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Sample records for aav inverted terminal

  1. Adeno-associated virus inverted terminal repeats stimulate gene editing.

    PubMed

    Hirsch, M L

    2015-02-01

    Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

  2. Hybrid adeno-associated virus bearing nonhomologous inverted terminal repeats enhances dual-vector reconstruction of minigenes in vivo.

    PubMed

    Yan, Ziying; Lei-Butters, Diana C M; Zhang, Yulong; Zak, Roman; Engelhardt, John F

    2007-01-01

    We have previously demonstrated that hybrid adeno-associated viral (AAV) vectors bearing nonhomologous inverted terminal repeats (ITRs) enhance directional intermolecular recombination and the efficiency of dual-AAV vector trans-splicing in cultured cells. Using hybrid-ITR vectors carrying two exons of a lacZ minigene, we demonstrate that this dual-vector approach also mediates higher levels (3- to 6-fold) of gene reconstitution in mouse skeletal muscle, liver, and heart. Inhibition of the proteasome by systemic administration of Doxil (Food and Drug Administration-approved lipid-formulated doxorubicin) further enhanced dual-vector trans-splicing 6- to 12-fold in two mouse strains. Hence, using hybrid-ITR AAV vectors in combination with proteasome modulation enhanced dual-vector delivery of a transgene approximately 36-fold over the current dual-vector trans-splicing approaches. These data provide in vivo evidence that ITR sequence-dependent homologous recombination, rather than nonhomologous end joining, is the predominant mechanism for AAV genome heterodimerization. Hence, enhanced directional recombination provided by hybrid-ITR vectors may be a useful in vivo strategy for improving dual-vector delivery of transgenes larger than the AAV packaging limit.

  3. Site-specific nicking within the adenovirus inverted terminal repetition.

    PubMed Central

    Chow, K C; Pearson, G D

    1984-01-01

    Site-specific nicking occurs on the l-strand, but not on the r-strand, of the adenovirus left inverted terminal repeat. Nicks are presumably introduced into double- or single-stranded DNA by a cellular endonuclease in an ATP-independent reaction. The consensus nick site has the sequence: (sequence in text). Images PMID:6322107

  4. Terminal differentiation of cardiac and skeletal myocytes induces permissivity to AAV transduction by relieving inhibition imposed by DNA damage response proteins.

    PubMed

    Lovric, Jasmina; Mano, Miguel; Zentilin, Lorena; Eulalio, Ana; Zacchigna, Serena; Giacca, Mauro

    2012-11-01

    Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.

  5. 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

  6. 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

  7. 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.

  8. High-performance inkjet-printed four-terminal microelectromechanical relays and inverters.

    PubMed

    Chung, Seungjun; Ul Karim, Muhammed Ahosan; Kwon, Hyuk-Jun; Subramanian, Vivek

    2015-05-13

    We report the first demonstration of inkjet-printed 4-terminal microelectromechanical (MEM) relays and inverters with hyper-abrupt switching that exhibit excellent electrical and mechanical characteristics. This first implementation of a printed 4-terminal device is critically important, since it allows for the realization of full complementary logic functions. The floated fourth terminal (body electrode), which allows the gate switching voltage to be adjusted, is bonded to movable channel beams via a printed epoxy layer in a planar structure, which can move downward together via the electrostatic force between the gate electrodes and body such that the channel can also actuate downward and touch the drain electrode. Because the body, channel, and drain electrodes are completely electrically separated, no detectable leakage or electrical interference between the electrodes is observed. The printed MEM relay exhibited an on-state resistance of only 3.48 Ω, immeasurable off-state leakage, subthreshold swing <1 mV/dec, and a stable operation over 10(4) cycles with a switching delay of 47 μs, and the relay inverter exhibits abrupt transitions between on/off states. The operation of the printed 4-terminal MEM relay was also verified against the results of a 3-dimensional (3D) finite element simulation.

  9. Intraspecific Variability of the Terminal Inverted Repeats of the Linear Chromosome of Streptomyces ambofaciens

    PubMed Central

    Choulet, Frédéric; Gallois, Alexandre; Aigle, Bertrand; Mangenot, Sophie; Gerbaud, Claude; Truong, Chantal; Francou, François-Xavier; Borges, Frédéric; Fourrier, Céline; Guérineau, Michel; Decaris, Bernard; Barbe, Valérie; Pernodet, Jean-Luc; Leblond, Pierre

    2006-01-01

    The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities. PMID:16952952

  10. Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens.

    PubMed

    Choulet, Frédéric; Gallois, Alexandre; Aigle, Bertrand; Mangenot, Sophie; Gerbaud, Claude; Truong, Chantal; Francou, François-Xavier; Borges, Frédéric; Fourrier, Céline; Guérineau, Michel; Decaris, Bernard; Barbe, Valérie; Pernodet, Jean-Luc; Leblond, Pierre

    2006-09-01

    The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

  11. Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

    PubMed

    Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell'Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

    2008-03-01

    We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

  12. Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

    PubMed Central

    Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell’Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

    2010-01-01

    We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 × 1010 vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations. PMID:18209734

  13. Inverted duplication with terminal deletion of 5p and no cat-like cry.

    PubMed

    Wang, Jia-Chi; Coe, Bradley P; Lomax, Brenda; MacLeod, Patrick M; Parslow, Malcolm I; Schein, Jacqueline E; Lam, Wan L; Eydoux, Patrice

    2008-05-01

    We report on a 6-year-old boy referred for cytogenetics study. A few non-specific features were observed in the newborn: hypotonia, failure to thrive, seizures, pre-auricular skin tags. Cat-like cry was not identified. No remarkable facial dysmorphism, gastrointestinal, respiratory or cardiac abnormalities were identified. At age 4 years, speech and motor skill delays were apparent. Karyotyping and FISH analysis revealed a de novo rearranged chromosome 5p, with subtelomeric deletion of 5p and a duplication of the cri-du-chat critical region. Array CGH using sub-megabase resolution tiling-set (SMRT) array followed by FISH analysis with labeled BACs showed a deletion of 5pter to 5p15.31 (0-6.9 Mb) and an inverted duplication of the greater part of 5p15.31 to the distal end of 5p14.3 (6.9-19.9 Mb). Although very rare, inverted duplications with terminal deletion (inv dup del) have been reported at different chromosomal ends. Our finding adds a second patient of inv dup del 5p to this growing list, and the potential causative mechanisms for this rearrangement are discussed. Review of the mapping information of cri-du-chat patients and the comparison with a previously reported patient suggested that the critical region for cat-like cry is located within a 0.6 Mb region.

  14. [Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus].

    PubMed

    Li, Taiming; Pan, Junjie; Qi, Jing; Zhang, Chun

    2015-08-01

    AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.

  15. Construction and packaging of herpes simplex virus/adeno-associated virus (HSV/AAV) Hybrid amplicon vectors.

    PubMed

    Saydam, Okay; Glauser, Daniel L; Fraefel, Cornel

    2012-03-01

    Herpes simplex virus type 1 (HSV-1)-based amplicon vectors conserve most properties of the parental virus: broad host range, the ability to transduce dividing and nondiving cells, and a large transgene capacity. This permits incorporation of genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell-specific expression, multiple transgene cassettes, or genetic elements from other viruses. Hybrid vectors use elements from HSV-1 that allow replication and packaging of large-vector DNA into highly infectious particles, and elements from other viruses that confer genetic stability to vector DNA in the transduced cell. For example, adeno-associated virus (AAV) has the unique ability to integrate its genome into a specific site on human chromosome 19. The viral rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. However, AAV-based vectors have a very small transgene capacity and do not conventionally contain the rep gene to support site-specific genomic integration. HSV/AAV hybrid vectors contain both HSV-1 replication and packaging functions and the AAV rep gene and a transgene cassette flanked by the AAV ITRs. This combines the large transgene capacity of HSV-1 with the capability of site-specific genomic transgene integration and long-term transgene expression of AAV. This protocol describes the preparation of HSV/AAV hybrid vectors using a replication-competent/conditional, packaging-defective HSV-1 genome cloned as a bacterial artificial chromosome (BAC) to provide helper functions for vector replication and packaging. The advantages and limitations of such vectors compared to standard HSV-1 amplicon vectors are also discussed.

  16. Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats.

    PubMed

    Ling, Alison; Cordaux, Richard

    2010-12-20

    Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements.

  17. Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae.

    PubMed

    Besansky, N J; Mukabayire, O; Bedell, J A; Lusz, H

    1996-10-01

    Pegasus, a novel transposable element, was discovered as a length polymorphism in the white gene of Anopheles gambiae. Sequence analysis revealed that this 535 bp element was flanked by 8 bp target site duplications and 8 bp perfect terminal inverted repeats similar to those found in many members of the Tc1 family. Its small size and lack of long open reading frames preclude protein coding capacity. Southern analysis and in situ hybridization to polytene chromosomes demonstrated that Pegasus occurs in approximately 30 copies in the genomes of An. gambiae and its sibling species and is homogenous in structure but polymorphic in chromosomal location. Characterization of five additional elements by sequencing revealed nucleotide identities of 95% to 99%. Of 30 Pegasus-containing phage clones examined by PCR, only one contained an element exceeding 535 bp in length, due to the insertion of another transposable element-like sequence. Thus, the majority, if not all, extant Pegasus elements may be defective copies of a complete element whose contemporary existence in An. gambiae is uncertain. No Pegasus-hybridizing sequences were detected in nine other anophelines and three culicines examined, suggesting a very limited taxonomic distribution.

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

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-02

    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.

  19. Mutator-Like Elements with Multiple Long Terminal Inverted Repeats in Plants

    PubMed Central

    Ferguson, Ann A.; Jiang, Ning

    2012-01-01

    Mutator-like transposable elements (MULEs) are widespread in plants and the majority have long terminal inverted repeats (TIRs), which distinguish them from other DNA transposons. It is known that the long TIRs of Mutator elements harbor transposase binding sites and promoters for transcription, indicating that the TIR sequence is critical for transposition and for expression of sequences between the TIRs. Here, we report the presence of MULEs with multiple TIRs mostly located in tandem. These elements are detected in the genomes of maize, tomato, rice, and Arabidopsis. Some of these elements are present in multiple copies, suggesting their mobility. For those elements that have amplified, sequence conservation was observed for both of the tandem TIRs. For one MULE family carrying a gene fragment, the elements with tandem TIRs are more prevalent than their counterparts with a single TIR. The successful amplification of this particular MULE demonstrates that MULEs with tandem TIRs are functional in both transposition and duplication of gene sequences. PMID:22474413

  20. 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.

  1. Anti-human α-synuclein N-terminal peptide antibody protects against dopaminergic cell death and ameliorates behavioral deficits in an AAV-α-synuclein rat model of Parkinson's disease.

    PubMed

    Shahaduzzaman, Md; Nash, Kevin; Hudson, Charles; Sharif, Masroor; Grimmig, Bethany; Lin, Xiaoyang; Bai, Ge; Liu, Hui; Ugen, Kenneth E; Cao, Chuanhai; Bickford, Paula C

    2015-01-01

    The protein α-synuclein (α-Syn) has a central role in the pathogenesis of Parkinson's disease (PD) and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN), while control rats received an AAV vector expressing green fluorescent protein (GFP). Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1), or central region (AB2) of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II) immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2) significantly inhibited α-Syn-induced dopaminergic cell (DA) and NeuN+ cell loss (one-way ANOVA (F (3, 30) = 5.8, p = 0.002 and (F (3, 29) = 7.92, p = 0.002 respectively), as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003). Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37) = 9.786; p = 0.0001) and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD.

  2. Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

    PubMed Central

    Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

    2004-01-01

    Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. PMID:14742212

  3. Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome.

    PubMed

    Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

    2004-02-01

    Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.

  4. 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

  5. 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

  6. 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

  7. 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

  8. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors.

    PubMed

    Liu, Q; Huang, W; Zhang, H; Wang, Y; Zhao, J; Song, A; Xie, H; Zhao, C; Gao, D; Wang, Y

    2014-08-01

    Adeno-associated viruses (AAV) have attracted attention as potential vectors for gene therapy and vaccines against several diseases, including HIV-1 infection. However, the presence of neutralizing antibodies (NAbs) after natural AAV infections inhibits their transfection in re-exposed subjects. To identify candidate AAV vectors for therapeutic or prophylactic HIV vaccines, NAbs against AAV2, AAV5 and AAV8 were screened in the sera of healthy individuals in China and 10 developed countries and an HIV-1-infected Chinese population. Seroprevalence was higher for AAV2 (96.6%) and AAV8 (82.0%) than for AAV5 (40.2%) in normal Chinese subjects. Among individuals seropositive for AAV5, >80% had low NAb titers (<1:90). The prevalence and titers of NAbs against the three AAVs were significantly higher in China than in developed countries (P<0.01). The prevalence of NAbs against AAV5 did not differ significantly between healthy and HIV-1-infected Chinese subjects (P=0.39). Co-occurrence of NAbs against AAV2, AAV5, and AAV8 was observed in the healthy population, and 15, 41, and 41% of individuals were AAV2(+), AAV2(+)/AAV8(+), and AAV2(+)/AAV5(+)/AAV8(+), respectively. Therefore, AAV5 exposure is low in healthy and HIV-1-infected populations Chinese individuals, and vectors based on AAV5 may be appropriate for human gene therapy or vaccines.

  9. Humoral Immune Response to AAV

    PubMed Central

    Calcedo, Roberto; Wilson, James M.

    2013-01-01

    Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy. PMID:24151496

  10. Humoral Immune Response to AAV.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2013-10-18

    Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.

  11. Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer.

    PubMed

    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

    2011-07-01

    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 (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) 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 Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-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-Tet(bidir)-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.

  12. "Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry.

    PubMed

    Simeonova, Diliana Dancheva; Susnea, Iuliana; Moise, Adrian; Schink, Bernhard; Przybylski, Michael

    2009-01-01

    We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

  13. Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

    PubMed Central

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith

    2011-01-01

    Abstract Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy. PMID:21453126

  14. Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation

    PubMed Central

    Musayev, Faik N.; Zarate-Perez, Francisco; Bardelli, Martino; Bishop, Clayton; Saniev, Emil F.; Linden, R. Michael; Henckaerts, Els; Escalante, Carlos R.

    2015-01-01

    Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ~20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA. PMID:26314310

  15. Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

    PubMed

    Musayev, Faik N; Zarate-Perez, Francisco; Bardelli, Martino; Bishop, Clayton; Saniev, Emil F; Linden, R Michael; Henckaerts, Els; Escalante, Carlos R

    2015-09-29

    Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.

  16. 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.

  17. Hydroxyl-Terminated CuInS2-Based Quantum Dots: Potential Cathode Interfacial Modifiers for Efficient Inverted Polymer Solar Cells.

    PubMed

    Chen, Hui; Chao, Pengjie; Han, Dengbao; Wang, Huan; Miao, Jingsheng; Zhong, Haizheng; Meng, Hong; He, Feng

    2017-03-01

    The use of interfacial modifiers on cathode or anode layers can effectively reduce the recombination loss and thus have potential to enhance the device performance of polymer solar cells. In this work, we demonstrated that hydroxyl-terminated CuInS2-based quantum dots could be potential cathode interfacial modifiers on ZnO layer for inverted polymer solar cells. By casting of a thin film of CuInS2-based quantum dots onto ZnO layer, the controlled devices show obvious enhancements of open-circuit voltage, short-circuit current, and fill factor. With an optimized interfacial layer with ∼7 nm thickness, an improvement of power conversion efficiency up to 16% is obtained and the optimized power conversion efficiency of PTB7-based (PTB7: poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl) carbonyl] thieno[3,4-b] thiophenediyl

  18. 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.

  19. Engineering AAV receptor footprints for gene therapy.

    PubMed

    Madigan, Victoria J; Asokan, Aravind

    2016-06-01

    Adeno-associated viruses (AAV) are currently at the forefront of human gene therapy clinical trials as recombinant vectors. Significant progress has been made in elucidating the structure, biology and tropisms of different naturally occurring AAV isolates in the past decade. In particular, a spectrum of AAV capsid interactions with host receptors have been identified and characterized. These studies have enabled a better understanding of key determinants of AAV cell recognition and entry in different hosts. This knowledge is now being applied toward engineering new, lab-derived AAV capsids with favorable transduction profiles. The current review conveys a structural perspective of capsid-glycan interactions and provides a roadmap for generating synthetic strains by engineering AAV receptor footprints.

  20. 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

  1. 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.

  2. Innate Immune Responses to AAV Vectors.

    PubMed

    Rogers, Geoffrey L; Martino, Ashley T; Aslanidi, George V; Jayandharan, Giridhara R; Srivastava, Arun; Herzog, Roland W

    2011-01-01

    Gene replacement therapy by in vivo delivery of adeno-associated virus (AAV) is attractive as a potential treatment for a variety of genetic disorders. However, while AAV has been used successfully in many models, other experiments in clinical trials and in animal models have been hampered by undesired responses from the immune system. Recent studies of AAV immunology have focused on the elimination of transgene-expressing cells by the adaptive immune system, yet the innate immune system also has a critical role, both in the initial response to the vector and in prompting a deleterious adaptive immune response. Responses to AAV vectors are primarily mediated by the TLR9-MyD88 pathway, which induces the production of pro-inflammatory cytokines by activating the NF-κB pathways and inducing type I IFN production; self-complementary AAV vectors enhance these inflammatory processes. Additionally, the alternative NF-κB pathway influences transgene expression in cells transduced by AAV. This review highlights these recent discoveries regarding innate immune responses to AAV and discusses strategies to ablate these potentially detrimental signaling pathways.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26412589

  4. AAV-8 is more efficient than AAV-9 in transducing neonatal dog heart.

    PubMed

    Pan, Xiufang; Yue, Yongping; Zhang, Keqing; Hakim, Chady H; Kodippili, Kasun; McDonald, Thomas; Duan, Dongsheng

    2015-04-01

    Adeno-associated virus serotype-8 and 9 (AAV-8 and 9) are the leading candidate vectors to test bodywide neonatal muscle gene therapy in large mammals. We have previously shown that systemic injection of 2-2.5×10(14) viral genome (vg) particles/kg of AAV-9 resulted in widespread skeletal muscle gene transfer in newborn dogs. However, nominal transduction was observed in the heart. In contrast, robust expression was achieved in both skeletal muscle and heart in neonatal dogs with 7.14-9.06×10(14) vg particles/kg of AAV-8. To determine whether superior cardiac transduction of AAV-8 is because of the higher vector dose, we delivered 6.14×10(14) and 9.65×10(14) vg particles/kg of AAV-9 to newborn puppies via the jugular vein. Transduction was examined 2.5 months later. Consistent with our previous reports, we observed robust bodywide transduction in skeletal muscle. However, increased AAV dose only moderately improved heart transduction. It never reached the level achieved by AAV-8. Our results suggest that differential cardiac transduction by AAV-8 and AAV-9 is likely because of the intrinsic property of the viral capsid rather than the vector dose.

  5. AAV natural infection induces broad cross-neutralizing antibody responses to multiple AAV serotypes in chimpanzees.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2016-06-01

    Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to sero-negative nonhuman primates which is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified three distinct groups of animals: those which never sero-converted to AAV (naïve); those which were persistently seropositive (chronic); and those that seroconverted during the 10 year period (acute). For the chronic group we found a broad sero-response characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes.

  6. AAV Natural Infection Induces Broad Cross-Neutralizing Antibody Responses to Multiple AAV Serotypes in Chimpanzees.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2016-06-01

    Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to seronegative nonhuman primates that is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified 3 distinct groups of animals: those that never seroconverted to AAV (naïve), those that were persistently seropositive (chronic), and those that seroconverted during the 10-year period (acute). For the chronic group we found a broad seroresponse characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes.

  7. 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.

  8. 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

  9. Direct interaction of human serum proteins with AAV virions to enhance AAV transduction: immediate impact on clinical applications.

    PubMed

    Wang, M; Sun, J; Crosby, A; Woodard, K; Hirsch, M L; Samulski, R J; Li, C

    2017-01-01

    Recent hemophilia B clinical trials using adeno-associated virus (AAV) gene delivery have demonstrated much lower coagulation factor IX (FIX) production in patients compared with the high levels observed in animal models and AAV capsid-specific cytotoxic T lymphocyte response elicited at high doses of AAV vectors. These results emphasize the necessity to explore effective approaches for enhancement of AAV transduction. Initially, we found that incubation of all AAV vectors with human serum enhanced AAV transduction. Complementary analytical experiments demonstrated that human serum albumin (HSA) directly interacted with the AAV capsid and augmented AAV transduction. The enhanced transduction was observed with clinical grade HSA. Mechanistic studies suggest that HSA increases AAV binding to target cells, and that the interaction of HSA with AAV does not interfere with the AAV infection pathway. Importantly, HSA incubation during vector dialysis also increased transduction. Finally, HSA enhancement of AAV transduction in a model of hemophilia B displayed greater than a fivefold increase in vector-derived circulating FIX, which improved the bleeding phenotype correction. In conclusion, incubation of HSA with AAV vectors supports a universal augmentation of AAV transduction and, more importantly, this approach can be immediately transitioned to the clinic for the treatment of hemophilia and other diseases.

