerault, Florence; Mingozzi, Federico

    2015-01-01

    Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors.

  19. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi

    PubMed Central

    Kaulich, Manuel; Lee, Yeon J.; Lönn, Peter; Springer, Aaron D.; Meade, Bryan R.; Dowdy, Steven F.

    2015-01-01

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. PMID:25586224

  20. AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

    PubMed Central

    Deng, Wen-Tao; Dyka, Frank M.; Min, Seok-Hong; Boye, Sanford L.; Chiodo, Vince A.; Abrahan, Carolina E.; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W. Clay; Hauswirth, William W.

    2016-01-01

    Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder. PMID:26881841

  1. The Skeletal Muscle Environment and Its Role in Immunity and Tolerance to AAV Vector-Mediated Gene Transfer

    PubMed Central

    Boisgérault, Florence; Mingozzi, Federico

    2015-01-01

    Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors. PMID:26122097

  2. Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain

    PubMed Central

    Alves, Sandro; Bode, Julia; Bemelmans, Alexis-Pierre; von Kalle, Christof; Cartier, Nathalie; Tews, Björn

    2016-01-01

    Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer’s disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain. PMID:27320056

  3. In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis.

    PubMed

    Rezvani, Milad; Español-Suñer, Regina; Malato, Yann; Dumont, Laure; Grimm, Andrew A; Kienle, Eike; Bindman, Julia G; Wiedtke, Ellen; Hsu, Bernadette Y; Naqvi, Syed J; Schwabe, Robert F; Corvera, Carlos U; Grimm, Dirk; Willenbring, Holger

    2016-06-01

    Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis. PMID:27257763

  4. Integrated Inverter For Driving Multiple Electric Machines

    DOEpatents

    Su, Gui-Jia [Knoxville, TN; Hsu, John S [Oak Ridge, TN

    2006-04-04

    An electric machine drive (50) has a plurality of inverters (50a, 50b) for controlling respective electric machines (57, 62), which may include a three-phase main traction machine (57) and two-phase accessory machines (62) in a hybrid or electric vehicle. The drive (50) has a common control section (53, 54) for controlling the plurality of inverters (50a, 50b) with only one microelectronic processor (54) for controlling the plurality of inverters (50a, 50b), only one gate driver circuit (53) for controlling conduction of semiconductor switches (S1-S10) in the plurality of inverters (50a, 50b), and also includes a common dc bus (70), a common dc bus filtering capacitor (C1) and a common dc bus voltage sensor (67). The electric machines (57, 62) may be synchronous machines, induction machines, or PM machines and may be operated in a motoring mode or a generating mode.

  5. Inverted organic electronic and optoelectronic devices

    NASA Astrophysics Data System (ADS)

    Small, Cephas E.

    The research and development of organic electronics for commercial application has received much attention due to the unique properties of organic semiconductors and the potential for low-cost high-throughput manufacturing. For improved large-scale processing compatibility and enhanced device stability, an inverted geometry has been employed for devices such as organic light emitting diodes and organic photovoltaic cells. These improvements are attributed to the added flexibility to incorporate more air-stable materials into the inverted device geometry. However, early work on organic electronic devices with an inverted geometry typically showed reduced device performance compared to devices with a conventional structure. In the case of organic light emitting diodes, inverted devices typically show high operating voltages due to insufficient carrier injection. Here, a method for enhancing hole injection in inverted organic electronic devices is presented. By incorporating an electron accepting interlayer into the inverted device, a substantial enhancement in hole injection efficiency was observed as compared to conventional devices. Through a detailed carrier injection study, it is determined that the injection efficiency enhancements in the inverted devices are due to enhanced charge transfer at the electron acceptor/organic semiconductor interface. A similar situation is observed for organic photovoltaic cells, in which devices with an inverted geometry show limited carrier extraction in early studies. In this work, enhanced carrier extraction is demonstrated for inverted polymer solar cells using a surface-modified ZnO-polymer composite electron-transporting layer. The insulating polymer in the composite layer inhibited aggregation of the ZnO nanoparticles, while the surface-modification of the composite interlayer improved the electronic coupling with the photoactive layer. As a result, inverted polymer solar cells with power conversion efficiencies of over 8

  6. Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

    PubMed

    Karumuthil-Melethil, Subha; Marshall, Michael S; Heindel, Clifford; Jakubauskas, Benas; Bongarzone, Ernesto R; Gray, Steven J

    2016-11-01

    Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc. PMID:27638599

  7. Work Station For Inverting Solar Cells

    NASA Technical Reports Server (NTRS)

    Feder, H.; Frasch, W.

    1982-01-01

    Final work station along walking-beam conveyor of solar-array assembly line turns each pretabbed solar cell over, depositing it back-side-up onto landing pad, which centers cell without engaging collector surface. Solar cell arrives at inverting work station collector-side-up with two interconnect tabs attached to collector side. Cells are inverted so that second soldering operation takes place in plain view of operator. Inversion protects collector from damage when handled at later stages of assembly.

  8. Unstable high molecular weight inverted repetitive DNA in human lymphocytes.

    PubMed Central

    Rogers, J C; Rucinsky, T E

    1982-01-01

    About 1% of newly synthesized DNA from PHA-stimulated human lymphocytes can be isolated as large (up to 90 kilobase pairs) double stranded fragments that resist sequential alkali and heat denaturation steps but are not closed circular. By electron microscopy about 1% have single-strand hairpin loops at one end and therefore present inverted repetitive sequences (IR-DNA). Most of the remainder have a blunt-appearing double-strand terminus at both ends (78%) or one end (18%). Indirect evidence indicates that these also are inverted complementary structures with terminal hairpin loops too small to be visualized: (1) Treatment with either a 5' or 3' single-strand exonuclease generates essentially only fragments with a single strand at one end; (2) with partial denaturation, the number of fragments with identifiable single-strand hairpin loops increases (to about 20%); (3) after S1 nuclease digestion, greater than 95% can be fully heat denatured. Cot analysis indicates that these fragments are derived from dispersed sites throughout the genome. Up to 25% of DNA released from lymphocytes during growth similarly resists denaturation, and released DNA and IR-DNA are both enriched in the same set of repetitive sequences. Thus at least a portion of IR-DNA appears to be unstable. Images PMID:7145706

  9. Invertible authentication for 3D meshes

    NASA Astrophysics Data System (ADS)

    Dittmann, Jana; Benedens, Oliver

    2003-06-01

    Digital watermarking has become an accepted technology for enabling multimedia protection schemes. Based on the introduced media independent protocol schemes for invertible data authentication in references 2, 4 and 5 we discuss the design of a new 3D invertible labeling technique to ensure and require high data integrity. We combine digital signature schemes and digital watermarking to provide a public verifiable integrity. Furthermore the protocol steps in the other papers to ensure that the original data can only be reproduced with a secret key is adopted for 3D meshes. The goal is to show how the existing protocol can be used for 3D meshes to provide solutions for authentication watermarking. In our design concept and evaluation we see that due to the nature of 3D meshes the invertible function are different from the image and audio concepts to achieve invertibility to guaranty reversibility of the original. Therefore we introduce a concept for distortion free invertibility and a concept for adjustable minimum distortion invertibility.

  10. Dynamic Inverted Quadtree: A Structure for Pictorial Databases.

    ERIC Educational Resources Information Center

    Vassilakopoulos, Michael; Manolopoulos, Yannis

    1995-01-01

    Presents an improved inverted region quadtree variation called Dynamic Inverted Quadtree that is able to support and index a pictorial database. Pattern-searching algorithms are described, and the space needs of Fully Inverted and Dynamic Inverted Quadtrees are analytically compared based on a model of image randomness expressed as a branching…

  11. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy.

    PubMed

    Zhang, Yadong; Yue, Yongping; Li, Liang; Hakim, Chady H; Zhang, Keqing; Thomas, Gail D; Duan, Dongsheng

    2013-09-15

    Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restore nNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia and injury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectors that expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9 and co-injected (5 × 10(12) viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-null mice. Four months later, we observed 30-50% mini-dystrophin positive myofibers in limb muscles. Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle force drop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in contracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-associated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors is warranted in dystrophic dogs and eventually in human patients.

  12. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy

    PubMed Central

    Zhang, Yadong; Yue, Yongping; Li, Liang; Hakim, Chady H.; Zhang, Keqing; Thomas, Gail D.; Duan, Dongsheng

    2013-01-01

    Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restore nNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia and injury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectors that expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9 and co-injected (5 × 1012 viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-null mice. Four months later, we observed 30–50% mini-dystrophin positive myofibers in limb muscles. Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle force drop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in contracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-associated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors is warranted in dystrophic dogs and eventually in human patients. PMID:23681067

  13. Termination unit

    DOEpatents

    Traeholt, Chresten [Frederiksberg, DK; Willen, Dag [Klagshamn, SE; Roden, Mark [Newnan, GA; Tolbert, Jerry C [Carrollton, GA; Lindsay, David [Carrollton, GA; Fisher, Paul W [Heiskell, TN; Nielsen, Carsten Thidemann [Jaegerspris, DK

    2014-01-07

    This invention relates to a termination unit comprising an end-section of a cable. The end section of the cable defines a central longitudinal axis and comprising end-parts of N electrical phases, an end-part of a neutral conductor and a surrounding thermally insulation envelope adapted to comprising a cooling fluid. The end-parts of the N electrical phases and the end-part of the neutral conductor each comprising at least one electrical conductor and being arranged in the cable concentrically around a core former with a phase 1 located relatively innermost, and phase N relatively outermost in the cable, phase N being surrounded by the neutral conductor, electrical insulation being arrange between neighboring electrical phases and between phase N and the neutral conductor, and wherein the end-parts of the neutral conductor and the electrical phases each comprise a contacting surface electrically connected to at least one branch current lead to provide an electrical connection: The contacting surfaces each having a longitudinal extension, and being located sequentially along the longitudinal extension of the end-section of the cable. The branch current leads being individually insulated from said thermally insulation envelope by individual electrical insulators.

  14. Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors.

    PubMed

    Chen, Sifeng; Kapturczak, Matthias; Loiler, Scott A; Zolotukhin, Sergei; Glushakova, Olena Y; Madsen, Kirsten M; Samulski, Richard J; Hauswirth, William W; Campbell-Thompson, Martha; Berns, Kenneth I; Flotte, Terence R; Atkinson, Mark A; Tisher, C Craig; Agarwal, Anupam

    2005-02-01

    Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.

  15. Flexible, AAV-equipped Genetic Modules for Inducible Control of Gene Expression in Mammalian Brain

    PubMed Central

    Dogbevia, Godwin K; Roβmanith, Martin; Sprengel, Rolf; Hasan, Mazahir T

    2016-01-01

    Controlling gene expression in mammalian brain is of utmost importance to causally link the role of gene function to cell circuit dynamics under normal conditions and disease states. We have developed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches for inducible and reversible control of gene expression in a cell type specific and brain subregion selective manner. Here, we characterize a two-virus approach to efficiently and reliably switch gene expression on and off, repetitively, both in vitro and in vivo. Our recombinant adeno-associated virus (rAAV)-Tet approach is highly flexible and it has great potential for application in basic and biomedical neuroscience research and gene therapy. PMID:27070301

  16. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer

    PubMed Central

    Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia; Chen, Shan; Zeng, Yong; Marangoni, Dario; Pope, Jodie G.; Bush, Ronald A.; Wu, Zhijian; Li, Wei; Sieving, Paul A.

    2015-01-01

    Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor–depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1–signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology. PMID:26098217

  17. EMPLACEMENT DRIFT INVERT-LOW STEEL EVALUATION

    SciTech Connect

    M. E. Taylor and D. H. Tang

    2000-09-29

    This technical report evaluates and develops options for reducing the amount of steel in the emplacement drift invert. Concepts developed in the ''Invert Configuration and Drip Shield Interface'' were evaluated to determine material properties required for the proposed invert concepts. Project requirements documents prescribe the use of a carbon steel frame for the invert with a granular material of crushed tuff as ballast. The ''Invert Configuration and Drip Shield Interface'' developed three concepts: (1) All-Ballast Invert; (2) Modified Steel Invert with Ballast; and (3) Steel Tie with Ballast Invert. Analysis of the steel frame members, runway beams, and guide beams, for the modified steel invert with ballast, decreased the quantity of steel in the emplacement drift invert, however a substantial steel support frame for the gantry and waste package/pallet assembly is still required. Use of one of the other two concepts appears to be an alternative to the steel frame and each of the concepts uses considerably less steel materials. Analysis of the steel tie with ballast invert shows that the bearing pressure on the ballast under the single steel tie, C 9 x 20, loaded with the waste package/pallet assembly, drip shield, and backfill exceeds the upper bound of the allowable bearing capacity for tuff used in this study. The single tie, C 10 x 20, will also fail for the same loading condition except for the tie length of 4.2 meters and longer. Analysis also shows that with two ties, C 9 or 10 x 20's, the average ballast pressure is less than the allowable bearing capacity. Distributing the waste package/pallet, drip shield, and backfill loads to two steel ties reduces the contact bearing pressure. Modifying the emplacement pallet end beams to a greater width, reducing the tie spacing, and increasing the width of the ties would ensure that the pallet beams are always supported by two steel ties. Further analysis is required to determine compatible tie size and spacing

  18. Inverting sets and the packing problem

    SciTech Connect

    Faber, V.; Goldberg, M.K.; Knill, E.; Spencer, T.H.

    1992-12-01

    Given a set V, a subset S, and a permutation {pi} of V, we say that {pi} permutes S if {pi}(S) {intersection} S = {theta}. Given a collection S = (V; S{sub 1}..., S{sub m}), where S{sub i} {improper_subset} V (i = 1,...,m), we say that S is invertible if there is a permutation {pi} of V such that {pi}(S{sub i}) {improper_subset} V -- S{sub i}. In this paper, we present necessary and sufficient conditions for the invertibility of a collection and construct a polynomial algorithm which determines whether a given collection is invertible. For an arbitrary collection, we give a lower bound for the maximum number of sets that can be inverted. Finally, we consider the problem of constructing a collection of sets such that no sub-collection of size three is invertible. Our constructions of such collections come from solutions to the packing problem with unbounded block sizes. We prove several new lower and upper bounds for the packing problem and present a new explicit construction of packing.

  19. Inverting sets and the packing problem

    SciTech Connect

    Faber, V. ); Goldberg, M.K. . Dept. of Computer Science); Knill, E. . School of Computer Science); Spencer, T.H. . Dept. of Mathematics and Computer Science)

    1992-01-01

    Given a set V, a subset S, and a permutation [pi] of V, we say that [pi] permutes S if [pi](S) [intersection] S = [theta]. Given a collection S = (V; S[sub 1]..., S[sub m]), where S[sub i] [improper subset] V (i = 1,...,m), we say that S is invertible if there is a permutation [pi] of V such that [pi](S[sub i]) [improper subset] V -- S[sub i]. In this paper, we present necessary and sufficient conditions for the invertibility of a collection and construct a polynomial algorithm which determines whether a given collection is invertible. For an arbitrary collection, we give a lower bound for the maximum number of sets that can be inverted. Finally, we consider the problem of constructing a collection of sets such that no sub-collection of size three is invertible. Our constructions of such collections come from solutions to the packing problem with unbounded block sizes. We prove several new lower and upper bounds for the packing problem and present a new explicit construction of packing.

  20. Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy.

    PubMed

    Jayandharan, Giridhara R; Aslanidi, George; Martino, Ashley T; Jahn, Stephan C; Perrin, George Q; Herzog, Roland W; Srivastava, Arun

    2011-03-01

    Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

  1. AAV2/8 vectors purified from culture medium with a simple and rapid protocol transduce murine liver, muscle, and retina efficiently.

    PubMed

    Doria, Monica; Ferrara, Antonella; Auricchio, Alberto

    2013-12-01

    During the production of some adeno-associated virus (AAV) serotypes, a large amount of vectors is found in the medium of producing cells. For their purification, previous protocols used tangential flow filtration (TFF) of the medium followed by iodixanol gradient centrifugation. Taking advantage of the higher purity of the medium than the cell-derived material as the source of AAV, we tested a simple method that combines production of large culture medium volumes containing AAV from cell stacks with medium clarification+TFF without further time-consuming and nonscalable centrifugation. To test this, we selected AAV2/8, which is emerging as a favored serotype for transduction of liver, muscle, and retina and abundantly found in the extracellular medium. We show that yields and in vitro infectivity of AAV2/8 vectors produced from the culture medium using this method are higher than those of vectors purified from the same cell lysate using a conventional CsCl2 gradient ultracentrifugation-based method, although purity appears inferior. In addition, we found that the transduction efficiency of AAV2/8 purified from medium was similar to that of AAV2/8 purified from the same cell lysate in the murine liver, muscle, and retina. Considering that the purification protocol from the medium we describe requires 3 hr as opposed to the 63 hr of a conventional two-round CsCl2-gradient ultracentrifugation+desalting, we conclude that TFF of the medium containing AAV2/8 represents a quick and scalable method to purify research-grade vectors for use in animal models.

  2. Power inverter implementing phase skipping control

    DOEpatents

    Somani, Utsav; Amirahmadi, Ahmadreza; Jourdan, Charles; Batarseh, Issa

    2016-10-18

    A power inverter includes a DC/AC inverter having first, second and third phase circuitry coupled to receive power from a power source. A controller is coupled to a driver for each of the first, second and third phase circuitry (control input drivers). The controller includes an associated memory storing a phase skipping control algorithm, wherein the controller is coupled to receive updating information including a power level generated by the power source. The drivers are coupled to control inputs of the first, second and third phase circuitry, where the drivers are configured for receiving phase skipping control signals from the controller and outputting mode selection signals configured to dynamically select an operating mode for the DC/AC inverter from a Normal Control operation and a Phase Skipping Control operation which have different power injection patterns through the first, second and third phase circuitry depending upon the power level.

  3. An SCR inverter for electric vehicles

    NASA Technical Reports Server (NTRS)

    Latos, T.; Bosack, D.; Ehrlich, R.; Jahns, T.; Mezera, J.; Thimmesch, D.

    1980-01-01

    An inverter for an electric vehicle propulsion application has been designed and constructed to excite a polyphase induction motor from a fixed propulsion battery source. The inverter, rated at 35kW peak power, is fully regenerative and permits vehicle operation in both the forward and reverse directions. Thyristors are employed as the power switching devices arranged in a dc bus commutated topology. This paper describes the major role the controller plays in generating the motor excitation voltage and frequency to deliver performance similar to dc systems. Motoring efficiency test data for the controller are presented. It is concluded that an SCR inverter in conjunction with an ac induction motor is a viable alternative to present dc vehicle propulsion systems on the basis of performance and size criteria.

  4. Intra-amniotic rAAV-mediated microdystrophin gene transfer improves canine X-linked muscular dystrophy and may induce immune tolerance.

    PubMed

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

    2015-04-01

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

  5. High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

    PubMed Central

    Ling, Chen; Bhukhai, Kanit; Yin, Zifei; Tan, Mengqun; Yoder, Mervin C.; Leboulch, Philippe; Payen, Emmanuel; Srivastava, Arun

    2016-01-01

    We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced less efficiently with the WT AAV6 vectors. The first strategy involved modifications of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705 + Y731F + T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs at significantly reduced vector doses. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. PMID:27759036

  6. Effect of SERCA2a overexpression in the pericardium mediated by the AAV1 gene transfer on rapid atrial pacing in rabbits.

    PubMed

    Kuken, B N; Aikemu, A N W E; Xiang, S Y; Wulasihan, M H Y T

    2015-10-29

    To study the effects of overexpression of the sarcoplasmic reticulum ATPase 2a (SERCA2a) gene on the activity and protein expression of SERCA2a after rapid atrial pacing (RAP) in New Zealand white rabbits. New Zealand white rabbits were randomly divided into a sham-operated group (group A), adeno-associated virus 1 (AAV1)/EGFP + atrial fibrillation (AF) model group (group B), or AVV1/SERCA2a + AF group (group C). The sham-operated group was used as a negative control. Each group consisted of 10 animals. Groups B and C were injected with 500 μL of the AAV1-EGFP reporter gene and 500 μL of the AAV1-SERCA2a target gene, respectively. Four weeks after AAV1-mediated gene transfer, the rabbits underwent 24 h of RAP to the right atrium. The animals were sacrificed and protein activity and protein expression in the myocardium were measured using the westernblot method. Four weeks after AAV1-mediated gene transfer, SERCA2a protein activity and expression were significantly higher in Group C than in Groups A and B (P < 0.05). RAP of the right atrium induced atrial fibrillation in rabbits, resulting in decreases in the activity and protein expression of SERCA2a. Pericardial AAV-1 mediated SERCA2a gene transfer resulted in the overexpression of SERCA2a, restoring SERCA2a activity and protein expression.

  7. Effect of SERCA2a overexpression in the pericardium mediated by the AAV1 gene transfer on rapid atrial pacing in rabbits.

    PubMed

    Kuken, B N; Aikemu, A N W E; Xiang, S Y; Wulasihan, M H Y T

    2015-01-01

    To study the effects of overexpression of the sarcoplasmic reticulum ATPase 2a (SERCA2a) gene on the activity and protein expression of SERCA2a after rapid atrial pacing (RAP) in New Zealand white rabbits. New Zealand white rabbits were randomly divided into a sham-operated group (group A), adeno-associated virus 1 (AAV1)/EGFP + atrial fibrillation (AF) model group (group B), or AVV1/SERCA2a + AF group (group C). The sham-operated group was used as a negative control. Each group consisted of 10 animals. Groups B and C were injected with 500 μL of the AAV1-EGFP reporter gene and 500 μL of the AAV1-SERCA2a target gene, respectively. Four weeks after AAV1-mediated gene transfer, the rabbits underwent 24 h of RAP to the right atrium. The animals were sacrificed and protein activity and protein expression in the myocardium were measured using the westernblot method. Four weeks after AAV1-mediated gene transfer, SERCA2a protein activity and expression were significantly higher in Group C than in Groups A and B (P < 0.05). RAP of the right atrium induced atrial fibrillation in rabbits, resulting in decreases in the activity and protein expression of SERCA2a. Pericardial AAV-1 mediated SERCA2a gene transfer resulted in the overexpression of SERCA2a, restoring SERCA2a activity and protein expression. PMID:26535677

  8. Intranasal Delivery of Recombinant AAV Containing BDNF Fused with HA2TAT: a Potential Promising Therapy Strategy for Major Depressive Disorder.