  10. OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

    PubMed

    Mietzsch, Mario; Hering, Henrik; Hammer, Eva-Maria; Agbandje-McKenna, Mavis; Zolotukhin, Sergei; Heilbronn, Regine

    2017-02-01

    Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates. OneBac 2.0 represents a 2-fold advancement: First, enhanced VP1 proportions in AAV5 capsids lead to vastly increased per-particle infectivity rates. Second, collateral packaging of foreign DNA is suppressed by removal of the Rep-binding element (RBE). In this study we show that this advancement of AAV5 packaging can be translated to OneBac 2.0-derived packaging systems for alternative AAV serotypes. By removal of the RBE, collateral packaging of nonvector DNA was drastically reduced in all newly tested serotypes (AAV1, AAV2, and AAV8). However, the splicing-based strategy to enhance VP1 expression in order to increase AAV5 infectivity hardly improved infectivity rates of AAV-1, -2, or -8 compared with the original OneBac cell lines. Our results emphasize that OneBac 2.0 represents an advancement for scalable, high-titer production of various AAV serotypes, leading to AAV particles with minimal packaging of foreign DNA.

  11. Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

    PubMed Central

    Seyffert, Michael; Glauser, Daniel L.; Schraner, Elisabeth M.; de Oliveira, Anna-Paula; Mansilla-Soto, Jorge; Vogt, Bernd; Büning, Hildegard; Linden, R. Michael; Ackermann, Mathias; Fraefel, Cornel

    2017-01-01

    As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity. PMID:28125695

  12. Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors.

    PubMed

    Lu, Yuanqing; Song, Sihong

    2009-10-06

    Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.

  13. [Inverted nipples].

    PubMed

    Saltvig, Iselin; Sjøstrand, Helle; Oldenburg, Mette Holmqvist; Matzen, Steen Henrik

    2016-10-17

    Inverted nipples is an anatomical variation which can be uni- or bilateral, congenital or acquired. The degree of inversion can vary from slight to severe. Treatment can be surgical or non-surgical and should depend on the degree of functional problems. Non-surgical treatment can be beneficial, does not risk affecting sensibility, spares the lactiferous ducts, and therefore does not risk any interference with breast-feeding. Surgical options should only be considered when non-surgical treatment is insufficient.

  14. Establishment of an AAV Reverse Infection-Based Array

    PubMed Central

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

    2010-01-01

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

  15. Packaging of an AAV vector encoding human acid alpha-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector.

    PubMed

    Sun, Baodong; Chen, Y-T; Bird, Andrew; Xu, Fang; Hou, Yang-Xun; Amalfitano, Andrea; Koeberl, Dwight D

    2003-04-01

    We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid alpha-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10(10) AAV vector particles. These modifications resulted in approximately 30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid alpha-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.

  16. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1.

    PubMed

    Musayev, Faik N; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W; Escalante, Carlos R

    2015-11-13

    Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains.

  17. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

    PubMed Central

    Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

    2015-01-01

    Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

  18. AAV-Mediated Liver-Directed Gene Therapy

    PubMed Central

    Sands, Mark S.

    2014-01-01

    The liver is directly or indirectly involved in many essential processes and is affected by numerous inherited diseases. Therefore, many inherited diseases could be effectively treated by targeting the liver using gene transfer approaches. The challenges associated with liver-directed gene therapy are efficient targeting of hepatocytes, stability of the vector genome, and persistent high level expression. Many of these obstacles can be overcome with adeno-associated viral (AAV) gene transfer vectors. The first AAV gene transfer vector developed for in vivo use was based on the AAV2 serotype. AAV2 has a broad tropism and transduces many cell types, including hepatocytes, relatively efficiently in vivo. The capsid protein confers the serological profile and at least 12 primate AAV serotypes have already been characterized. Importantly, pseudotyping a recombinant AAV vector with different capsid proteins can dramatically alter the tropism. Both AAV8 and AAV9 have higher affinities for hepatocytes when compared to AAV2. In particular, AAV8 can transduce 3–4 fold more hepatocytes and deliver 3–4 fold more genomes per transduced cell when compared to AAV2. Depending on the dose, AAV8 can transduce up to 90–95% of hepatocytes in the mouse liver following intraportal vein injection. Interestingly, comparable levels of transduction can be achieved following intravenous injection. Direct intraparenchymal injection of an AAV vector also mediates relatively high level long term expression. Additional specificity can be conferred by using liver-specific promoters in conjunction with AAV8 capsid proteins. In addition to treating primary hepatocyte defects, immune reactions to transgene products can be minimized by circumventing the fixed tissue macrophages of the liver, Kupffer cells, and limiting expression to hepatocytes. The ability to target hepatocytes by virtue of the AAV serotype and the use of liver-specific promoters allows investigators to test novel therapeutic

  19. Vaccinia virus as a subhelper for AAV replication and packaging.

    PubMed

    Moore, Andrea R; Dong, Biao; Chen, Lingxia; Xiao, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV) has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

  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. Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical.

    PubMed

    He, Yi; Weinberg, Marc S; Hirsch, Matt; Johnson, Mark C; Tisch, Roland; Samulski, R Jude; Li, Chengwen

    2013-05-01

    Adeno-associated viral (AAV) vectors 2 and 8 have been used in clinical trials for patients with hemophilia, and data suggest that the capsid-specific CD8⁺ T cell response has had a negative impact on therapeutic success. To date the pattern of capsid cross-presentation from AAV2 and AAV8 transduction in vivo has not been elucidated. Previously, we have demonstrated that an engineered AAV2 virus carrying the immune-dominant SIINFEKL peptide in the capsid backbone was indistinguishable from wild type with respect to titer, tropism, and the ability to induce capsid-specific CD8⁺ T cell responses in vivo. In this study, we used the same strategy to engineer an AAV8 vector and demonstrated that antigen from SIINFEKL peptide-integrated AAV8 capsid was effectively presented via either plasmid transfection or AAV8 transduction in vitro. The tissue tropism and transgene expression kinetics of the engineered AAV8 vector in vivo were identical to that of wild-type AAV8. Animal studies show that capsid antigen presentation from AAV transduction was dose dependent, and more importantly, the proliferation of capsid-specific CD8⁺ T cells had similar kinetics (detectable before 30 days and undetectable after 40 days) for both AAV2 and AAV8 vectors. Elucidation of the kinetics of capsid antigen presentation from AAV transduction by various serotypes provides new insight into the potential impact CD8⁺ T cells can have during clinical trials and may help with rational design of effective strategies to prevent capsid-specific CD8⁺ T cell-mediated elimination of AAV-transduced target cells.

  2. High-throughput screening and biophysical interrogation of hepatotropic AAV.

    PubMed

    Murphy, Samuel L; Bhagwat, Anand; Edmonson, Shyrie; Zhou, Shangzhen; High, Katherine A

    2008-12-01

    We set out to analyze the fundamental biological differences between AAV2 and AAV8 that may contribute to their different performances in vivo. High-throughput protein interaction screens were used to identify binding partners for each serotype. Of the >8,000 proteins probed, 115 and 134 proteins were identified that interact with AAV2 and AAV8, respectively. Notably, 76 of these protein interactions were shared between the two serotypes. CDK2/cyclinA kinase was identified as a binding partner for both serotypes in the screen. Subsequent analysis confirmed direct binding of CDK2/cyclinA by AAV2 and AAV8. Inhibition of CDK2/cyclinA resulted in increased levels of vector transduction. Biophysical study of vector particle stability and genome uncoating demonstrated slightly greater thermostability for AAV8 than for AAV2. Heat-induced genome uncoating occurred at the same temperature as particle degradation, suggesting that these two processes may be intrinsically related for adeno-associated virus (AAV). Together, these analyses provide insight into commonalities and divergences in the biology of functionally distinct hepatotropic AAV serotypes.

  3. Properly scaled and targeted AAV2-NRTN (neurturin) to the substantia nigra is safe, effective and causes no weight loss: support for nigral targeting in Parkinson's disease.

    PubMed

    Bartus, Raymond T; Brown, Lamar; Wilson, Alistair; Kruegel, Brian; Siffert, Joao; Johnson, Eugene M; Kordower, Jeffrey H; Herzog, Christopher D

    2011-10-01

    Recent analyses of autopsied brains from subjects previously administered AAV2-neurturin (NRTN) gene transfer argues that optimizing the effects of neurotrophic factors in Parkinson's disease (PD) likely requires delivery to both the degenerating cell bodies (in substantia nigra) and their terminals (in striatum). Prior to implementing this novel dosing paradigm in humans, we conducted eight nonclinical experiments with three general objectives: (1) evaluate the feasibility, safety and effectiveness of targeting the substantia nigra (SN) with AAV2-NRTN, (2) better understand and appraise recent warnings of serious weight loss that might occur with targeting the SN with neurotrophic factors, and (3) define an appropriate dose of AAV2-NRTN that should safely and effectively cover the SN in PD patients. Toward these ends, we first determined SN volume for rats, monkeys and humans, and employed these values to calculate comparable dose equivalents for each species by scaling each dose, based on relative SN volume. Using this information, we next injected AAV2-GFP to monkey SN to quantify AAV2-vector distribution and confirm reasonable SN coverage. We then selected and administered a ~200-fold range of AAV2-NRTN doses (and a single AAV2-GDNF dose) to rat SN, producing a wide range of protein expression. In contrast to recent warnings regarding nigra targeting, no dose produced any serious side effects or toxicity, though we replicated the modest reduction in weight gain reported by others with the highest AAV2-NRTN and the AAV2-GDNF dose. A dose-related increase in NRTN expression was seen, with the lower doses limiting NRTN to the peri-SN and the highest dose producing mistargeted NRTN well outside the SN. We then demonstrated that the reduction in weight gain following excessive-doses can be dissociated from NRTN in the targeted SN, and is linked to mistargeted NRTN in the diencephalon. We also showed that prior destruction of the dopaminergic SN neurons via 6-OHDA

  4. High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence.

    PubMed

    Hüser, Daniela; Khalid, Dina; Lutter, Timo; Hammer, Eva-Maria; Weger, Stefan; Heßler, Melanie; Kalus, Ulrich; Tauchmann, Yvonne; Hensel-Wiegel, Karin; Lassner, Dirk; Heilbronn, Regine

    2017-02-15

    Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3(+) T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation.

  5. Adeno-associated virus (AAV) vectors in cancer gene therapy.

    PubMed

    Santiago-Ortiz, Jorge L; Schaffer, David V

    2016-10-28

    Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field.

  6. Preferred transduction with AAV8 and AAV9 via thalamic administration in the MPS IIIB model: A comparison of four rAAV serotypes

    PubMed Central

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

    2015-01-01

    Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV) vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS) and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP) bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery. PMID:27014573

  7. Assaying the Stability and Inactivation of AAV Serotype 1 Vectors.

    PubMed

    Howard, Douglas B; Harvey, Brandon K

    2017-02-01

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

  8. Clinically Relevant Effects of Convection-Enhanced Delivery of AAV2-GDNF on the Dopaminergic Nigrostriatal Pathway in Aged Rhesus Monkeys

    PubMed Central

    Johnston, Louisa C.; Eberling, Jamie; Pivirotto, Philip; Hadaczek, Piotr; Federoff, Howard J.; Forsayeth, John

    2009-01-01

    Abstract Growth factor therapy for Parkinson's disease offers the prospect of restoration of dopaminergic innervation and/or prevention of neurodegeneration. Safety and efficacy of an adeno-associated virus (AAV2) encoding human glial cell-derived neurotrophic factor (GDNF) was investigated in aged nonhuman primates. Positron emission tomography with 6-[18F]-fluoro-l-m-tyrosine (FMT-PET) in putamen was assessed 3 months before and after AAV2 infusion. In the right putamen, monkeys received either phosphate-buffered saline or low-dose (LD) or high-dose (HD) AAV2-GDNF. Monkeys that had received putaminal phosphate-buffered saline (PBS) infusions additionally received either PBS or HD AAV2-GDNF in the right substantia nigra (SN). The convection-enhanced delivery method used for infusion of AAV2-GDNF vector resulted in robust volume of GDNF distribution within the putamen. AAV2-GDNF increased FMT-PET uptake in the ipsilateral putamen as well as enhancing locomotor activity. Within the putamen and caudate, the HD gene transfer mediated intense GDNF fiber and extracellular immunoreactivity (IR). Retrograde and anterograde transport of GDNF to other brain regions was observed. AAV2-GDNF did not significantly affect dopamine in the ipsilateral putamen or caudate, but increased dopamine turnover in HD groups. HD putamen treatment increased the density of dopaminergic terminals in these regions. HD treatments, irrespective of the site of infusion, increased the number of nonpigmented TH-IR neurons in the SN. AAV2-GDNF gene transfer does not appear to elicit adverse effects, delivers therapeutic levels of GDNF within target brain areas, and enhances utilization of striatal dopamine and dopaminergic nigrostriatal innervation. PMID:19203243

  9. Stability and Safety of an AAV Vector for Treating RPGR-ORF15 X-Linked Retinitis Pigmentosa.

    PubMed

    Deng, Wen-Tao; Dyka, Frank M; Dinculescu, Astra; Li, Jie; Zhu, Ping; Chiodo, Vince A; Boye, Sanford L; Conlon, Thomas J; Erger, Kirsten; Cossette, Travis; Hauswirth, William W

    2015-09-01

    Our collaborative successful gene replacement therapy using AAV vectors expressing a variant of human RPGR-ORF15 in two canine models provided therapeutic proof of concept for translation into human treatment. The ORF15 sequence contained within this AAV vector, however, has ORF15 DNA sequence variations compared to the published sequence that are likely due to its unusual composition of repetitive purine nucleotides. This mutability is a concern for AAV vector production and safety when contemplating a human trial. In this study, we establish the safety profile of AAV-hIRBP-hRPGR and AAV-hGRK1-hRPGR vectors used in the initial canine proof-of-principle experiments by demonstrating hRPGR-ORF15 sequence stability during all phases of manipulation, from plasmid propagation to vector production to its stability in vivo after subretinal administration to animals. We also evaluate potential toxicity in vivo by investigating protein expression, retinal structure and function, and vector biodistribution. Expression of hRPGR is detected in the inner segments and synaptic terminals of photoreceptors and is restricted to the connecting cilium when the vector is further diluted. Treated eyes exhibit no toxicity as assessed by retinal histopathology, immunocytochemistry, optical coherence tomography, fundoscopy, electroretinogram, and vector biodistribution. Therefore, the hRPGR-ORF15 variant in our AAV vectors appears to be a more stable form than the endogenous hRPGR cDNA when propagated in vitro. Its safety profile presented here in combination with its proven efficacy supports future gene therapy clinical trials.

  10. AAV genome loss from dystrophic mouse muscles during AAV-U7 snRNA-mediated exon-skipping therapy.

    PubMed

    Le Hir, Maëva; Goyenvalle, Aurélie; Peccate, Cécile; Précigout, Guillaume; Davies, Kay E; Voit, Thomas; Garcia, Luis; Lorain, Stéphanie

    2013-08-01

    In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment.

  11. Characterization of naturally-occurring humoral immunity to AAV in sheep.

    PubMed

    Tellez, Joseph; Van Vliet, Kim; Tseng, Yu-Shan; Finn, Jonathan D; Tschernia, Nick; Almeida-Porada, Graça; Arruda, Valder R; Agbandje-McKenna, Mavis; Porada, Christopher D

    2013-01-01

    AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.

  12. Characterization of Naturally-Occurring Humoral Immunity to AAV in Sheep

    PubMed Central

    Tellez, Joseph; Van Vliet, Kim; Tseng, Yu-Shan; Finn, Jonathan D.; Tschernia, Nick; Almeida-Porada, Graça; Arruda, Valder R.; Agbandje-McKenna, Mavis; Porada, Christopher D.

    2013-01-01

    AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity. PMID:24086458

  13. 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 ...

  14. The AAV Vector Toolkit: Poised at the Clinical Crossroads.

    PubMed

    Asokan, Aravind; Schaffer, David V; Jude Samulski, R

    2012-04-01

    The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.

  15. The AAV vector toolkit: poised at the clinical crossroads.

    PubMed

    Asokan, Aravind; Schaffer, David V; Samulski, R Jude

    2012-04-01

    The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.

  16. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    PubMed Central

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues. PMID:10954565

  17. 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

  18. Improved adeno-associated virus (AAV) serotype 1 and 5 vectors for gene therapy.

    PubMed

    Sen, Dwaipayan; Balakrishnan, Balaji; Gabriel, Nishanth; Agrawal, Prachi; Roshini, Vaani; Samuel, Rekha; Srivastava, Alok; Jayandharan, Giridhara R

    2013-01-01

    Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1-10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia.

  19. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

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

  20. Targeted gene delivery to the enteric nervous system using AAV: a comparison across serotypes and capsid mutants.

    PubMed

    Benskey, Matthew J; Kuhn, Nathan C; Galligan, James J; Garcia, Joanna; Boye, Shannon E; Hauswirth, William W; Mueller, Christian; Boye, Sanford L; Manfredsson, Fredric P

    2015-03-01

    Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS.

  1. Immunology of AAV-Mediated Gene Transfer in the Eye

    PubMed Central

    Willett, Keirnan; Bennett, Jean

    2013-01-01

    The eye has been at the forefront of translational gene therapy largely owing to suitable disease targets, anatomic accessibility, and well-studied immunologic privilege. These advantages have fostered research culminating in several clinical trials and adeno-associated virus (AAV) has emerged as the vector of choice for many ocular therapies. Pre-clinical and clinical investigations have assessed the humoral and cellular immune responses to a variety of naturally occurring and engineered AAV serotypes as well as their delivered transgenes and these data have been correlated to potential clinical sequelae. Encouragingly, AAV appears safe and effective with clinical follow-up surpassing 5 years in some studies. As disease targets continue to expand for AAV in the eye, thorough and deliberate assessment of immunologic safety is critical. With careful study, the development of these technologies should concurrently inform the biology of the ocular immune response. PMID:24009613

  2. Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy.

    PubMed

    Louis Jeune, Vedell; Joergensen, Jakob A; Hajjar, Roger J; Weber, Thomas

    2013-04-01

    Adeno-associated virus (AAV)-based vectors are promising tools for gene therapeutic applications, in part because AAVs are nonpathogenic viruses, and vectors derived from them can drive long-term transgene expression without integration of the vector DNA into the host genome. AAVs are not strongly immunogenic, but they can, nonetheless, give rise to both a cellular and humoral immune response. As a result, a significant fraction of potential patients for AAV-based gene therapy harbors pre-existing antibodies against AAV. Because even very low levels of antibodies can prevent successful transduction, antecedent anti-AAV antibodies pose a serious obstacle to the universal application of AAV gene therapy. In this review, we discuss the current knowledge of the role of anti-AAV antibodies in AAV-based gene therapy with a particular emphasis on approaches to overcome the hurdle that they pose.

  3. Retinal gene delivery by adeno-associated virus (AAV) vectors: Strategies and applications.

    PubMed

    Schön, Christian; Biel, Martin; Michalakis, Stylianos

    2015-09-01

    Adeno-associated virus (AAV) vectors are the most widely used vehicle systems for neuronal gene transfer. This popularity is based on the non-pathogenic nature of AAVs and their versatility making them a multifunctional vector system for basic research and clinical applications. AAVs are successfully applied in clinical and pre-clinical gene therapy studies for inherited retinal disorders. Their excellent transduction profile and efficiency also boosted the use of AAV vectors in basic research. The AAV vector system can be easily modified and adjusted at multiple levels to allow for optimized and specific gene expression in target cells. Here, we will provide an overview on the AAV vector system and its applications focusing on gene transfer into retinal cells. Furthermore, we will outline and discuss strategies for the optimization of AAV gene transfer by modifications to the AAV vector expression cassette, the AAV capsid or the routes of vector administration.