    PubMed

    Ma, Xian-cang; Liu, Peng; Zhang, Xiao-ling; Jiang, Wen-hui; Jia, Min; Wang, Cai-xia; Dong, Ying-ying; Dang, Yong-hui; Gao, Cheng-ge

    2016-01-01

    Depression is a disturbing psychiatric disease with unsatisfied therapy. Not all patients are sensitive to anti-depressants currently in use, side-effects are unavoidable during therapy, and the cases with effectiveness are always accompanied with delayed onset of clinical efficacy. Delivering brain-derived neurotrophic factor (BDNF) to brain seems to be a promising therapy. However, a better approach to delivery is still rudimentary. The purpose of our present work is to look for a rapid-onset and long-lasting therapeutic strategy for major depressive disorder (MDD) by effectively delivering BDNF to brain. BDNF, fused with cell-penetrating peptides (TAT and HA2), was packaged in adenovirus associated virus (AAV) to construct the BDNF-HA2TAT/AAV for intranasally delivering BDNF to central nervous system (CNS) via nose-brain pathway. Intranasal administration of BDNF-HA2TAT/AAV to normal mice displayed anti-depression effect in forced swimming test when the delivery lasted relatively longer. The AAV applied to mice subjected to chronic mild stress (CMS) through intranasal administration for 10 days also alleviated depression-like behaviors. Western-blotting analysis revealed that BDNF-HA2TAT/AAV nasal administration enhanced hippocampal BDNF content. These results indicate intranasal administration of constructed BDNF-HA2TAT/AAV exerts anti-depression effect in CMS mice by increasing hippocampal BDNF, suggesting that this strategy holds a promising therapeutic potential for MDD. PMID:26935651

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

    PubMed Central

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

    2015-01-01

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

  10. AAV2/1 CD74 Gene Transfer Reduces β-amyloidosis and Improves Learning and Memory in a Mouse Model of Alzheimer's Disease

    PubMed Central

    Kiyota, Tomomi; Zhang, Gang; Morrison, Christine M; Bosch, Megan E; Weir, Robert A; Lu, Yaman; Dong, Weiguo; Gendelman, Howard E

    2015-01-01

    Modulation of the amyloid-β (Aβ) trafficking pathway heralds a new therapeutic frontier for Alzheimer's disease (AD). As CD74 binds to the amyloid-β precursor protein (APP) and can suppresses Aβ processing, we investigated whether recombinant adeno-associated virus (AAV) delivery of CD74 could reduce Aβ production and affect disease outcomes. This idea was tested in a mouse AD model. Cotransduction of AAV-tetracycline-controlled transactivator (tTA) and AAV-tet-response element (TRE)-CD74 resulted in CD74 expression, reduced Aβ production in mouse neurons containing the human APP with familial AD-linked mutations. Stereotaxic injection of AAV-TRE-GFP or CD74 into the hippocampi of an AD mouse, defined as a TgCRND8 × calmodulin-dependent protein kinase II derived promoter-tTA double-transgenic, reduced Aβ loads and pyramidal neuronal Aβ accumulation in the hippocampus. Immunofluorescent studies showed that APP colocalization with Lamp1 was increased in CD74-expressing neurons. Moreover, Morris water maze tasks demonstrated that mice treated with AAV-TRE-CD74 showed improved learning and memory compared to AAV-TRE-GFP control animals. These results support the idea that CD74-induced alteration of Aβ processing could improve AD-associated memory deficits as shown in mouse models of human disease. PMID:26227349

  11. Intranasal Delivery of Recombinant AAV Containing BDNF Fused with HA2TAT: a Potential Promising Therapy Strategy for Major Depressive Disorder

    PubMed Central

    Ma, Xian-cang; Liu, Peng; Zhang, Xiao-ling; Jiang, Wen-hui; Jia, Min; Wang, Cai-xia; Dong, Ying-ying; Dang, Yong-hui; Gao, Cheng-ge

    2016-01-01

    Depression is a disturbing psychiatric disease with unsatisfied therapy. Not all patients are sensitive to anti-depressants currently in use, side-effects are unavoidable during therapy, and the cases with effectiveness are always accompanied with delayed onset of clinical efficacy. Delivering brain-derived neurotrophic factor (BDNF) to brain seems to be a promising therapy. However, a better approach to delivery is still rudimentary. The purpose of our present work is to look for a rapid-onset and long-lasting therapeutic strategy for major depressive disorder (MDD) by effectively delivering BDNF to brain. BDNF, fused with cell-penetrating peptides (TAT and HA2), was packaged in adenovirus associated virus (AAV) to construct the BDNF-HA2TAT/AAV for intranasally delivering BDNF to central nervous system (CNS) via nose-brain pathway. Intranasal administration of BDNF-HA2TAT/AAV to normal mice displayed anti-depression effect in forced swimming test when the delivery lasted relatively longer. The AAV applied to mice subjected to chronic mild stress (CMS) through intranasal administration for 10 days also alleviated depression-like behaviors. Western-blotting analysis revealed that BDNF-HA2TAT/AAV nasal administration enhanced hippocampal BDNF content. These results indicate intranasal administration of constructed BDNF-HA2TAT/AAV exerts anti-depression effect in CMS mice by increasing hippocampal BDNF, suggesting that this strategy holds a promising therapeutic potential for MDD. PMID:26935651

  12. Biochemical and Physiological Improvement in a Mouse Model of Smith-Lemli-Opitz Syndrome (SLOS) Following Gene Transfer with AAV Vectors.

    PubMed

    Ying, Lee; Matabosch, Xavier; Serra, Montserrat; Watson, Berna; Shackleton, Cedric; Watson, Gordon

    2014-01-01

    Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of cholesterol synthesis resulting from a defect in 7-dehydrocholesterol reductase (DHCR7), the enzyme that produces cholesterol from its immediate precursor 7-dehydrocholesterol. Current therapy employing dietary cholesterol is inadequate. As SLOS is caused by a defect in a single gene, restoring enzyme functionality through gene therapy may be a direct approach for treating this debilitating disorder. In the present study, we first packaged a human DHCR7 construct into adeno-associated virus (AAV) vectors having either type-2 (AAV2) or type-8 (AAV2/8) capsid, and administered treatment to juvenile mice. While a positive response (assessed by increases in serum and liver cholesterol) was seen in both groups, the improvement was greater in the AAV2/8-DHCR7 treated mice. Newborn mice were then treated with AAV2/8-DHCR7 and these mice, compared to mice treated as juveniles, showed higher DHCR7 mRNA expression in liver and a greater improvement in serum and liver cholesterol levels. Systemic treatment did not affect brain cholesterol in any of the experimental groups. Both juvenile and newborn treatments with AAV2/8-DHCR7 resulted in increased rates of weight gain indicating that gene transfer had a positive physiological effect.

  13. Genome of brown tide virus (AaV), the little giant of the Megaviridae, elucidates NCLDV genome expansion and host-virus coevolution.

    PubMed

    Moniruzzaman, Mohammad; LeCleir, Gary R; Brown, Christopher M; Gobler, Christopher J; Bidle, Kay D; Wilson, William H; Wilhelm, Steven W

    2014-10-01

    Aureococcus anophagefferens causes economically and ecologically destructive "brown tides" in the United States, China and South Africa. Here we report the 370,920bp genomic sequence of AaV, a virus capable of infecting and lysing A. anophagefferens. AaV is a member of the nucleocytoplasmic large DNA virus (NCLDV) group, harboring 377 putative coding sequences and 8 tRNAs. Despite being an algal virus, AaV shows no phylogenetic affinity to the Phycodnaviridae family, to which most algae-infecting viruses belong. Core gene phylogenies, shared gene content and genome-wide similarities suggest AaV is the smallest member of the emerging clade "Megaviridae". The genomic architecture of AaV demonstrates that the ancestral virus had an even smaller genome, which expanded through gene duplication and assimilation of genes from diverse sources including the host itself - some of which probably modulate important host processes. AaV also harbors a number of genes exclusive to phycodnaviruses - reinforcing the hypothesis that Phycodna- and Mimiviridae share a common ancestor. PMID:25035289

  14. A diagonally inverted LU implicit multigrid scheme

    NASA Technical Reports Server (NTRS)

    Yokota, Jeffrey W.; Caughey, David A.; Chima, Rodrick V.

    1988-01-01

    A new Diagonally Inverted LU Implicit scheme is developed within the framework of the multigrid method for the 3-D unsteady Euler equations. The matrix systems that are to be inverted in the LU scheme are treated by local diagonalizing transformations that decouple them into systems of scalar equations. Unlike the Diagonalized ADI method, the time accuracy of the LU scheme is not reduced since the diagonalization procedure does not destroy time conservation. Even more importantly, this diagonalization significantly reduces the computational effort required to solve the LU approximation and therefore transforms it into a more efficient method of numerically solving the 3-D Euler equations.

  15. Maskless inverted pyramid texturization of silicon.

    PubMed

    Wang, Yan; Yang, Lixia; Liu, Yaoping; Mei, Zengxia; Chen, Wei; Li, Junqiang; Liang, Huili; Kuznetsov, Andrej; Xiaolong, Du

    2015-06-02

    We discovered a technical solution of such outstanding importance that it can trigger new approaches in silicon wet etching processing and, in particular, photovoltaic cell manufacturing. The so called inverted pyramid arrays, outperforming conventional pyramid textures and black silicon because of their superior light-trapping and structure characteristics, can currently only be achieved using more complex techniques involving lithography, laser processing, etc. Importantly, our data demonstrate a feasibility of inverted pyramidal texturization of silicon by maskless Cu-nanoparticles assisted etching in Cu(NO3)2 / HF / H2O2 / H2O solutions and as such may have significant impacts on communities of fellow researchers and industrialists.

  16. Simplified space vector PWM algorithm for five-level inverter

    NASA Astrophysics Data System (ADS)

    Lalili, D.; Berkouk, E. M.; Boudjema, F.; Lourci, N.; Taleb, T.; Petzold, J.

    2007-12-01

    In this work, we present an algorithm for the space vector pulse width modulation (SVPWM) applied to five-level diode clamped inverter. In this algorithm, the space vector diagram of the five-level inverter is decomposed into six space vector diagrams of three-level inverters. In turn, each of these six space vector diagrams of three-level inverter is decomposed into six space vector diagrams of two-level inverters. This idea allows us to generalize the two-level SVPWM algorithm into the case of five-level inverter.

  17. High voltage series resonant inverter ion engine screen supply. [SCR series resonant inverter for space applications

    NASA Technical Reports Server (NTRS)

    Biess, J. J.; Inouye, L. Y.; Shank, J. H.

    1974-01-01

    A high-voltage, high-power LC series resonant inverter using SCRs has been developed for an Ion Engine Power Processor. The inverter operates within 200-400Vdc with a maximum output power of 2.5kW. The inverter control logic, the screen supply electrical and mechanical characteristics, the efficiency and losses in power components, regulation on the dual feedback principle, the SCR waveforms and the component weight are analyzed. Efficiency of 90.5% and weight density of 4.1kg/kW are obtained.

  18. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons.

    PubMed

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P

    2015-11-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to

  19. Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan.

    PubMed

    Weismann, Cara M; Ferreira, Jennifer; Keeler, Allison M; Su, Qin; Qui, Linghua; Shaffer, Scott A; Xu, Zuoshang; Gao, Guangping; Sena-Esteves, Miguel

    2015-08-01

    GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid β-galactosidase (βgal) activity. βgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mβgal vector infused systemically in adult GM1 mice (βGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high βGal activity in liver and serum. Moderate βGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of βgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of βgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

  20. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.

    PubMed

    Yang, Yang; Wang, Lili; Bell, Peter; McMenamin, Deirdre; He, Zhenning; White, John; Yu, Hongwei; Xu, Chenyu; Morizono, Hiroki; Musunuru, Kiran; Batshaw, Mark L; Wilson, James M

    2016-03-01

    Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

  1. A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses.

    PubMed

    Chtarto, Abdelwahed; Humbert-Claude, Marie; Bockstael, Olivier; Das, Atze T; Boutry, Sébastien; Breger, Ludivine S; Klaver, Bep; Melas, Catherine; Barroso-Chinea, Pedro; Gonzalez-Hernandez, Tomas; Muller, Robert N; DeWitte, Olivier; Levivier, Marc; Lundberg, Cecilia; Berkhout, Ben; Tenenbaum, Liliane

    2016-01-01

    Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses. PMID:27069954

  2. AAV-mediated and pharmacological induction of Hsp70 expression stimulates survival of retinal ganglion cells following axonal injury

    PubMed Central

    Kwong, Jacky MK; Gu, Lei; Nassiri, Nariman; Bekerman, Vlad; Kumar-Singh, Rajendra; Rhee, Kun Do; Yang, Xian-Jie; Hauswirth, William W.; Caprioli, Joseph; Piri, Natik

    2014-01-01

    We evaluated the effect of AAV2- and 17-AAG (17-N -allylamino-17-demethoxygeldanamycin)-mediated upregulation of Hsp70 expression on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). AAV2-Hsp70 expression in the retina was primarily observed in the ganglion cell layer. Approximately 75% of all transfected cells were RGCs. RGC survival in AAV2-Hsp70 injected animals was increased by an average of 110% 2 weeks after the axonal injury compared to the control. The increase in cell numbers was not even across the retinas with a maximum effect of approximately 306% observed in the inferior quadrant. 17-AAG-mediated expression of Hsp70 has been associated with cell protection in various models of neurodegenerative diseases. We show here that a single intravitreal injection of 17-AAG (0.2 ug/ul) results in an increased survival of ONC injured RGCs by approximately 49% compared to the vehicle-treated animals. Expression of Hsp70 in retinas of 17-AAG-treated animals was upregulated approximately by 2-fold compared to control animals. Our data support the idea that the upregulation of Hsp70 has a beneficial effect on the survival of injured RGCs, and the induction of this protein could be viewed as a potential neuroprotective strategy for optic neuropathies. PMID:25427613

  3. Rapid/sustained anti-anthrax passive immunity mediated by co-administration of Ad/AAV.

    PubMed

    De, Bishnu P; Hackett, Neil R; Crystal, Ronald G; Boyer, Julie L

    2008-01-01

    Achieving both immediate and sustained protection against diseases caused by bacterial toxins and extracellular pathogens is a challenge in developing biodefense therapeutics. We hypothesized that a single co-administration of an adenovirus (Ad) vector and an adeno-associated virus (AAV) vector, both expressing a pathogen-specific monoclonal antibody, would provide rapid, persistent passive immunotherapy against the pathogen. In order to test this strategy, we used the lethal toxin of Bacillus anthracis as a target of a monoclonal antibody directed against the protective antigen (PA) component of the toxin, using co-administration of an Ad vector encoding an anti-PA monoclonal antibody (AdalphaPA) and an AAV vector encoding an anti-PA monoclonal antibody (AAVrh.10alphaPA). As early as 1 day after co-administration of AdalphaPA and AAVrh.10alphaPA to mice, serum anti-PA antibody levels were detectable, and were sustained through 6 months. Importantly, animals that received both vectors were protected against toxin challenge as early as 1 day after administration and throughout the 6 month duration of the experiment. These data provide a new paradigm of genetic passive immunotherapy by co-administration of Ad and AAV vectors, each encoding a pathogen-specific monoclonal antibody, as an effective approach for both rapid and sustained protection against a bio-terror attack.

  4. A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses

    PubMed Central

    Chtarto, Abdelwahed; Humbert-Claude, Marie; Bockstael, Olivier; Das, Atze T; Boutry, Sébastien; Breger, Ludivine S; Klaver, Bep; Melas, Catherine; Barroso-Chinea, Pedro; Gonzalez-Hernandez, Tomas; Muller, Robert N; DeWitte, Olivier; Levivier, Marc; Lundberg, Cecilia; Berkhout, Ben; Tenenbaum, Liliane

    2016-01-01

    Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 109 viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses. PMID:27069954

  5. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice

    PubMed Central

    Yang, Yang; Wang, Lili; Bell, Peter; McMenamin, Deirdre; He, Zhenning; White, John; Yu, Hongwei; Xu, Chenyu; Morizono, Hiroki; Musunuru, Kiran; Batshaw, Mark L.; Wilson, James M.

    2016-01-01

    Many genetic liver diseases present in newborns with repeated, often lethal, metabolic crises. Gene therapy using non-integrating viruses such as AAV is not optimal in this setting because the non-integrating genome is lost as developing hepatocytes proliferate1,2. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7% – 20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet. PMID:26829317

  6. In vivo cellular imaging of various stress/response pathways using AAV following axonal injury in mice

    PubMed Central

    Fujita, Kosuke; Nishiguchi, Koji M; Yokoyama, Yu; Tomiyama, Yusuke; Tsuda, Satoru; Yasuda, Masayuki; Maekawa, Shigeto; Nakazawa, Toru

    2015-01-01

    Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-κB and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells. PMID:26670005

  7. Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex.

    PubMed

    Jara, J H; Stanford, M J; Zhu, Y; Tu, M; Hauswirth, W W; Bohn, M C; DeVries, S H; Özdinler, P H

    2016-03-01

    Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken β-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases. PMID:26704722

  8. Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex

    PubMed Central

    Jara, J H; Stanford, M J; Zhu, Y; Tu, M; Hauswirth, W W; Bohn, M C; DeVries, S H; Özdinler, P H

    2016-01-01

    Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1G93A transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken β-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1G93A transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases. PMID:26704722

  9. Efficacy and Safety of rAAV2-ND4 Treatment for Leber’s Hereditary Optic Neuropathy

    PubMed Central

    Wan, Xing; Pei, Han; Zhao, Min-jian; Yang, Shuo; Hu, Wei-kun; He, Heng; Ma, Si-qi; Zhang, Ge; Dong, Xiao-yan; Chen, Chen; Wang, Dao-wen; Li, Bin

    2016-01-01

    Leber’s hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness. A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit 4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety of a recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carrying the G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eye and then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and optical coherence tomography were performed. Physical examinations included routine blood and urine. The visual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months of follow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remained relatively stable. No other outcome measure was significantly changed. None of the nine patients had local or systemic adverse events related to the vector during the 9-month follow-up period. These findings support the feasible use of gene therapy for LHON. PMID:26892229

  10. Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

    PubMed Central

    Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

    2016-01-01

    The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

  11. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    PubMed Central

    Leclerc, Xavier; Danos, Olivier; Scherman, Daniel; Kichler, Antoine

    2009-01-01

    Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments. PMID:19379497

  12. The Internet and the Inverted Classroom.

    ERIC Educational Resources Information Center

    Lage, Maureen J.; Platt, Glenn

    2000-01-01

    Describes a Web site that is for an undergraduate principles of microeconomics course and a main component of "The Inverted Classroom" in which lectures take place outside of class. Explains that the Web site is divided into four sections: (1) the classroom; (2) the desk; (3) the coffee shop; and (4) the library. (CMK)

  13. Pulse width modulation inverter with battery charger

    DOEpatents

    Slicker, James M.

    1985-01-01

    An inverter is connected between a source of DC power and a three-phase AC induction motor, and a microprocessor-based circuit controls the inverter using pulse width modulation techniques. In the disclosed method of pulse width modulation, both edges of each pulse of a carrier pulse train are equally modulated by a time proportional to sin .theta., where .theta. is the angular displacement of the pulse center at the motor stator frequency from a fixed reference point on the carrier waveform. The carrier waveform frequency is a multiple of the motor stator frequency. The modulated pulse train is then applied to each of the motor phase inputs with respective phase shifts of 120.degree. at the stator frequency. Switching control commands for electronic switches in the inverter are stored in a random access memory (RAM) and the locations of the RAM are successively read out in a cyclic manner, each bit of a given RAM location controlling a respective phase input of the motor. The DC power source preferably comprises rechargeable batteries and all but one of the electronic switches in the inverter can be disabled, the remaining electronic switch being part of a "flyback" DC-DC converter circuit for recharging the battery.

  14. Pulse width modulation inverter with battery charger

    NASA Technical Reports Server (NTRS)

    Slicker, James M. (Inventor)

    1985-01-01

    An inverter is connected between a source of DC power and a three-phase AC induction motor, and a microprocessor-based circuit controls the inverter using pulse width modulation techniques. In the disclosed method of pulse width modulation, both edges of each pulse of a carrier pulse train are equally modulated by a time proportional to sin .theta., where .theta. is the angular displacement of the pulse center at the motor stator frequency from a fixed reference point on the carrier waveform. The carrier waveform frequency is a multiple of the motor stator frequency. The modulated pulse train is then applied to each of the motor phase inputs with respective phase shifts of 120.degree. at the stator frequency. Switching control commands for electronic switches in the inverter are stored in a random access memory (RAM) and the locations of the RAM are successively read out in a cyclic manner, each bit of a given RAM location controlling a respective phase input of the motor. The DC power source preferably comprises rechargeable batteries and all but one of the electronic switches in the inverter can be disabled, the remaining electronic switch being part of a flyback DC-DC converter circuit for recharging the battery.

  15. A Prefix Trie Index for Inverted Files.

    ERIC Educational Resources Information Center

    Nelson, Michael J.

    1997-01-01

    Applies a prefix trie index, or trie hashing, to the problem of providing fast search times, fast load times, and fast update properties in a bibliographic or full-text retrieval system with inverted files. Statistics are given for a test database consisting of an online catalog at the University of Western Ontario. (Author/LRW)

  16. Inverted drop testing and neck injury potential.

    PubMed

    Forrest, Stephen; Herbst, Brian; Meyer, Steve; Sances, Anthony; Kumaresan, Srirangam

    2003-01-01

    Inverted drop testing of vehicles is a methodology that has long been used by the automotive industry and researchers to test roof integrity and is currently being considered by the National Highway Traffic Safety Administration as a roof strength test. In 1990 a study was reported which involved 8 dolly rollover tests and 5 inverted drop tests. These studies were conducted with restrained Hybrid III instrumented Anthropometric Test Devices (ATD) in production and rollcaged vehicles to investigate the relationship between roof strength and occupant injury potential. The 5 inverted drop tests included in the study provided a methodology producing "repeatable roof impacts" exposing the ATDs to the similar impact environment as those seen in the dolly rollover tests. Authors have conducted two inverted drop test sets as part of an investigation of two real world rollover accidents. Hybrid-III ATD's were used in each test with instrumented head and necks. Both test sets confirm that reduction of roof intrusion and increased headroom can significantly enhance occupant protection. In both test pairs, the neck force of the dummy in the vehicle with less crush and more survival space was significantly lower. Reduced roof crush and dynamic preservation of the occupant survival space resulted in only minor occupant contact and minimal occupant loading, establishing a clear causal relationship between roof crush and neck injuries.

  17. View of Inverted Siphon crossing Hot Water (or White) Canyon. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of Inverted Siphon crossing Hot Water (or White) Canyon. Looking northeast - Childs-Irving Hydroelectric Project, Irving System, Inverted Siphon, Forest Service Road 708/502, Camp Verde, Yavapai County, AZ

  18. PEO-PPO-PEO Carriers for rAAV-Mediated Transduction of Human Articular Chondrocytes in Vitro and in a Human Osteochondral Defect Model.

    PubMed

    Rey-Rico, Ana; Frisch, Janina; Venkatesan, Jagadesh Kumar; Schmitt, Gertrud; Rial-Hermida, Isabel; Taboada, Pablo; Concheiro, Angel; Madry, Henning; Alvarez-Lorenzo, Carmen; Cucchiarini, Magali

    2016-08-17

    Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA.

  19. Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model.

    PubMed

    Pleger, Sven T; Shan, Changguang; Ksienzyk, Jan; Bekeredjian, Raffi; Boekstegers, Peter; Hinkel, Rabea; Schinkel, Stefanie; Leuchs, Barbara; Ludwig, Jochen; Qiu, Gang; Weber, Christophe; Raake, Philip; Koch, Walter J; Katus, Hugo A; Müller, Oliver J; Most, Patrick

    2011-07-20

    As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

  20. Gene therapy using self-complementary Y733F capsid mutant AAV2/8 restores vision in a model of early onset Leber congenital amaurosis.

    PubMed

    Ku, Cristy A; Chiodo, Vince A; Boye, Sanford L; Goldberg, Andrew F X; Li, Tiansen; Hauswirth, William W; Ramamurthy, Visvanathan

    2011-12-01

    Defects in the photoreceptor-specific gene aryl hydrocarbon receptor interacting protein-like 1 (Aipl1) are associated with Leber congenital amaurosis (LCA), a childhood blinding disease with early-onset retinal degeneration and vision loss. Furthermore, Aipl1 defects are characterized at the most severe end of the LCA spectrum. The rapid photoreceptor degeneration and vision loss observed in the LCA patient population are mimicked in a mouse model lacking AIPL1. Using this model, we evaluated if gene replacement therapy using recent advancements in adeno-associated viral vectors (AAV) provides advantages in preventing rapid retinal degeneration. Specifically, we demonstrated that the novel self-complementary Y733F capsid mutant AAV2/8 (sc-Y733F-AAV) provided greater preservation of photoreceptors and functional vision in Aipl1 null mice compared with single-stranded AAV2/8. The benefits of sc-Y733F-AAV were evident following viral administration during the active phase of retinal degeneration, where only sc-Y733F-AAV treatment achieved functional vision rescue. This result was likely due to higher and earlier onset of Aipl1 expression. Based on our studies, we conclude that the sc-Y733F-AAV2/8 viral vector, to date, achieves the best rescue for rapid retinal degeneration in Aipl1 null mice. Our results provide important considerations for viral vectors to be used in future gene therapy clinical trials targeting a wider severity spectrum of inherited retinal dystrophies.

  1. Mobile Inverted Constructivism: Education of Interaction Technology in Social Media

    ERIC Educational Resources Information Center

    Chai, Jia-Xiang; Fan, Kuo-Kuang

    2016-01-01

    The combination of social media and invert teaching is a new path to inverting interation technology education and reconstructing the curriculum of context. In this paper, based on the theory of constructivism learning, a model named Mobile Inverted Constructivism (MIC) is provided. Moreover, in view of the functional quality of social media in…

  2. Primary and recurrent inverting papilloma: appearances with magnetic resonance imaging.

    PubMed

    Roobottom, C A; Jewell, F M; Kabala, J

    1995-07-01

    Inverting papilloma of the sinonasal cavity is of importance because of its association with squamous cell carcinoma and its tendency to local recurrence. Appearances of inverting papilloma have only been reported infrequently, and never in recurrent cases. We present the magnetic resonance imaging (MRI) appearances of four cases of inverting papilloma, three of which are recurrent.

  3. Efficient/reliable dc-to-dc inverter circuit

    NASA Technical Reports Server (NTRS)

    Pasciutti, E. R.

    1970-01-01

    Feedback loop, which contains an inductor in series with a saturable reactor, is added to a standard inverter circuit to permit the inverter power transistors to be switched in a controlled and efficient manner. This inverter is applicable where the power source has either high or low impedance properties.

  4. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    PubMed

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2014-10-30

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene.

  5. AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6β-deficient Dogs.

    PubMed

    Pichard, Virginie; Provost, Nathalie; Mendes-Madeira, Alexandra; Libeau, Lyse; Hulin, Philippe; Tshilenge, Kizito-Tshitoko; Biget, Marine; Ameline, Baptiste; Deschamps, Jack-Yves; Weber, Michel; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

    2016-05-01

    We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

  6. Continuous DOPA synthesis from a single AAV: dosing and efficacy in models of Parkinson's disease

    PubMed Central

    Cederfjäll, Erik; Nilsson, Nathalie; Sahin, Gurdal; Chu, Yaping; Nikitidou, Elisabeth; Björklund, Tomas; Kordower, Jeffrey H.; Kirik, Deniz

    2013-01-01

    We used a single adeno-associated viral (AAV) vector co-expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) to investigate the relationship between vector dose, and the magnitude and rate of recovery in hemi-parkinsonian rats. Intrastriatal injections of >1E10 genomic copies (gc) of TH-GCH1 vector resulted in complete recovery in drug-naïve behavior tests. Lower vector dose gave partial to no functional improvement. Stereological quantification revealed no striatal NeuN+ cell loss in any of the groups, whereas a TH-GCH1 dose of >1E11 gc resulted in cell loss in globus pallidus. Thus, a TH-GCH1 dose of 1E10 gc gave complete recovery without causing neuronal loss. Safety and efficacy was also studied in non-human primates where the control vector resulted in co-expression of the transgenes in caudate-putamen. In the TH-GCH1 group, GCH1 expression was robust but TH was not detectable. Moreover, TH-GCH1 treatment did not result in functional improvement in non-human primates. PMID:23831692

  7. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    PubMed

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2015-03-01

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene. PMID:25359548

  8. AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i.

    PubMed

    Koornneef, A; van Logtenstein, R; Timmermans, E; Pisas, L; Blits, B; Abad, X; Fortes, P; Petry, H; Konstantinova, P; Ritsema, T

    2011-09-01

    RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.

  9. Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods.

    PubMed

    Fagone, Paolo; Wright, J Fraser; Nathwani, Amit C; Nienhuis, Arthur W; Davidoff, Andrew M; Gray, John T

    2012-02-01

    Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities.

  10. Electronic Document Management Using Inverted Files System

    NASA Astrophysics Data System (ADS)

    Suhartono, Derwin; Setiawan, Erwin; Irwanto, Djon

    2014-03-01

    The amount of documents increases so fast. Those documents exist not only in a paper based but also in an electronic based. It can be seen from the data sample taken by the SpringerLink publisher in 2010, which showed an increase in the number of digital document collections from 2003 to mid of 2010. Then, how to manage them well becomes an important need. This paper describes a new method in managing documents called as inverted files system. Related with the electronic based document, the inverted files system will closely used in term of its usage to document so that it can be searched over the Internet using the Search Engine. It can improve document search mechanism and document save mechanism.

  11. Invertible linear transformations and the Lie algebras

    NASA Astrophysics Data System (ADS)

    Zhang, Yufeng; Tam, Honwah; Guo, Fukui

    2008-07-01

    With the help of invertible linear transformations and the known Lie algebras, a way to generate new Lie algebras is given. These Lie algebras obtained have a common feature, i.e. integrable couplings of solitary hierarchies could be obtained by using them, specially, the Hamiltonian structures of them could be worked out. Some ways to construct the loop algebras of the Lie algebras are presented. It follows that some various loop algebras are given. In addition, a few new Lie algebras are explicitly constructed in terms of the classification of Lie algebras proposed by Ma Wen-Xiu, which are bases for obtaining new Lie algebras by using invertible linear transformations. Finally, some solutions of a (2 + 1)-dimensional partial-differential equation hierarchy are obtained, whose Hamiltonian form-expressions are manifested by using the quadratic-form identity.

  12. Maskless inverted pyramid texturization of silicon

    PubMed Central

    Wang, Yan; Yang, Lixia; Liu, Yaoping; Mei, Zengxia; Chen, Wei; Li, Junqiang; Liang, Huili; Kuznetsov, Andrej; Xiaolong, Du

    2015-01-01

    We discovered a technical solution of such outstanding importance that it can trigger new approaches in silicon wet etching processing and, in particular, photovoltaic cell manufacturing. The so called inverted pyramid arrays, outperforming conventional pyramid textures and black silicon because of their superior light-trapping and structure characteristics, can currently only be achieved using more complex techniques involving lithography, laser processing, etc. Importantly, our data demonstrate a feasibility of inverted pyramidal texturization of silicon by maskless Cu-nanoparticles assisted etching in Cu(NO3)2 / HF / H2O2 / H2O solutions and as such may have significant impacts on communities of fellow researchers and industrialists. PMID:26035520

  13. The Invertible Double of Elliptic Operators

    NASA Astrophysics Data System (ADS)

    Booss-Bavnbek, Bernhelm; Lesch, Matthias

    2009-02-01

    First, we review the Dirac operator folklore about basic analytic and geometrical properties of operators of Dirac type on compact manifolds with smooth boundary and on closed partitioned manifolds and show how these properties depend on the construction of a canonical invertible double and are related to the concept of the Calderón projection. Then we summarize a recent construction of a canonical invertible double for general first order elliptic differential operators over smooth compact manifolds with boundary. We derive a natural formula for the Calderón projection which yields a generalization of the famous Cobordism Theorem. We provide a list of assumptions to obtain a continuous variation of the Calderón projection under smooth variation of the coefficients. That yields various new spectral flow theorems. Finally, we sketch a research program for confining, respectively closing, the last remaining gaps between the geometric Dirac operator type situation and the general linear elliptic case.

  14. Maskless inverted pyramid texturization of silicon.

    PubMed

    Wang, Yan; Yang, Lixia; Liu, Yaoping; Mei, Zengxia; Chen, Wei; Li, Junqiang; Liang, Huili; Kuznetsov, Andrej; Xiaolong, Du

    2015-01-01

    We discovered a technical solution of such outstanding importance that it can trigger new approaches in silicon wet etching processing and, in particular, photovoltaic cell manufacturing. The so called inverted pyramid arrays, outperforming conventional pyramid textures and black silicon because of their superior light-trapping and structure characteristics, can currently only be achieved using more complex techniques involving lithography, laser processing, etc. Importantly, our data demonstrate a feasibility of inverted pyramidal texturization of silicon by maskless Cu-nanoparticles assisted etching in Cu(NO3)2 / HF / H2O2 / H2O solutions and as such may have significant impacts on communities of fellow researchers and industrialists. PMID:26035520

  15. Intelligent Control of an Inverted Pendulum

    NASA Astrophysics Data System (ADS)

    Rekdalsbakken, Webjorn

    An inverted pendulum represents an unstable system which is excellent for demonstrating the use of feedback control with different kinds of control strategies. In this work state feedback of the inverted pendulum is examined. First a pole placement algorithm is explored. After that artificial intelligence (AI) methods are investigated to better cope with the nonlinearities of the physical model. The technique used is based on a hybrid system combining a neural network (NN) with a genetic algorithm (GA). The NN controller is trained by the GA against the behaviour of the physical model. The results of the training process show that the chromosome population tends to station at a suboptimal level, and that changes in the environmental parameters have to take place to reach a new optimal level. By systematically changing these parameters the NN controller will gradually adapt to the pendulum behaviour.

  16. Micro-inverter solar panel mounting

    DOEpatents

    Morris, John; Gilchrist, Phillip Charles

    2016-02-02

    Processes, systems, devices, and articles of manufacture are provided. Each may include adapting micro-inverters initially configured for frame-mounting to mounting on a frameless solar panel. This securement may include using an adaptive clamp or several adaptive clamps secured to a micro-inverter or its components, and using compressive forces applied directly to the solar panel to secure the adaptive clamp and the components to the solar panel. The clamps can also include compressive spacers and safeties for managing the compressive forces exerted on the solar panels. Friction zones may also be used for managing slipping between the clamp and the solar panel during or after installation. Adjustments to the clamps may be carried out through various means and by changing the physical size of the clamps themselves.

  17. scAAV9-VEGF prolongs the survival of transgenic ALS mice by promoting activation of M2 microglia and the PI3K/Akt pathway.

    PubMed

    Wang, Ying; Duan, Weisong; Wang, Wan; Di Wen; Liu, Yaling; Liu, Yakun; Li, Zhongyao; Hu, Haojie; Lin, Huiqian; Cui, Can; Li, Dongxiao; Dong, Hui; Li, Chunyan

    2016-10-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads to paralysis and death three to five years after diagnosis in most patients. The disease is incurable, and the mechanism of motoneuron degeneration remains unknown, although research has demonstrated that activated microglia are involved in motor neuron death. Here, we used a simple method to deliver AAV9 virus by direct intrathecal injection and found that scAAV9-VEGF-165 improved the motor performance and prolonged the life span of SOD1-G93A mice. Furthermore, scAAV9-VEGF-165 activated the PI3K/Akt survival pathway and increased the level of Bcl-2, which contributed to the protection of motor neurons. Additionally, scAAV9-VEGF-165 attenuated the expression of classically activated (M1) microglial markers and enhanced the expression of alternatively activated (M2) microglial markers. Taken together, the results of our study suggest that simple, direct intrathecal injection of scAAV9-VEGF-165 may have a curative effect for ALS. PMID:27392886

  18. AAV2/8-humanFOXP3 gene therapy shows robust anti-atherosclerosis efficacy in LDLR-KO mice on high cholesterol diet.

    PubMed

    Cao, M; Theus, S A; Straub, K D; Figueroa, J A; Mirandola, L; Chiriva-Internati, M; Hermonat, P L

    2015-07-18

    Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.

  19. Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

    PubMed

    Tshilenge, Kizito-Tshitoko; Ameline, Baptiste; Weber, Michel; Mendes-Madeira, Alexandra; Nedellec, Steven; Biget, Marine; Provost, Nathalie; Libeau, Lyse; Blouin, Véronique; Deschamps, Jack-Yves; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Pichard, Virginie; Rolling, Fabienne

    2016-06-01

    Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons. PMID:27229628

  20. Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

    PubMed

    Mizutani, Takeomi; Li, Rui; Haga, Hisashi; Kawabata, Kazushige

    2015-09-18

    The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

  1. Rhenium Disulfide Depletion-Load Inverter

    NASA Astrophysics Data System (ADS)

    McClellan, Connor; Corbet, Chris; Rai, Amritesh; Movva, Hema C. P.; Tutuc, Emanuel; Banerjee, Sanjay K.

    2015-03-01

    Many semiconducting Transition Metal Dichalcogenide (TMD) materials have been effectively used to create Field-Effect Transistor (FET) devices but have yet to be used in logic designs. We constructed a depletion-load voltage inverter using ultrathin layers of Rhenium Disulfide (ReS2) as the semiconducting channel. This ReS2 inverter was fabricated on a single micromechanically-exfoliated flake of ReS2. Electron beam lithography and physical vapor deposition were used to construct Cr/Au electrical contacts, an Alumina top-gate dielectric, and metal top-gate electrodes. By using both low (Aluminum) and high (Palladium) work-function metals as two separate top-gates on a single ReS2 flake, we create a dual-gated depletion mode (D-mode) and enhancement mode (E-mode) FETs in series. Both FETs displayed current saturation in the output characteristics as a result of the FET ``pinch-off'' mechanism and On/Off current ratios of 105. Field-effect mobilities of 23 and 17 cm2V-1s-1 and subthreshold swings of 97 and 551 mV/decade were calculated for the E-mode and D-mode FETs, respectively. With a supply voltage of 1V, at low/negative input voltages the inverter output was at a high logic state of 900 mV. Conversely with high/positive input voltages, the inverter output was at a low logic state of 500 mV. The inversion of the input signal demonstrates the potential for using ReS2 in future integrated circuit designs and the versatility of depletion-load logic devices for TMD research. NRI SWAN Center and ARL STTR Program.

  2. Coaxial inverted geometry transistor having buried emitter

    NASA Technical Reports Server (NTRS)

    Hruby, R. J.; Cress, S. B.; Dunn, W. R. (Inventor)

    1973-01-01

    The invention relates to an inverted geometry transistor wherein the emitter is buried within the substrate. The transistor can be fabricated as a part of a monolithic integrated circuit and is particularly suited for use in applications where it is desired to employ low actuating voltages. The transistor may employ the same doping levels in the collector and emitter, so these connections can be reversed.

  3. Base drive circuit for a four-terminal power Darlington

    DOEpatents

    Lee, Fred C.; Carter, Roy A.

    1983-01-01

    A high power switching circuit which utilizes a four-terminal Darlington transistor block to improve switching speed, particularly in rapid turn-off. Two independent reverse drive currents are utilized during turn off in order to expel the minority carriers of the Darlington pair at their own charge sweep-out rate. The reverse drive current may be provided by a current transformer, the secondary of which is tapped to the base terminal of the power stage of the Darlington block. In one application, the switching circuit is used in each power switching element in a chopper-inverter drive of an electric vehicle propulsion system.

  4. FET commutated current-FED inverter

    NASA Technical Reports Server (NTRS)

    Rippel, Wally E. (Inventor); Edwards, Dean B. (Inventor)

    1983-01-01

    A shunt switch comprised of a field-effect transistor (Q.sub.1) is employed to commutate a current-fed inverter (10) using thyristors (SCR1, SCR2) or bijunction transistors (Q.sub.2, Q.sub.3) in a full bridge (1, 2, 3, 4) or half bridge (5, 6) and transformer (T.sub.1) configuration. In the case of thyristors, a tapped inverter (12) is employed to couple the inverter to a dc source to back bias the thyristors during commutation. Alternatively, a commutation power supply (20) may be employed for that purpse. Diodes (D.sub.1, D.sub.2) in series with some voltage dropping element (resistor R.sub.12 or resistors R.sub.1, R.sub.2 or Zener diodes D.sub.4, D.sub.5) are connected in parallel with the thyristors in the half bridge and transformer configuration to assure sharing the back bias voltage. A clamp circuit comprised of a winding (18) negatively coupled to the inductor and a diode (D.sub.3) return stored energy from the inductor to the power supply for efficient operation with buck or boost mode.

  5. Electrodynamics of AN Ion Inverted V.

    NASA Astrophysics Data System (ADS)

    Burgess, Georgette Olive

    Particle precipitation around the earth's polar regions may be the footprint of various energizing phenomena in the magnetosphere. Satellite-observed electron fluxes whose peak energy increases then decreases are called inverted V's. The Atmosphere Explorer-D Low Energy Electron (LEE) data for January 11, 1976 indicates that the precipitating ions have been accelerated. In this event the spectrograms of the ion flux shows the change of the peak energy with time characteristic of an inverted V. The electron population is decelerated as the ion population is accelerated, consistent with a downward electric field. The Birkeland current at an inverted V may be calculated in two ways: from the divergence of the electric field or from the observed particle fluxes. We found that the two methods agree on the location of Birkeland current throughout the event, but the magnitudes are not the same. This is not surprising, since the component of (DEL)((')(SIGMA)(.)(')E) perpendicular to the trajectory can not be determined. The electric potential along the spacecraft's trajectory (790-650 km altitude) was calculated from the measured electric fields. The sum of the parallel potential drop (inferred from the ion distribution function) and the ionospheric potential gives the potential profile at the magnetosphere. The parallel electric field thus partially decoupled the ionospheric flow from the magnetospheric flow. The electric field pattern in the magnetosphere -ionosphere system demands field-aligned currents. When the thermal current is insufficient, a field-aligned potential drop can accelerate particles to satisfy the requirements. The thermal electron current from the ionosphere is much greater than that from the magnetosphere. Thus, it is more common to observe the signatures of an upward electric field: the electron "inverted V". In the ion inverted V observed during AE-D orbit 1141, the postulated parallel potential has reduced the required parallel current. This high

  6. The PLATO V Terminal.

    ERIC Educational Resources Information Center

    Stifle, J. E.

    This report provides a detailed description of the architecture and programming of the PLATO V terminal, which contains an 8080 microprocessor and is capable of being operated by programs located in a host computer. The terminal contains 8k of memory for storing local programs, a 4k ROM resident program which supervises terminal operation, a 2k…

  7. CAI Terminal Characteristics.

    ERIC Educational Resources Information Center

    Braun, Peter

    The bewildering number of available terminals which are offered to CAI users presents a rather formidable problem of which one to choose. This article surveys what appear to be evolving standards for terminals. The usefulness of these terminals for CAI purposes is discussed, together with the best known prototype exhibiting the particular feature.…

  8. AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia

    PubMed Central

    Somanathan, Suryanarayan; Jacobs, Frank; Wang, Qiang; Hanlon, Alexandra L; Wilson, James M; Rader, Daniel J

    2014-01-01

    Rationale Familial hypercholesterolemia (FH) is a genetic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous FH (hoFH) is a candidate for gene therapy using adeno-associated viral (AAV) vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen AAV encoded LDLR expression. Objective We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. Methods and Results Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and hIDOL. A panel of mutant hLDLRs was initially screened in vitro for escape from PCSK9. The variant hLDLR-L318D was further evaluated using a mouse model of hoFH lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme, APOBEC-1 (DKO). Administration of wild type hLDLR to DKO mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9 mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\\C818A construct avoided hIDOL regulation and achieved stable reductions in serum cholesterol. An AAV8.LDLR-L318D\\K809R\\C818A vector that carried all three amino acid substitutions conferred partial resistance to both hPCSK9 and hIDOL mediated degradation. Conclusion Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve upon a potential gene therapeutic approach to treat homozygous FH subjects. PMID:25023731

  9. RH10 provides superior transgene expression in mice when compared with natural AAV serotypes for neonatal gene therapy

    PubMed Central

    Hu, Chuhong; Busuttil, Ronald W.; Lipshutz, Gerald S.