  4. Adeno-Associated Virus-2 (AAV-2) Causes Trophoblast Dysfunction, and Placental AAV-2 Infection Is Associated with Preeclampsia

    PubMed Central

    Arechavaleta-Velasco, Fabian; Ma, Yujie; Zhang, Jian; McGrath, Cindy M.; Parry, Samuel

    2006-01-01

    Shallow invasion by extravillous trophoblast cells into the uterine wall reduces placental perfusion and causes placental dysfunction, but the one or more causes of shallow placental invasion are unknown. We hypothesized that infection with adeno-associated virus-2 (AAV-2) inhibits trophoblast invasion and is associated with preeclampsia, which is a common obstetric complication resulting from placental dysfunction. We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002). These results indicate that AAV-2 infection is a previously unidentified cause of placental dysfunction. Additional studies to determine the susceptibility of extravillous trophoblast to other viruses, and the mechanisms by which viral infection impairs placental function, are warranted. PMID:16723710

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

    PubMed

    Pleticha, Josef; Heilmann, Lukas F; Evans, Christopher H; Asokan, Aravind; Samulski, Richard Jude; Beutler, Andreas S

    2014-09-02

    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.

  6. 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

  7. Inverted organic photosensitive devices

    DOEpatents

    Forrest, Stephen R.; Bailey-Salzman, Rhonda F.

    2016-12-06

    The present disclosure relates to organic photosensitive optoelectronic devices grown in an inverted manner. An inverted organic photosensitive optoelectronic device of the present disclosure comprises a reflective electrode, an organic donor-acceptor heterojunction over the reflective electrode, and a transparent electrode on top of the donor-acceptor heterojunction.

  8. Next Generation Inverter

    SciTech Connect

    Zhao, Zilai; Gough, Charles

    2016-04-22

    The goal of this Cooperative Agreement was the development of a Next Generation Inverter for General Motors’ electrified vehicles, including battery electric vehicles, range extended electric vehicles, plug-in hybrid electric vehicles and hybrid electric vehicles. The inverter is a critical electronics component that converts battery power (DC) to and from the electric power for the motor (AC).

  9. 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

  10. [Gene expression of AAV-ITR ssDNA mini vector in skeletal muscle of mice].

    PubMed

    Zhu, Dongqin; Zhang, Yun; Liu, Xiaomei; Zhang, Chun

    2014-11-01

    AAV-ITR single strand DNA mini vector (AAV-ITR ssDNA mini vector) is a novel gene expression vector based on AAV-ITR. We have shown efficient gene expression of AAV-ITR ssDNA mini vector in HEK 293T. Here, we studied the efficacy of gene expression of AAV-ITR ssDNA mini vector in vivo. We injected the skeletal muscle of ICR mice separately with equal molars of AAV-ITR ssDNA mini vector, ITR mutated AAV-ITR single strand DNA mini vector (AAV-ITRmm ssDNA mutant vector), AAV-ITR dsDNA and pUC57-minivector-GFP, combined with TurboFect. Florescence microscope analysis of skeletal muscle section shows that AAV-ITR ssDNA mini vector had higher expression efficiency and longer expression period. We extracted DNA from the muscle three months after injection and quantified three vectors by Real-time PCR. RT-PCR analysis shows that there were highest copy numbers of AAV-ITR ssDNA mini vector existing in muscle. Stable existing of AAV- TR ssDNA mini vector in muscle could be the molecular basis of long term gene expression of the vector. The results suggest that AAV-ITR ssDNA mini vector might be a promising vector for gene therapy.

  11. Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

    PubMed

    Bowles, Dawn E; McPhee, Scott W J; Li, Chengwen; Gray, Steven J; Samulski, Jade J; Camp, Angelique S; Li, Juan; Wang, Bing; Monahan, Paul E; Rabinowitz, Joseph E; Grieger, Joshua C; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Xiao, Xiao; Samulski, R Jude

    2012-02-01

    Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

  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.

  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

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

    2016-02-22

    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.

  15. 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

  16. 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.

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

    PubMed

    Tseng, Yu-Shan; Agbandje-McKenna, Mavis

    2014-01-01

    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.

  18. 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

  19. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo.

    PubMed

    György, Bence; Fitzpatrick, Zachary; Crommentuijn, Matheus H W; Mu, Dakai; Maguire, Casey A

    2014-08-01

    Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector.

  20. 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

  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. Improved inverted Stepanov apparatus

    NASA Technical Reports Server (NTRS)

    Berkman, S.; Temple, H. E.

    1979-01-01

    Modifications in inverted Stepanov process improve heat transfer and energy efficiency in growing silicon ribbon crystals. Using system, silicon is directly heated by induction, minimizing heat transfer and contamination problems.

  3. 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.

  4. Reliable inverter systems

    NASA Technical Reports Server (NTRS)

    Nagano, S.

    1979-01-01

    Base driver with common-load-current feedback protects paralleled inverter systems from open or short circuits. Circuit eliminates total system oscillation that can occur in conventional inverters because of open circuit in primary transformer winding. Common feedback signal produced by functioning modules forces operating frequency of failed module to coincide with clock drive so module resumes normal operating frequency in spite of open circuit.

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

    PubMed Central

    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

  6. 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

  7. Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

    PubMed Central

    Keeler, Allison M; Conlon, Thomas; Walter, Glenn; Zeng, Huadong; Shaffer, Scott A; Dungtao, Fu; Erger, Kirsten; Cossette, Travis; Tang, Qiushi; Mueller, Christian; Flotte, Terence R

    2012-01-01

    Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans. PMID:22395529

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

    PubMed

    Hudry, E; Martin, C; Gandhi, S; György, B; Scheffer, D I; Mu, D; Merkel, S F; Mingozzi, F; Fitzpatrick, Z; Dimant, H; Masek, M; Ragan, T; Tan, S; Brisson, A R; Ramirez, S H; Hyman, B T; Maguire, C A

    2016-04-01

    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 the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with 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 three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the 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.

  9. 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

  10. 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

  11. Local administration of AAV-BDNF to subventricular zone induces functional recovery in stroke rats.

    PubMed

    Yu, Seong-Jin; Tseng, Kuan-Yin; Shen, Hui; Harvey, Brandon K; Airavaara, Mikko; Wang, Yun

    2013-01-01

    Migration of new neuroprogenitor cells (NPCs) from the subventricular zone (SVZ) plays an important role in neurorepair after injury. Previous studies have shown that brain derived neurotrophic factor (BDNF) enhances the migration of NPCs from SVZ explants in neonatal mice in vitro. The purpose of this study was to identify the role of BDNF in SVZ cells using AAV-BDNF in an animal model of stroke. BDNF protein production after AAV-BDNF infection was verified in primary neuronal culture. AAV-BDNF or AAV-RFP was injected into the left SVZ region of adult rats at 14 days prior to right middle cerebral artery occlusion (MCAo). SVZ tissues were collected from the brain and placed in Metrigel cultures 1 day after MCAo. Treatment with AAV-BDNF significantly increased the migration of SVZ cells in the stroke brain in vitro. In another set of animals, AAV-GFP was co-injected with AAV-BDNF or AAV-RFP to label cells in left SVZ prior to right MCAo. Local administration of AAV-BDNF significantly enhanced recovery of locomotor function and migration of GFP-positive cells from the SVZ toward the lesioned hemisphere in stroke rats. Our data suggest that focal administration of AAV-BDNF to the SVZ increases behavioral recovery post stroke, possibly through the enhancement of migration of cells from SVZ in stroke animals. Regional manipulation of BDNF expression through AAV may be a novel approach for neurorepair in stroke brains.

  12. 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.

  13. Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses.

    PubMed

    Ling, Chen; Li, Baozheng; Ma, Wenqin; Srivastava, Arun

    2016-08-01

    We have described the development of capsid-modified next-generation AAV vectors for both AAV2 and AAV3 serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses. We have also described the development of genome-modified AAV vectors, in which the transcriptionally inactive, single-stranded AAV genome was modified to achieve improved transgene expression. Here, we describe that combination of capsid modifications and genome modifications leads to the generation of optimized AAV serotype vectors, which transduce cells and tissues more efficiently, both in vitro and in vivo, at ∼20-30-fold reduced doses. These studies have significant implications in the potential use of the optimized AAV serotype vectors in human gene therapy.

  14. 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.

  15. Resonant snubber inverter

    DOEpatents

    Lai, J.S.; Young, R.W. Sr.; Chen, D.; Scudiere, M.B.; Ott, G.W. Jr.; White, C.P.; McKeever, J.W.

    1997-06-24

    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. 14 figs.

  16. 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.

  17. 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

  18. The prevalence of neutralizing antibodies against AAV serotype 1 in healthy subjects in China: implications for gene therapy and vaccines using AAV1 vector.

    PubMed

    Liu, Qiang; Huang, Weijin; Zhao, Chenyan; Zhang, Li; Meng, Shufang; Gao, Dongying; Wang, Youchun

    2013-09-01

    Recombinant adeno-associated virus serotype 1 (AAV1) has attracted tremendous interest as a promising vector for gene therapy and vaccine applications. However, the presence of AAV1 neutralizing antibodies as a consequence of exposure to wild type AAV1 can limit significantly effective gene transfer for biologics based AAV1 vector. Prior studies have reported that a prevalence of AAV1 neutralizing antibodies ranged from 10% to 50% in different countries around the world, and up to 79% in Dutch subjects. However, few studies have reported on the AAV1 neutralizing antibody prevalence in Chinese subjects. In this study, a high-throughput luciferase-based virus neutralization assay was established and standardized for critical parameters, including the appropriate cell line, and the optimal viral infection dose, and the infection time with homologous AAV1 vaccinated mice and guinea pig sera. Then, a total of 500 healthy individual serum samples from two separate regions of China were screened for the AAV1 neutralizing antibodies by conducting a non-randomized, cross-sectional analysis. Interestingly, a high prevalence of AAV1 neutralizing antibody (69.8%) was found in all individuals. There was significant difference observed for prevalence by gender (P = 0.042), age range (P = 0.011) and geographic origin (P < 0.001). The percentage of positive AAV1 neutralizing antibodies (NT50  > 10) in teenagers (year <18, as of 2012) was significant lower than that of adults (19-56, as of 2012) (P = 0.011), indicating the optimal vaccination period of childhood. The current study provides a useful insight for the future development of AAV1-based vaccination and gene therapy strategies in Beijing and Anhui provinces of China.

  19. 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

  20. Efficacy of codelivery of dual AAV2/5 vectors in the murine retina and hippocampus.

    PubMed

    Palfi, Arpad; Chadderton, Naomi; McKee, Alex G; Blanco Fernandez, Alfonso; Humphries, Peter; Kenna, Paul F; Farrar, G Jane

    2012-08-01

    Recombinant adeno-associated virus (AAV) represents an efficient system for neuronal transduction. However, a potential drawback of AAV is its restricted packaging capacity of approximately 5 kb. To bypass this limitation, a number of dual- and triple-vector strategies divide the transgene(s) between two or three AAVs. The success of these approaches relies directly on efficient cotransduction of the component AAVs. Although proof of concept for these stratagems has been demonstrated, the underlying cotransduction rate has not been analyzed quantitatively. In this study, cotransduction efficiencies in both retina and hippocampus have been investigated, using two reporter AAVs expressing either a green (GFP) or red (DsR) fluorescent protein. Transduction efficiencies were monitored via microscopy, flow cytometry, and quantitative PCR. After viral transduction with 1.5×10(9) viral particles of each of the reporter AAVs, approximately one-third of the retinal cells expressed one or both transgenes at levels detectable by native fluorescence. Notably, the majority of the remaining retinal cells were also transduced and expressed the reporters at lower levels, which were detectable only by immunolabeling. Flow cytometric analysis demonstrated cotransduction rates of up to 55% with the two reporter AAVs in retinal cells. Modifying the ratio of the two coadministered AAVs resulted in altered mRNA expression levels of the two reporter genes in cotransduced cell populations. The study suggests that codelivery of AAV is an efficient means of expanding the therapeutic application of AAV in neurons.

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

    PubMed

    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

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. 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, AAV9 (refs. 14,15,16,17), 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.

  2. Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer.

    PubMed

    Li, Chengwen; Wu, Shuqing; Albright, Blake; Hirsch, Matthew; Li, Wuping; Tseng, Yu-Shan; Agbandje-McKenna, Mavis; McPhee, Scott; Asokan, Aravind; Samulski, R Jude

    2016-02-01

    A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

  3. Energy-Saving Inverter

    NASA Technical Reports Server (NTRS)

    Rippel, W. E.; Edwards, D. B.

    1984-01-01

    Commutation by field-effect transistor allows more efficient operation. High voltage field-effect transistor (FET) controls silicon controlled rectifiers (SCR's). Circuit requires only one capacitor and one inductor in commutation circuit: simpler, more efficient, and more economical than conventional inverters. Adaptable to dc-to-dc converters.

  4. A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery.

    PubMed

    Yu, C-Y; Yuan, Z; Cao, Z; Wang, B; Qiao, C; Li, J; Xiao, X

    2009-08-01

    Adeno-associated virus (AAV) has become a leading gene transfer vector for striated muscles. However, the AAV vectors also exhibit broad tropisms after systemic delivery. In an attempt to improve muscle tropism, we inserted a 7-amino-acid (ASSLNIA) muscle-targeting peptide (MTP) in the capsids of AAV2 at residue 587 or 588, generating AAV(587)MTP and AAV(588)MTP. In vitro studies showed that both viruses diminished their infectivity on non-muscle cell lines as well as on un-differentiated myoblasts; however, preserved or enhanced their infectivity on differentiated myotubes. AAV(587)MTP, but not AAV(588)MTP, also abolished its heparin-binding capacity and infected myotubes in a heparin-independent manner. Furthermore, in vivo studies by intravenous vector administration in mice showed that AAV(587)MTP enhanced its tropism to various muscles and particularly to the heart (24.3-fold of unmodified AAV2), whereas reduced its tropism to the non-muscle tissues such as the liver, lungs, spleen and so on. This alteration of tissue tropism is not simply because of the loss of heparin-binding, as a mutant AAV2 (AAVHBSMut) containing heparin-binding site mutations lost infectivity on both non-muscle and muscle cells. Furthermore, free MTP peptide, but not the scrambled control peptide, competitively inhibited AAV(587)MTP infection on myotubes. These results suggest that AAV2 could be re-targeted to the striated muscles by a MTP inserted after residue 587 of the capsids. This proof of principle study showed first evidence of peptide-directed muscle targeting on systemic administration of AAV vectors.

  5. 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.

  6. The impact of AAV capsid-specific T cell responses on design and outcome of clinical gene transfer trials with recombinant AAV vectors - an evolving controversy.

    PubMed

    Ertl, Hildegund Cj; High, Katherine A

    2017-01-02

    Recombinant adenovirus-associated (rAAV) vectors due to their ease of construction, wide tissue tropism and lack of pathogenicity remain at the forefront for long-term gene replacement therapy. In spite of very encouraging pre-clinical results, clinical trials were initially unsuccessful; expression of the rAAV vector-delivered therapeutic protein was transient. Loss of expression was linked to an expansion of AAV capsid-specific T cell responses, leading to the hypothesis that rAAV vectors recall pre-existing memory T cells that had been induced by natural infections with AAV together with a helper virus. Although this was hotly debated at first, AAV capsid-specific T cell responses were observed in several gene transfer trials that used high doses of rAAV vectors. Subsequent trials designed to circumvent these T cell responses through the use of immunosuppressive drugs, rAAV vectors based on rare serotypes or modified to allow for therapeutic levels of the transgene product at low, non-immunogenic vector doses are now successful in correcting debilitating diseases.

  7. Inverter communications using output signal

    DOEpatents

    Chapman, Patrick L.

    2017-02-07

    Technologies for communicating information from an inverter configured for the conversion of direct current (DC) power generated from an alternative source to alternating current (AC) power are disclosed. The technologies include determining information to be transmitted from the inverter over a power line cable connected to the inverter and controlling the operation of an output converter of the inverter as a function of the information to be transmitted to cause the output converter to generate an output waveform having the information modulated thereon.

  8. 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…

  9. 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.

  10. 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.

  11. Significant changes in endogenous retinal gene expression assessed 1 year after a single intraocular injection of AAV-CNTF or AAV-BDNF

    PubMed Central

    LeVaillant, Chrisna J; Sharma, Anil; Muhling, Jill; Wheeler, Lachlan PG; Cozens, Greg S; Hellström, Mats; Rodger, Jennifer; Harvey, Alan R

    2016-01-01

    Use of viral vectors to deliver therapeutic genes to the central nervous system holds promise for the treatment of neurodegenerative diseases and neurotrauma. Adeno-associated viral (AAV) vectors encoding brain-derived neurotrophic factor (BDNF) or ciliary derived neurotrophic factor (CNTF) promote the viability and regeneration of injured adult rat retinal ganglion cells. However, these growth-inducing transgenes are driven by a constitutively active promoter, thus we examined whether long-term AAV-mediated secretion of BDNF or CNTF affected endogenous retinal gene expression. One year after the intravitreal injection of AAV-green fluorescent protein (GFP), bi-cistronic AAV-BDNF-GFP or AAV-CNTF-GFP, mRNA was extracted and analyzed using custom 96 well polymerase chain reaction arrays. Of 93 test genes, 56% showed significantly altered expression in AAV-BDNF-GFP and/or AAV-CNTF-GFP retinas compared with AAV-GFP controls. Of these genes, 73% showed differential expression in AAV-BDNF versus AAV-CNTF injected eyes. To focus on retinal ganglion cell changes, quantitative polymerase chain reaction was undertaken on mRNA (16 genes) obtained from fixed retinal sections in which the ganglion cell layer was enriched. The sign and extent of fold changes in ganglion cell layer gene expression differed markedly from whole retinal samples. Sustained and global alteration in endogenous mRNA expression after gene therapy should be factored into any interpretation of experimental/clinical outcomes, particularly when introducing factors into the central nervous system that require secretion to evoke functionality. PMID:27933306

  12. Significant changes in endogenous retinal gene expression assessed 1 year after a single intraocular injection of AAV-CNTF or AAV-BDNF.

    PubMed

    LeVaillant, Chrisna J; Sharma, Anil; Muhling, Jill; Wheeler, Lachlan Pg; Cozens, Greg S; Hellström, Mats; Rodger, Jennifer; Harvey, Alan R

    2016-01-01

    Use of viral vectors to deliver therapeutic genes to the central nervous system holds promise for the treatment of neurodegenerative diseases and neurotrauma. Adeno-associated viral (AAV) vectors encoding brain-derived neurotrophic factor (BDNF) or ciliary derived neurotrophic factor (CNTF) promote the viability and regeneration of injured adult rat retinal ganglion cells. However, these growth-inducing transgenes are driven by a constitutively active promoter, thus we examined whether long-term AAV-mediated secretion of BDNF or CNTF affected endogenous retinal gene expression. One year after the intravitreal injection of AAV-green fluorescent protein (GFP), bi-cistronic AAV-BDNF-GFP or AAV-CNTF-GFP, mRNA was extracted and analyzed using custom 96 well polymerase chain reaction arrays. Of 93 test genes, 56% showed significantly altered expression in AAV-BDNF-GFP and/or AAV-CNTF-GFP retinas compared with AAV-GFP controls. Of these genes, 73% showed differential expression in AAV-BDNF versus AAV-CNTF injected eyes. To focus on retinal ganglion cell changes, quantitative polymerase chain reaction was undertaken on mRNA (16 genes) obtained from fixed retinal sections in which the ganglion cell layer was enriched. The sign and extent of fold changes in ganglion cell layer gene expression differed markedly from whole retinal samples. Sustained and global alteration in endogenous mRNA expression after gene therapy should be factored into any interpretation of experimental/clinical outcomes, particularly when introducing factors into the central nervous system that require secretion to evoke functionality.

  13. HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production.

    PubMed

    Cao, M; Zhu, H; Bandyopadhyay, S; You, H; Hermonat, P L

    2012-04-01

    Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large-scale recombinant (r) AAV production remains problematic because of low particle yield. The adenovirus (Ad) and herpes (simplex) virus helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild-type (wt) AAV productive infection in differentiating keratinocytes, however, HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild-to-moderate changes in rep- and cap-encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, although rep mRNA was upregulated, cap mRNA was upregulated more. Higher virus yields did correlate most consistently with increased Rep52-, VP3- and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets.

  14. HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production

    PubMed Central

    Cao, M.; Zhu, H.; Bandyopadhyay, S; You, H; Hermonat, P.L.

    2011-01-01

    Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large scale recombinant (r)AAV production remains problematic due to low particle yield. The adenovirus (Ad) and herpes (simplex) virus (HSV) helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild type (wt) AAV productive infection in differentiating keratinocytes, however HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild to moderate changes in rep- and cap–encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, while rep mRNA was up-regulated, cap mRNA was up-regulated more. Higher virus yields did correlate most consistently with increased Rep52, VP3 and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets. PMID:21850053

  15. Production and Characterization of Vectors Based on the Cardiotropic AAV Serotype 9.

    PubMed

    Kohlbrenner, Erik; Weber, Thomas

    2017-01-01

    Vectors based on adeno-associated virus serotype 9 (AAV9) efficiently transduce cardiomyocytes in both rodents and large animal models upon either systemic or regional vector delivery. In this chapter, we describe the most widely used production and purification method of AAV9. This production approach does not depend on the use of a helpervirus but instead on transient transfection of HEK293T cells with a plasmid containing the recombinant AAV genome and a second plasmid encoding the AAV9 capsid proteins, the AAV Rep proteins and the adenoviral helper functions. The recombinant AAV is then purified by iodixanol density gradient centrifugation. This chapter also describes in detail the characterization and quality control methods required for assuring high quality vector preparations, which is of particular importance for experiments in large animal models.

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

    PubMed

    Shen, F; Mao, L; Zhu, W; Lawton, M T; Pechan, P; Colosi, P; Wu, Z; Scaria, A; Su, H

    2015-11-01

    The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, 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. AAVs 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. IV 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. Inverter ratio failure detector

    NASA Technical Reports Server (NTRS)

    Wagner, A. P.; Ebersole, T. J.; Andrews, R. E. (Inventor)

    1974-01-01

    A failure detector which detects the failure of a dc to ac inverter is disclosed. The inverter under failureless conditions is characterized by a known linear relationship of its input and output voltages and by a known linear relationship of its input and output currents. The detector includes circuitry which is responsive to the detector's input and output voltages and which provides a failure-indicating signal only when the monitored output voltage is less by a selected factor, than the expected output voltage for the monitored input voltage, based on the known voltages' relationship. Similarly, the detector includes circuitry which is responsive to the input and output currents and provides a failure-indicating signal only when the input current exceeds by a selected factor the expected input current for the monitored output current based on the known currents' relationship.

  2. AAV retinal transduction in a large animal model species: Comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector

    PubMed Central

    Bartoe, J.T.; Fischer, A.J.; Scott, M.; Boye, S.L.; Chiodo, V.; Hauswirth, W.W.

    2009-01-01

    Purpose To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection. Methods ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity. Results GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced. Conclusions When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors. PMID:19756181

  3. rAAV vector product characterization and stability studies.

    PubMed

    Snyder, Richard O; Audit, Muriel; Francis, Joyce D

    2011-01-01

    Recombinant adeno-associated viral (rAAV) vectors mediate the safe and long-term correction of genetic diseases following a single administration. Preclinical studies in animal models and human trials have shown rAAV vector persistence and safety. In some trials, sustained or transient transgene expression has been demonstrated in humans treated for alpha-1 antitrypsin deficiency, LPL deficiency, hemophilia B and cystic fibrosis, and sustained correction of inherited blindness has been reported by three groups. For human use, rAAV vectors are manufactured and tested in compliance with current Good Manufacturing Practices as outlined in the Code of Federal Regulations (21CFR) or European Good Manufacturing Practices (Eudralex, Volume 4, GMP Guidelines, 2003/94/CE and 91/356/EEC). Manufacturing control, as well as product quality is evaluated by quality control testing and all manufacturing, facilities, and testing activities are reviewed by the quality assurance department. In-process specifications are set and in-process testing is conducted to confirm that the manufacturing process is controlled, aseptic, and performs consistently. Final product is tested to ensure release specifications are met for identity, safety, purity, potency, and stability.

  4. AAV Hybrid Serotypes: Improved Vectors for Gene Delivery

    PubMed Central

    Choi, Vivian W.; McCarty, Douglas M.; Samulski, R. Jude

    2006-01-01

    In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus. PMID:15975007

  5. Inverted organic photosensitive device

    DOEpatents

    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.

  6. 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

  7. Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them.

    PubMed

    Hareendran, Sangeetha; Balakrishnan, Balaji; Sen, Dwaipayan; Kumar, Sanjay; Srivastava, Alok; Jayandharan, Giridhara R

    2013-11-01

    AAV-based gene transfer protocols have shown remarkable success when directed to immune-privileged sites such as for retinal disorders like Lebers congenital amaurosis. In contrast, AAV-mediated gene transfer into liver or muscle tissue for diseases such as hemophilia B, α1 anti-trypsin deficiency and muscular dystrophy has demonstrated a decline in gene transfer efficacy over time. It is now known that in humans, AAV triggers specific pathways that recruit immune sensors. These factors initiate an immediate reaction against either the viral capsid or the vector encoded protein as part of innate immune response or to produce a more specific adaptive response that generates immunological memory. The vector-transduced cells are then rapidly destroyed due to this immune activation. However, unlike other viral vectors, AAV is not immunogenic in murine models. Its immunogenicity becomes apparent only in large animal models and human subjects. Moreover, humans are natural hosts to AAV and exhibit a high seroprevalence against AAV vectors. This limits the widespread application of AAV vectors into patients with pre-existing neutralising antibodies or memory T cells. To address these issues, various strategies are being tested. Alternate serotype vectors (AAV1-10), efficient expression cassettes, specific tissue targeting, immune-suppression and engineered capsid variants are some approaches proposed to minimise this immune stimulation. In this review, we have summarised the nature of the immune response documented against AAV in various pre-clinical and clinical settings and have further discussed the strategies to evade them.

  8. Advances in AAV vector development for gene therapy in the retina.

    PubMed

    Day, Timothy P; Byrne, Leah C; Schaffer, David V; Flannery, John G

    2014-01-01

    Adeno-associated virus (AAV) is a small, non-pathogenic dependovirus that has shown great potential for safe and long-term expression of a genetic payload in the retina. AAV has been used to treat a growing number of animal models of inherited retinal degeneration, though drawbacks-including a limited carrying capacity, slow onset of expression, and a limited ability to transduce some retinal cell types from the vitreous-restrict the utility of AAV for treating some forms of inherited eye disease. Next generation AAV vectors are being created to address these needs, through rational design efforts such as the creation of self-complementary AAV vectors for faster onset of expression and specific mutations of surface-exposed residues to increase transduction of viral particles. Furthermore, directed evolution has been used to create, through an iterative process of selection, novel variants of AAV with newly acquired, advantageous characteristics. These novel AAV variants have been shown to improve the therapeutic potential of AAV vectors, and further improvements may be achieved through rational design, directed evolution, or a combination of these approaches, leading to broader applicability of AAV and improved treatments for inherited retinal degeneration.

  9. Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

    PubMed Central

    Finn, Jonathan D; Hui, Daniel; Downey, Harre D; Dunn, Danielle; Pien, Gary C; Mingozzi, Federico; Zhou, Shangzhen; High, Katherine A

    2009-01-01

    Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans. PMID:19904235

  10. Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

    PubMed

    Earley, Lauriel F; Powers, John M; Adachi, Kei; Baumgart, Joshua T; Meyer, Nancy L; Xie, Qing; Chapman, Michael S; Nakai, Hiroyuki

    2017-02-01

    Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.

  11. Synthesizing Virtual Oscillators to Control Islanded Inverters

    SciTech Connect

    Johnson, Brian B.; Sinha, Mohit; Ainsworth, Nathan G.; Dorfler, Florian; Dhople, Sairaj V.

    2016-08-01

    Virtual oscillator control (VOC) is a decentralized control strategy for islanded microgrids where inverters are regulated to emulate the dynamics of weakly nonlinear oscillators. Compared to droop control, which is only well defined in sinusoidal steady state, VOC is a time-domain controller that enables interconnected inverters to stabilize arbitrary initial conditions to a synchronized sinusoidal limit cycle. However, the nonlinear oscillators that are elemental to VOC cannot be designed with conventional linear-control design methods. We address this challenge by applying averaging- and perturbation-based nonlinear analysis methods to extract the sinusoidal steady-state and harmonic behavior of such oscillators. The averaged models reveal conclusive links between real- and reactive-power outputs and the terminal-voltage dynamics. Similarly, the perturbation methods aid in quantifying higher order harmonics. The resultant models are then leveraged to formulate a design procedure for VOC such that the inverter satisfies standard ac performance specifications related to voltage regulation, frequency regulation, dynamic response, and harmonic content. Experimental results for a single-phase 750 VA, 120 V laboratory prototype demonstrate the validity of the design approach. They also demonstrate that droop laws are, in fact, embedded within the equilibria of the nonlinear-oscillator dynamics. This establishes the backward compatibility of VOC in that, while acting on time-domain waveforms, it subsumes droop control in sinusoidal steady state.

  12. 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.

  13. Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques.

    PubMed

    Gao, Guangping; Bish, Lawrence T; Sleeper, Meg M; Mu, Xin; Sun, Lan; Lou, You; Duan, Jiachuan; Hu, Chunyan; Wang, Li; Sweeney, H Lee

    2011-08-01

    Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.

  14. 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.

  15. Gas cooled traction drive inverter

    SciTech Connect

    Chinthavali, Madhu Sudhan

    2016-04-19

    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.

  16. 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.

  17. 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...

  18. Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

    PubMed

    Colella, P; Trapani, I; Cesi, G; Sommella, A; Manfredi, A; Puppo, A; Iodice, C; Rossi, S; Simonelli, F; Giunti, M; Bacci, M L; Auricchio, A

    2014-04-01

    Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

  19. Gene transfer properties and structural modeling of human stem cell-derived AAV.

    PubMed

    Smith, Laura J; Ul-Hasan, Taihra; Carvaines, Sarah K; Van Vliet, Kim; Yang, Ethel; Wong, Kamehameha K; Agbandje-McKenna, Mavis; Chatterjee, Saswati

    2014-09-01

    Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34(+) hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34(+) human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34(+) cells. Every AAV isolated from CD34(+) cells mapped to AAV Clade F. Gene transfer vectors derived from these novel AAVs efficiently underwent entry and postentry processing in human cord blood stem cells and supported stable gene transfer into long-term, in vivo engrafting human HSCs significantly better than other serotypes. AAVHSC-transduced human CD34(+) cells engrafted in vivo and gave rise to differentiated transgene-expressing progeny. Importantly, gene-marked CD34(+) stem cells persisted long term in xenograft recipients, indicating transduction of primitive progenitors. Notably, correlation of structure with function permitted identification of potential capsid components important for HSC transduction. Thus, AAVHSCs represent a new class of genetic vectors for the manipulation of HSC genomes.

  20. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    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.

  1. 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

  2. Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.

    PubMed

    Mingozzi, F; Chen, Y; Edmonson, S C; Zhou, S; Thurlings, R M; Tak, P P; High, K A; Vervoordeldonk, M J

    2013-04-01

    Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing antibody (NAb) titer against AAV serotypes 2, 5, 6 and 8 in the serum and matched synovial fluid (SF) from rheumatoid arthritis patients. The titer in the SF was lower than that in the matched plasma samples, indicating a difference in distribution of NAb to AAV depending on the body fluid compartment. This difference was more evident for AAV2, against which higher titers were measured. Of all serotypes, anti-AAV5 antibodies were the least prevalent in both the serum and SF. We next evaluated the impact of B-cell depletion on anti-AAV antibodies in rheumatoid arthritis patients who received one or two courses of the anti-CD20 antibody rituximab as part of their disease management. A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5. This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

  3. Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.

    PubMed

    Chicoine, L G; Montgomery, C L; Bremer, W G; Shontz, K M; Griffin, D A; Heller, K N; Lewis, S; Malik, V; Grose, W E; Shilling, C J; Campbell, K J; Preston, T J; Coley, B D; Martin, P T; Walker, C M; Clark, K R; Sahenk, Z; Mendell, J R; Rodino-Klapac, L R

    2014-02-01

    Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

  4. 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

    ... Thereof'' ; and U.S. Patent 6, 855, 314 entitled ``AAV5 Vector for Transducing Brain Cells and Lung Cells... sale of AAV5 based therapeutic products to be delivered to the brain, eyes and liver for treatment of... particles. The specific brain cells that are targeted by AAV5 belong to both non-neuronal/glial cells...

  5. Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo.

    PubMed

    Geoghegan, James C; Keiser, Nicholas W; Okulist, Anna; Martins, Inês; Wilson, Matthew S; Davidson, Beverly L

    2014-10-14

    Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium.

  6. Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins.

    PubMed

    Klein, Ronald L; Dayton, Robert D; Leidenheimer, Nancy J; Jansen, Karen; Golde, Todd E; Zweig, Richard M

    2006-03-01

    Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.

  7. 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.

  8. AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease.

    PubMed

    Xie, Chang; Gong, Xue-Min; Luo, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-03-01

    Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1(-/-) mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1(-/-) mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

  9. AAV2 gene therapy readministration in three adults with congenital blindness.

    PubMed

    Bennett, Jean; Ashtari, Manzar; Wellman, Jennifer; Marshall, Kathleen A; Cyckowski, Laura L; Chung, Daniel C; McCague, Sarah; Pierce, Eric A; Chen, Yifeng; Bennicelli, Jeannette L; Zhu, Xiaosong; Ying, Gui-Shuang; Sun, Junwei; Wright, J Fraser; Auricchio, Alberto; Simonelli, Francesca; Shindler, Kenneth S; Mingozzi, Federico; High, Katherine A; Maguire, Albert M

    2012-02-08

    Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant adeno-associated virus (AAV) carrying the RPE65 gene (AAV2-hRPE65v2) prompted us to determine whether it was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis due to mutations in the RPE65 gene 1.7 to 3.3 years after they had received their initial subretinal injection of AAV2-hRPE65v2. Results (through 6 months) including evaluations of immune response, retinal and visual function testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious after previous exposure to AAV2-hRPE65v2.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26421998

  11. 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.

  12. Predictors of Poor Outcome in ANCA-Associated Vasculitis (AAV).

    PubMed

    Vega, Luis E; Espinoza, Luis R

    2016-12-01

    It is important to recognize factors that might predict poor outcome and prognosis in patients with AAV. The predictors reported in the literature encompass genetic, histopathological, and clinical ones. Genetic studies (genetic predictors) have found genes that are associated with prediction of poor response to treatment, deterioration of renal function, and risk of mortality. Histopathological studies (histopathological predictors) have shown that sclerotic renal lesions are associated with increased risk of progression to end-stage renal disease and death. Lastly, scores (clinical predictors) obtained with tool as FFS, Maldini risk score, VDI, and emerging new biomarkers could potentially be helpful in assessment of prognosis in the future.

  13. 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

  14. Humoral immunity to AAV vectors in gene therapy: challenges and potential solutions.

    PubMed

    Masat, Elisa; Pavani, Giulia; Mingozzi, Federico

    2013-06-01

    Gene transfer trials with adeno-associated virus (AAV) vectors have initiated to unveil the therapeutic potential of this approach, with some of the most exciting results coming from clinical studies of gene transfer for hemophilia B, congenital blindness, and the recent market approval of the first AAV-based gene therapy in Europe. With clinical development, however, some of the limitations of in vivo gene transfer have emerged; in particular the host immune system represents an important obstacle to be overcome in terms of both safety and efficacy of gene transfer in vivo with AAV vectors. Results in humans undergoing gene transfer indicate that capsid-specific T cell responses directed against transduced cells may limit the duration of transgene expression following AAV gene transfer, and similarly anti-AAV neutralizing antibodies can completely prevent transduction of a target tissue, resulting in lack of efficacy. Anti-AAV neutralizing antibodies are highly prevalent in humans, and the frequency of subjects with detectable titers can reach up to two thirds of the population. The approach to the problem of preexisting humoral immunity to AAV so far has been the exclusion of seropositive subjects, but this solution is far from being optimal. Several additional strategies have been proposed and tested in a variety of preclinical animal models. Future studies will help defining the optimal strategy, or combination of strategies, to successfully treat subjects with preexisting antibodies to AAV due to natural infection or to prior administration of AAV vectors. These advancements will likely have a significant impact on the field of gene transfer with AAV vectors.

  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. Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway.

    PubMed

    Hirsch, Matthew L; Li, Chengwen; Bellon, Isabella; Yin, Chaoying; Chavala, Sai; Pryadkina, Marina; Richard, Isabelle; Samulski, Richard Jude

    2013-12-01

    A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

  17. Simplified High-Power Inverter

    NASA Technical Reports Server (NTRS)

    Edwards, D. B.; Rippel, W. E.

    1984-01-01

    Solid-state inverter simplified by use of single gate-turnoff device (GTO) to commutate multiple silicon controlled rectifiers (SCR's). By eliminating conventional commutation circuitry, GTO reduces cost, size and weight. GTO commutation applicable to inverters of greater than 1-kilowatt capacity. Applications include emergency power, load leveling, drives for traction and stationary polyphase motors, and photovoltaic-power conditioning.

  18. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential

    PubMed Central

    Miller, Rehae; Han, Ping-Yang; Pang, Jijing; Dinculescu, Astra; Chiodo, Vince; Hauswirth, William W.

    2008-01-01

    Purpose Safety and efficiency are critical for successful gene therapy. Adeno-associated viral (AAV) vectors are commonly used for gene transfer in both human and animal studies. However, administration of AAV vectors can lead to development of neutralizing antibodies against the vector capsid, thus decreasing the efficiency of therapeutic gene transfer and preventing effective vector readministration. We investigated immune responses to different routes of ocular administration and readministration of AAV vectors, and the effect of previous exposure of AAV vector in one eye on the transduction efficacy of subsequent intraocular AAV-mediated gene delivery to the partner eye. Methods We tested two vector systems. One contained a cDNA encoding a secreted pigment epithelial derived factor (PEDF) cDNA under the control of a Cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CBA; AAV2-CBA-PEDF) and was tested in a murine model of laser-induced choroidal neovascularization (CNV). The other vector contained a cDNA encoding the intracellular reporter green fluorescent protein (GFP) under the control of the same promoter (AAV2-CBA-GFP). Animals were divided into groups and received sequential injections at different combinations of either intravitreal or subretinal routes. CNV was evaluated by fluorescein angiographic choroidal flat-mount image analysis. The expression of GFP was analyzed in retinal sections by direct fluorescence imaging. Antibodies against AAV2 capsid and transgenes were analyzed by ELISA using serum samples collected before injection and different time points after the injection. Neutralizing antibodies were characterized by in vitro assays. Results Various ocular compartments responded to AAV administration differently. Intravitreal administration of AAV vectors, which resulted in transduction of inner retina (primarily retinal ganglion cells), generated a humoral immune response against AAV capsid that blocked vector expression upon

  19. Potential of AAV vectors in the treatment of metabolic disease.

    PubMed

    Alexander, I E; Cunningham, S C; Logan, G J; Christodoulou, J

    2008-06-01

    Inborn errors of metabolism are collectively common, frequently severe and in many instances difficult or impossible to treat. Accordingly, there is a compelling need to explore novel therapeutic modalities, including gene therapy, and examine multiple phenotypes where the risks of experimental therapy are outweighed by potential benefits to trial participants. Among available gene delivery systems recombinant AAV shows special promise for the treatment of metabolic disease given the unprecedented efficiencies achieved in transducing key target tissues, such as liver and muscle, in small animal models. To date over 30 metabolic disease phenotypes have been investigated in small animal studies with complete phenotype correction being achieved in a substantial proportion. Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.

  20. Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

    PubMed Central

    Rapti, Kleopatra; Louis-Jeune, Vedell; Kohlbrenner, Erik; Ishikawa, Kiyotake; Ladage, Dennis; Zolotukhin, Sergei; Hajjar, Roger J; Weber, Thomas

    2012-01-01

    Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models. PMID:21915102

  1. Systemic gene transfer reveals distinctive muscle transduction profile of tyrosine mutant AAV-1, -6, and -9 in neonatal dogs.

    PubMed

    Hakim, Chady H; Yue, Yongping; Shin, Jin-Hong; Williams, Regina R; Zhang, Keqing; Smith, Bruce F; Duan, Dongsheng

    2014-03-05

    The muscular dystrophies are a group of devastating genetic disorders that affect both skeletal and cardiac muscle. An effective gene therapy for these diseases requires bodywide muscle delivery. Tyrosine mutant adeno-associated virus (AAV) has been considered as a class of highly potent gene transfer vectors. Here, we tested the hypothesis that systemic delivery of tyrosine mutant AAV can result in bodywide muscle transduction in newborn dogs. Three tyrosine mutant AAV vectors (Y445F/Y731F AAV-1, Y445F AAV-6, and Y731F AAV-9) were evaluated. These vectors expressed the alkaline phosphatase reporter gene under transcriptional regulation of either the muscle-specific Spc5-12 promoter or the ubiquitous Rous sarcoma virus promoter. Robust skeletal and cardiac muscle transduction was achieved with Y445F/Y731F AAV-1. However, Y731F AAV-9 only transduced skeletal muscle. Surprisingly, Y445F AAV-6 resulted in minimal muscle transduction. Serological study suggests that the preexisting neutralization antibody may underlie the limited transduction of Y445F AAV-6. In summary, we have identified Y445F/Y731F AAV-1 as a potentially excellent systemic gene transfer vehicle to target both skeletal muscle and the heart in neonatal puppies. Our findings have important implications in exploring systemic neonatal gene therapy in canine models of muscular dystrophy.