    2010-01-01

    Background Neonatal gene therapy is a promising strategy for treating diseases diagnosed before or shortly after birth. Early and long-term expression of therapeutic proteins may limit the consequences of genetic mutations and result in a potential ‘cure’. Adeno-associated viral vectors have shown promise in many areas of adult gene therapy but their properties have not been systematically investigated in the neonate. Methods In these studies using a constitutive promoter expressing luciferase, animals were administered one of ten serotypes of AAV on the second day of life. Examination of expression, organ growth and vector distribution, maintenance of expression and copy number were examined. Results All serotypes demonstrated expression and, in general, transduction of all organs within 3 days, albeit with different biodistribution patterns and expression levels. Highest expression was detected with AAVrh10 while lowest was with AAV4. Expression and genomes declined with growth over the first 10 weeks of life; thereafter, to day 100, expression and genomes remained relatively stable. With the highest expressing vectors, whole animal expression at 100 days declined to ~10% of that detected on the fifth day. AAVrh10 maintained the highest expression level and copy number throughout these studies. Conclusion The impact of tissue and organ growth on the stability of AAV expression will be important if neonatal gene transfer is to be considered as a modality for human gene therapy. While all vectors did demonstrate expression, rh10 holds the greater promise of the vectors tested to maintain copy number in both mitotic and post-mitotic tissues. PMID:20821747

  10. An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α

    PubMed Central

    Ojano-Dirain, Carolyn; Glushakova, Lyudmyla G.; Zhong, Li; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun; Stacpoole, Peter W.

    2010-01-01

    We evaluated the feasibility of self-complementary adeno-associated virus (scAAV) vector-mediated knockdown of the pyruvate dehydrogenase complex using small interfering RNAs directed against the E1α subunit gene (PDHA1). AAV serotype 8 was used to stereotaxically deliver scAAV8-si3-PDHA1-Enhanced Green Fluorescent Protein (knockdown) or scAAV8-EGFP (control) vectors into the right striatum and substantia nigra of rats. Rotational asymmetry was employed to quantify abnormal rotation following neurodegeneration in the nigrostriatal system. By 20 weeks after surgery, the siRNA-injected rats exhibited higher contralateral rotation during the first 10 min following amphetamine administration and lower 90-min total rotations (p≤0.05). Expression of PDC E1α, E1β and E2 subunits in striatum was decreased (p≤0.05) in the siRNA-injected striatum after 14 weeks. By week 25, both PDC activity and expression of E1α were lower (p≤0.05) in siRNA-injected striata compared to controls. E1α expression was associated with PDC activity (R2=0.48; p=0.006) and modestly associated with counterclockwise rotation (R2=0.51;p=0.07). The use of tyrosine-mutant scAAV8 vectors resulted in ~17-fold increase in transduction efficiency of rat striatal neurons in vivo. We conclude that scAAV8-siRNA vector-mediated knockdown of PDC E1α in brain regions typically affected in humans with PDC deficiency results in a reproducible biochemical and clinical phenotype in rats that may be further enhanced with the use of tyrosine-mutant vectors. PMID:20685142

  11. Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

    PubMed

    Pañeda, Astrid; Lopez-Franco, Esperanza; Kaeppel, Christine; Unzu, Carmen; Gil-Royo, Ana Gloria; D'Avola, Delia; Beattie, Stuart G; Olagüe, Cristina; Ferrero, Roberto; Sampedro, Ana; Mauleon, Itsaso; Hermening, Stephan; Salmon, Florence; Benito, Alberto; Gavira, Juan Jose; Cornet, María Eugenia; del Mar Municio, María; von Kalle, Christof; Petry, Harald; Prieto, Jesus; Schmidt, Manfred; Fontanellas, Antonio; González-Aseguinolaza, Gloria

    2013-12-01

    Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients. PMID:24070415

  12. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin

    PubMed Central

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Miller, Paul E.; Sharma, Alok K.; Ver Hoeve, James N.; Howard, Kellie; Knop, David R.; Neuringer, Martha; McGill, Trevor; Stoddard, Jonathan; Chulay, Jeffrey D.

    2015-01-01

    Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 1010 or 4 × 1011 vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS. PMID:26390090

  13. Treatment of hypophosphatasia by muscle-directed expression of bone-targeted alkaline phosphatase via self-complementary AAV8 vector

    PubMed Central

    Nakamura-Takahashi, Aki; Miyake, Koichi; Watanabe, Atsushi; Hirai, Yukihiko; Iijima, Osamu; Miyake, Noriko; Adachi, Kumi; Nitahara-Kasahara, Yuko; Kinoshita, Hideaki; Noguchi, Taku; Abe, Shinichi; Narisawa, Sonoko; Millán, Jose Luis; Shimada, Takashi; Okada, Takashi

    2016-01-01

    Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2−/−) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2−/− mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP. PMID:26904710

  14. Volunteer Losing Balance Wearing Inverted Glasses

    NASA Technical Reports Server (NTRS)

    2002-01-01

    Brad McLain for the Space Biology Museum Network puts a volunteer back on balance as he tries to adjust to a world inverted by a special pair of glasses. This helps illustrate how dependent the human vestibular system is on visual cues. A volunteer is The activity was part of the Space Research and You education event held by NASA's Office of Biological and Physical Research on June 25, 2002, in Arlington, VA, to highlight the research that will be conducted on STS-107.

  15. Linear control of the flywheel inverted pendulum.

    PubMed

    Olivares, Manuel; Albertos, Pedro

    2014-09-01

    The flywheel inverted pendulum is an underactuated mechanical system with a nonlinear model but admitting a linear approximation around the unstable equilibrium point in the upper position. Although underactuated systems usually require nonlinear controllers, the easy tuning and understanding of linear controllers make them more attractive for designers and final users. In a recent paper, a simple PID controller was proposed by the authors, leading to an internally unstable controlled plant. To achieve global stability, two options are developed here: first by introducing an internal stabilizing controller and second by replacing the PID controller by an observer-based state feedback control. Simulation and experimental results show the effectiveness of the design.

  16. Novel multilevel inverter carrier-based PWM method

    SciTech Connect

    Tolbert, L.M.; Habetler, T.G.

    1999-10-01

    The advent of the transformerless multilevel inverter topology has brought forth various pulsewidth modulation (PWM) schemes as a means to control the switching of the active devices in each of the multiple voltage levels in the inverter. An analysis of how existing multilevel carrier-based PWM affects switch utilization for the different levels of a diode-clamped inverter is conducted. Two novel carrier-based multilevel PWM schemes are presented which help to optimize or balance the switch utilization in multilevel inverters. A 10-kW prototype six-level diode-clamped inverter has been built and controlled with the novel PWM strategies proposed in this paper to act as a voltage-source inverter for a motor drive.

  17. On Inverting the Heat Flow with Engineering Materials

    NASA Astrophysics Data System (ADS)

    Zhou, Li

    2016-02-01

    Transformation thermodynamics enriches our understanding of heat flow and makes it possible to manipulate the heat flow at will, like shielding, concentrating and inverting. The inverting of heat flow is the extreme one, which has not been studied specifically yet. In this study we firstly inverted the heat flow by transformation thermodynamics and provided the formula for the transformed thermal conductivity. Finite element simulations were conducted to realize the steady and non-steady inverting of heat flow, based on the eccentric-semi-ring structures with natural materials. To do the inverting of heat flow, a simple "L"-shape conductive structure was proposed and verified with an infrared camera. It is concluded that inverting heat flow can be done by both complex engineering materials and some simple structures.

  18. Multilevel cascade voltage source inverter with seperate DC sources

    DOEpatents

    Peng, Fang Zheng; Lai, Jih-Sheng

    1997-01-01

    A multilevel cascade voltage source inverter having separate DC sources is described herein. This inverter is applicable to high voltage, high power applications such as flexible AC transmission systems (FACTS) including static VAR generation (SVG), power line conditioning, series compensation, phase shifting and voltage balancing and fuel cell and photovoltaic utility interface systems. The M-level inverter consists of at least one phase wherein each phase has a plurality of full bridge inverters equipped with an independent DC source. This inverter develops a near sinusoidal approximation voltage waveform with only one switching per cycle as the number of levels, M, is increased. The inverter may have either single-phase or multi-phase embodiments connected in either wye or delta configurations.

  19. Multilevel cascade voltage source inverter with separate DC sources

    DOEpatents

    Peng, F.Z.; Lai, J.S.

    1997-06-24

    A multilevel cascade voltage source inverter having separate DC sources is described herein. This inverter is applicable to high voltage, high power applications such as flexible AC transmission systems (FACTS) including static VAR generation (SVG), power line conditioning, series compensation, phase shifting and voltage balancing and fuel cell and photovoltaic utility interface systems. The M-level inverter consists of at least one phase wherein each phase has a plurality of full bridge inverters equipped with an independent DC source. This inverter develops a near sinusoidal approximation voltage waveform with only one switching per cycle as the number of levels, M, is increased. The inverter may have either single-phase or multi-phase embodiments connected in either wye or delta configurations. 15 figs.

  20. Multilevel cascade voltage source inverter with seperate DC sources

    DOEpatents

    Peng, Fang Zheng; Lai, Jih-Sheng

    2002-01-01

    A multilevel cascade voltage source inverter having separate DC sources is described herein. This inverter is applicable to high voltage, high power applications such as flexible AC transmission systems (FACTS) including static VAR generation (SVG), power line conditioning, series compensation, phase shifting and voltage balancing and fuel cell and photovoltaic utility interface systems. The M-level inverter consists of at least one phase wherein each phase has a plurality of full bridge inverters equipped with an independent DC source. This inverter develops a near sinusoidal approximation voltage waveform with only one switching per cycle as the number of levels, M, is increased. The inverter may have either single-phase or multi-phase embodiments connected in either wye or delta configurations.

  1. Multilevel cascade voltage source inverter with seperate DC sources

    DOEpatents

    Peng, Fang Zheng; Lai, Jih-Sheng

    2001-04-03

    A multilevel cascade voltage source inverter having separate DC sources is described herein. This inverter is applicable to high voltage, high power applications such as flexible AC transmission systems (FACTS) including static VAR generation (SVG), power line conditioning, series compensation, phase shifting and voltage balancing and fuel cell and photovoltaic utility interface systems. The M-level inverter consists of at least one phase wherein each phase has a plurality of full bridge inverters equipped with an independent DC source. This inverter develops a near sinusoidal approximation voltage waveform with only one switching per cycle as the number of levels, M, is increased. The inverter may have either single-phase or multi-phase embodiments connected in either wye or delta configurations.

  2. Apollo 14 inverted pigeonites - Possible samples of lunar plutonic rocks.

    NASA Technical Reports Server (NTRS)

    Papike, J. J.; Bence, A. E.

    1972-01-01

    Analysis of 'inverted pigeonites' found in Apollo 14 samples 14082 and 14083 (a polymict breccia, the 'white rock') by a combination of optical, electron probe, and single-crystal X-ray diffraction techniques. These 'inverted pigeonites' are regarded as samples of plutonic rocks that have been blasted out of the Imbrium Basin. It is also concluded that lunar pigeonites will invert to orthopyroxenes, given sufficiently slow cooling histories even in very anhydrous environments.

  3. Integral inverter/battery charger for use in electric vehicles

    NASA Technical Reports Server (NTRS)

    Thimmesch, D.

    1983-01-01

    The design and test results of a thyristor based inverter/charger are discussed. A battery charger is included integral to the inverter by using a subset of the inverter power circuit components. The resulting charger provides electrical isolation between the vehicle propulsion battery and ac line and is capable of charging a 25 kWh propulsion battery in 8 hours from a 220 volt ac line. The integral charger employs the inverter commutation components at a resonant ac/dc isolated converter rated at 3.6 kW. Charger efficiency and power factor at an output power of 3.6 kW are 86% and 95% respectively. The inverter, when operated with a matching polyphase ac induction motor and nominal 132 volt propulsion battery, can provide a peak shaft power of 34 kW (45 ph) during motoring operation and 45 kW (60 hp) during regeneration. Thyristors are employed for the inverter power switching devices and are arranged in an input-commutated topology. This configuration requires only two thyristors to commutate the six main inverter thyristors. Inverter efficiency during motoring operation at motor shaft speeds above 450 rad/sec (4300 rpm) is 92-94% for output power levels above 11 KW (15 hp). The combined ac inverter/charger package weighs 47 kg (103 lbs).

  4. An inverter/controller subsystem optimized for photovoltaic applications

    NASA Technical Reports Server (NTRS)

    Pickrell, R. L.; Osullivan, G.; Merrill, W. C.

    1978-01-01

    Conversion of solar array dc power to ac power stimulated the specification, design, and simulation testing of an inverter/controller subsystem tailored to the photovoltaic power source characteristics. Optimization of the inverter/controller design is discussed as part of an overall photovoltaic power system designed for maximum energy extraction from the solar array. The special design requirements for the inverter/ controller include: a power system controller (PSC) to control continuously the solar array operating point at the maximum power level based on variable solar insolation and cell temperatures; and an inverter designed for high efficiency at rated load and low losses at light loadings to conserve energy.

  5. Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery.

    PubMed

    Mitrani-Rosenbaum, Stella; Yakovlev, Lena; Becker Cohen, Michal; Telem, Michal; Elbaz, Moran; Yanay, Nurit; Yotvat, Hagit; Ben Shlomo, Uri; Harazi, Avi; Fellig, Yakov; Argov, Zohar; Sela, Ilan

    2012-11-01

    GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

  6. AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse.

    PubMed

    Lee, E K; Hu, C; Bhargava, R; Ponnusamy, R; Park, H; Novicoff, S; Rozengurt, N; Marescau, B; De Deyn, P; Stout, D; Schlichting, L; Grody, W W; Cederbaum, S D; Lipshutz, G S

    2013-08-01

    Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

  7. One-time injection of AAV8 encoding urocortin 2 provides long-term resolution of insulin resistance

    PubMed Central

    Gao, Mei Hua; Giamouridis, Dimosthenis; Lai, N. Chin; Walenta, Evelyn; Paschoal, Vivian Almeida; Kim, Young Chul; Miyanohara, Atsushi; Guo, Tracy; Liao, Min; Liu, Li; Ciaraldi, Theodore P.; Bhargava, Aditi; Oh, Da Young; Hammond, H. Kirk

    2016-01-01

    Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2’s cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes. PMID:27699250

  8. Cell-Type Specific Oxytocin Gene Expression from AAV Delivered Promoter Deletion Constructs into the Rat Supraoptic Nucleus in vivo

    PubMed Central

    Kawasaki, Makoto; Gainer, Harold

    2012-01-01

    The magnocellular neurons (MCNs) in the hypothalamus selectively express either oxytocin (OXT) or vasopressin (AVP) neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV) vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON). Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located −216 to −100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs. PMID:22363799

  9. AAV-Mediated Gene Delivery in a Feline Model of Sandhoff Disease Corrects Lysosomal Storage in the Central Nervous System

    PubMed Central

    Rockwell, Hannah E.; McCurdy, Victoria J.; Eaton, Samuel C.; Wilson, Diane U.; Johnson, Aime K.; Randle, Ashley N.; Bradbury, Allison M.; Gray-Edwards, Heather L.; Baker, Henry J.; Hudson, Judith A.; Cox, Nancy R.; Sena-Esteves, Miguel; Seyfried, Thomas N.

    2015-01-01

    Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients. PMID:25873306

  10. One-time injection of AAV8 encoding urocortin 2 provides long-term resolution of insulin resistance

    PubMed Central

    Gao, Mei Hua; Giamouridis, Dimosthenis; Lai, N. Chin; Walenta, Evelyn; Paschoal, Vivian Almeida; Kim, Young Chul; Miyanohara, Atsushi; Guo, Tracy; Liao, Min; Liu, Li; Ciaraldi, Theodore P.; Bhargava, Aditi; Oh, Da Young; Hammond, H. Kirk

    2016-01-01

    Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2’s cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes.

  11. Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.

    PubMed

    Oceguera-Yanez, Fabian; Kim, Shin-Il; Matsumoto, Tomoko; Tan, Ghee Wan; Xiang, Long; Hatani, Takeshi; Kondo, Takayuki; Ikeya, Makoto; Yoshida, Yoshinori; Inoue, Haruhisa; Woltjen, Knut

    2016-05-15

    The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping. PMID:26707206

  12. AAV8-mediated in vivo overexpression of miR-155 enhances the protective capacity of genetically attenuated malarial parasites.

    PubMed

    Hentzschel, Franziska; Hammerschmidt-Kamper, Christiane; Börner, Kathleen; Heiss, Kirsten; Knapp, Bettina; Sattler, Julia M; Kaderali, Lars; Castoldi, Mirco; Bindman, Julia G; Malato, Yann; Willenbring, Holger; Mueller, Ann-Kristin; Grimm, Dirk

    2014-12-01

    Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.

  13. Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.

    PubMed

    Oceguera-Yanez, Fabian; Kim, Shin-Il; Matsumoto, Tomoko; Tan, Ghee Wan; Xiang, Long; Hatani, Takeshi; Kondo, Takayuki; Ikeya, Makoto; Yoshida, Yoshinori; Inoue, Haruhisa; Woltjen, Knut

    2016-05-15

    The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

  14. AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa.

    PubMed

    Mao, Haoyu; James, Thomas; Schwein, Alison; Shabashvili, Arseniy E; Hauswirth, William W; Gorbatyuk, Marina S; Lewin, Alfred S

    2011-05-01

    Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

  15. Intrathecal administration of AAV vectors for the treatment of lysosomal storage in the brains of MPS I mice.

    PubMed

    Watson, G; Bastacky, J; Belichenko, P; Buddhikot, M; Jungles, S; Vellard, M; Mobley, W C; Kakkis, E

    2006-06-01

    Mucopolysaccharidosis type I (MPS I) is caused by an inherited deficiency of alpha-L-iduronidase (IDUA). The result is a progressive, lysosomal storage disease with central nervous system (CNS) as well as systemic involvement. To target gene therapy to the CNS, recombinant adeno-associated virus (AAV) vectors carrying IDUA sequence were administered to MPS I mice via injection into cerebrospinal fluid. In contrast to intravenous administration, this intrathecal administration was effective in generating widespread IDUA activity in the brain, with the cerebellum and olfactory bulbs having highest activities. In general, IDUA levels correlated with vector dose, although this correlation was obscured in cerebellum by particularly high variability. High doses of vector (4 x 10(10) particles) provided IDUA levels approaching or exceeding normal levels in the brain. Histopathology indicated that the number of cells with storage vacuoles was reduced extensively or was eliminated entirely. Elimination of storage material in Purkinje cells was particularly dramatic. A lower vector dose (2 x 10(9) particles) reduced both the number of storage cells and the extent of storage per cell, but the effect was not complete. Some perivascular cells with storage persisted, and this cell type appeared to be more resistant to treatment than neurons or glial cells. We conclude that intrathecal administration of AAV-IDUA delivers vector to brain cells, and that this route of administration is both minimally invasive and effective.

  16. Lightweight PV Inverters: Dual Bi-Directional IGBTs Modules Enables Breakthrough PV Inverter Using Current Modulation Topology

    SciTech Connect

    2012-01-30

    Solar ADEPT Project: PV inverters convert DC power generated by modules into usable AC power. IPC’s initial 30kW 94lb. PV inverter reduces the weight of comparable 30kW PV inverters by 90%—reducing the cost of materials, manufacturing, shipping, and installation. With ARPA-E support, new bi-directional silicon power switches will be developed, commercialized, and utilized in IPC’s next-generation PV inverter. With these components, IPC will produce 100kW inverters that weight less than 100lb., reducing the weight of conventional 3,000lb. 100kW inverters by more than 95%. The new power switches will cut IPC’s $/W manufacturing cost in half, as well as further reduce indirect shipping and installation costs.

  17. Towards high performance inverted polymer solar cells

    NASA Astrophysics Data System (ADS)

    Gong, Xiong

    2013-03-01

    Bulk heterojunction polymer solar cells that can be fabricated by solution processing techniques are under intense investigation in both academic institutions and industrial companies because of their potential to enable mass production of flexible and cost-effective alternative to silicon-based electronics. Despite the envisioned advantages and recent technology advances, so far the performance of polymer solar cells is still inferior to inorganic counterparts in terms of the efficiency and stability. There are many factors limiting the performance of polymer solar cells. Among them, the optical and electronic properties of materials in the active layer, device architecture and elimination of PEDOT:PSS are the most determining factors in the overall performance of polymer solar cells. In this presentation, I will present how we approach high performance of polymer solar cells. For example, by developing novel materials, fabrication polymer photovoltaic cells with an inverted device structure and elimination of PEDOT:PSS, we were able to observe over 8.4% power conversion efficiency from inverted polymer solar cells.

  18. Dual Mode Inverter Control Test Verification

    SciTech Connect

    Bailey, J.M.

    2001-04-25

    Permanent Magnet Motors with either sinusoidal back emf (permanent magnet synchronous motor [PMSM]) or trapezoidal back emf (brushless dc motor [BDCM]) do not have the ability to alter the air gap flux density (field weakening). Since the back emf increases with speed, the system must be designed to operate with the voltage obtained at its highest speed. Oak Ridge National Laboratory's (ORNL) Power Electronics and Electric Machinery Research Center (PEEMRC) has developed a dual mode inverter controller (DMIC) that overcomes this disadvantage. This report summarizes the results of tests to verify its operation. The standard PEEMRC 75 kW hard-switched inverter was modified to implement the field weakening procedure (silicon controlled rectifier enabled phase advance). A 49.5 hp motor rated at 2800 rpm was derated to a base of 400 rpm and 7.5 hp. The load developed by a Kahn Industries hydraulic dynamometer, was measured with a MCRT9-02TS Himmelstein and Company torque meter. At the base conditions a current of 212 amperes produced the 7.5 hp. Tests were run at 400, 1215, and 2424 rpm. In each run, the current was no greater than 214 amperes. The horsepower obtained in the three runs were 7.5, 9.3, and 8.12. These results verified the basic operation of the DMIC in producing a Constant Power Speed Ratios (CPSR) of six.