  2. The MRI contrast agent gadoteridol enhances distribution of rAAV1 in the rat hippocampus.

    PubMed

    Hullinger, R; Ugalde, J; Purón-Sierra, L; Osting, S; Burger, C

    2013-12-01

    Contrast agents are commonly used in combination with magnetic resonance imaging (MRI) to monitor the distribution of molecules in the brain. Recent experiments conducted in our laboratory have shown that co-infusion of recombinant Adeno-associated virus serotype 5 (rAAV5) and the MRI contrast agent gadoteridol (Gd) enhances vector transduction in the rat striatum. The goal of this study was to determine whether gadoteridol may also be used as a tool to enhance transduction efficiency of rAAV1 and rAAV5 within the rat hippocampus. We show that Gd/rAAV1-GFP but not Gd/rAAV5-GFP co-infusion results in significantly higher distribution of the transgene both in the injected hemisphere as well as in the contralateral side and adjacent areas of cortex along the injection track. We also show that Gd/rAAV1-GFP co-infusion has no deleterious effect on hippocampal function as assessed by two tests of spatial memory formation. This work indicates that Gd can be exploited as a method to increase transduction efficiency of AAV1 in the hippocampus for animal studies.

  3. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.

    PubMed

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-04-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

  4. High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution

    PubMed Central

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-01-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the “natural” AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid–DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid–genome identity correlation. Overall, our observations demonstrate that production of “natural” AAVs results in low capsid mosaicism and high capsid–genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype–genotype correlation. PMID:25586687

  5. Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy.

    PubMed

    Yi, Haiqing; Zhang, Quan; Brooks, Elizabeth D; Yang, Chunyu; Thurberg, Beth L; Kishnani, Priya S; Sun, Baodong

    2016-11-10

    Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1(ys/ys)). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1(ys/ys) mice at a dose of 5 × 10(11) vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1(ys/ys) mice.

  6. Systematic Evaluation of AAV Vectors for Liver directed Gene Transfer in Murine Models

    PubMed Central

    Wang, Lili; Wang, Huan; Bell, Peter; McCarter, Robert J; He, Jianping; Calcedo, Roberto; Vandenberghe, Luk H; Morizono, Hiroki; Batshaw, Mark L; Wilson, James M

    2009-01-01

    Vectors based on adeno-associated viruses (AAVs) are being evaluated for use in liver-directed gene therapy. Candidates have been preselected on the basis of capsid structure that plays an important role in determining performance profiles. We describe a comprehensive and statistically powered set of mouse studies designed to compare the performance of vectors based on seven novel AAV capsids. The key criteria used to select candidates for successful gene therapy are high level and stable transgene expression in the absence of toxicity. Based on these criteria, the best performing vectors, AAV8, AAVhu.37, and AAVrh.8, will be further evaluated in nonhuman primates (NHPs). PMID:19861950

  7. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

    PubMed Central

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-01-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application. PMID:26134901

  8. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

    PubMed

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-10-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

  9. C-reactive protein (CRP) is essential for efficient systemic transduction of recombinant adeno-associated virus vector 1 (rAAV-1) and rAAV-6 in mice.

    PubMed

    Denard, Jerome; Marolleau, Beatrice; Jenny, Christine; Rao, Tata Nageswara; Fehling, Hans Jörg; Voit, Thomas; Svinartchouk, Fedor

    2013-10-01

    The clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a species-specific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.

  10. 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.

  11. 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.

  12. Robust Cardiomyocyte-Specific Gene Expression Following Systemic Injection of AAV: In Vivo Gene Delivery Follows a Poisson Distribution

    PubMed Central

    Prasad, Konkal-Matt R.; Xu, Yaqin; Yang, Zequan; Acton, Scott T.; French, Brent A

    2010-01-01

    Newly-isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T promoter (cTnT). Using luciferase and eGFP, the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1 week old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared to the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15×1010 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution; requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency. PMID:20703310

  13. 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

  14. 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...

  15. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes.

    PubMed

    Kang, W; Wang, L; Harrell, H; Liu, J; Thomas, D L; Mayfield, T L; Scotti, M M; Ye, G J; Veres, G; Knop, D R

    2009-02-01

    Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

  16. A multi-functional AAV-CRISPR-Cas9 and its host response

    PubMed Central

    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.

    2017-01-01

    CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. 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. PMID:27595405

  17. A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo.

    PubMed

    Lu, Jiamiao; Williams, James A; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A

    2017-01-01

    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.

  18. Plectin-1 Targeted AAV Vector for the Molecular Imaging of Pancreatic Cancer.

    PubMed

    Konkalmatt, Prasad R; Deng, Defeng; Thomas, Stephanie; Wu, Michael T; Logsdon, Craig D; French, Brent A; Kelly, Kimberly A

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1-2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer.

  19. AAV vector-mediated reversal of hypoglycemia in canine and murine glycogen storage disease type Ia.

    PubMed

    Koeberl, Dwight D; Pinto, Carlos; Sun, Baodong; Li, Songtao; Kozink, Daniel M; Benjamin, Daniel K; Demaster, Amanda K; Kruse, Meghan A; Vaughn, Valerie; Hillman, Steven; Bird, Andrew; Jackson, Mark; Brown, Talmage; Kishnani, Priya S; Chen, Yuan-Tsong

    2008-04-01

    Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

  20. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates

    PubMed Central

    Bell, Peter; Wang, Lili; Gao, Guangping; Haskins, Mark E.; Tarantal, Alice F.; McCarter, Robert J.; Zhu, Yanqing; Yu, Hongwei; Wilson, James M.

    2011-01-01

    Gene transfer vectors based on adeno-associated virus 8 (AAV8) are highly efficient in liver transduction and can be easily administered by intravenous injection. In mice, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in periportal regions. This transduction bias has important implications for gene therapy that aims to correct metabolic liver enzymes because metabolic zonation along the porto-central axis requires the expression of therapeutic proteins within the zone where they are normally localized. In the present study we compared the expression pattern of AAV8 expressing green fluorescent protein (GFP) in liver between mice, dogs, and non-human primates. We confirmed the pericentral dominance in transgene expression in mice with AAV8 when the liver-specific thyroid hormone-binding globulin (TBG) promoter was used but also observed the same expression pattern with the ubiquitous chicken β-actin (CB) and cytomegalovirus (CMV) promoters, suggesting that transduction zonation is not caused by promoter specificity. Predominantly pericentral expression was also found in dogs injected with AAV8. In contrast, in cynomolgus and rhesus macaques the expression pattern from AAV vectors was reversed, i.e. transgene expression was most intense around portal areas and less intense or absent around central veins. Infant rhesus macaques as well as newborn mice injected with AAV8 however showed a random distribution of transgene expression with neither portal nor central transduction bias. Based on the data in monkeys, adult humans treated with AAV vectors are predicted to also express transgenes predominantly in periportal regions whereas infants are likely to show a uniform transduction pattern in liver. PMID:21778099

  1. 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

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

    PubMed

    Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A; Song, Juan; Asokan, Aravind

    2016-09-08

    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.

  3. Intramuscular administration of AAV overcomes pre-existing neutralizing antibodies in rhesus macaques.

    PubMed

    Greig, Jenny A; Calcedo, Roberto; Grant, Rebecca L; Peng, Hui; Medina-Jaszek, C Angelica; Ahonkhai, Omua; Qin, Qiuyue; Roy, Soumitra; Tretiakova, Anna P; Wilson, James M

    2016-12-07

    The seroprevalence of neutralizing antibodies (NAbs) to adeno-associated viral (AAV) vector capsids may preclude a percentage of the population from receiving gene therapy, particularly following systemic vector administration. We hypothesized that the use of intramuscular (IM) administration of AAV vectors might circumvent this issue. IM injections were used to administer AAV8 vectors expressing either secreted or non-secreted transgenes into mice and the influence of NAbs supplied by pre-administration of pooled human IgG on transgene expression was evaluated. We then studied the impact of naturally occurring pre-existing AAV8 NAbs on expression of a secreted transgene following IM vector delivery in rhesus macaques. Finally, we evaluated the ability to readminister AAV vectors via IM injections in rhesus macaques. In mice, the presence of AAV8 NAbs had no effect on gene expression in the injected skeletal muscle. However, liver transgene expression following hepatic distribution of the vector was ablated. In rhesus macaques, naturally occurring pre-existing AAV8 NAb titers of ⩽1:160 had no effect on expression levels of a secreted transgene after IM delivery of the vector. Additionally, readministration of AAV vectors was possible by IM injection into the previously injected muscle groups, with no effect on transgene expression by the original vector. Therefore, the presence of pre-existing NAbs in the human population should not preclude subjects from receiving gene therapy by IM administration of the vector so long as sufficient levels of secreted transgene expression can be produced without the involvement of liver.

  4. 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.

  5. 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; Abou Alaiwa, Mahmoud H.; 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

  6. Triptolide T10 enhances AAV-mediated gene transfer in mice striatum.

    PubMed

    Ren, Xinmiao; Zhang, Ting; Hu, Jing; Ding, Wei; Wang, Xiaomin

    2010-08-02

    Adeno-associated virus (AAV) mediated gene transfer has been demonstrated to be an effective approach for treating Parkinson's disease (PD). Triptolide T10 is a monomeric compound isolated from tripterygium wilfordii Hook.f. (Thunder God vine), a traditional Chinese herb for anti-inflammatory medications. In the present study, we co-administered T10 with recombinant AAV2 in SH-SY5Y human neuroblastoma cells and in the striatum of C57BL/6 mice, and then evaluated the AAV-mediated gene expression levels. The results have shown that T10 significantly augmented the expression of AAV-mediated gene in a dose-dependent fashion without detectable cytotoxicity. As growing evidence indicated that inflammation contributed to the progression of PD, and the anti-inflammatory effect of T10 was shown in our previous studies, our data of T10 to enhance AAV transduction suggest that T10 might be potentially used as a facilitating reagent for the AAV gene therapy applications in neurodegenerative diseases.

  7. 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

  8. AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses.

    PubMed

    Mendoza, Skyler D; El-Shamayleh, Yasmine; Horwitz, Gregory D

    2017-02-15

    Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously.

  9. Locally Targeted Cardiac Gene Delivery by AAV Microbubble Destruction in a Large Animal Model.

    PubMed

    Schlegel, Philipp; Huditz, Regina; Meinhardt, Eric; Rapti, Kleopatra; Geis, Nicolas; Most, Patrick; Katus, Hugo A; Müller, Oliver J; Bekeredjian, Raffi; Raake, Philip W

    2016-04-01

    Cardiac gene therapy is a promising approach for treating heart diseases. Although clinical studies are ongoing, effective and targeted transgene delivery is still a major obstacle. We sought to improve and direct transgene expression in myocardium by ultrasound-targeted microbubble destruction (UTMD). In pigs, adeno-associated virus-derived (AAV) vectors harboring the luciferase reporter gene were delivered via retroinfusion into the anterior interventricular coronary vein (AIV). AAV vectors were either loaded to lipid microbubbles before injection or injected unmodified. Upon injection of AAV/microbubble solution, UTMD was performed. After 4 weeks, reporter gene expression levels in the anterior wall (target area), in the posterior wall (control area), and in noncardiac organs were analyzed. Retroinfusion of AAV-luciferase vectors loaded to lipid microbubbles led to a significant increase in transgene expression, with an increase in UTMD targeted areas of the anterior wall. Moreover, off-target expression was reduced in comparison to control animals, receiving AAV-luciferase without microbubbles. Besides an increase in overall target area transgene expression, UTMD alters the spatial expression of the luciferase transgene, focusing expression to ultrasound-targeted left ventricular wall. These data suggest UTMD as a promising approach for directing AAV to specific cardiac segments.

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

    PubMed

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

    2015-09-08

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

  11. A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning.

    PubMed

    Guo, Ping; Xiao, Xiangwei; El-Gohary, Yousef; Paredes, Jose; Prasadan, Krishna; Shiota, Chiyo; Wiersch, John; Welsh, Carey; Gittes, George K

    2013-01-01

    Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH(4))(2)SO(4) aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.

  12. Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs

    PubMed Central

    Boyd, RF; Sledge, DG; Boye, SL; Boye, SE; Hauswirth, WW; Komáromy, AM; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~ 4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog. PMID:26467396

  13. AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta).

    PubMed

    Wang, Lili; Bell, Peter; Lin, Jianping; Calcedo, Roberto; Tarantal, Alice F; Wilson, James M

    2011-11-01

    Many genetic metabolic diseases manifest in infancy, therefore, it is important to develop effective treatments that could be implemented at this time. Adeno-associated virus serotype 8 (AAV8) gene transfer has been studied in neonatal mouse, cat, and dog models and shown some efficacy with a single hepatic injection at birth. AAV8-mediated liver gene transfer has also generated sustained therapeutic effects in feline and canine models of lysosomal storage disorders. In these models, delaying the age of vector treatment increased gene transfer stability. The growth rate of infant nonhuman primates is more similar to the growth trajectory of humans, thus infant monkeys provide an excellent model to study AAV gene transfer efficiency, stability, and safety. In this study, we report for the first time that AAV8-mediated hepatic gene transfer in infant monkeys is safe and efficient but less stable when compared to adolescent animals. Infant monkeys administered AAV8 intravenously at 1 week postnatal age achieved up to 98% transduction of hepatocytes within 7 days of injection; however, there was significant dilution of genomes and loss of transgene expression 35 days postadministration. Delaying the injection to 1 month postnatal age did not improve stability of transduction but decreased the antibody response to AAV8 capsid.

  14. Computational and molecular tools for scalable rAAV-mediated genome editing

    PubMed Central

    Stoimenov, Ivaylo; Ali, Muhammad Akhtar; Pandzic, Tatjana; Sjöblom, Tobias

    2015-01-01

    The rapid discovery of potential driver mutations through large-scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV)-mediated gene targeting is suited for knock-in of single nucleotide substitutions and to a lesser degree for gene knock-outs. However, the generation of gene targeting constructs and the targeting process is time-consuming and labor-intense. To facilitate rAAV-mediated gene targeting, we developed the first software and complementary automation-friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ∼71% of bases in protein-coding exons were designed. Similarly, ∼81% of genes were predicted to be targetable by rAAV-mediated knock-out. A Gateway-based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations. PMID:25488813

  15. Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

    PubMed

    Boyd, R F; Sledge, D G; Boye, S L; Boye, S E; Hauswirth, W W; Komáromy, A M; Petersen-Jones, S M; Bartoe, J T

    2016-02-01

    Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

  16. 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,…

  17. 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.

  18. 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

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

    PubMed Central

    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

  20. AAV-mediated gene delivery of BDNF or GDNF is neuroprotective in a model of Huntington disease.

    PubMed

    Kells, Adrian P; Fong, Dahna M; Dragunow, Mike; During, Matthew J; Young, Deborah; Connor, Bronwen

    2004-05-01

    Huntington disease (HD) is a neurodegenerative disorder that results in the progressive loss of GABAergic medium spiny projection neurons in the striatum. Neurotrophic factors have demonstrated neuroprotective actions on striatal neurons, suggesting that increased neurotrophic factor expression may prevent or reduce neuronal loss in the HD brain. We investigated whether enhanced expression of brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), achieved by adeno-associated viral (AAV) vector-mediated gene delivery, could protect striatal neurons in the quinolinic acid (QA) rodent model of HD. Adult Wistar rats received unilateral intrastriatal injections of AAV-BDNF, AAV-GDNF, AAV-GFP, or PBS. Three weeks later, the rats were lesioned with QA, a toxin that induces striatal neuron death by an excitotoxic process. Both AAV-BDNF and AAV-GDNF significantly reduced the loss of both NeuN- and calbindin-immunopositive striatal neurons 2 weeks after lesion compared to controls. AAV-BDNF also provided significant neurotrophic support to NOS-immunopositive striatal interneurons, while AAV-GDNF-treated rats demonstrated significant protection of parvalbumin-immunopositive striatal interneurons compared to controls. These results indicate that AAV-mediated gene transfer of BDNF or GDNF into the striatum provides neuronal protection in a rodent model of HD.

  1. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    PubMed

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong

    2013-07-01

    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  2. 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.

  3. Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV.

    PubMed

    György, Bence; Sage, Cyrille; Indzhykulian, Artur A; Scheffer, Deborah I; Brisson, Alain R; Tan, Sisareuth; Wu, Xudong; Volak, Adrienn; Mu, Dakai; Tamvakologos, Panos I; Li, Yaqiao; Fitzpatrick, Zachary; Ericsson, Maria; Breakefield, Xandra O; Corey, David P; Maguire, Casey A

    2017-02-01

    Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs(-/-)]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.

  4. 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.

  5. 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.

  6. Inverter design for high frequency power distribution

    NASA Technical Reports Server (NTRS)

    King, R. J.

    1985-01-01

    A class of simple resonantly commutated inverters are investigated for use in a high power (100 KW - 1000 KW) high frequency (10 KHz - 20 KHz) AC power distribution system. The Mapham inverter is found to provide a unique combination of large thyristor turn-off angle and good utilization factor, much better than an alternate 'current-fed' inverter. The effects of loading the Mapham inverter entirely with rectifier loads are investigated by simulation and with an experimental 3 KW 20 KHz inverter. This inverter is found to be well suited to a power system with heavy rectifier loading.

  7. Pre-Clinical Assessment of Immune Responses to Adeno-Associated Virus (AAV) Vectors.

    PubMed

    Basner-Tschakarjan, Etiena; Bijjiga, Enoch; Martino, Ashley T

    2014-01-01

    Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also

  8. Cell-Mediated Immunity to AAV Vectors, Evolving Concepts and Potential Solutions.

    PubMed

    Basner-Tschakarjan, Etiena; Mingozzi, Federico

    2014-01-01

    Adeno-associated virus (AAV) vectors are one of the most efficient in vivo gene delivery platforms. Over the past decade, clinical trials of AAV vector-mediated gene transfer led to some of the most exciting results in the field of gene therapy and, recently, to the market approval of an AAV-based drug in Europe. With clinical development, however, it became obvious that the host immune system represents an important obstacle to successful gene transfer with AAV vectors. In this review article, we will discuss the issue of cytotoxic T cell responses directed against the AAV capsid encountered on human studies. While over the past several years the field has acquired a tremendous amount of information on the interactions of AAV vectors with the immune system, a lot of questions are still unanswered. Novel concepts are emerging, such as the relationship between the total capsid dose and the T cell-mediated clearance of transduced cells, the potential role of innate immunity in vector immunogenicity highlighted in preclinical studies, and the cross talk between regulatory and effector T cells in the determination of the outcome of gene transfer. There is still a lot to learn about immune responses in AAV gene transfer, for example, it is not well understood what are the determinants of the kinetics of activation of T cells in response to vector administration, why not all subjects develop detrimental T cell responses following gene transfer, and whether the intervention strategies currently in use to block T cell-mediated clearance of transduced cells will be safe and effective for all gene therapy indications. Results from novel preclinical models and clinical studies will help to address these points and to reach the important goal of developing safe and effective gene therapy protocols to treat human diseases.

  9. 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

  10. 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

  11. Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system.

    PubMed

    Liu, Yu-Kuo; Yang, Ching-Jen; Liu, Chao-Lin; Shen, Chia-Rui; Shiau, Lie-Ding

    2010-08-01

    Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.

  12. Overload protection system for power inverter

    NASA Technical Reports Server (NTRS)

    Nagano, S. (Inventor)

    1977-01-01

    An overload protection system for a power inverter utilized a first circuit for monitoring current to the load from the power inverter to detect an overload and a control circuit to shut off the power inverter, when an overload condition was detected. At the same time, a monitoring current inverter was turned on to deliver current to the load at a very low power level. A second circuit monitored current to the load, from the monitoring current inverter, to hold the power inverter off through the control circuit, until the overload condition was cleared so that the control circuit may be deactivated in order for the power inverter to be restored after the monitoring current inverter is turned off completely.

  13. Biodistribution and safety assessment of AAV2-GAD following intrasubthalamic injection in the rat

    PubMed Central

    Fitzsimons, Helen L.; Riban, Veronique; Bland, Ross J.; Wendelken, Jennifer L.; Sapan, Christine V.; During, Matthew J.

    2010-01-01

    Background The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy there are unique aspects that need to be addressed in biodistribution studies. Notably, spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events, thus it is critical to evaluate risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. Methods Here we present a rodent study comprising of a comprehensive assessment of vector biodistribution through the brain, blood and major organs of rats injected into the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. Results AAV genomes were not detected in blood or CSF, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral STN. There were no discernable effects of AAV2-GAD on general health and behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. Conclusions This study met FDA requirements, in addition to efficacy and toxicity studies in rodents and non-human primates, to support and supplement a Phase II clinical trial for gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson’s disease. PMID:20352617

  14. Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors.

    PubMed

    Uhrig-Schmidt, Silke; Geiger, Matthias; Luippold, Gerd; Birk, Gerald; Mennerich, Detlev; Neubauer, Heike; Grimm, Dirk; Wolfrum, Christian; Kreuz, Sebastian

    2014-01-01

    In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A - a lipid-droplet-associated protein - resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo.

  15. 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.

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

    PubMed

    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.

  17. 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

  18. 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.

  19. Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

    PubMed Central

    Liu, Xianglan; Magee, Daniel; Wang, Chuansong; McMurphy, Travis; Slater, Andrew; During, Matthew; Cao, Lei

    2014-01-01

    Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue. PMID:25383359

  20. Future of rAAV Gene Therapy: Platform for RNAi, Gene Editing, and Beyond.

    PubMed

    Valdmanis, Paul N; Kay, Mark A

    2017-01-10

    The use of recombinant adeno-associated viruses (rAAVs) ushered in a new millennium of gene transfer for therapeutic treatment of a number of conditions, including congenital blindness, hemophilia, and spinal muscular atrophy. rAAV vectors have remarkable staying power from a therapeutic standpoint, withstanding several ebbs and flows. As new technologies such as clustered regularly interspaced short palindromic repeat genome editing emerge, it is now the delivery tool-the AAV vector-that is the stalwart. The long-standing safety of this vector in a multitude of clinical settings makes rAAV a selling point in the advancement of approaches for gene replacement, gene knockdown, gene editing, and genome modification/engineering. The research community is building on these advances to develop more tailored delivery approaches and to tweak the genome in new and unique ways. Intertwining these approaches with newly engineered rAAV vectors is greatly expanding the available tools to manipulate gene expression with a therapeutic intent.

  1. AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson's disease in vitro and in vivo.

    PubMed

    Saal, Kim-Ann; Koch, Jan C; Tatenhorst, Lars; Szegő, Eva M; Ribas, Vinicius Toledo; Michel, Uwe; Bähr, Mathias; Tönges, Lars; Lingor, Paul

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and

  2. The Length of the Terminal Repetition in Adenovirus-2 DNA

    PubMed Central

    Roberts, Richard J.; Arrand, John R.; Keller, Walter

    1974-01-01

    Adenovirus-2 DNA was end-labeled by partial digestion with Escherichia coli exonuclease III and resynthesis with the DNA polymerase from avian myeloblastosis virus and α-32P-labeled deoxyribonucleoside triphosphates. This end-labeled DNA was cleaved with several specific endonucleases and the terminal fragments were characterized by gel electrophoresis and pyrimidine tract analysis. Two endonucleases gave identical fragments from both ends, presumably from cleavage within the inverted terminal repetition, while all other endonucleases gave dissimilar fragments from the two ends. From the sizes of these fragments it is estimated that the inverted terminal repetition is between 100 and 140 nucleotide pairs long. Images PMID:4139703

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. Inverted meiosis: the true bugs as a model to study.

    PubMed

    Viera, A; Page, J; Rufas, J S

    2009-01-01

    Most of the meiotic literature is based on species with monocentric chromosomes, however meiosis in protoctist, plant and animal species with holocentric chromosomes is less characterized. In some cases, an inverted meiotic sequence is claimed to occur, in which segregation of homologs is postponed until the second meiotic division. Additionally, other features also deserve interest, namely: (i) the different behavior of sex chromosomes if compared to that of the autosomes; (ii) the absence of a canonical kinetochore structure; (iii) the restriction of the kinetic activity to the chromosomal ends; (iv) the variations in the orientation of bivalents at the division plate, and (v) the possible occurrence of chiasma terminalization. Here we summarize the current knowledge on these topics in the meiosis of Hemiptera (Heteroptera) and present novel results that illustrate some of the special features mentioned above. We also point out the necessity of reviewing the term 'inverted meiosis' and propose some future prospects to study this peculiar meiosis.

  8. 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.

  9. Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye

    PubMed Central

    Li, Wensheng; Kong, Fansheng; Li, Xia; Dai, Xufeng; Liu, Xiaoqiang; Zheng, Qinxiang; Wu, Ronghan; Zhou, Xiangtian; Lü, Fan; Chang, Bo; Li, Qiuhong; Hauswirth, William W.; Pang, Ji-jing

    2009-01-01

    Purpose In an earlier study we found normal adeno-associated viral vector type 2 (AAV2)-mediated GFP expression after intravitreal injection to one eye of normal C57BL/6J mice. However, GFP expression was very poor in the partner eye of the same mouse if this eye received an intravitreal injection of the same vector one month after the initial intravitreal injection. We also found both injections worked well if they were subretinal. In this study, we tested whether the efficiency of subretinal AAV vector transduction is altered by a previous intravitreal injection in the partner eye and more importantly whether therapeutic efficiency is altered in the rd12 mouse (with a recessive RPE65 mutation) after the same injection series. Methods One μl of scAAV5-smCBA-GFP (1x1013 genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old C57BL/6J mice and 1 μl of scAAV5-smCBA-hRPE65 (1x1013 genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old rd12 mice Four weeks later, the same vectors were subretinally injected into the left eyes of the same C57BL/6J and rd12 mice. Left eyes of another cohort of eight-week-old rd12 mice received a single subretinal injection of the same scAAV5-smCBA-hRPE65 vector as the positive control. Dark-adapted electroretinograms (ERGs) were recorded five months after the subretinal injections. AAV-mediated GFP expression in C57BL/6J mice and RPE65 expression and ERG restoration in rd12 mice were evaluated five months after the second subretinal injection. Frozen section analysis was performed for GFP fluorescence in C57BL/6J mice and immunostaining for RPE65 in rd12 eyes. Results In rd12 mice, dark-adapted ERGs were minimal following the first intravitreal injection of scAAV5-smCBA-RPE65. Following subsequent subretinal injection in the partner eye, dramatic ERG restoration was recorded in that eye. In fact, ERG b-wave amplitudes were

  10. Comparative Transduction Efficiency of AAV Vector Serotypes 1–6 in the Substantia Nigra and Striatum of the Primate Brain

    PubMed Central

    Markakis, Eleni A; Vives, Kenneth P; Bober, Jeremy; Leichtle, Stefan; Leranth, Csaba; Beecham, Jeff; Elsworth, John D; Roth, Robert H; Samulski, R Jude; Redmond, D Eugene

    2009-01-01

    Vectors derived from adeno-associated virus (AAV) are promising candidates for neural cell transduction in vivo because they are nonpathogenic and achieve long-term transduction in the central nervous system. AAV serotype 2 (AAV2) is the most widely used AAV vector in clinical trials based largely on its ability to transduce neural cells in the rodent and primate brain. Prior work in rodents suggests that other serotypes might be more efficient; however, a systematic evaluation of vector transduction efficiency has not yet been performed in the primate brain. In this study, AAV viral vectors of serotypes 1–6 with an enhanced green-fluorescent protein (GFP) reporter gene were generated at comparable titers, and injected in equal amounts into the brains of Chlorocebus sabaeus. Vector injections were placed in the substantia nigra (SN) and the caudate nucleus (CD). One month after injection, immunohistochemistry for GFP was performed and the total number of GFP+ cells was calculated using unbiased stereology. AAV5 was the most efficient vector, not only transducing significantly more cells than any other serotype, but also transducing both NeuN+ and glial-fibrillary-acidic protein positive (GFAP+) cells. These results suggest that AAV5 is a more effective vector than AAV2 at delivering potentially therapeutic transgenes to the nigrostriatal system of the primate brain. PMID:20010918

  11. Preexisting Immunity and Low Expression in Primates Highlight Translational Challenges for Liver-directed AAV8-mediated Gene Therapy

    PubMed Central

    Hurlbut, Gregory D; Ziegler, Robin J; Nietupski, Jennifer B; Foley, Joseph W; Woodworth, Lisa A; Meyers, Elizabeth; Bercury, Scott D; Pande, Nilesh N; Souza, David W; Bree, Mark P; Lukason, Michael J; Marshall, John; Cheng, Seng H; Scheule, Ronald K

    2010-01-01

    Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients. PMID:20736932

  12. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection.

    PubMed

    Wassmer, Sarah J; Carvalho, Livia S; György, Bence; Vandenberghe, Luk H; Maguire, Casey A

    2017-03-31

    Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye.

  13. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection

    PubMed Central

    Wassmer, Sarah J.; Carvalho, Livia S.; György, Bence; Vandenberghe, Luk H.; Maguire, Casey A.

    2017-01-01

    Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye. PMID:28361998

  14. 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

  15. Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

    PubMed

    Jackson, Kasey L; Dayton, Robert D; Deverman, Benjamin E; Klein, Ronald L

    2016-01-01

    Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

  16. 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 ...

  17. Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

    PubMed Central

    Jackson, Kasey L.; Dayton, Robert D.; Deverman, Benjamin E.; Klein, Ronald L.

    2016-01-01

    Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats. PMID:27867348

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  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. Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

    PubMed Central

    Doerfler, Phillip A.; Todd, Adrian G.; Clément, Nathalie; Falk, Darin J.; Nayak, Sushrusha; Herzog, Roland W.; Byrne, Barry J.

    2016-01-01

    Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa−/− mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders. PMID:26603344

  6. 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

    2016-02-04

    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.

  7. Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9

    PubMed Central

    Dashkoff, Jonathan; Lerner, Eli P; Truong, Nhi; Klickstein, Jacob A; Fan, Zhanyun; Mu, Dakai; Maguire, Casey A; Hyman, Bradley T; Hudry, Eloise

    2016-01-01

    The capacity of certain adeno-associated virus (AAV) vectors to cross the blood–brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9) expressing the green fluorescent protein (GFP) either under an astrocyte (glial fibrillary acidic (GFA) protein) or neuronal (Synapsin (Syn)) promoter, after intravenous injection of adult mice (2 × 1013 vg/kg). ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11%) and neurons (17%), respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter), significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive) and resting astrocytes (GFAP-negative) express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS) gene therapy approaches in which peripheral expression of transgene is a concern. PMID:27933308

  8. Invertible linear ordinary differential operators

    NASA Astrophysics Data System (ADS)

    Chetverikov, Vladimir N.

    2017-03-01

    We consider invertible linear ordinary differential operators whose inversions are also differential operators. To each such operator we assign a numerical table. These tables are described in the elementary geometrical language. The table does not uniquely determine the operator. To define this operator uniquely some additional information should be added, as it is described in detail in this paper. The possibility of generalization of these results to partial differential operators is also discussed.

  9. Integrated Inverter And Battery Charger

    NASA Technical Reports Server (NTRS)

    Rippel, Wally E.

    1988-01-01

    Circuit combines functions of dc-to-ac inversion (for driving ac motor in battery-powered vehicle) and ac-to-dc conversion (for charging battery from ac line when vehicle not in use). Automatically adapts to either mode. Design of integrated inverter/charger eliminates need for duplicate components, saves space, reduces weight and cost of vehicle. Advantages in other applications : load-leveling systems, standby ac power systems, and uninterruptible power supplies.

  10. 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.

  11. Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.

    PubMed

    Bruewer, Ashlee R; Mowat, Freya M; Bartoe, Joshua T; Boye, Sanford L; Hauswirth, William W; Petersen-Jones, Simon M

    2013-01-01

    Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these

  12. Whole Body Skeletal Muscle Transduction in Neonatal Dogs with AAV-9

    PubMed Central

    Yue, Yongping; Shin, Jin-Hong; Duan, Dongsheng

    2011-01-01

    Gene therapy of muscular dystrophy requires systemic gene delivery to all muscles in the body. Adeno-associated viral (AAV) vectors have been shown to lead to body-wide muscle transduction after a single intravascular injection. Proof-of-principle has been demonstrated in mouse models of Duchenne muscular dystrophy and limb girdle muscular dystrophy. Before initiating clinical trials, it is important to validate these promising results in large animal models. More than a dozen canine muscular dystrophy models have been developed. Here, we outline a protocol for performing systemic AAV gene transfer in neonatal dogs. Implementing this technique in dystrophic dogs will accelerate translational muscular dystrophy research. PMID:21194038

  13. 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.

  14. 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.

  15. 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.

  16. 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

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

    PubMed

    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

    2015-10-27

    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.

  18. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors.

    PubMed

    Sabatino, Denise E; Lange, Amy M; Altynova, Ekaterina S; Sarkar, Rita; Zhou, Shangzhen; Merricks, Elizabeth P; Franck, Helen G; Nichols, Timothy C; Arruda, Valder R; Kazazian, Haig H

    2011-03-01

    Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

  19. AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress.

    PubMed

    Liang, Katharine J; Woodard, Kenton T; Weaver, Mark A; Gaylor, John Paul; Weiss, Ellen R; Samulski, R Jude

    2017-03-01

    NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.

  20. 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…

  1. Functional Effects of AAV2-GDNF on the Dopaminergic Nigrostriatal Pathway in Parkinsonian Rhesus Monkeys

    PubMed Central

    Eberling, Jamie L.; Kells, Adrian P.; Pivirotto, Philip; Beyer, Janine; Bringas, John; Federoff, Howard J.; Forsayeth, John

    2009-01-01

    Abstract We investigated the safety and neuroregenerative potential of an adeno-associated virus (AAV2) containing human glial cell line-derived neurotrophic factor (GDNF) in an MPTP primate model of Parkinson's disease. Dopaminergic function was evaluated by positron emission tomography with 6-[18F]fluoro-l-m-tyrosine (FMT) before and after AAV2-GDNF or phosphate-buffered saline infusion bilaterally into the putamen. FMT uptake was significantly increased bilaterally in the putamen of AAV2-GDNF but not phosphate-buffered saline-treated animals 6 months after infusion, indicating increased dopaminergic activity in the nigrostriatal pathways. AAV2-GDNF-treated animals also showed clinical improvement without adverse effects. These findings are consistent with our previous report in aged nonhuman primates that showed evidence of enhanced use of striatal dopamine and dopaminergic nigrostriatal innervation. Clinical improvement and evidence of functional recovery in the nigrostriatal pathway, and the absence of adverse effects, support the safety of this approach for the delivery of GDNF over a 6-month period. PMID:19254173

  2. AAV ANCESTRAL RECONSTRUCTION LIBRARY ENABLES SELECTION OF BROADLY INFECTIOUS VIRAL VARIANTS

    PubMed Central

    Santiago-Ortiz, Jorge; Ojala, David S.; Westesson, Oscar; Weinstein, John R.; Wong, Sophie Y.; Steinsapir, Andrew; Kumar, Sanjay; Holmes, Ian; Schaffer, David V.

    2015-01-01

    Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, 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 utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy. PMID:26186661

  3. Antidepressant effect of recombinant NT4-NAP/AAV on social isolated mice through intranasal route.

    PubMed

    Liu, Fei; Liu, You-Ping; Lei, Gang; Liu, Peng; Chu, Zheng; Gao, Cheng-Ge; Dang, Yong-Hui

    2017-02-07

    The purpose of the present study was to observe the depression-like behavior induced by social isolation; detect the antidepressant effect of a recombinant adeno-associated virus (AAV) expressing NAP on social isolation mice by intranasal delivery. After construction of NT4-NAP/AAV, expression of NAP was confirmed in vitro. 3-week-old C57/BL mice were bred individually in cages as social isolation-rearing. Six weeks later, the first subset of mice underwent behavioral tests and western blot; the second was for enzyme-linked immunosorbent assay. NT4-NAP/AAV was delivered quaque die by nasal administration for consecutive 10 days before behavioral test. Several depression-like behaviors were observed in social isolation mice, including decreased relative sucrose preference, longer immobility time in forced swimming test, lower plasma corticosterone and decreased brain-derived neurotrophic factor in hippocampus. Thus, social isolation procedure appears to be an animal model of depression with good face and construct validity. What's more, the antidepressant effect in social isolation-rearing mice was observed after intranasal administration of NT4-NAP/AAV, suggesting that this might be a promising therapeutic strategy for depressive disorder.

  4. Sites in the AAV5 capsid tolerant to deletions and tandem duplications

    PubMed Central

    Hida, Kaoru; Won, Sang Y.; Di Pasquale, Giovanni; Hanes, Justin; Chiorini, John A.; Ostermeier, Marc

    2010-01-01

    Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >105 members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands. PMID:20102698

  5. Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo

    PubMed Central

    Gupta, Suneel; Rodier, Jason T.; Sharma, Ajay; Giuliano, Elizabeth A.; Sinha, Prashant R.; Hesemann, Nathan P.; Ghosh, Arkasubhra; Mohan, Rajiv R.

    2017-01-01

    Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision. PMID:28339457

  6. Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

    PubMed

    GuhaSarkar, Dwijit; Neiswender, James; Su, Qin; Gao, Guangping; Sena-Esteves, Miguel

    2017-02-01

    The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-β) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-β eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-β vectors with different promoters to drive safe expression of mouse IFN-β in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-β vector. We also assessed the therapeutic effect of combining AAV-IFN-β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency.

  7. 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.

  8. New type of transformerless high efficiency inverter

    NASA Astrophysics Data System (ADS)

    Naaijer, G. J.

    Inverter architectures are presented which allow economical ac/dc switching for solar cell array and battery power use in domestic and industrial applications. The efficiencies of currently available inverters are examined and compared with a new 2.2 kW transformerless stepped wave inverter. The inverter has low no-load losses, amounting to 200 Wh/24 hr, and features voltage steps occurring 15-30 times/sine wave period. An example is provided for an array/battery/inverter assembly with the inverter control electronics activating or disconnecting the battery subassemblies based on the total number of activated subassemblies in relation to a reference sinewave, and the need to average the battery subassembly discharge rates. A total harmonic distortion of 6 percent was observed, and the system is noted to be usable as a battery charger.

  9. 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.

  10. 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.

  11. Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease.

    PubMed

    Sun, Lijun; Hao, Yuewen; Nie, Xiaowei; Zhang, Xuexin; Yang, Guangxiao; Wang, Quanying

    2012-11-01

    The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the

  12. Intramuscular AAV delivery of NT-3 alters synaptic transmission to motoneurons in adult rats

    PubMed Central

    Petruska, Jeffrey C.; Kitay, Brandon; Boyce, Vanessa S.; Kaspar, Brian; Pearse, Damien; Gage, Fred H.; Mendell, Lorne M.

    2010-01-01

    We examined whether elevating levels of neurotrophin-3 (NT-3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno-associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT-3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT-3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT-3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT-3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT-3 expression, the decline in EPSP amplitude was reversed indicating that NT-3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT-3 concentration in the DRG. Treatment with control viruses could elevate NT-3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT-3 level. These novel correlations between modified NT-3 expression and single-cell electrophysiological parameters indicate that intramuscular administration of AAV(NT-3) can exert long lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury. PMID:20849530

  13. AAV1/2-mediated BDNF gene therapy in a transgenic rat model of Huntington's disease.

    PubMed

    Connor, B; Sun, Y; von Hieber, D; Tang, S K; Jones, K S; Maucksch, C

    2016-03-01

    Reduced expression and disrupted corticostriatal transportation of brain-derived neurotrophic factor (BDNF) is proposed to contribute to the selective vulnerability of medium spiny striatal projection neurons (MSNs) in Huntington's disease (HD). We have previously demonstrated that BDNF overexpression in the quinolinic acid lesioned rat striatum attenuates motor impairment and reduces the extent of MSN cell loss. To further investigate the potential therapeutic properties of BDNF for HD, the current study examines the effect of bilateral AAV1/2-mediated BDNF expression in the striatum of a transgenic rat model of HD. Transfer of the BDNF gene to striatal neurons using an AAV1/2 serotype vector enhanced BDNF protein levels in the striatum. Bilateral BDNF expression attenuated the impairment of both motor and cognitive function when compared with AAV1/2-vehicle- or YFP-treated transgenic HD rats. Interestingly, a gender effect was apparent with female transgenic HD rats exhibiting less functional impairment than males. Quantification of NeuN and DARRP32 immunoreactivity and striatal volume revealed limited disease phenotype between wild type and transgenic HD animals. However, AAV1/2-BDNF-treated transgenic HD rats showed evidence of greater striatal volume and increased NeuN+ cell numbers compared with wild-type vehicle- and AAV1/2-vehicle- or YFP-treated transgenic HD rats. We propose BDNF holds considerable therapeutic potential for alleviating behavioral dysfunction and neuronal degeneration in HD, with further work required to examine the role of BDNF-TrkB signaling and the preservation of axonal and synaptic function.

  14. 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.