  19. Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.

    PubMed

    Song, Yuhu; Lou, Howard H; Boyer, Julie L; Limberis, Maria P; Vandenberghe, Luk H; Hackett, Neil R; Leopold, Philip L; Wilson, James M; Crystal, Ronald G

    2009-03-01

    Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.

  20. Distinct Expression Patterns of AAV8 Vectors with Broadly Active Promoters from Subretinal Injections of Neonatal Mouse Eyes at Two Different Ages.

    PubMed

    Xiong, Wenjun; Cepko, Constance

    2016-01-01

    The retinal expression patterns were analyzed following the injection of serotype 8 adeno-associated virus (AAV8) vectors that utilize two broadly active and commonly used sets of transcription regulatory sequences. These include the human cytomegalovirus (CMV) immediate early (IE) enhancer/promoter and the hybrid CAG element (also known as CAGGS or CBA) composed of a partial human CMV IE enhancer and the chicken β-actin promoter and intron. Subretinal delivery to postnatal day 0 (P0) or 6 (P6) mouse eyes resulted in efficient labeling of retinal cells, but with very distinct patterns. With P0 delivery, AAV8-CMV-GFP selectively labelled photoreceptors, while AAV8-CAG-GFP efficiently labeled both outer and inner retinal neurons, including photoreceptors, horizontal cells, amacrine cells and retinal ganglion cells. With P6 delivery, both vectors led to efficient labeling of photoreceptors and Müller glia cells, but not of inner retinal neurons. Our results suggest that the cell types that express the genes encoded by subretinally delivered AAV8 vectors are determined by both the timing of the injection and the regulatory sequences.

  1. AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease

    PubMed Central

    Katz, Martin L.; Tecedor, Luis; Chen, Yonghong; Williamson, Baye G.; Lysenko, Elena; Wininger, Fred A.; Young, Whitney M.; Johnson, Gayle C.; Whiting, Rebecca E. H.; Coates, Joan R.; Davidson, Beverly L.

    2016-01-01

    The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit. PMID:26560358

  2. Vitreal delivery of AAV vectored Cnga3 restores cone function in CNGA3−/−/Nrl−/− mice, an all-cone model of CNGA3 achromatopsia†

    PubMed Central

    Du, Wei; Tao, Ye; Deng, Wen-Tao; Zhu, Ping; Li, Jie; Dai, Xufeng; Zhang, Yuxin; Shi, Wei; Liu, Xuan; Chiodo, Vince A.; Ding, Xi-Qin; Zhao, Chen; Michalakis, Stylianos; Biel, Martin; Zhang, Zuoming; Qu, Jia; Hauswirth, William W.; Pang, Ji-jing

    2015-01-01

    The CNGA3−/−/Nrl−/− mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function–structure could be rescued in CNGA3−/−/Nrl−/− mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function. PMID:25855802

  3. Targeting Channelrhodopsin-2 to ON-bipolar Cells With Vitreally Administered AAV Restores ON and OFF Visual Responses in Blind Mice

    PubMed Central

    Macé, Emilie; Caplette, Romain; Marre, Olivier; Sengupta, Abhishek; Chaffiol, Antoine; Barbe, Peggy; Desrosiers, Mélissa; Bamberg, Ernst; Sahel, Jose-Alain; Picaud, Serge; Duebel, Jens; Dalkara, Deniz

    2015-01-01

    Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell–specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration. PMID:25095892

  4. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

    PubMed Central

    Cereso, Nicolas; Pequignot, Marie O; Robert, Lorenne; Becker, Fabienne; De Luca, Valerie; Nabholz, Nicolas; Rigau, Valerie; De Vos, John; Hamel, Christian P; Kalatzis, Vasiliki

    2014-01-01

    Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC–derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model. PMID:26015956

  5. View of the upper part of the Inverted Siphon showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of the upper part of the Inverted Siphon showing sandbox at the top of the slope before dropping into the Hot Water (or White) Canyon. Looking northeast - Childs-Irving Hydroelectric Project, Irving System, Inverted Siphon, Forest Service Road 708/502, Camp Verde, Yavapai County, AZ

  6. Three-phase inverter for small high speed motors

    NASA Technical Reports Server (NTRS)

    Mccormick, John A.; Valenzuela, Javier A.

    1991-01-01

    A high-frequency three-phase inverter is being developed to drive a miniature centrifugal compressor which is a key component in a long-life space-borne cryocooler. The inverter is a unique transformer-coupled design, tailored to the low-voltage high-current characteristic of the compressor's induction motor. The design and performance demonstration of a breadboard model of the inverter are described. The cryocooler uses a reverse-Brayton cycle with turbomachines to provide 5 watt of cooling at 70 K. The design target for input power to the compressor motor is 175 watts. Line-to-neutral phase voltage waveforms to be supplied by the inverter have an amplitude of 15 volt-rms at a frequency of 8 kHz. DC power at 28 volt is supplied to the inverter. The breadboard inverter was tested with a preliminary development model of the compressor. It drove the compressor over a range of operating conditions encompassing frequencies of 5 to 9 kHz at powers of 56 to 437 watt. Inverter efficiencies, calculated from experimentally verified loss models, ranged from 89 to 95 percent over the tests. The design target on efficiency is 90 percent. The inverter was demonstrated to supply starting current adequate to overcome the starting friction of the compressor's self-acting gas bearings by a safe margin.

  7. Novel optoelectronic RS flipflop based on optically coupled inverters

    NASA Astrophysics Data System (ADS)

    Chino, T.; Matsuda, K.; Adachi, H.; Shibata, J.

    1992-03-01

    An optoelectronic RS flipflop emitting differential output has been proposed and demonstrated. It consists of two optical inverters. Optical interconnections are used to couple these inverters. Stable operation for the variation of bias voltage is demonstrated, which exhibits the possibility for 2-dimensional integration.

  8. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

  9. Quasicompactons in inverted nonlinear photonic crystals

    SciTech Connect

    Li Yongyao; Malomed, Boris A.; Wu Jianxiong; Pang Wei; Wang Sicong; Zhou Jianying

    2011-10-15

    We study large-amplitude one-dimensional solitary waves in photonic crystals featuring competition between linear and nonlinear lattices, with minima of the linear potential coinciding with maxima of the nonlinear pseudopotential, and vice versa (inverted nonlinear photonic crystals, INPCs), in the case of the saturable self-focusing nonlinearity. Such crystals were recently fabricated using a mixture of SU-8 and Rhodamine-B optical materials. By means of numerical methods and analytical approximations, we find that large-amplitude solitons are broad sharply localized stable pulses (quasicompactons, QCs). With the increase of the total power, P, the QC's centroid performs multiple switchings between minima and maxima of the linear potential. Unlike cubic INPCs, the large-amplitude solitons are mobile in the medium with the saturable nonlinearity. The threshold value of the kick necessary to set the soliton in motion is found as a function of P. Collisions between moving QCs are considered too.

  10. HOLLOTRON switch for megawatt lightweight space inverters

    NASA Technical Reports Server (NTRS)

    Poeschel, R. L.; Goebel, D. M.; Schumacher, R. W.

    1991-01-01

    The feasibility of satisfying the switching requirements for a megawatt ultralight inverter system using HOLLOTRON switch technology was determined. The existing experimental switch hardware was modified to investigate a coaxial HOLLOTRON switch configuration and the results were compared with those obtained for a modified linear HOLLOTRON configuration. It was concluded that scaling the HOLLOTRON switch to the current and voltage specifications required for a megawatt converter system is indeed feasible using a modified linear configuration. The experimental HOLLOTRON switch operated at parameters comparable to the scaled coaxial HOLLOTRON. However, the linear HOLLOTRON data verified the capability for meeting all the design objectives simultaneously including current density (greater than 2 A/sq cm), voltage (5 kV), switching frequency (20 kHz), switching time (300 ns), and forward voltage drop (less than or equal to 20 V). Scaling relations were determined and a preliminary design was completed for an engineering model linear HOLLOTRON switch to meet the megawatt converter system specifications.

  11. Scalar field cosmologies with inverted potentials

    SciTech Connect

    Boisseau, B.; Giacomini, H.

    2015-10-01

    Regular bouncing solutions in the framework of a scalar-tensor gravity model were found in a recent work. We reconsider the problem in the Einstein frame (EF) in the present work. Singularities arising at the limit of physical viability of the model in the Jordan frame (JF) are either of the Big Bang or of the Big Crunch type in the EF. As a result we obtain integrable scalar field cosmological models in general relativity (GR) with inverted double-well potentials unbounded from below which possess solutions regular in the future, tending to a de Sitter space, and starting with a Big Bang. The existence of the two fixed points for the field dynamics at late times found earlier in the JF becomes transparent in the EF.

  12. Linear control of the flywheel inverted pendulum.

    PubMed

    Olivares, Manuel; Albertos, Pedro

    2014-09-01

    The flywheel inverted pendulum is an underactuated mechanical system with a nonlinear model but admitting a linear approximation around the unstable equilibrium point in the upper position. Although underactuated systems usually require nonlinear controllers, the easy tuning and understanding of linear controllers make them more attractive for designers and final users. In a recent paper, a simple PID controller was proposed by the authors, leading to an internally unstable controlled plant. To achieve global stability, two options are developed here: first by introducing an internal stabilizing controller and second by replacing the PID controller by an observer-based state feedback control. Simulation and experimental results show the effectiveness of the design. PMID:24480638

  13. Terminal Air Flow Planning

    NASA Technical Reports Server (NTRS)

    Denery, Dallas G.; Erzberger, Heinz; Edwards, Thomas A. (Technical Monitor)

    1998-01-01

    The Center TRACON Automation System (CTAS) will be the basis for air traffic planning and control in the terminal area. The system accepts arriving traffic within an extended terminal area and optimizes the flow based on current traffic and airport conditions. The operational use of CTAS will be presented together with results from current operations.

  14. Superconducting Cable Termination

    DOEpatents

    Sinha, Uday K.; Tolbert, Jerry

    2005-08-30

    Disclosed is a termination that connects high temperature superconducting (HTS) cable immersed in pressurized liquid nitrogen to high voltage and neutral (shield) external bushings at ambient temperature and pressure. The termination consists of a splice between the HTS power (inner) and shield (outer) conductors and concentric copper pipes which are the conductors in the termination. There is also a transition from the dielectric tape insulator used in the HTS cable to the insulators used between and around the copper pipe conductors in the termination. At the warm end of the termination the copper pipes are connected via copper braided straps to the conventional warm external bushings which have low thermal stresses. This termination allows for a natural temperature gradient in the copper pipe conductors inside the termination which enables the controlled flashing of the pressurized liquid coolant (nitrogen) to the gaseous state. Thus the entire termination is near the coolant supply pressure and the high voltage and shield cold bushings, a highly stressed component used in most HTS cables, are eliminated. A sliding seal allows for cable contraction as it is cooled from room temperature to ˜72-82 K. Seals, static vacuum, and multi-layer superinsulation minimize radial heat leak to the environment.

  15. Highly efficient fully transparent inverted OLEDs

    NASA Astrophysics Data System (ADS)

    Meyer, J.; Winkler, T.; Hamwi, S.; Schmale, S.; Kröger, M.; Görrn, P.; Johannes, H.-H.; Riedl, T.; Lang, E.; Becker, D.; Dobbertin, T.; Kowalsky, W.

    2007-09-01

    One of the unique selling propositions of OLEDs is their potential to realize highly transparent devices over the visible spectrum. This is because organic semiconductors provide a large Stokes-Shift and low intrinsic absorption losses. Hence, new areas of applications for displays and ambient lighting become accessible, for instance, the integration of OLEDs into the windshield or the ceiling of automobiles. The main challenge in the realization of fully transparent devices is the deposition of the top electrode. ITO is commonly used as transparent bottom anode in a conventional OLED. To obtain uniform light emission over the entire viewing angle and a low series resistance, a TCO such as ITO is desirable as top contact as well. However, sputter deposition of ITO on top of organic layers causes damage induced by high energetic particles and UV radiation. We have found an efficient process to protect the organic layers against the ITO rf magnetron deposition process of ITO for an inverted OLED (IOLED). The inverted structure allows the integration of OLEDs in more powerful n-channel transistors used in active matrix backplanes. Employing the green electrophosphorescent material Ir(ppy) 3 lead to IOLED with a current efficiency of 50 cd/A and power efficiency of 24 lm/W at 100 cd/m2. The average transmittance exceeds 80 % in the visible region. The on-set voltage for light emission is lower than 3 V. In addition, by vertical stacking we achieved a very high current efficiency of more than 70 cd/A for transparent IOLED.

  16. Repetition probability effects for inverted faces.

    PubMed

    Grotheer, Mareike; Hermann, Petra; Vidnyánszky, Zoltán; Kovács, Gyula

    2014-11-15

    It has been shown, that the repetition related reduction of the blood-oxygen level dependent (BOLD) signal is modulated by the probability of repetitions (P(rep)) for faces (Summerfield et al., 2008), providing support for the predictive coding (PC) model of visual perception (Rao and Ballard, 1999). However, the stage of face processing where repetition suppression (RS) is modulated by P(rep) is still unclear. Face inversion is known to interrupt higher level configural/holistic face processing steps and if modulation of RS by P(rep) takes place at these stages of face processing, P(rep) effects are expected to be reduced for inverted when compared to upright faces. Therefore, here we aimed at investigating whether P(rep) effects on RS observed for face stimuli originate at the higher-level configural/holistic stages of face processing by comparing these effects for upright and inverted faces. Similarly to previous studies, we manipulated P(rep) for pairs of stimuli in individual blocks of fMRI recordings. This manipulation significantly influenced repetition suppression in the posterior FFA, the OFA and the LO, independently of stimulus orientation. Our results thus reveal that RS in the ventral visual stream is modulated by P(rep) even in the case of face inversion and hence strongly compromised configural/holistic face processing. An additional whole-brain analysis could not identify any areas where the modulatory effect of probability was orientation specific either. These findings imply that P(rep) effects on RS might originate from the earlier stages of face processing.

  17. Transistorized PWM inverter-induction motor drive system

    NASA Technical Reports Server (NTRS)

    Peak, S. C.; Plunkett, A. B.

    1982-01-01

    This paper describes the development of a transistorized PWM inverter-induction motor traction drive system. A vehicle performance analysis was performed to establish the vehicle tractive effort-speed requirements. These requirements were then converted into a set of inverter and motor specifications. The inverter was a transistorized three-phase bridge using General Electric power Darlington transistors. The description of the design and development of this inverter is the principal object of this paper. The high-speed induction motor is a design which is optimized for use with an inverter power source. The primary feedback control is a torque angle control with voltage and torque outer loop controls. A current-controlled PWM technique is used to control the motor voltage. The drive has a constant torque output with PWM operation to base motor speed and a constant horsepower output with square wave operation to maximum speed. The drive system was dynamometer tested and the results are presented.

  18. Evaluation of a high power inverter for potential space applications

    NASA Technical Reports Server (NTRS)

    Guynes, B. V.; Lanier, J. R., Jr.

    1976-01-01

    The ADM-006 inverter discussed utilizes a unique method of using power switching circuits to produce three-phase low harmonic content voltages without any significant filtering. This method is referred to as the power center approach to inverter design and is explained briefly. The results are presented of tests performed by MSFC to evaluate inverter performance, especially when required to provide power to nonlinear loads such as half or full wave rectified loads with capacitive filtering. Test preocedures and results are described. These tests show that the power center inverter essentially met or exceeded all of claims excluding voltage regulation (3.9 percent versus specified 3.3 percent) and would be a good candidate for high power inverter applications such as may be found on Space Station, Spacelab, etc.

  19. A ZnO nanowire-based photo-inverter with pulse-induced fast recovery

    NASA Astrophysics Data System (ADS)

    Ali Raza, Syed Raza; Lee, Young Tack; Hosseini Shokouh, Seyed Hossein; Ha, Ryong; Choi, Heon-Jin; Im, Seongil

    2013-10-01

    We demonstrate a fast response photo-inverter comprised of one transparent gated ZnO nanowire field-effect transistor (FET) and one opaque FET respectively as the driver and load. Under ultraviolet (UV) light the transfer curve of the transparent gate FET shifts to the negative side and so does the voltage transfer curve (VTC) of the inverter. After termination of UV exposure the recovery of photo-induced current takes a long time in general. This persistent photoconductivity (PPC) is due to hole trapping on the surface of ZnO NWs. Here, we used a positive voltage short pulse after UV exposure, for the first time resolving the PPC issue in nanowire-based photo-detectors by accumulating electrons at the ZnO/dielectric interface. We found that a pulse duration as small as 200 ns was sufficient to reach a full recovery to the dark state from the UV induced state, realizing a fast UV detector with a voltage output.We demonstrate a fast response photo-inverter comprised of one transparent gated ZnO nanowire field-effect transistor (FET) and one opaque FET respectively as the driver and load. Under ultraviolet (UV) light the transfer curve of the transparent gate FET shifts to the negative side and so does the voltage transfer curve (VTC) of the inverter. After termination of UV exposure the recovery of photo-induced current takes a long time in general. This persistent photoconductivity (PPC) is due to hole trapping on the surface of ZnO NWs. Here, we used a positive voltage short pulse after UV exposure, for the first time resolving the PPC issue in nanowire-based photo-detectors by accumulating electrons at the ZnO/dielectric interface. We found that a pulse duration as small as 200 ns was sufficient to reach a full recovery to the dark state from the UV induced state, realizing a fast UV detector with a voltage output. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03801g

  20. Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro

    NASA Astrophysics Data System (ADS)

    Lange, Wienke; Jin, Lei; Maybeck, Vanessa; Meisenberg, Annika; Baumann, Arnd; Offenhäusser, Andreas

    2014-03-01

    Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

  1. AAV-mediated, optogenetic ablation of Müller Glia leads to structural and functional changes in the mouse retina.

    PubMed

    Byrne, Leah C; Khalid, Fakhra; Lee, Trevor; Zin, Emilia A; Greenberg, Kenneth P; Visel, Meike; Schaffer, David V; Flannery, John G

    2013-01-01

    Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies. PMID:24086689

  2. AAV-Mediated, Optogenetic Ablation of Müller Glia Leads to Structural and Functional Changes in the Mouse Retina

    PubMed Central

    Byrne, Leah C.; Khalid, Fakhra; Lee, Trevor; Zin, Emilia A.; Greenberg, Kenneth P.; Visel, Meike; Schaffer, David V.; Flannery, John G.

    2013-01-01

    Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies. PMID:24086689

  3. Human melanopsin-AAV2/8 transfection to retina transiently restores visual function in rd1 mice

    PubMed Central

    Liu, Ming-Ming; Dai, Jia-Man; Liu, Wen-Yi; Zhao, Cong-Jian; Lin, Bin; Yin, Zheng-Qin

    2016-01-01

    AIM To explore whether ectopic expression of human melanopsin can effectively and safely restore visual function in rd1 mice. METHODS Hematoxylin-eosin staining of retinal sections from rd1 mice was used to detect the thickness of the outer nuclear layer to determine the timing of surgery. We constructed a human melanopsin-AAV2/8 viral vector and injected it into the subretinal space of rd1 mice. The Phoenix Micron IV system was used to exclude the aborted injections, and immunohistochemistry was used to validate the ectopic expression of human melanopsin. Furthermore, visual electrophysiology and behavioral tests were used to detect visual function 30 and 45d after the injection. The structure of the retina was compared between the human melanopsin-injected group and phosphate buffer saline (PBS)-injected group. RESULTS Retinas of rd1 mice lost almost all of their photoreceptors on postnatal day 28 (P28). We therefore injected the human melanopsin-adeno-associated virus (AAV) 2/8 viral vector into P30 rd1 mice. After excluding aborted injections, we used immunohistochemistry of the whole mount retina to confirm the ectopic expression of human melanopsin by co-expression of human melanopsin and YFP that was carried by a viral vector. At 30d post-injection, visual electrophysiology and the behavioral test significantly improved. However, restoration of vision disappeared 45d after human melanopsin injection. Notably, human melanopsin-injected mice did not show any structural differences in their retinas compared with PBS-injected mice. CONCLUSION Ectopic expression of human melanopsin effectively and safely restores visual function in rd1 mice. PMID:27275417

  4. Phenylethynyl terminated imide oligomers

    NASA Technical Reports Server (NTRS)

    Hergenrother, Paul M. (Inventor); Bryant, Robert G. (Inventor); Jensen, Brian J. (Inventor); Havens, Stephen J. (Inventor)

    1995-01-01

    Four phenylethynyl amine compounds - 3 and 4-aminophenoxy-4'-phenylethynylbenzophenone, and 3 and 4-amino-4'-phenylethynylbenzophenone - were readily prepared and were used to endcap imide oligomers. Phenylethynyl-terminated amide acid oligomers and phenylethynyl-terminated imide oligomers with various molecular weights and compositions were prepared and characterized. These oligomers were cured at 300 to 400 C to provide crosslinked polyimides with excellent solvent resistance, high strength and modulus, and good high temperature properties. Adhesive panels, composites, films, and moldings from these phenylethynyl terminated imide oligomers gave excellent mechanical performance.

  5. Sail intelligent terminal evaluation

    NASA Technical Reports Server (NTRS)

    Pruitt, J. L.

    1977-01-01

    Engineering assessments, recommendations, and equipment necessary to solve the operational problems are described, and operational flexibility of the intelligent terminal facility are extended. The following capabilities were considered: (1) the operation of at least two D/D stations and one remote graphics terminal simultaneously; (2) the capability to run plotter, AIDS and FORTRAN programs simultaneously; (3) simultaneous use of system utility routines of D/D stations and remote graphics terminal; (4) the capability to provide large volume hardcopy of data and graphics; and (5) the capability to eliminate or at least ease the current operation/programming problems with related labor costs. The overall intelligent terminal development, and plans guiding the analysis and equipment acquisitions were studied, and the assessments and analyses performed are also summarized.

  6. Nearest Alignment Space Termination

    2006-07-13

    Near Alignment Space Termination (NAST) is the Greengenes algorithm that matches up submitted sequences with the Greengenes database to look for similarities and align the submitted sequences based on those similarities.

  7. An iterative method to invert the LTSn matrix

    SciTech Connect

    Cardona, A.V.; Vilhena, M.T. de

    1996-12-31

    Recently Vilhena and Barichello proposed the LTSn method to solve, analytically, the Discrete Ordinates Problem (Sn problem) in transport theory. The main feature of this method consist in the application of the Laplace transform to the set of Sn equations and solve the resulting algebraic system for the transport flux. Barichello solve the linear system containing the parameter s applying the definition of matrix invertion exploiting the structure of the LTSn matrix. In this work, it is proposed a new scheme to invert the LTSn matrix, decomposing it in blocks and recursively inverting this blocks.

  8. A PWM transistor inverter for an ac electric vehicle drive

    NASA Technical Reports Server (NTRS)

    Slicker, J. M.

    1981-01-01

    A prototype system consisting of closely integrated motor, inverter, and transaxle has been built in order to demonstrate the feasibility of a three-phase ac transistorized inverter for electric vehicle applications. The microprocessor-controlled inverter employs monolithic power transistors to drive an oil-cooled, three-phase induction traction motor at a peak output power of 30 kW from a 144 V battery pack. Transistor safe switching requirements are discussed, and a circuit is presented for recovering trapped snubber inductor energy at transistor turn-off.

  9. Electric machine and current source inverter drive system

    DOEpatents

    Hsu, John S

    2014-06-24

    A drive system includes an electric machine and a current source inverter (CSI). This integration of an electric machine and an inverter uses the machine's field excitation coil for not only flux generation in the machine but also for the CSI inductor. This integration of the two technologies, namely the U machine motor and the CSI, opens a new chapter for the component function integration instead of the traditional integration by simply placing separate machine and inverter components in the same housing. Elimination of the CSI inductor adds to the CSI volumetric reduction of the capacitors and the elimination of PMs for the motor further improve the drive system cost, weight, and volume.

  10. Ru(II)-based metallosurfactant forming inverted aggregates.

    PubMed

    Domínguez-Gutiérrez, David; Surtchev, Marko; Eiser, Erika; Elsevier, Cornelis J

    2006-02-01

    Knowing the advantages of incorporating a transition metal into interfaces, we report on the first inverted aggregates formed using metallosurfactants. The metallosurfactant possesses four long linear tails that account for the shielding of the polar headgroup in apolar solvents. The nature of the so-formed aggregates changes dramatically from inverted vesicles (toluene) to inverted micelles (hexane). The size of the aggregates was determined using dynamic light scattering. Atomic force microscopy allowed us to study the dry structure of the vesicles on a glass surface.

  11. ACTS broadband aeronautical terminal

    NASA Technical Reports Server (NTRS)

    Agan, M. J.; Densmore, A. C.

    1995-01-01

    This paper discusses the design of, and experiments with, the ACTS Broadband Aeronautical Terminal. As part of the ongoing effort to investigate commercial applications of ACTS technologies, NASA's Jet Propulsion Laboratory and various industry/government partners are developing a broadband mobile terminal for aeronautical applications. The ACTS Broadband Aeronautical Terminal is designed to explore the use of K/Ka-band for high data rate aeronautical satellite communications. Currently available commercial aeronautical satellite communications systems are only capable of achieving data rates on the order of tens of kilobits per second. The broadband terminal used in conjunction with the ACTS mechanically steerable antenna, can achieve data rates of 384 kilobits per second, while use of an ACTS spot beam antenna with this terminal will allow up to T1 data rates (1.544 megabits per second). The aeronautical terminal will be utilized to test a variety of applications that require a high data rate communications link. The use of the K/Ka-band for wideband aeronautical communications has the advantages of spectrum availability and smaller antennas, while eliminating the one major drawback of this frequency band, rain attenuation, by flying above the clouds the majority of the time.

  12. Direct Comparison of Inverted and Non-Inverted Growths of GaInP Solar Cells: Preprint

    SciTech Connect

    Steiner, M. A.; Geisz, J. F.; Reedy Jr, R.C.; Kurtz, S.

    2008-05-01

    The inverted growth of III-V solar cells presents some specific challenges that are not present in regular, non-inverted growths. Because the highly doped top contact layer is grown first, followed by the lengthy high-temperature growth of the remainder of the structure, there is ample time for the dopants in the contact layer to diffuse away. This leads to an increase in the contact resistance to the top layer, and a corresponding drop in voltage. The diffusion of dopants in other layers is similarly altered with respect to the non-inverted configuration because of the change in growth sequence. We compare the dopant profiles of inverted and non-inverted structures by using secondary ion mass spectroscopy and correlate the results with the observed performance of the devices. We also describe a technique for growing a GaInAsN contact layer in the inverted configuration and show that it achieves a specific contact resistance comparable to what is normally observed in non-inverted cells.

  13. Skull Base Inverted Papilloma: A Comprehensive Review

    PubMed Central

    Wassef, Shafik N.; Batra, Pete S.; Barnett, Samuel

    2012-01-01

    Skull base inverted papilloma (IP) is an unusual entity for many neurosurgeons. IP is renowned for its high rate of recurrence, its ability to cause local destruction, and its association with malignancy. This paper is a comprehensive review of the reports, studies, and reviews published in the current biomedical literature from 1947 to September 2010 and synthesize this information to focus on its potential invasion to the base of the skull and possible intradural extension. The objective is to familiarize the clinician with the different aspects of this unusual disease. The role of modern diagnostic tools in medical imaging in order to assess clearly the limits of the tumors and to enhance the efficiency and the safety in the choice of a surgical approach is pointed out. The treatment guidelines for IP have undergone a complex evolution that continues today. Radical excision of the tumour is technically difficult and often incomplete. Successful management of IP requires resection of the affected mucosa which could be achieved with open surgery, endoscopic, or combined approach. Radio and chemotherapy were used for certain indications. More optimally research would be a multicenter randomized trials with large size cohorts. PMID:23346418

  14. Inverted Metamorphic Multijunction (IMM) Cell Processing Instructions

    SciTech Connect

    Duda, A.; Ward, S.; Young, M.

    2012-02-01

    This technical report details the processing schedule used to fabricate Inverted Metamorphic Multijunction (IMM) concentrator solar cells at The National Renewable Energy Laboratory (NREL). These devices are used as experimental test structures to support the research at NREL that is focused on increasing the efficiency of photovoltaic power conversion. They are not intended to be devices suitable for deployment in working concentrator systems primarily because of heat sinking issues. The process schedule was developed to be compatible with small sample sizes and to afford relatively rapid turn-around times, in support of research efforts. The report describes the use of electro deposition of gold for both the back and front contacts. Electro-deposition is used because of its rapid turn around time and because it is a benign metallization technique that is seldom responsible for damage to the semiconductors. The layer transfer technique is detailed including the use of a commercially available adhesive and the etching away of the parent gallium arsenide substrate. Photolithography is used to define front contact grids as well as the mesa area of the cell. Finally, the selective wet chemical etchant system is introduced and its use to reveal the back contact is described.

  15. Performance of an inverted ion source

    SciTech Connect

    Salvadori, M. C.; Teixeira, F. S.; Sgubin, L. G.; Araujo, W. W. R.; Spirin, R. E.; Oks, E. M.; Brown, I. G.

    2013-02-15

    Whereas energetic ion beams are conventionally produced by extracting ions (say, positive ions) from a plasma that is held at high (positive) potential, with ion energy determined by the potential drop through which the ions fall in the beam formation electrode system, in the device described here the plasma and its electronics are held at ground potential and the ion beam is formed and injected energetically into a space maintained at high (negative) potential. We refer to this configuration as an 'inverted ion source.' This approach allows considerable savings both technologically and economically, rendering feasible some ion beam applications, in particular small-scale ion implantation, that might otherwise not be possible for many researchers and laboratories. We have developed a device of this kind utilizing a metal vapor vacuum arc plasma source, and explored its operation and beam characteristics over a range of parameter variation. The downstream beam current has been measured as a function of extraction voltage (5-35 kV), arc current (50-230 A), metal ion species (Ti, Nb, Au), and extractor grid spacing and beamlet aperture size (3, 4, and 5 mm). The downstream ion beam current as measured by a magnetically-suppressed Faraday cup was up to as high as 600 mA, and with parametric variation quite similar to that found for the more conventional metal vapor vacuum arc ion source.

  16. Silicon controlled rectifier polyphase bridge inverter commutated with gate-turn-off thyristor

    NASA Technical Reports Server (NTRS)

    Edwards, Dean B. (Inventor); Rippel, Wally E. (Inventor)

    1986-01-01

    A polyphase SCR inverter (10) having N switching poles, each comprised of two SCR switches (1A, 1B; 2A, 2B . . . NA, NB) and two diodes (D1B; D1B; D2A, D2B . . . DNA, DNB) in series opposition with saturable reactors (L1A, L1B; L2A, L2B . . . LNA, LNB) connecting the junctions between the SCR switches and diodes to an output terminal (1, 2 . . . 3) is commutated with only one GTO thyristor (16) connected between the common negative terminal of a dc source and a tap of a series inductor (14) connected to the positive terminal of the dc source. A clamp winding (22) and diode (24) are provided, as is a snubber (18) which may have its capacitance (c) sized for maximum load current divided into a plurality of capacitors (C.sub.1, C.sub.2 . . . C.sub.N), each in series with an SCR switch S.sub.1, S.sub.2 . . . S.sub.N). The total capacitance may be selected by activating selected switches as a function of load current. A resistor 28 and SCR switch 26 shunt reverse current when the load acts as a generator, such as a motor while braking.

  17. Triple inverter pierce oscillator circuit suitable for CMOS

    DOEpatents

    Wessendorf; Kurt O.

    2007-02-27

    An oscillator circuit is disclosed which can be formed using discrete field-effect transistors (FETs), or as a complementary metal-oxide-semiconductor (CMOS) integrated circuit. The oscillator circuit utilizes a Pierce oscillator design with three inverter stages connected in series. A feedback resistor provided in a feedback loop about a second inverter stage provides an almost ideal inverting transconductance thereby allowing high-Q operation at the resonator-controlled frequency while suppressing a parasitic oscillation frequency that is inherent in a Pierce configuration using a "standard" triple inverter for the sustaining amplifier. The oscillator circuit, which operates in a range of 10 50 MHz, has applications for use as a clock in a microprocessor and can also be used for sensor applications.

  18. 8. Inverted siphon structure carrying ditch flow under Willow Creek, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Inverted siphon structure carrying ditch flow under Willow Creek, looking southwest - Natomas Ditch System, Blue Ravine Segment, Juncture of Blue Ravine & Green Valley Roads, Folsom, Sacramento County, CA

  19. 7. Inverted siphon structure carrying ditch flow under Willow Creek, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Inverted siphon structure carrying ditch flow under Willow Creek, looking east - Natomas Ditch System, Blue Ravine Segment, Juncture of Blue Ravine & Green Valley Roads, Folsom, Sacramento County, CA

  20. 145. Credit JE. Steel section of Coleman Canal inverted siphon ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    145. Credit JE. Steel section of Coleman Canal inverted siphon #2, crossing Baldwin Creek. (JE, v. 27 1911 p. 413). - Battle Creek Hydroelectric System, Battle Creek & Tributaries, Red Bluff, Tehama County, CA

  1. The Generation of an Organic Inverted Chemical Garden.

    PubMed

    Pampalakis, Georgios

    2016-05-10

    A chemical garden based on iron salt that grows in organic solvents and ions is demonstrated for the first time. This prototype chemical garden develops in an inverted orientation, thus providing evidence that downward growth is feasible.

  2. Stability of Brillouin flow in planar, conventional, and inverted magnetrons

    SciTech Connect

    Simon, D. H.; Lau, Y. Y.; Greening, G.; Wong, P.; Gilgenbach, R. M.; Hoff, B. W.

    2015-08-15

    The Brillouin flow is the prevalent flow in crossed-field devices. We systematically study its stability in the conventional, planar, and inverted magnetron geometry. To investigate the intrinsic negative mass effect in Brillouin flow, we consider electrostatic modes in a nonrelativistic, smooth bore magnetron. We found that the Brillouin flow in the inverted magnetron is more unstable than that in a planar magnetron, which in turn is more unstable than that in the conventional magnetron. Thus, oscillations in the inverted magnetron may startup faster than the conventional magnetron. This result is consistent with simulations, and with the negative mass property in the inverted magnetron configuration. Inclusion of relativistic effects and electromagnetic effects does not qualitatively change these conclusions.

  3. Performance model for grid-connected photovoltaic inverters.

    SciTech Connect

    Boyson, William Earl; Galbraith, Gary M.; King, David L.; Gonzalez, Sigifredo

    2007-09-01

    This document provides an empirically based performance model for grid-connected photovoltaic inverters used for system performance (energy) modeling and for continuous monitoring of inverter performance during system operation. The versatility and accuracy of the model were validated for a variety of both residential and commercial size inverters. Default parameters for the model can be obtained from manufacturers specification sheets, and the accuracy of the model can be further refined using measurements from either well-instrumented field measurements in operational systems or using detailed measurements from a recognized testing laboratory. An initial database of inverter performance parameters was developed based on measurements conducted at Sandia National Laboratories and at laboratories supporting the solar programs of the California Energy Commission.

  4. 15. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 2 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 2 ON THE GROUND FLOOR OF THE FURNACE AISLE IN THE BOP SHOP. - U.S. Steel Duquesne Works, Basic Oxygen Steelmaking Plant, Along Monongahela River, Duquesne, Allegheny County, PA

  5. 13. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 2 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 2 ON THE OPERATING FLOOR OF THE FURNACE AISLE IN THE BOP SHOP. - U.S. Steel Duquesne Works, Basic Oxygen Steelmaking Plant, Along Monongahela River, Duquesne, Allegheny County, PA

  6. 14. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 1 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. WESTERN VIEW OF INVERTED BASIC OXYGEN FURNACE No. 1 ON THE OPERATING FLOOR OF THE FURNACE AISLE IN THE BOP SHOP. - U.S. Steel Duquesne Works, Basic Oxygen Steelmaking Plant, Along Monongahela River, Duquesne, Allegheny County, PA

  7. Patterns of inverted reading and subgroups in dyslexia.

    PubMed

    Larsen, S; Parlenvi, P

    1984-01-01

    Reading performance of 46 poor readers was compared with that of 20 normal control readers. All subjects were second grade children. In Experiment 1 two matching word lists were presented under two conditions: one version of the test was read in the upright position and the other inverted. In Experiment 2 the eye movements of all subjects were recorded during reading of two meaningful sentences in the normal and inverted position. While the controls were negatively influenced by inversion of the text, the poor readers showed a variety of responses. Overall, the poor readers showed a slight tendency to be better at reading in the inverted position when the text must be scanned from right to left. An individual analysis of the data revealed that in 28.3% of the poor readers inverted reading improved performance at least 15 %, a phenomenon found in none of the controls.

  8. 2. DETAIL OF INVERTED MINE CARS, LOOKING SOUTHWEST; NOTE SHORT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. DETAIL OF INVERTED MINE CARS, LOOKING SOUTHWEST; NOTE SHORT WHEEL BASE NEEDED FOR DUMPING CONTENTS - Nuttallburg Mine Complex, Main Mine, North side of New River, 2.7 miles upstream from Fayette Landing, Lookout, Fayette County, WV

  9. Documents from malicious terminals

    NASA Astrophysics Data System (ADS)

    Berta, Istvan Z.; Vajda, Istvan

    2003-04-01

    The user wishes to communicate with a remote partner over an insecure network. Since the user is a human being, a terminal is needed for communication. Cryptographic algorithms running on the terminal may provide authenticity for the user's messages. In this paper the problem of sending authentic messages from insecure or untrusted terminals is analyzed. In this case attackers are able to gain total control over the terminal, so the user must consider the terminal a potential attacker. Smart cards are often considered the ultimate tool for secure messaging from untrusted terminals. However, their lack of user interface enables man-in-the middle attack from the terminal. The authors assume, that user is a human being with limited memory and computational power, and also makes mistakes in his calculations. They demnostrate, that only exceptional useres are able to authenticate messages without a trusted device. Several biometric media encapsulate the content of the message and the identity of the sender, such as speech, video and handwriting. The authors suggest, that such media is far more difficult to counterfeit than plaintext. Thus, the user must rely on his other resources, like biometric ones. In the protocol proposed by the authors, the user sends messages in a biometric format, strengthened by simple algorithmic authenticators. The smart card functions as a secure time gate ensuring, that the attacker has extremely little time to counterfeit both the biometric and the algorithmic protection on the message. The authors claim, that with the proper calibration of the biometric method and the time gate of the smart card, their protocol is strong enough for practical use.

  10. Control of Two Permanent Magnet Machines Using a Five-Leg Inverter for Automotive Applications

    SciTech Connect

    Su, Gui-Jia; Tang, Lixin; Huang, Xianghui

    2006-01-01

    This paper presents digital control schemes for control of two permanent magnet (PM) machines in an integrated traction and air-conditioning compressor drive system for automotive applications. The integrated drive system employs a five-leg inverter to power a three-phase traction PM motor and a two-phase compressor PM motor by tying the common terminal of the two-phase motor to the neutral point of the three-phase motor. Compared to a three-phase or a standalone two-phase inverter, it eliminates one phase leg and shares the control electronics between the two drives, thus significantly reducing the component count of the compressor drive. To demonstrate that the speed and torque of the two PM motors can be controlled independently, a control strategy was implemented in a digital signal processor, which includes a rotor flux field orientation based control (RFOC) for the three-phase motor, a similar RFOC and a position sensorless control in the brushless dc (BLDC) mode for the two-phase motor. Control implementation issues unique to a two-phase PM motor are also discussed. Test results with the three-phase motor running in the ac synchronous (ACS) mode while the two-phase motor either in the ACS or the BLDC mode are included to verify the independent speed and torque control capability of the integrated drive.

  11. Border Collision of Three-Phase Voltage-Source Inverter System with Interacting Loads

    NASA Astrophysics Data System (ADS)

    Li, Zhen; Liu, Bin; Li, Yining; Wong, Siu-Chung; Liu, Xiangdong; Huang, Yuehui

    As a commercial interface, three-phase voltage-source inverters (VSI) are commonly equipped for energy conversion to export DC power from most distributed generation (DG) to the AC utility. Not only do voltage-source converters take charge of converting the power to the loads but support the grid voltage at the point of common connection (PCC) as well, which is dependent on the condition of the grid-connected loads. This paper explores the border collision and its interacting mechanism among the VSI, resistive interacting loads and grids, which manifests as the alternating emergence of the inverting and rectifying operations, where the normal operation is terminated and a new one is assumed. Their mutual effect on the power quality under investigation will cause the circuital stability issue and further deteriorate the voltage regulation capability of VSI by dramatically raising the grid voltage harmonics. It is found in a design-oriented view that the border collision operation will be induced within the unsuitable parameter space with respect to transmission lines of AC grid, resistive loads and internal resistance of VSI. The physical phenomenon is also identified by the theoretical analysis. With numerical simulations for various circuit conditions, the corresponding bifurcation boundaries are collected, where the stability of the system is lost via border collision.

  12. Distortion and regulation characterization of a Mapham inverter

    NASA Technical Reports Server (NTRS)

    Sundberg, Richard C.; Brush, Andrew S.; Button, Robert M.; Patterson, Alexander G.

    1989-01-01

    Output voltage Total Harmonic Distortion (THD) of a 20kHz, 6kVA Mapham resonant inverter is characterized as a function of its switching-to-resonant frequency ratio, f sub s/f sub r, using the EASY5 engineering analysis system. EASY5 circuit simulation results are compared with hardware test results to verify the accuracy of the simulations. The effects of load on the THD versus f sub s/f sub r ratio is investigated for resistive, leading, and lagging power factor load impedances. The effect of the series output capacitor on the Mapham inverter output voltage distortion and inherent load regulation is characterized under loads of various power factors and magnitudes. An optimum series capacitor value which improves the inherent load regulation to better than 3 percent is identified. The optimum series capacitor value is different than the value predicted from a modeled frequency domain analysis. An explanation is proposed which takes into account the conduction overlap in the inductor pairs during steady-state inverter operation, which decreases the effective inductance of a Mapham inverter. A fault protection and current limit method is discussed which allows the Mapham inverter to operate into a short circuit, even when the inverter resonant circuit becomes overdamped.

  13. Multifocal inverted papillomas in the head and neck.

    PubMed

    Sharma, Jyoti; Goldenberg, David; Crist, Henry; McGinn, Johnathan

    2015-03-01

    Inverted papilloma is a rare benign neoplasm that usually originates in the lateral nasal wall. It can be a locally aggressive lesion and invade nearby structures. While primarily a nasal neoplasm, cases of an inverted papilloma involving the temporal bone, pharynx, nasopharynx, and lacrimal sac have been reported. We describe the case of a 67-year-old man with a history of nasal inverted papilloma who presented with a recurrent nasal mass and a large mass on the left side of his upper neck. The patient's history included inverted papillomas in multiple locations: the temporal bone, the sinonasal tract, and the nasopharynx. The new neck mass raised a concern for malignant degeneration and metastasis, but pathology demonstrated that it was a benign inverted papilloma. No clear etiology for the new neck lesion was evident except for an origin in salivary gland tissue. However, there was no physical connection between the neck mass and the submandibular gland identifiable on pathologic evaluation. This case illustrates the need for an aggressive primary resection to minimize local recurrence, as well as adequate surveillance to address recurrences early. Given the potential for multicentricity, patients with a typical sinonasal inverted papilloma should undergo a complete head and neck examination as part of their follow-up.