  15. Self-oscillating inverter with bipolar transistors

    NASA Astrophysics Data System (ADS)

    Baciu, I.; Cunţan, C. D.; Floruţa, M.

    2016-02-01

    The paper presents a self-oscillating inverter manufactured with bipolar transistors that supplies a high-amplitude alternating voltage to a fluorescent tube with a burned filament. The inverter is supplied from a low voltage accumulator that can be charged from a photovoltaic panel through a voltage regulator.

  16. Radiotherapy for inverted papilloma: a case report.

    PubMed

    Levendag, P C; Annyas, A A; Escajadillo, J R; Elema, J D

    1984-06-01

    Inverted papilloma is an infrequent tumour of the nasal cavity and paranasal sinuses associated with controversy. The incidence of carcinoma in situ associated with inverted papilloma, has not been very well documented until now. Therefore, we present a case report characterized by an aggressive clinical behaviour, treated by extensive surgery and ultimately controlled by radiotherapy.

  17. Phoenix Scoop Inverted Showing Rasp

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This image taken by the Surface Stereo Imager on Sol 49, or the 49th Martian day of the mission (July 14, 2008), shows the silver colored rasp protruding from NASA's Phoenix Mars Lander's Robotic Arm scoop. The scoop is inverted and the rasp is pointing up.

    Shown with its forks pointing toward the ground is the thermal and electrical conductivity probe, at the lower right. The Robotic Arm Camera is pointed toward the ground.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is led by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  18. 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

  19. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin Vector

    DTIC Science & Technology

    2015-09-01

    1 AWARD NUMBER: W81XWH-14-1-0302 TITLE: A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin...COVERED 1 Sep 2014 - 31 Aug 2015 4. TITLE AND SUBTITLE A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin...phophotase) or are not clinically meaningful (Table 2). 4   In our previous study, we demonstrated bodywide skeletal muscle transduction after

  20. Negation-Limited Inverters of Linear Size

    NASA Astrophysics Data System (ADS)

    Morizumi, Hiroki; Suzuki, Genki

    An inverter is a circuit which outputs ¬x1, ¬x2, …, ¬xn for any Boolean inputs x1, x2, …, xn. We consider constructing an inverter with AND gates and OR gates and a few NOT gates. Beals, Nishino and Tanaka have given a construction of an inverter which has size O(nlog n) and depth O(log n) and uses ⌈ log(n+1) ⌉ NOT gates. In this paper we give a construction of an inverter which has size O(n) and depth log1+o(1)n and uses log1+o(1)n NOT gates. This is the first negation-limited inverter of linear size using only o(n) NOT gates. We also discuss implications of our construction for negation-limited circuit complexity.

  1. 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

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

    PubMed

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

    2014-05-01

    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.

  3. 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

  4. Intratumoral decorin gene delivery by AAV vector inhibits brain glioblastomas and prolongs survival of animals by inducing cell differentiation.

    PubMed

    Ma, Hsin-I; Hueng, Dueng-Yuan; Shui, Hao-Ai; Han, Jun-Ming; Wang, Chi-Hsien; Lai, Ying-Hsiu; Cheng, Shi-Yuan; Xiao, Xiao; Chen, Ming-Teh; Yang, Yi-Ping

    2014-03-12

    Glioblastoma multiforme (GBM) is the most malignant cancer in the central nervous system with poor clinical prognosis. In this study, we investigated the therapeutic effect of an anti-cancer protein, decorin, by delivering it into a xenograft U87MG glioma tumor in the brain of nude mice through an adeno-associated viral (AAV2) gene delivery system. Decorin expression from the AAV vector in vitro inhibited cultured U87MG cell growth by induction of cell differentiation. Intracranial injection of AAV-decorin vector to the glioma-bearing nude mice in vivo significantly suppressed brain tumor growth and prolonged survival when compared to control non-treated mice bearing the same U87MG tumors. Proteomics analysis on protein expression profiles in the U87MG glioma cells after AAV-mediated decorin gene transfer revealed up- and down-regulation of important proteins. Differentially expressed proteins between control and AAV-decorin-transduced cells were identified through MALDI-TOF MS and database mining. We found that a number of important proteins that are involved in apoptosis, transcription, chemotherapy resistance, mitosis, and fatty acid metabolism have been altered as a result of decorin overexpression. These findings offer valuable insight into the mechanisms of the anti-glioblastoma effects of decorin. In addition, AAV-mediated decorin gene delivery warrants further investigation as a potential therapeutic approach for brain tumors.

  5. Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).

    PubMed

    Athanasopoulos, T; Graham, I R; Foster, H; Dickson, G

    2004-10-01

    Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene. The size of the gene (2.4 Mb) and mRNA (14 kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of efficient gene therapy approaches. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems (nonpathogenicity and minimal immunogenicity, extensive cell and tissue tropism) but accommodate limited transgene capacity (<5 kb). As a result of these observations, a number of laboratories worldwide have engineered a series of microdystrophin cDNAs based on genotype-phenotype relationship in Duchenne (DMD) and Becker (BMD) dystrophic patients, and transgenic studies in mdx mice. Recent progress in characterization of AAV serotypes from various species has demonstrated that alternative AAV serotypes are far more efficient in transducing muscle than the traditionally used AAV2. This article summarizes the current progress in the field of recombinant adeno-associated viral (rAAV) delivery for DMD, including optimization of recombinant AAV-microdystrophin vector systems/cassettes targeting the skeletal and cardiac musculature.

  6. 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.

  7. 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.

  8. Terminal Ballistics

    DTIC Science & Technology

    1976-02-01

    ballistics doe, little to c~plaini the workings of explosike pro jectiles. The termni,iaf phase (of di esplodling tbalfftic weapon is comiposed of esents...citterior, and terminal baillistic phases, and the study of this n’!w systemn hius useful parallel% it) the proiecoile ballistic sysStem. The...charge liner collapse Vi Jet velocity V, Slug velocity W Work Wp Weight of projecti!e Z A length variable a, bi Empirical constants. (In Chap. 5, Eq

  9. 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.

  10. Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing.

    PubMed

    Kaulich, Manuel; Dowdy, Steven F

    2015-12-01

    Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Recent work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. Here we review the current state of CRISPR/Cas-based gene editing and provide a practical guide to applying the CRISPR/Cas and rAAV system for highly efficient, time- and cost-effective gene targeting.

  11. Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing

    PubMed Central

    Kaulich, Manuel

    2015-01-01

    Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Recent work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. Here we review the current state of CRISPR/Cas-based gene editing and provide a practical guide to applying the CRISPR/Cas and rAAV system for highly efficient, time- and cost-effective gene targeting. PMID:26540648

  12. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges.

    PubMed

    Mingozzi, Federico; High, Katherine A

    2011-05-01

    In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.

  13. 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

  14. 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

  15. AAV-IL-22 Modifies Liver Chemokine Activity and Ameliorates Portal Inflammation in Murine Autoimmune Cholangitis

    PubMed Central

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

    2015-01-01

    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. PMID:26537567

  16. Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives.

    PubMed

    Rolling, F

    2004-10-01

    Retinal degenerative diseases such as retinal macular degeneration and retinitis pigmentosa constitute a broad group of diseases that all share one critical feature, the progressive apoptotic loss of cells in the retina. There is currently no effective treatment available by which the course of these disorders can be modified, and visual dysfunction often progresses to total blindness. Gene therapy represents an attractive approach to treating retinal degeneration because the eye is easily accessible and allows local application of therapeutic vectors with reduced risk of systemic effects. Furthermore, transgene expression within the retina and effects of treatments may be monitored by a variety of noninvasive examinations. An increasing number of strategies for molecular treatment of retinal disease rely on recombinant adeno-associated virus (rAAV) as a therapeutic gene delivery vector. Before rAAV-mediated gene therapy for retinal degeneration becomes a reality, there are a number of important requirements that include: (1) evaluation of different rAAV serotypes, (2) screening of vectors in large animals in order to ensure that they mediate safe and long-term gene expression, (3) appropriate regulation of therapeutic gene expression, (4) evaluation of vectors carrying a therapeutic gene in relevant animal models, (5) identification of suitable patients, and finally (6) manufacture of clinical grade vector. All these steps towards gene therapy are still being explored. Outcomes of these studies will be discussed in the order in which they occur, from vector studies to preclinical assessment of the therapeutic potential of rAAV in animal models of retinal degeneration.

  17. AAV2-mediated gene delivery to monkey putamen: Evaluation of an infusion device and delivery parameters

    PubMed Central

    Sanftner, Laura M.; Sommer, Jurg M.; Suzuki, Brian M.; Smith, Peter H.; Vijay, Sharmila; Vargas, Joseph A.; Forsayeth, John R.; Cunningham, Janet; Bankiewicz, Krys S.; Kao, Haihwa; Bernal, Jan; Pierce, Glenn F.; Johnson, Kirk W.

    2013-01-01

    In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell® clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic L-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson&apos ;s disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 μL/min to 1μL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 μL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated. PMID:16022872

  18. AAV-mediated transfer of RhoA shRNA and CNTF promotes retinal ganglion cell survival and axon regeneration.

    PubMed

    Cen, Ling-Ping; Liang, Jia-Jian; Chen, Jian-Huan; Harvey, Alan R; Ng, Tsz Kin; Zhang, Mingzhi; Pang, Chi Pui; Cui, Qi; Fan, You-Ming

    2017-02-20

    The aim of the present study was to determine whether adeno-associated viral vector (AAV) mediated transfer of ciliary neurotrophic factor (CNTF) and RhoA shRNA has additive effects on promoting the survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve crush (ONC). Silencing effects of AAV-RhoA shRNA were confirmed by examining neurite outgrowth in PC12 cells, and by quantifying RhoA expression levels with western blotting. Young adult Fischer rats received an intravitreal injection of (i) saline, (ii) AAV green fluorescent protein (GFP), (iii) AAV-CNTF, (iv) AAV-RhoA shRNA, or (v) a combination of both AAV-CNTF and AAV-RhoA shRNA. Two weeks later, the ON was completely crushed. Three weeks after ONC, RGC survival was estimated by counting βIII-tubulin-positive neurons in retinal whole mounts. Axon regeneration was evaluated by counting GAP-43-positive axons in the crushed ON. It was found that AAV-RhoA shRNA decreased RhoA expression levels and promoted neurite outgrowth in vitro. In the ONC model, AAV-RhoA shRNA by itself had only weak beneficial effects on RGC axon regeneration. However, when combined with AAV-CNTF, AAV-RhoA shRNA significantly improved the therapeutic effect of AAV-CNTF on axon regeneration by nearly two fold, even though there was no significant change in RGC viability. In sum, this combination of vectors increases the regenerative response and can lead to more successful therapeutic outcomes following neurotrauma.

  19. AAV-mediated gene therapy for retinal disorders in large animal models.

    PubMed

    Stieger, Knut; Lhériteau, Elsa; Lhéariteau, Elsa; Moullier, Phillip; Rolling, Fabienne

    2009-01-01

    Retinal gene therapy holds great promise for the treatment of inherited and noninherited blinding diseases such as retinitis pigmentosa and age-related macular degeneration. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because they elicit minimal immune responses and mediated long-term transgene expression in a variety of retinal cell types. Extensive preclinical evaluation of new strategies in large animal models is key to the development of successful gene-based therapies for the retina. Because of differences in the retinal structures among species and unique structures such as the macula and fovea in the primate retina, nonhuman primates are widely used as preclinical animal models. But the observation of inherited retinal degenerations in dogs, which share a number of clinical and pathologic similarities with humans, has led to the characterization of several canine models for retinal diseases, one of which has already responded successfully to AAV-mediated gene therapy. This article presents a review and detailed discussion of the various large animal models available for the study of AAV-mediated gene-based therapies in the retina.

  20. AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

    PubMed

    Bradbury, Allison M; Peterson, Tiffany A; Gross, Amanda L; Wells, Stephen Z; McCurdy, Victoria J; Wolfe, Karen G; Dennis, John C; Brunson, Brandon L; Gray-Edwards, Heather; Randle, Ashley N; Johnson, Aime K; Morrison, Edward E; Cox, Nancy R; Baker, Henry J; Sena-Esteves, Miguel; Martin, Douglas R

    2017-01-06

    Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

  1. 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.

  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. Recombinant AAV-PR39-mediated hypoxia-inducible factor 1α gene expression attenuates myocardial infarction.

    PubMed

    Sun, Lijun; Hao, Yuewen; Nie, Xiaowei; Xu, Jian; Li, Zhenwu; Zhang, Wei; Liu, Ying; Zhang, Xuexin

    2014-01-01

    PR39 is an angiogenic masterswitch protein, belonging to the second generation of angiogenic growth factors. However, the role of recombinant adeno-associated virus (AAV) carrying the PR39 fusion gene (AAV-PR39) in acute myocardial infarction remains unclear. Therefore, in this study, we investigated the role of AAV-PR39 in an experimental animal model of acute myocardial infarction. The PR39 gene was fused with the transmembrane peptide, TAT, 6xHis‑tag and NT4 signal sequences. AAV-PR39 was then obtained by calcium phosphate co-precipitation. A total of 18 healthy Chinese mini pigs were randomly divided into an experimental groups (the AAV-PR39-treated group) and a control group [phosphated-buffered saline (PBS)-treated group]. Following the induction of myocardial infarction, enhanced 3.0T MR imaging was performed to observe the changes in myocardial signal intensity at 0 h, 1, 2 and 3 weeks. The expression of hypoxia-inducible factor‑1α (HIF-1α) in the myocardial tissues was determined by SABC immunohistochemistry. In addition, in vitro experiments using CRL-1730 endothelial cells transfected with AAV vector containing NT4-TAT-His-PR39 revealed that the AAV-PR39-treated group had a significantly higher expression of HIF-1α compared with the control group. Moreover, PR39 regulated the HIF-1α-induced expression of angiogenic growth factors. Under hypoxic conditions, the anti-apoptotic effects in the AAV-PR39 group were more pronounced than those observed in the control (PBS-treated) group. In vivo, the enforced expression of recombinant PR39 elevated the level of HIF-1α under hypoxic conditions and decreased the size of the infarcted areas by upregulating the expression of HIF-1α in the areas surrounding the infarct area. Taken together, our data demonstrate that the recombinant AAV-PR39-mediated HIF-1α expression attenuates myocardial infarction, indicating that AAV-PR39 may serve as a novel therapeutic agent for the treatment of myocardial infarction.

  4. Efficient intrathymic gene transfer following in situ administration of a rAAV serotype 8 vector in mice and nonhuman primates.

    PubMed

    Moreau, Aurélie; Vicente, Rita; Dubreil, Laurence; Adjali, Oumeya; Podevin, Guillaume; Jacquet, Chantal; Deschamps, Jack Yves; Klatzmann, David; Cherel, Yan; Taylor, Naomi; Moullier, Philippe; Zimmermann, Valérie S

    2009-03-01

    The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

  5. 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.

  6. 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

    McIntosh, J H; Cochrane, M; Cobbold, S; Waldmann, H; Nathwani, S A; Davidoff, A M; Nathwani, A C

    2012-01-01

    The ability of transient immunosuppression with a combination of a non-depleting anti-CD4 (NDCD4) antibody and cyclosporine (CyA) to abrogate immune reactivity to both adeno-associated viral 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 × 10(12) AAV8 vector particles per 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. AAV2-mediated in vivo immune gene therapy of solid tumours

    PubMed Central

    2010-01-01

    Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour vasculature and

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

    PubMed

    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

  13. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models.

    PubMed

    Albert, Katrina; Voutilainen, Merja H; Domanskyi, Andrii; Airavaara, Mikko

    2017-02-08

    Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-a-synuclein (a-syn) to target substantia nigra dopamine neurons to produce an α-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.

  14. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models

    PubMed Central

    Albert, Katrina; Voutilainen, Merja H.; Domanskyi, Andrii; Airavaara, Mikko

    2017-01-01

    Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson’s disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson’s disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson’s disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson’s disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-α-synuclein (α-syn) to target substantia nigra dopamine neurons to produce an α-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia

  15. Were carbon isotopic gradients in post-snowball oceans inverted?

    NASA Astrophysics Data System (ADS)

    Halverson, G. P.; Hoffman, P. F.; MacDonald, F. A.; Higgins, J. A.; Schrag, D. P.

    2008-12-01

    In the Otavi Group of Namibia, 900 m of platform carbonate strata above the 635-Ma post-glacial cap dolostone are consistently lighter in δ13C by up to 2.5 per mil than the adjacent 180-320 m of correlative, >0.6-km-deep, foreslope strat. Assuming the foreslope carbonate was equilibrated with deeper water, the normal isotopic gradient dynamically sustained by the biological 'pump' was inverted. Geochronology of isotopically correlative Doushantuo strata in South China suggests that the inverted gradient lasted for ~2 Myr after the glacial termination. Thereafter, the inverted gradient disappears. A similar transient inverse gradient follows the older Cryogenian glaciation in Arctic Alaska. We hypothesize that the inverse gradients reflect high pCO2 in the glacial aftermaths. This had two consequences. First, the size of the DIC pool was enlarged, reducing the isotopic effect of the biological pump. Second, isotopic fractionation was strongly temperature-dependent due to the large fractionation between CO2 and CO32- coupled with the dominance of CO2(gas) among carbon species at pH<7.2. A difference in SST of ~25° between the areas of air-sea equilibration is required to account for a 2.5 per mil gradient in δ13C. This is realized in non-upwelling zones of the southern hemisphere today, where warm subtropical surface waters are underlain by Antarctic Intermediate Water. For our hypothesis to be valid, a large meridional temperature gradient must have coexisted with strong CO2 radiative forcing.

  16. Possible Problems: Inverted, Flat, or Pierced Nipples

    MedlinePlus

    ... Breastfeeding Crying & Colic Diapers & Clothing Feeding & Nutrition Preemie Sleep Teething & Tooth Care Toddler Preschool Gradeschool Teen Young Adult Healthy Children > Ages & Stages > Baby > Breastfeeding > Possible Problems: Inverted, Flat, or Pierced ...

  17. 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.

  18. 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

  19. Automatic load sharing in inverter modules

    NASA Technical Reports Server (NTRS)

    Nagano, S.

    1979-01-01

    Active feedback loads transistor equally with little power loss. Circuit is suitable for balancing modular inverters in spacecraft, computer power supplies, solar-electric power generators, and electric vehicles. Current-balancing circuit senses differences between collector current for power transistor and average value of load currents for all power transistors. Principle is effective not only in fixed duty-cycle inverters but also in converters operating at variable duty cycles.

  20. Base drive for paralleled inverter systems

    NASA Technical Reports Server (NTRS)

    Nagano, S. (Inventor)

    1980-01-01

    In a paralleled inverter system, a positive feedback current derived from the total current from all of the modules of the inverter system is applied to the base drive of each of the power transistors of all modules, thereby to provide all modules protection against open or short circuit faults occurring in any of the modules, and force equal current sharing among the modules during turn on of the power transistors.

  1. 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.

  2. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver.

    PubMed

    Qiao, C; Yuan, Z; Li, J; He, B; Zheng, H; Mayer, C; Li, J; Xiao, X

    2011-04-01

    Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues

  3. rAAV/ABAD-DP-6His attenuates oxidative stress-induced injury of PC12 cells

    PubMed Central

    Jia, Mingyue; Wang, Mingyu; Yang, Yi; Chen, Yixin; Liu, Dujuan; Wang, Xu; Song, Lei; Wu, Jiang; Yang, Yu

    2014-01-01

    Our previous studies have revealed that amyloid β (Aβ)-binding alcohol dehydrogenase (ABAD) decoy peptide antagonizes Aβ42-induced neurotoxicity. However, whether it improves oxidative stress injury remains unclear. In this study, a recombinant adenovirus constitutively secreting and expressing Aβ-ABAD decoy peptide (rAAV/ABAD-DP-6His) was successfully constructed. Our results showed that rAAV/ABAD-DP-6His increased superoxide dismutase activity in hydrogen peroxide-induced oxidative stress-mediated injury of PC12 cells. Moreover, rAAV/ABAD-DP-6His decreased malondialdehyde content, intracellular Ca2+ concentration, and the level of reactive oxygen species. rAAV/ABAD-DP-6His maintained the stability of the mitochondrial membrane potential. In addition, the ATP level remained constant, and apoptosis was reduced. Overall, the results indicate that rAAV/ABAD-DP-6His generates the fusion peptide, Aβ-ABAD decoy peptide, which effectively protects PC12 cells from oxidative stress injury induced by hydrogen peroxide, thus exerting neuroprotective effects. PMID:25206842

  4. 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.

  5. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.