  14. Management and treatment of sinonasal inverted papilloma

    PubMed Central

    Ungari, Claudio; Riccardi, Emiliano; Reale, Gabriele; Agrillo, Alessandro; Rinna, Claudio; Mitro, Valeria; Filiaci, Fabio

    2015-01-01

    Summary Aims The aim of this paper is to describe the surgical experience of 35 patients with Inverted Papilloma (IP) of paranasal sinuses and its recurrence rate after a year of follow-up. Materials A retrospective chart review was performed on patients presenting with IP of paranasal sinuses. Thirty-five patients comprised the focus of this study. For all patients was performed a pre-surgery TC, and for more 5 patients it was necessary to perform a Magnetic Resonance (MR) with gadolinium. Results Among 35 patients selected, 18 patients underwent to open surgery, 4 patients had a combined approach with endoscopy and open surgery, while 13 patients were managed only with an endoscopic approach, with a minimum of 1 year of follow-up. Our results highlighted that the global percentage of success 12 months after the treatment was 93% and it not vary according to the tipology of the approach used if a radical excision of the lesion is achieved. More in depth, among 35 cases, only 2 patients were found to have recurrences and were treated with coronal and endoscopic approach. Conclusion It is fundamental to underline that surgery must be carried on in a radical manner to treat these tend to recur. A complete removal of the lesion and bone peripheral border filing are essential to perform a correct and definitive treatment. Also, endoscopic approach can be taken into account when tumors are localized median to a sagittal plan crossing the orbit median wall and when they did not massively compromised paranasal sinus walls. PMID:26941894

  15. Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

    PubMed

    Le Guiner, Caroline; Stieger, Knut; Toromanoff, Alice; Guilbaud, Mickaël; Mendes-Madeira, Alexandra; Devaux, Marie; Guigand, Lydie; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne; Adjali, Oumeya

    2014-01-01

    Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed. PMID:25248159

  16. AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer

    PubMed Central

    Pépin, David; Sosulski, Amanda; Zhang, Lihua; Wang, Dan; Vathipadiekal, Vinod; Hendren, Katherine; Coletti, Caroline M.; Yu, Aaron; Castro, Cesar M.; Birrer, Michael J.; Gao, Guangping; Donahoe, Patricia K.

    2015-01-01

    To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer. PMID:26216943

  17. Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy

    PubMed Central

    Armbruster, Nicole; Lattanzi, Annalisa; Jeavons, Matthieu; Van Wittenberghe, Laetitia; Gjata, Bernard; Marais, Thibaut; Martin, Samia; Vignaud, Alban; Voit, Thomas; Mavilio, Fulvio; Barkats, Martine; Buj-Bello, Ana

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery. PMID:27652289

  18. A comparison of AAV strategies distinguishes overlapping vectors for efficient systemic delivery of the 6.2 kb Dysferlin coding sequence

    PubMed Central

    Pryadkina, Marina; Lostal, William; Bourg, Nathalie; Charton, Karine; Roudaut, Carinne; Hirsch, Matthew L; Richard, Isabelle

    2015-01-01

    Recombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies. PMID:26029720

  19. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs

    PubMed Central

    Boyd, RF; Boye, SL; Conlon, TJ; Erger, KE; Sledge, DG; Langohr, IM; Hauswirth, WW; Komáromy, AM; Boye, SE; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions. PMID:27052802

  20. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size

    PubMed Central

    Hagg, Adam; Colgan, Timothy D.; Thomson, Rachel E.; Qian, Hongwei; Lynch, Gordon S.; Gregorevic, Paul

    2016-01-01

    Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease. PMID:26972746

  1. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks.

    PubMed

    Veldwijk, Marlon R; Topaly, Julian; Laufs, Stephanie; Hengge, Ulrich R; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan

    2002-08-01

    Despite the clinical application of adeno-associated virus (AAV) gene therapy, the titration of viral stocks has not yet been standardized. This complicates the comparison of viral stocks between laboratories. Functional titering of AAV is time-consuming, requires the manipulation of hazardous material, and often has a high degree of variability. We established an optimized real-time quantitative polymerase chain reaction (RQ-PCR) titration assay to determine viral titers and compared it with a functional green fluorescent protein (GFP)-based titration method. With a combination of improved lysis procedures and RQ-PCR protocols we could decrease the intraexperimental coefficient of variation (CV) from 0.24 +/- 0.03 to 0.042 +/- 0.004 and the interexperimental CV from 0.34 +/- 0.06 to 0.093 +/- 0.028 following functional and RQPCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQPCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.

  2. Distinct transduction profiles in the CNS via three injection routes of AAV9 and the application to generation of a neurodegenerative mouse model

    PubMed Central

    Huda, Fathul; Konno, Ayumu; Matsuzaki, Yasunori; Goenawan, Hanna; Miyake, Koichi; Shimada, Takashi; Hirai, Hirokazu

    2014-01-01

    Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS. The DC injection route robustly and exclusively transduced the whole cerebellum, whereas the IT injection route primarily transduced the cerebellar lobules 9 and 10 close to the injection site and the spinal cord. An IV injection in neonatal mice weakly and homogenously transduced broad CNS areas. In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells. To verify the usefulness of this method, we generated a mouse model of spinocerebellar ataxia type 1 (SCA1). Mice that received a DC injection of the ssAAV9 vector expressing mutant ATXN1, a protein responsible for SCA1, showed the intranuclear aggregation of mutant ATXN1 in Purkinje cells, significant atrophy of the Purkinje cell dendrites and progressive motor deficits, which are characteristics of SCA1. Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection. Moreover, this approach can be used for the generation of different mouse models of CNS/neurodegenerative diseases. PMID:26015973

  3. Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy

    PubMed Central

    Armbruster, Nicole; Lattanzi, Annalisa; Jeavons, Matthieu; Van Wittenberghe, Laetitia; Gjata, Bernard; Marais, Thibaut; Martin, Samia; Vignaud, Alban; Voit, Thomas; Mavilio, Fulvio; Barkats, Martine; Buj-Bello, Ana

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

  4. Design of a Single AAV Vector for Coexpression of TH and GCH1 to Establish Continuous DOPA Synthesis in a Rat Model of Parkinson's Disease

    PubMed Central

    Cederfjäll, Erik; Sahin, Gurdal; Kirik, Deniz; Björklund, Tomas

    2012-01-01

    Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach. PMID:22294150

  5. AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.

    PubMed

    Buj-Bello, Anna; Fougerousse, Françoise; Schwab, Yannick; Messaddeq, Nadia; Spehner, Danièle; Pierson, Christopher R; Durand, Muriel; Kretz, Christine; Danos, Olivier; Douar, Anne-Marie; Beggs, Alan H; Schultz, Patrick; Montus, Marie; Denèfle, Patrice; Mandel, Jean-Louis

    2008-07-15

    Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

  6. Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model.

    PubMed

    Sugano, E; Tabata, K; Takahashi, M; Nishiyama, F; Shimizu, H; Sato, M; Tamai, M; Tomita, H

    2016-02-01

    We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa. PMID:26440056

  7. Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy

    PubMed Central

    Borrás, Teresa; Buie, LaKisha K.; Spiga, Maria Grazia

    2016-01-01

    Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide that depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential re-inducible vector DNAs in the trabecular meshwork (0.4 vg/cell) and negligible copies in six major internal organs (0.00002-0.005 vg/cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid–induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted scAAV vectors in a large animal model. PMID:26855269

  8. Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery.

    PubMed

    Fu, Haiyan; Cataldi, Marcela P; Ware, Tierra A; Zaraspe, Kimberly; Meadows, Aaron S; Murrey, Darren A; McCarty, Douglas M

    2016-01-01

    The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach. PMID:27331076

  9. Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy.

    PubMed

    Armbruster, Nicole; Lattanzi, Annalisa; Jeavons, Matthieu; Van Wittenberghe, Laetitia; Gjata, Bernard; Marais, Thibaut; Martin, Samia; Vignaud, Alban; Voit, Thomas; Mavilio, Fulvio; Barkats, Martine; Buj-Bello, Ana

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery. PMID:27652289

  10. Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery

    PubMed Central

    Fu, Haiyan; Cataldi, Marcela P; Ware, Tierra A; Zaraspe, Kimberly; Meadows, Aaron S; Murrey, Darren A; McCarty, Douglas M

    2016-01-01

    The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach. PMID:27331076

  11. Phenylethynyl terminated reactive oligomer

    NASA Technical Reports Server (NTRS)

    Bryant, Robert G. (Inventor); Jensen, Brian J. (Inventor); Hergenrother, Paul M. (Inventor)

    1995-01-01

    A composition of matter having the general structure: ##STR1## (wherein X is F, Cl, or NO.sub.2, and Y is CO, SO.sub.2 or C(CF.sub.3).sub.2) is employed to terminate a nucleophilic reagent, resulting in the exclusive production of phenylethynyl terminated reactive oligomers which display unique thermal characteristics. A reactive diluent having the general structure: ##STR2## (wherein R is any aliphatic or aromatic moiety) is employed to decrease the melt viscosity of a phenylethynyl terminated reactive oligomer and to subsequently react therewith to provide a thermosetting material of enhanced density. These materials have features which make them attractive candidates for use as composite matrices and adhesives.

  12. Electrical termination techniques

    NASA Technical Reports Server (NTRS)

    Oakey, W. E.; Schleicher, R. R.

    1976-01-01

    A technical review of high reliability electrical terminations for electronic equipment was made. Seven techniques were selected from this review for further investigation, experimental work, and preliminary testing. From the preliminary test results, four techniques were selected for final testing and evaluation. These four were: (1) induction soldering, (2) wire wrap, (3) percussive arc welding, and (4) resistance welding. Of these four, induction soldering was selected as the best technique in terms of minimizing operator errors, controlling temperature and time, minimizing joint contamination, and ultimately producing a reliable, uniform, and reusable electrical termination.

  13. ALSEP termination report

    NASA Technical Reports Server (NTRS)

    Bates, J. R.; Lauderdale, W. W.; Kernaghan, H.

    1979-01-01

    The Apollo Lunar Surface Experiments Package (ALSEP) final report was prepared when support operations were terminated September 30, 1977, and NASA discontinued the receiving and processing of scientific data transmitted from equipment deployed on the lunar surface. The ALSEP experiments (Apollo 11 to Apollo 17) are described and pertinent operational history is given for each experiment. The ALSEP data processing and distribution are described together with an extensive discussion on archiving. Engineering closeout tests and results are given, and the status and configuration of the experiments at termination are documented. Significant science findings are summarized by selected investigators. Significant operational data and recommendations are also included.

  14. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  15. Breaking Barriers to Low-Cost Modular Inverter Production & Use

    SciTech Connect

    Bogdan Borowy; Leo Casey; Jerry Foshage; Steve Nichols; Jim Perkinson

    2005-05-31

    The goal of this cost share contract is to advance key technologies to reduce size, weight and cost while enhancing performance and reliability of Modular Inverter Product for Distributed Energy Resources (DER). Efforts address technology development to meet technical needs of DER market protection, isolation, reliability, and quality. Program activities build on SatCon Technology Corporation inverter experience (e.g., AIPM, Starsine, PowerGate) for Photovoltaic, Fuel Cell, Energy Storage applications. Efforts focused four technical areas, Capacitors, Cooling, Voltage Sensing and Control of Parallel Inverters. Capacitor efforts developed a hybrid capacitor approach for conditioning SatCon's AIPM unit supply voltages by incorporating several types and sizes to store energy and filter at high, medium and low frequencies while minimizing parasitics (ESR and ESL). Cooling efforts converted the liquid cooled AIPM module to an air-cooled unit using augmented fin, impingement flow cooling. Voltage sensing efforts successfully modified the existing AIPM sensor board to allow several, application dependent configurations and enabling voltage sensor galvanic isolation. Parallel inverter control efforts realized a reliable technique to control individual inverters, connected in a parallel configuration, without a communication link. Individual inverter currents, AC and DC, were balanced in the paralleled modules by introducing a delay to the individual PWM gate pulses. The load current sharing is robust and independent of load types (i.e., linear and nonlinear, resistive and/or inductive). It is a simple yet powerful method for paralleling both individual devices dramatically improves reliability and fault tolerance of parallel inverter power systems. A patent application has been made based on this control technology.

  16. Prematurely terminated slug tests

    SciTech Connect

    Karasaki, K. )

    1990-07-01

    A solution of the well response to a prematurely terminated slug test (PTST) is presented. The advantages of a PTST over conventional slug tests are discussed. A systematized procedure of a PTST is proposed, where a slug test is terminated in the midpoint of the flow point, and the subsequent shut-in data is recorded and analyzed. This method requires a downhole shut-in device and a pressure transducer, which is no more than the conventional deep-well slug testing. As opposed to slug tests, which are ineffective when a skin is present, more accurate estimate of formation permeability can be made using a PTST. Premature termination also shortens the test duration considerably. Because in most cases no more information is gained by completing a slug test to the end, the author recommends that conventional slug tests be replaced by the premature termination technique. This study is part of an investigation of the feasibility of geologic isolation of nuclear wastes being carried out by the US Department of Energy and the National Cooperative for the Storage of Radioactive Waste of Switzerland.

  17. The Versatile Terminal.

    ERIC Educational Resources Information Center

    Evans, C. D.

    This paper describes the experiences of the industrial research laboratory of Kodak Ltd. in finding and providing a computer terminal most suited to its very varied requirements. These requirements include bibliographic and scientific data searching and access to a number of worldwide computing services for scientific computing work. The provision…

  18. Shipboard regasification terminal

    SciTech Connect

    Campbell, G.; Zednik, J.

    1999-07-01

    Mobil Technology Company and Mobil Shipping and Transportation Company have jointly developed a new combination of existing proven equipment to regasify LNG. Advantages of this Shipboard Regasification Terminal (SRT) include accelerated initial gas delivery schedule, low capital cost, delivery of smaller quantities of LNG at a competitive price and shorter term of LNG purchase and improved financing options. These advantages benefit both the supplier of LNG and the purchaser. SRT can be used as an interim supply to developing markets allowing the demand to grow while developing downstream infrastructure. This concept does not involve offshore transfer of cryogenic fluids while delivering near-ambient temperature pipeline quality gas at typical pipeline pressures. During times when gas is not required, the SRT ship can easily be returned to the trade of transporting and delivering LNG to conventional land based terminals. This paper will discuss the merits of Shipboard Regasification Terminals in general, cover the development of this concept and review the factors guiding the use of SRT vs. an onshore terminal.

  19. Modeling Terminal Velocity

    ERIC Educational Resources Information Center

    Brand, Neal; Quintanilla, John A.

    2013-01-01

    Using a simultaneously falling softball as a stopwatch, the terminal velocity of a whiffle ball can be obtained to surprisingly high accuracy with only common household equipment. This classroom activity engages students in an apparently daunting task that nevertheless is tractable, using a simple model and mathematical techniques at their…

  20. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products. PMID:27229773

  1. Expression of an engineered soluble coxsackievirus and adenovirus receptor by a dimeric AAV9 vector inhibits adenovirus infection in mice.

    PubMed

    Röger, C; Pozzuto, T; Klopfleisch, R; Kurreck, J; Pinkert, S; Fechner, H

    2015-06-01

    Immunosuppressed (IS) patients, such as recipients of hematopoietic stem cell transplantation, occasionally develop severe and fatal adenovirus (Ad) infections. Here, we analyzed the potential of a virus receptor trap based on a soluble coxsackievirus and Ad receptor (sCAR) for inhibition of Ad infection. In vitro, a dimeric fusion protein, sCAR-Fc, consisting of the extracellular domain of CAR and the Fc portion of human IgG1 and a monomeric sCAR lacking the Fc domain, were expressed in cell culture. More sCAR was secreted into the cell culture supernatant than sCAR-Fc, but it had lower Ad neutralization activity than sCAR-Fc. Further investigations showed that sCAR-Fc reduced the Ad infection by a 100-fold and Ad-induced cytotoxicity by ~20-fold. Not only was Ad infection inhibited by sCAR-Fc applied prior to infection, it also inhibited infection when used to treat ongoing Ad infection. In vivo, sCAR-Fc was delivered to IS mice by an AAV9 vector, resulting in persistent and high (>40 μg ml(-1)) sCAR-Fc serum levels. The sCAR-Fc serum concentration was sufficient to significantly inhibit hepatic and cardiac wild-type Ad5 infection. Treatment with sCAR-Fc did not induce side effects. Thus, sCAR-Fc virus receptor trap may be a promising novel therapeutic for treatment of Ad infections.

  2. Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson’s Disease

    PubMed Central

    Oueslati, Abid; Lovisa, Blaise; Perrin, John; Wagnières, Georges; van den Bergh, Hubert; Tardy, Yanik; Lashuel, Hilal A.

    2015-01-01

    Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson’s disease (PD). In this model, daily exposure of both sides of the rat’s head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies. PMID:26484876

  3. Expression of an engineered soluble coxsackievirus and adenovirus receptor by a dimeric AAV9 vector inhibits adenovirus infection in mice.

    PubMed

    Röger, C; Pozzuto, T; Klopfleisch, R; Kurreck, J; Pinkert, S; Fechner, H

    2015-06-01

    Immunosuppressed (IS) patients, such as recipients of hematopoietic stem cell transplantation, occasionally develop severe and fatal adenovirus (Ad) infections. Here, we analyzed the potential of a virus receptor trap based on a soluble coxsackievirus and Ad receptor (sCAR) for inhibition of Ad infection. In vitro, a dimeric fusion protein, sCAR-Fc, consisting of the extracellular domain of CAR and the Fc portion of human IgG1 and a monomeric sCAR lacking the Fc domain, were expressed in cell culture. More sCAR was secreted into the cell culture supernatant than sCAR-Fc, but it had lower Ad neutralization activity than sCAR-Fc. Further investigations showed that sCAR-Fc reduced the Ad infection by a 100-fold and Ad-induced cytotoxicity by ~20-fold. Not only was Ad infection inhibited by sCAR-Fc applied prior to infection, it also inhibited infection when used to treat ongoing Ad infection. In vivo, sCAR-Fc was delivered to IS mice by an AAV9 vector, resulting in persistent and high (>40 μg ml(-1)) sCAR-Fc serum levels. The sCAR-Fc serum concentration was sufficient to significantly inhibit hepatic and cardiac wild-type Ad5 infection. Treatment with sCAR-Fc did not induce side effects. Thus, sCAR-Fc virus receptor trap may be a promising novel therapeutic for treatment of Ad infections. PMID:25786873

  4. Cirrus Crystal Terminal Velocities.

    NASA Astrophysics Data System (ADS)

    Heymsfield, Andrew J.; Iaquinta, Jean

    2000-04-01

    Cirrus crystal terminal velocities are of primary importance in determining the rate of transport of condensate from upper- to middle-tropospheric levels and profoundly influence the earth's radiation balance through their effect on the rate of buildup or decay of cirrus clouds. In this study, laboratory and field-based cirrus crystal drag coefficient data, as well as analytical descriptions of cirrus crystal shapes, are used to derive more physically based expressions for the velocities of cirrus crystals than have been available in the past.Polycrystals-often bullet rosettes-are shown to be the dominant crystal types in synoptically generated cirrus, with columns present in varying but relatively large percentages, depending on the cloud. The two critical parameters needed to calculate terminal velocity are the drag coefficient and the ratio of mass to cross-sectional area normal to their fall direction. Using measurements and calculations, it is shown that drag coefficients from theory and laboratory studies are applicable to crystals of the types found in cirrus. The ratio of the mass to area, which is shown to be relatively independent of the number of bullets in the rosette, is derived from an analytic model that represents bullet rosettes containing one to eight bullets in 19 primary geometric configurations. The ratio is also derived for columns. Using this information, a general set of equations is developed to calculate the terminal velocities and masses in terms of the aspect ratio (width divided by length), ice density, and rosette maximum dimension. Simple expressions for terminal velocity and mass as a function of bullet rosette maximum dimension are developed by incorporating new information on bullet aspect ratios.The general terminal velocity and mass relations are then applied to a case from the First International Satellite Cloud Climatology Project (ISCCP) Research Experiment (FIRE) 2, when size spectra from a balloon-borne ice crystal

  5. Graphics Software For VT Terminals

    NASA Technical Reports Server (NTRS)

    Wang, Caroline

    1991-01-01

    VTGRAPH graphics software tool for DEC/VT computer terminal or terminals compatible with it, widely used by government and industry. Callable in FORTRAN or C language, library program enabling user to cope with many computer environments in which VT terminals used for window management and graphic systems. Provides PLOT10-like package plus color or shade capability for VT240, VT241, and VT300 terminals. User can easily design more-friendly user-interface programs and design PLOT10 programs on VT terminals with different computer systems. Requires ReGis graphics set terminal and FORTRAN compiler.

  6. Direct Conversion from Terminal Borylene into Terminal Phosphinidene.

    PubMed

    Braunschweig, Holger; Jimenez-Halla, J Oscar C; Radacki, Krzysztof; Shang, Rong

    2016-10-01

    The first terminal manganese phosphinidene complex was quantitatively synthesized from a terminal alkylborylene complex. Its structure and bonding, as well as the reaction mechanism, were investigated through a combination of experimental and computational studies. PMID:27621216

  7. Direct Conversion from Terminal Borylene into Terminal Phosphinidene.

    PubMed

    Braunschweig, Holger; Jimenez-Halla, J Oscar C; Radacki, Krzysztof; Shang, Rong

    2016-10-01

    The first terminal manganese phosphinidene complex was quantitatively synthesized from a terminal alkylborylene complex. Its structure and bonding, as well as the reaction mechanism, were investigated through a combination of experimental and computational studies.

  8. The pathology of urinary bladder lesions with an inverted growth pattern.

    PubMed

    Guo, Aitao; Liu, Aijun; Teng, Xiaodong

    2016-02-01

    Inverted lesions in the urinary bladder have been the source of some difficulty in urological pathology. The two common ones are von Brunn's nests and cystitis cystic/cystitis glandularis, which are considered normal variants of urothelium. Apart from them, a number of other rare urothelial lesions with inverted growth pattern occur in the urinary bladder. Some of them are only reactive conditions, just as pseudocarcinomatous hyperplasia. Some are benign tumors, namely inverted papilloma. Whereas others are malignant neoplasms, including inverted papillary urothelial neoplasm of low malignant potential (PUNLMP), non-invasive inverted papillary urothelial carcinoma (low-grade and high-grade), and invasive urothelial carcinoma (inverted, nested and big nested variants). Because of the overlapping morphological features of all the inverted lesions mentioned above, even between high-grade invasive carcinoma and pseudocarcinomatous hyperplasia which are only a kind of reactive conditions, it is very important for the surgical pathologist to recognize and be familiar with these inverted lesions in urinary bladder. In this article, we review these spectrums of inverted lesions of the urinary bladder. Emphasis is placed on histogenesis, morphology, differential diagnosis of these lesions, and the pathologic grading of the non-invasive inverted neoplasms, such as inverted papilloma, inverted PUNLMP, non-invasive inverted papillary urothelial carcinoma with low-grade, and non-invasive inverted papillary urothelial carcinoma with high-grade.

  9. The pathology of urinary bladder lesions with an inverted growth pattern

    PubMed Central

    Liu, Aijun; Teng, Xiaodong

    2016-01-01

    Inverted lesions in the urinary bladder have been the source of some difficulty in urological pathology. The two common ones are von Brunn’s nests and cystitis cystic/cystitis glandularis, which are considered normal variants of urothelium. Apart from them, a number of other rare urothelial lesions with inverted growth pattern occur in the urinary bladder. Some of them are only reactive conditions, just as pseudocarcinomatous hyperplasia. Some are benign tumors, namely inverted papilloma. Whereas others are malignant neoplasms, including inverted papillary urothelial neoplasm of low malignant potential (PUNLMP), non-invasive inverted papillary urothelial carcinoma (low-grade and high-grade), and invasive urothelial carcinoma (inverted, nested and big nested variants). Because of the overlapping morphological features of all the inverted lesions mentioned above, even between high-grade invasive carcinoma and pseudocarcinomatous hyperplasia which are only a kind of reactive conditions, it is very important for the surgical pathologist to recognize and be familiar with these inverted lesions in urinary bladder. In this article, we review these spectrums of inverted lesions of the urinary bladder. Emphasis is placed on histogenesis, morphology, differential diagnosis of these lesions, and the pathologic grading of the non-invasive inverted neoplasms, such as inverted papilloma, inverted PUNLMP, non-invasive inverted papillary urothelial carcinoma with low-grade, and non-invasive inverted papillary urothelial carcinoma with high-grade. PMID:27041933

  10. Mobile Phone Terminal

    NASA Technical Reports Server (NTRS)

    1978-01-01

    In the photo, an employee of a real estate firm is contacting his office by means of HICOM, an advanced central terminal for mobile telephones. Developed by the Orlando Division of Martin Marietta Aerospace, Orlando, Florida, and manufactured by Harris Corporation's RF Division, Rochester, N.Y., HICOM upgrades service to users, provides better system management to telephone companies, and makes more efficient use of available mobile telephone channels through a computerized central control terminal. The real estate man, for example, was able to dial his office and he could also have direct-dialed a long distance number. Mobile phones in most areas not yet served by HICOM require an operator's assistance for both local and long distance calls. HICOM improves system management by automatically recording information on all calls for accurate billing, running continual performance checks on its own operation, and reporting any malfunctions to a central office.

  11. Shipboard fisheries management terminals

    NASA Technical Reports Server (NTRS)

    Nagler, R. G.; Sager, E. V.

    1980-01-01

    The needs of the National Marine Fisheries Service (NMGS), National Weather Service, and the U.S. Coast Guard for locational, biological, and environmental data were assessed. The fisheries conservation zones and the yellowfin tuna jurisdiction of the NMFS operates observer programs on foreign and domestic fishing vessels. Data input terminal and data transfer and processing technology are reviewed to establish available capability. A matrix of implementation options is generated to identify the benefits of each option, and preliminary cost estimates are made. Recommendations are made for incremental application of available off the shelf hardware to obtain improved performance and benefits within a well bounded cost. Terminal recommendations are made for three interdependent shipboard units emphasizing: (1) the determination of location and fishing activity; (2) hand held data inputting and formatting in the fishing work areas; and (3) data manipulation, merging, and editing.

  12. Factorization method and new potentials from the inverted oscillator

    SciTech Connect

    Bermudez, David Fernández C, David J.

    2013-06-15

    In this article we will apply the first- and second-order supersymmetric quantum mechanics to obtain new exactly-solvable real potentials departing from the inverted oscillator potential. This system has some special properties; in particular, only very specific second-order transformations produce non-singular real potentials. It will be shown that these transformations turn out to be the so-called complex ones. Moreover, we will study the factorization method applied to the inverted oscillator and the algebraic structure of the new Hamiltonians. -- Highlights: •We apply supersymmetric quantum mechanics to the inverted oscillator potential. •The complex second-order transformations allow us to build new non-singular potentials. •The algebraic structure of the initial and final potentials is analyzed. •The initial potential is described by a complex-deformed Heisenberg–Weyl algebra. •The final potentials are described by polynomial Heisenberg algebras.

  13. Endoscopic transnasal management of inverted papilloma involving frontal sinuses

    PubMed Central

    Krzeski, Antoni; Held-Ziółkowska, Marta; Niemczyk, Kazimierz

    2012-01-01

    Inverted papilloma is a benign locally aggressive tumor of paranasal sinuses which has been traditionally managed with external surgical approaches. Advances in tumor imaging, surgical instrumentation and intraoperative visualization have led to a gradual shift to endonasal attachment-oriented surgery. Involvement of both frontal sinuses by inverted papilloma is rare. There are scant reports in the literature regarding this topic. We present 2 cases of the tumor involving both frontal sinuses removed by median drainage (Draf III procedure) under endoscopic guidance without any additional external approach. The whole cavity of both frontal sinuses was easily inspected at the end of the procedure. No early or late complications were observed. No recurrence was seen in 1-year or 2-year follow-up. Management of frontal sinus inverted papilloma with the endoscopic median drainage approach is feasible and seems to be effective. PMID:23362431

  14. Suspended Patch Antenna Array With Electromagnetically Coupled Inverted Microstrip Feed

    NASA Technical Reports Server (NTRS)

    Simons, Rainee N.

    2000-01-01

    The paper demonstrates a four-element suspended patch antenna array, with a parasitic patch layer and an electromagnetically coupled inverted microstrip feed, for linear polarization at K-Band frequencies. This antenna has the following advantages over conventional microstrip antennas: First, the inverted microstrip has lower attenuation than conventional microstrip; hence, conductor loss associated with the antenna corporate feed is lower resulting in higher gain and efficiency. Second, conventional proximity coupled patch antennas require a substrate for the feed and a superstrate for the patch. However, the inverted microstrip fed patch antenna makes use of a single substrate, and hence, is lightweight and low cost. Third, electromagnetic coupling results in wider bandwidth. Details regarding the design and fabrication will be presented as well as measured results including return loss, radiation patterns and cross-polarization levels.

  15. Remote terminal system evaluation

    NASA Technical Reports Server (NTRS)

    Phillips, T. L.; Grams, H. L.; Lindenlaub, J. C.; Schwingendorf, S. K.; Swain, P. H.; Simmons, W. R.

    1975-01-01

    An Earth Resources Data Processing System was developed to evaluate the system for training, technology transfer, and data processing. In addition to the five sites included in this project two other sites were connected to the system under separate agreements. The experience of these two sites is discussed. The results of the remote terminal project are documented in seven reports: one from each of the five project sites, Purdue University, and an overview report summarizing the other six reports.

  16. "Terminal" Students Do Transfer.

    ERIC Educational Resources Information Center

    Townsend, Barbara K.

    This report discusses the transfer rate of community college students with A.S. or A.A.S. degrees from vocational or "terminal" programs in Missouri. The study shows that during the 1995-1996 academic year, 6,171 students received an associate degree from a Missouri public two-year college. Of these, 3,169 (51%) earned the A.A. degree and 3,002…

  17. Amine terminated bisaspartimide polymer

    NASA Technical Reports Server (NTRS)

    Kumar, D. (Inventor); Fohlen, G. M. (Inventor); Parker, J. A. (Inventor)

    1986-01-01

    Novel amine terminated bisaspartimides are prepared by a Michael-type reaction of an aromatic bismalteimide and an aromatic diamine in an aprotic solvent. These bisaspartimides are thermally polymerized to yield tough, resinous polymers cross-lined through -NH- groups. Such polymers are useful in applications requiring materials with resistance to change at elevated temperatures, e.g., as lightweight laminates with graphite cloth, molding material prepregs, adhesives and insulating material.

  18. Transparent film with inverted conical microholes array for reflection enhancement

    NASA Astrophysics Data System (ADS)

    Lei, Biao; Liu, Hongzhong; Jiang, Weitao; Chen, Bangdao; Shi, Yongsheng; Yin, Lei; Liu, Xiaokang

    2016-04-01

    PDMS has been widely utilized for microfluidic chips and microchannel detections, as its good optical properties are the prerequisite to achieve accurate and efficient detection. However, it is difficult to obtain effective information for opaque liquids. With the development of microchannel detection for wider fields, it is imperative to obtain more comprehensive information of the observed objects by integrating high transmission with enhanced reflection. This article investigates reflection enhancement by Polydimethylsiloxane (PDMS) film with inverted conical microholes array. PDMS film with inverted conical microholes array is fabricated by replication from the silicon mold with inverted microcones array which is prepared by Inductively Coupled Plasma (ICP) etch tool. The monolayer PDMS film with inverted conical microholes array shows a two-fold effectively increase in reflection, approximately up to 15%, at a broad wavelength range of 637-1131 nm and 1214-1350 nm, compared with bare PDMS film. In addition, the reflection can be further enhanced by multilayered lamination of PDMS film with inverted conical microholes array, and the enhancement is also dependent on the lamination way, i.e., for bilayer laminations, the maximum reflection enhancement occurs when with face-to-back lamination, and 32.79% larger than that with back-to-face lamination. From the experiments, the maximum reflectivity of 8-layered PDMS films can obtain 64.4% while the maximum reflectivity of monolayer PDMS film barely has 17.5%. The transparent film with inverted conical microholes array for reflection enhancement may find a variety of applications in optical devices, microchips, and energy conservation technologies etc.

  19. Inverter power module with distributed support for direct substrate cooling

    DOEpatents

    Miller, David Harold; Korich, Mark D.; Ward, Terence G.; Mann, Brooks S.

    2012-08-21

    Systems and/or methods are provided for an inverter power module with distributed support for direct substrate cooling. An inverter module comprises a power electronic substrate. A first support frame is adapted to house the power electronic substrate and has a first region adapted to allow direct cooling of the power electronic substrate. A gasket is interposed between the power electronic substrate and the first support frame. The gasket is configured to provide a seal between the first region and the power electronic substrate. A second support frame is adapted to house the power electronic substrate and joined to the first support frame to form the seal.

  20. Toroidal transformer design program with application to inverter circuitry

    NASA Technical Reports Server (NTRS)

    Dayton, J. A., Jr.

    1972-01-01

    Estimates of temperature, weight, efficiency, regulation, and final dimensions are included in the output of the computer program for the design of transformers for use in the basic parallel inverter. The program, written in FORTRAN 4, selects a tape wound toroidal magnetic core and, taking temperature, materials, core geometry, skin depth, and ohmic losses into account, chooses the appropriate wire sizes and number of turns for the center tapped primary and single secondary coils. Using the program, 2- and 4-kilovolt-ampere transformers are designed for frequencies from 200 to 3200 Hz and the efficiency of a basic transistor inverter is estimated.

  1. Optical-Mechanical System for Stabilizing an Inverted Pendulum

    NASA Astrophysics Data System (ADS)

    Kuzyakov, O. N.; Andreeva, M. A.

    2016-08-01

    Controlling open-loop unstable systems is a common benchmark for designing the algorithms to maintain the equilibrium state of anthropomorphous technical devices used within control theory. In this connection, considerable attention is currently being focused on the problem of stabilizing the inverted pendulum system. In this work, the execution of swinging-up the pendulum and, subsequently, maintaining its upward equilibrium state is presented with the help of the laboratory bench TP-802 by Festo Didactics and the movement control device. The configuration of dynamic system for stabilizing the inverted pendulum is offered. The algorithms to swing-up the pendulum and balance it around its upright position are offered as well.

  2. Inverted organic photovoltaic device with a new electron transport layer

    PubMed Central

    2014-01-01

    We demonstrate that there is a new solution-processed electron transport layer, lithium-doped zinc oxide (LZO), with high-performance inverted organic photovoltaic device. The device exhibits a fill factor of 68.58%, an open circuit voltage of 0.86 V, a short-circuit current density of −9.35 cm/mA2 along with 5.49% power conversion efficiency. In addition, we studied the performance of blend ratio dependence on inverted organic photovoltaics. Our device also demonstrates a long stability shelf life over 4 weeks in air. PMID:24674457

  3. ZCS High Frequency Inverter for Aluminum Vessel Induction Heating

    NASA Astrophysics Data System (ADS)

    Ogiwara, Hiroyuki; Nakaoka, Mutsuo

    Recent induction cooking apparatus are utilized for induction heating of ferromagnetic materials at 20-50kHz with a high efficiency. They can not, however, be applied for non-magnetic materials such as aluminum vessels. Here, we present a voltage-clamp reverse conducting ZCS high frequency inverter of half bridge type for induction heating of an aluminum vessel. The switching devices utilized for this inverter are SITs and its operating frequency is determined as 200kHz. This paper describes its circuit constitution and the obtained experimental results from a practical point of view.

  4. Testing of a single cell cylindrical inverted converter

    NASA Astrophysics Data System (ADS)

    Desplat, Jean-Louis

    2002-01-01

    A Cylindrical Inverted Converter (CIC), made by Lutch, with the emitter on the outside was tested in a vacuum furnace supplying radiant heat to the emitter outer surface. The collector, coaxial with the emitter, has an integral heat pipe with sodium as the working fluid, which carries the heat dissipated in the collector to a radiating area with a coating of alumina and sub-stoichiometric TiO2. The CIC is a proof-of-principle device which will lead to the development of multi-cell inverted converter assemblies for space solar power systems. The thermionic performance at emitter temperatures of 1800 and 1900 K is presented. .

  5. Addressable Inverter Matrix Tests Integrated-Circuit Wafer

    NASA Technical Reports Server (NTRS)

    Buehler, Martin G.

    1988-01-01

    Addressing elements indirectly through shift register reduces number of test probes. With aid of new technique, complex test structure on silicon wafer tested with relatively small number of test probes. Conserves silicon area by reduction of area devoted to pads. Allows thorough evaluation of test structure characteristics and of manufacturing process parameters. Test structure consists of shift register and matrix of inverter/transmission-gate cells connected to two-by-ten array of probe pads. Entire pattern contained in square area having only 1.6-millimeter sides. Shift register is conventional static CMOS device using inverters and transmission gates in master/slave D flip-flop configuration.

  6. Non-invertible transformations of differential–difference equations

    NASA Astrophysics Data System (ADS)

    Garifullin, R. N.; Yamilov, R. I.; Levi, D.

    2016-09-01

    We discuss aspects of the theory of non-invertible transformations of differential–difference equations and, in particular, the notion of Miura type transformation. We introduce the concept of non-Miura type linearizable transformation and we present techniques that allow one to construct simple linearizable transformations and might help one to solve classification problems. This theory is illustrated by the example of a new integrable differential–difference equation depending on five lattice points, interesting from the viewpoint of the non-invertible transformation, which relate it to an Itoh–Narita–Bogoyavlensky equation.

  7. Design and field performance of the KENETECH photovoltaic inverter system

    SciTech Connect

    Behnke, M.R.

    1995-11-01

    KENETECH Windpower has recently adapted the power conversion technology developed for the company`s variable speed wind turbine to grid-connected photovoltaic applications. KENETECH PV inverter systems are now in successful operation at the Sacramento Municipal Utility District`s (SMUD) Hedge Substation and the PVUSA-Davis site, with additional systems scheduled to be placed into service by the end of 1995 at SMUD, the New York Power Authority, Xerox Corporation`s Clean Air Now project, and the Georgia Tech Aquatic Center. The features of the inverter are described.

  8. Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal

    PubMed Central

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan

    2015-01-01

    We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3′ end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA. PMID:26297494

  9. Recombination-restarted replication makes inverted chromosome fusions at inverted repeats.

    PubMed

    Mizuno, Ken'Ichi; Miyabe, Izumi; Schalbetter, Stephanie A; Carr, Antony M; Murray, Johanne M

    2013-01-10

    Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.

  10. Terminal weather information management

    NASA Technical Reports Server (NTRS)

    Lee, Alfred T.

    1990-01-01

    Since the mid-1960's, microburst/windshear events have caused at least 30 aircraft accidents and incidents and have killed more than 600 people in the United States alone. This study evaluated alternative means of alerting an airline crew to the presence of microburst/windshear events in the terminal area. Of particular interest was the relative effectiveness of conventional and data link ground-to-air transmissions of ground-based radar and low-level windshear sensing information on microburst/windshear avoidance. The Advanced Concepts Flight Simulator located at Ames Research Center was employed in a line oriented simulation of a scheduled round-trip airline flight from Salt Lake City to Denver Stapleton Airport. Actual weather en route and in the terminal area was simulated using recorded data. The microburst/windshear incident of July 11, 1988 was re-created for the Denver area operations. Six experienced airline crews currently flying scheduled routes were employed as test subjects for each of three groups: (1) A baseline group which received alerts via conventional air traffic control (ATC) tower transmissions; (2) An experimental group which received alerts/events displayed visually and aurally in the cockpit six miles (approx. 2 min.) from the microburst event; and (3) An additional experimental group received displayed alerts/events 23 linear miles (approx. 7 min.) from the microburst event. Analyses of crew communications and decision times showed a marked improvement in both situation awareness and decision-making with visually displayed ground-based radar information. Substantial reductions in the variability of decision times among crews in the visual display groups were also found. These findings suggest that crew performance will be enhanced and individual differences among crews due to differences in training and prior experience are significantly reduced by providing real-time, graphic display of terminal weather hazards.

  11. Emotional memory impairments induced by AAV-mediated overexpression of human α-synuclein in dopaminergic neurons of the ventral tegmental area.

    PubMed

    Alvarsson, A; Caudal, D; Björklund, A; Svenningsson, P

    2016-01-01

    Parkinson's disease (PD) is associated with extensive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta, but neuronal loss is also found in the ventral tegmental area (VTA). The VTA projects to areas involved in cognitive and emotional processes, including hippocampus, amygdala, nucleus accumbens and prefrontal cortex, and has thus been proposed to play a role in emotional memory impairments in PD. Since the formation of α-synuclein inclusions throughout the central nervous system is a pathological hallmark of PD, we studied the progressive effects of α-synuclein overexpression in the VTA on motor functions, emotional behaviour and emotional memory. Adeno-associated viral (AAV) vectors encoding either human α-synuclein or green fluorescent protein (GFP) were injected stereotactically into the VTA, and behaviour was monitored 3 and 8 weeks following AAV injection. At week 8, there was a 22% reduction of TH+ neurons in the VTA. We demonstrate that α-synuclein overexpression in dopaminergic neurons of the VTA induced mild motor deficits that appeared 3 weeks following AAV-α-synuclein injection and were aggravated at week 8. No depressive- or anxiety-like behaviours were found. To address emotional memory, we used the passive avoidance test, a one-trial associative learning paradigm based on contextual conditioning which requires minimal training. Interestingly, emotional memory impairments were found in α-synuclein overexpressing animals at week 8. These findings indicate that α-synuclein overexpression induces progressive memory impairments likely caused by a loss of function of mesolimbic dopaminergic projections.

  12. Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

    PubMed Central

    Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

    2013-01-01

    Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

  13. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice

    PubMed Central

    Marangoni, Dario; Bush, Ronald A; Zeng, Yong; Wei, Lisa L; Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bartoe, Joshua T; Palyada, Kiran; Santos, Maria; Hiriyanna, Suja; Wu, Zhijian; Colosi, Peter; Sieving, Paul A

    2016-01-01

    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

  14. Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness.

    PubMed

    Scalabrino, Miranda L; Boye, Sanford L; Fransen, Kathryn M H; Noel, Jennifer M; Dyka, Frank M; Min, Seok Hong; Ruan, Qing; De Leeuw, Charles N; Simpson, Elizabeth M; Gregg, Ronald G; McCall, Maureen A; Peachey, Neal S; Boye, Shannon E

    2015-11-01

    Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1. PMID:26310623

  15. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice

    PubMed Central

    Marangoni, Dario; Bush, Ronald A; Zeng, Yong; Wei, Lisa L; Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bartoe, Joshua T; Palyada, Kiran; Santos, Maria; Hiriyanna, Suja; Wu, Zhijian; Colosi, Peter; Sieving, Paul A

    2016-01-01

    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS. PMID:27626041

  16. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

    PubMed

    Marangoni, Dario; Bush, Ronald A; Zeng, Yong; Wei, Lisa L; Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bartoe, Joshua T; Palyada, Kiran; Santos, Maria; Hiriyanna, Suja; Wu, Zhijian; Colosi, Peter; Sieving, Paul A

    2016-01-01

    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS. PMID:27626041

  17. Safety and Tolerability of Magnetic Resonance Imaging-Guided Convection-Enhanced Delivery of AAV2-hAADC with a Novel Delivery Platform in Nonhuman Primate Striatum

    PubMed Central

    San Sebastian, Waldy; Richardson, R. Mark; Kells, Adrian P.; Lamarre, Clementine; Bringas, John; Pivirotto, Philip; Salegio, Ernesto A.; DeArmond, Stephen J.; Forsayeth, John

    2012-01-01

    Abstract Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-18F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC. PMID:22017504

  18. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

    PubMed Central

    Sather, Blythe D.; Romano Ibarra, Guillermo S.; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F.; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A.; Scharenberg, Andrew M.; Rawlings, David J.

    2016-01-01

    Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV) – mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP – modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. PMID:26424571

  19. Intra-Arterial Delivery of AAV Vectors to the Mouse Brain After Mannitol Mediated Blood Brain Barrier Disruption

    PubMed Central

    Santillan, Alejandro; Sondhi, Dolan; Dyke, Jonathan P.; Crystal, Ronald G.; Gobin, Y. Pierre; Ballon, Douglas J.

    2014-01-01

    The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single

  20. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

    PubMed

    Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

    2015-09-30

    Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.