    PubMed

    Ge, Xianglian; Xi, Haitao; Yang, Fayu; Zhi, Xiao; Fu, Yanghua; Chen, Ding; Xu, Ren-He; Lin, Ge; Qu, Jia; Zhao, Junzhao; Gu, Feng

    2016-01-01

    Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  6. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.

    PubMed

    Ge, Xianglian; Xi, Haitao; Yang, Fayu; Zhi, Xiao; Fu, Yanghua; Chen, Ding; Xu, Ren-He; Lin, Ge; Qu, Jia; Zhao, Junzhao; Gu, Feng

    2016-11-29

    Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  7. Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.

    PubMed

    Brantly, Mark L; Chulay, Jeffrey D; Wang, Lili; Mueller, Christian; Humphries, Margaret; Spencer, L Terry; Rouhani, Farshid; Conlon, Thomas J; Calcedo, Roberto; Betts, Michael R; Spencer, Carolyn; Byrne, Barry J; Wilson, James M; Flotte, Terence R

    2009-09-22

    Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.

  8. Preclinical safety evaluation of AAV2-sFLT01- a gene therapy for age-related macular degeneration.

    PubMed

    Maclachlan, Timothy K; Lukason, Michael; Collins, Margaret; Munger, Robert; Isenberger, Elisabete; Rogers, Cindy; Malatos, Shana; Dufresne, Elizabeth; Morris, James; Calcedo, Roberto; Veres, Gabor; Scaria, Abraham; Andrews, Laura; Wadsworth, Samuel

    2011-02-01

    AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.4 × 10(10) vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ~1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression.

  9. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

    PubMed Central

    Ge, Xianglian; Xi, Haitao; Yang, Fayu; Zhi, Xiao; Fu, Yanghua; Chen, Ding; Xu, Ren-He; Lin, Ge; Qu, Jia; Zhao, Junzhao; Gu, Feng

    2016-01-01

    Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome. PMID:27898094

  10. 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

  11. 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.

  12. 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

  13. Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B.

    PubMed

    Mingozzi, Federico; Chen, Yifeng; Murphy, Samuel L; Edmonson, Shyrie C; Tai, Alex; Price, Sandra D; Metzger, Mark E; Zhou, Shangzhen; Wright, J Fraser; Donahue, Robert E; Dunbar, Cynthia E; High, Katherine A

    2012-07-01

    Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

  14. Therapeutic Efficacy of Bone Marrow Transplant, Intracranial AAV-mediated Gene Therapy, or Both in the Mouse Model of MPS IIIB

    PubMed Central

    Heldermon, Coy D; Ohlemiller, Kevin K; Herzog, Erik D; Vogler, Carole; Qin, Elizabeth; Wozniak, David F; Tan, Yun; Orrock, John L; Sands, Mark S

    2010-01-01

    Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. In an attempt to correct the disease in the murine model of MPS IIIB, neonatal mice were treated with intracranial AAV2/5-NAGLU (AAV), syngeneic bone marrow transplant (BMT), or both (AAV/BMT). All treatments resulted in some improvement in clinical phenotype. Adeno-associated viral (AAV) treatment resulted in improvements in lifespan, motor function, hearing, time to activity onset, and daytime activity level, but no reduction of lysosomal storage. BMT resulted in improved hearing by 9 months, and improved circadian measures, but had no effect on lifespan, motor function, or central nervous system (CNS) lysosomal storage. AAV/BMT treatment resulted in improvements in hearing, time to activity onset, motor function, and reduced CNS lysosomal storage, but had no effect on lifespan. Combination therapy compared to either therapy alone resulted in synergistic effects on hearing and CNS lysosomal inclusions but antagonistic effects on motor function and lifespan. AAV alone is more efficacious than BMT or AAV/BMT treatment for lifespan. BMT was the least efficacious treatment by all measures. CNS-directed AAV treatment alone appears to be the preferred treatment, combining the most efficacy with the least toxicity of the approaches assessed. PMID:20179679

  15. Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

    PubMed Central

    Monahan, Paul E; Lothrop, Clinton D; Sun, Junjiang; Hirsch, Matthew L; Kafri, Tal; Kantor, Boris; Sarkar, Rita; Tillson, D Michael; Elia, Joseph R; Samulski, R Jude

    2010-01-01

    Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration–approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 1013 vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy. PMID:20700109

  16. Transduction efficiency of neurons and glial cells by AAV-1, -5, -9, -rh10 and -hu11 serotypes in rat spinal cord following contusion injury.

    PubMed

    Petrosyan, H A; Alessi, V; Singh, V; Hunanyan, A S; Levine, J M; Arvanian, V L

    2014-12-01

    Adeno-associated viruses (AAVs) are a promising system for therapeutic gene delivery to neurons in a number of neurodegenerative conditions including spinal cord injuries (SCIs). Considering the role of macrophages and glia in the progression of 'secondary damage', we searched for the optimal vectors for gene transfer to both neurons and glia following contusion SCI in adult rats. Contusion models share many similarities to most human spinal cord traumas. Several AAV serotypes known for their neuronal tropism expressing enhanced green-fluorescent protein (GFP) were injected intraspinally following thoracic T10 contusion. We systematically compared the transduction efficacy and cellular tropism of these vectors for neurons, macrophages/microglia, oligodendrocytes, astrocytes and NG2-positive glial cells following contusion SCI. No additional changes in inflammatory responses or behavioral performance were observed for any of the vectors. We identified that AAV-rh10 induced robust transduction of both neuronal and glial cells. Even though efficacy to transduce neurons was comparable to already established AAV-1, AAV-5 and AAV-9, AAV-rh10 transduced significantly higher number of macrophages/microglia and oligodendrocytes in damaged spinal cord compared with other serotypes tested. Thus, AAV-rh10 carries promising potential as a gene therapy vector, particularly if both the neuronal and glial cell populations in damaged spinal cord are targeted.

  17. Biodistribution of rAAV vectors following intraocular administration: evidence for the presence and persistence of vector DNA in the optic nerve and in the brain.

    PubMed

    Provost, Nathalie; Le Meur, Guylène; Weber, Michel; Mendes-Madeira, Alexandra; Podevin, Guillaume; Cherel, Yan; Colle, Marie-Anne; Deschamps, Jack-Yves; Moullier, Philippe; Rolling, Fabienne

    2005-02-01

    The purpose of our study was to evaluate the biodistribution of rAAV vectors following subretinal or intravitreal injection. In rats, we performed subretinal or intravitreal injections of rAAV-2/2.CMV.gfp. In large animals, rAAV-2/4.CMV.gfp or rAAV-2/5.CMV.gfp was delivered into the subretinal space while rAAV-2/2.CMV.gfp was delivered either to the subretinal space or to the vitreous. In euthanized animals, we undertook a complete necropsy. In animals maintained alive, we collected blood and tissue samples from the submandibular lymph node, liver, and gonads. We analyzed total DNA, extracted from various tissue samples and peripheral blood mononuclear cells (PBMC), by PCR. Following subretinal or intravitreal injections in rats and in large animals, vector sequences were not detected in the liver or in the gonads but were occasionally found in PBMC. An unexpected result was the detection of rAAV sequences in the optic nerve following subretinal injection. The most striking finding was the detection of vector sequences in the brain, along the visual pathway, in rAAV-2/2 intravitreally injected dogs. These findings raise safety concerns regarding intraocular administration of rAAV vectors and will have an impact on the development of future gene therapy trials for retinal diseases.

  18. Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.

    PubMed

    Luo, Yongquan; Rao, Mahendra; Zou, Jizhong

    2014-05-16

    Generation of a fluorescent GFP reporter line in human induced pluripotent stem cells (hiPSCs) provides enormous potentials in both basic stem cell research and regenerative medicine. A protocol for efficiently generating such an engineered reporter line by gene targeting is highly desired. Transcription activator-like effector nucleases (TALENs) are a new class of artificial restriction enzymes that have been shown to significantly promote homologous recombination by >1000-fold. The AAVS1 (adeno-associated virus integration site 1) locus is a "safe harbor" and has an open chromatin structure that allows insertion and stable expression of transgene. Here, we describe a step-by-step protocol from determination of TALENs activity, hiPSC culture, and delivery of a donor into AAVS1 targeting site, to validation of targeted integration by PCR and Southern blot analysis using hiPSC line, and a pair of open-source AAVS1 TALENs.

  19. AAV-Mediated angiotensin 1-7 overexpression inhibits tumor growth of lung cancer in vitro and in vivo

    PubMed Central

    Mao, Yingying; Wang, Shengyao; Yan, Renhe; Bai, Na; Li, Andrew; Zhang, Yanling; Du, Hongyan; Chen, Baihong; Sumners, Colin; Li, Jinlong; Li, Hongwei

    2017-01-01

    Ang-(1-7) inhibits lung cancer cell growth both in vitro and in vivo. However, the molecular mechanism of action is unclear and also the rapid degradation of Ang-(1-7) in vivo limits its clinical application. Here, we have demonstrated that Ang- (1-7) inhibits lung cancer cell growth by interrupting pre-replicative complex assembly and restrains epithelial-mesenchymal transition via Cdc6 inhibition. Furthermore, we constructed a mutant adeno-associated viral vector AAV8 (Y733F) that produced stable and high efficient Ang-(1-7) expression in a xenograft tumor model. The results show that AAV8-mediated Ang-(1-7) over-expression can remarkably suppress tumor growth in vivo by down-regulating Cdc6 and anti-angiogenesis. Ang-(1-7) over-expression via the AAV8 method may be a promising strategy for lung cancer treatment. PMID:27861149

  20. 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

  1. Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome

    PubMed Central

    Carvalho, Claudia M. B.; Ramocki, Melissa B.; Pehlivan, Davut; Franco, Luis M.; Gonzaga-Jauregui, Claudia; Fang, Ping; McCall, Alanna; Pivnick, Eniko Karman; Hines-Dowell, Stacy; Seaver, Laurie; Friehling, Linda; Lee, Sansan; Smith, Rosemarie; del Gaudio, Daniela; Withers, Marjorie; Liu, Pengfei; Cheung, Sau Wai; Belmont, John W.; Zoghbi, Huda Y.; Hastings, P. J.; Lupski, James R.

    2011-01-01

    We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at both the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 12 unrelated subjects. Interestingly, only two novel breakpoint junctions were generated during each rearrangement formation. Remarkably, all the complex rearrangement products share the common genomic organization duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) wherein the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by over 300 kb; a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology driven, via inverted repeats, and microhomologous/nonhomologous events. PMID:21964572

  2. 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

  3. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

    PubMed

    Gardner, Matthew R; Kattenhorn, Lisa M; Kondur, Hema R; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J; Haworth, Kevin G; Decker, Julie M; Alpert, Michael D; Bailey, Charles C; Neale, Ernest S; Fellinger, Christoph H; Joshi, Vinita R; Fuchs, Sebastian P; Martinez-Navio, Jose M; Quinlan, Brian D; Yao, Annie Y; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C; Burton, Dennis R; Kwong, Peter D; Piatak, Michael; Lifson, Jeffrey D; Gao, Guangping; Desrosiers, Ronald C; Evans, David T; Hahn, Beatrice H; Ploss, Alexander; Cannon, Paula M; Seaman, Michael S; Farzan, Michael

    2015-03-05

    Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

  4. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges

    PubMed Central

    Gardner, Matthew R.; Kattenhorn, Lisa M.; Kondur, Hema R.; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J.; Haworth, Kevin G.; Decker, Julie M.; Alpert, Michael D.; Bailey, Charles C.; Neale, Ernest S.; Fellinger, Christoph H.; Joshi, Vinita R.; Fuchs, Sebastian P.; Martinez-Navio, Jose M.; Quinlan, Brian D.; Yao, Annie Y.; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C.; Burton, Dennis R.; Kwong, Peter D.; Piatak, Michael; Lifson, Jeffrey D.; Gao, Guangping; Desrosiers, Ronald C.; Evans, David T.; Hahn, Beatrice H.; Ploss, Alexander; Cannon, Paula M.; Seaman, Michael S.; Farzan, Michael

    2015-01-01

    Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

  5. Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.

    PubMed

    Vulin, Adeline; Barthélémy, Inès; Goyenvalle, Aurélie; Thibaud, Jean-Laurent; Beley, Cyriaque; Griffith, Graziella; Benchaouir, Rachid; le Hir, Maëva; Unterfinger, Yves; Lorain, Stéphanie; Dreyfus, Patrick; Voit, Thomas; Carlier, Pierre; Blot, Stéphane; Garcia, Luis

    2012-11-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

  6. Nipple Retractor to Correct Inverted Nipples.

    PubMed

    Long, Xiao; Zhao, Ru

    2011-12-01

    BACKGROUND: Inverted nipples are a common problem and a challenging clinical condition to repair. Multiple methods have been reported to correct inverted nipples, most of which will destroy breastfeeding function. PATIENTS AND METHODS: We have designed a simple nipple retractor to correct inverted nipples. A total of 53 patients with 95 inverted nipples underwent an operation in which the nipples were retracted into a normal position and fixated with the nipple retractor and wires under local anesthesia. Nipple retractors were to be worn for 6 months. Postoperatively, the patients were invited to follow-up on the 1st day, the 7th day, after 1 month, 3 months and 6 months, and yearly thereafter. Wire adjustments were performed as needed. Mean follow-up was 11.9 months (range 8-18 months). RESULTS: Improvement occurred in all patients and was sustained in all cases throughout the follow-up period. The total complication rate was 5.26% (5/95). The main complications included depigmentation (2.11%, 2/95), areolar ulcer (2.11%, 2/95), and wire dislocation (1.05%, 1/95). CONCLUSION: The nipple retractor is a simple tool with which severely inverted nipples can be successfully corrected with a low complication rate. Close follow-up and careful postoperative care are important to avoid complications.

  7. Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

    PubMed Central

    Brewer, Bonita J.; Payen, Celia; Di Rienzi, Sara C.; Higgins, Megan M.; Ong, Giang; Dunham, Maitreya J.; Raghuraman, M. K.

    2015-01-01

    DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of

  8. 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

  9. Long-Term Correction of Sandhoff Disease Following Intravenous Delivery of rAAV9 to Mouse Neonates

    PubMed Central

    Walia, Jagdeep S; Altaleb, Naderah; Bello, Alexander; Kruck, Christa; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Chowdhury, Biswajit; Hurlbut, David; Hemming, Richard; Kobinger, Gary P; Triggs-Raine, Barbara

    2015-01-01

    GM2 gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb−/−) of the GM2 gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or –LacZ and monitored for serum β-hexosaminidase activity, motor function, and survival. Brain GM2 ganglioside, β-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB–treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB–treated mice had a significant increase in brain β-hexosaminidase activity, and a reduction in GM2 ganglioside storage and neuroinflammation compared to adult SD-HexB– and SD-LacZ–treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other GM2 gangliosidoses through early rAAV9 based systemic gene therapy. PMID:25515709

  10. Long-term follow-up of a randomized AAV2-GAD gene therapy trial for Parkinson’s disease

    PubMed Central

    Niethammer, Martin; Tang, Chris C.; LeWitt, Peter A.; Rezai, Ali R.; Leehey, Maureen A.; Ojemann, Steven G.; Eskandar, Emad N.; Kostyk, Sandra K.; Sarkar, Atom; Siddiqui, Mustafa S.; Schwalb, Jason M.; Poston, Kathleen L.; Kurlan, Roger M.; Richard, Irene H.; Sapan, Christine V.; Eidelberg, David; During, Matthew J.; Kaplitt, Michael G.

    2017-01-01

    BACKGROUND. We report the 12-month clinical and imaging data on the effects of bilateral delivery of the glutamic acid decarboxylase gene into the subthalamic nuclei (STN) of advanced Parkinson’s disease (PD) patients. METHODS. 45 PD patients were enrolled in a 6-month double-blind randomized trial of bilateral AAV2-GAD delivery into the STN compared with sham surgery and were followed for 12 months in open-label fashion. Subjects were assessed with clinical outcome measures and 18F-fluorodeoxyglucose (FDG) PET imaging. RESULTS. Improvements under the blind in Unified Parkinson’s Disease Rating Scale (UPDRS) motor scores in the AAV2-GAD group compared with the sham group continued at 12 months [time effect: F(4,138) = 11.55, P < 0.001; group effect: F(1,35) = 5.45, P < 0.03; repeated-measures ANOVA (RMANOVA)]. Daily duration of levodopa-induced dyskinesias significantly declined at 12 months in the AAV2-GAD group (P = 0.03; post-hoc Bonferroni test), while the sham group was unchanged. Analysis of all FDG PET images over 12 months revealed significant metabolic declines (P < 0.001; statistical parametric mapping RMANOVA) in the thalamus, striatum, and prefrontal, anterior cingulate, and orbitofrontal cortices in the AAV2-GAD group compared with the sham group. Across all time points, changes in regional metabolism differed for the two groups in all areas, with significant declines only in the AAV2-GAD group (P < 0.005; post-hoc Bonferroni tests). Furthermore, baseline metabolism in the prefrontal cortex (PFC) correlated with changes in motor UPDRS scores; the higher the baseline PFC metabolism, the better the clinical outcome. CONCLUSION. These findings show that clinical benefits after gene therapy with STN AAV2-GAD in PD patients persist at 12 months. TRIAL REGISTRATION. ClinicalTrials.gov NCT00643890. FUNDING. Neurologix Inc.

  11. 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

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

    PubMed

    Chen, Peter Gin-Fu; Sun, Zhongjie

    2017-02-01

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

  13. Addressable inverter matrix for process and device characterization

    NASA Technical Reports Server (NTRS)

    Buehler, M. G.; Sayah, H. R.

    1985-01-01

    The addressable inverter matrix consists of 222 inverters each accessible with the aid of a shift register. The structure has proven useful in characterizing the variability of inverter transfer curves and in diagnosing processing faults. For good 3-micron CMOS bulk inverters investigated in this study, the percent standard deviation of the inverter threshold voltage was less than one percent and the inverter gain (the slope of the inverter transfer curve at the inverter threshold voltage) was less than 3 percent. The average noise margin for the inverters was near 2 volts for a power supply voltage of 5 volts. The specific faults studied included undersize pull-down transistor widths and various open contacts in the matrix.

  14. Addressable inverter matrix for process and device characterization

    NASA Technical Reports Server (NTRS)

    Buehler, M. G.; Sayah, H. R.

    1985-01-01

    The addressable inverter matrix consists of 222 inverters each accessible with the aid of a shift register. The structure has proven useful in characterizing the variability of inverter transfer curves and in diagnosing processing faults. For good 3-micron CMOS bulk inverters investigated, the percent standard deviation of the inverter threshold voltage was less than one percent and the inverter gain (the slope of the inverter transfer curve at the inverter threshold vltage) was less than 3 percent. The average noise margin for the inverters was near 2 volts for a power supply voltage of 5 volts. The specific faults studied included undersize pull-down transistor widths and various open contacts in the matrix.

  15. Engineering and evolution of synthetic adeno-associated virus (AAV) gene therapy vectors via DNA family shuffling.

    PubMed

    Kienle, Eike; Senís, Elena; Börner, Kathleen; Niopek, Dominik; Wiedtke, Ellen; Grosse, Stefanie; Grimm, Dirk

    2012-04-02

    Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties(1). These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors(2). A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol(1,2). Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling(3), i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically >80% for most AAV serotypes), or peptide display(4,5), i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Patterned semiconductor inverted quantum dot photonic devices

    NASA Astrophysics Data System (ADS)

    Coleman, J. J.

    2016-03-01

    A novel inverted quantum dot structure is presented, which consists of an InGaAs quantum well that has been periodically perforated and then filled with the higher bandgap GaAs barrier material. This structure exhibits a unique quantized energy structure something like a planar atomic bond structure and formation of allowed and forbidden energy bands instead of highly localized, fully discrete states. We describe the growth, processing and characteristics of inverted quantum dot structures and outline interesting and potentially important effects arising from the introduction of nanoscale features (<50 nm) in the active medium.

  1. Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

    PubMed Central

    Schober, Alexandra L.; Gagarkin, Dmitriy A.; Chen, Ying; Gao, Guangping; Jacobson, Lauren; Mongin, Alexander A.

    2016-01-01

    Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for neuronal cells. Glial cells, specifically astrocytes, appear to be infected at low rates. To overcome this limitation, previous studies utilized rAAVs with astrocyte-specific promoters or assorted rAAV serotypes and pseudotypes with purported selectivity for astrocytes. Yet, the reported glial infection rates are not consistent from study to study. In the present work, we tested seven commercially available recombinant serotypes– rAAV1, 2, and 5 through 9, for their ability to transduce primary rat astrocytes [visualized via viral expression of green fluorescent protein (GFP)]. In cell cultures, rAAV6 consistently demonstrated the highest infection rates, while rAAV2 showed astrocytic transduction in some, but not all, of the tested viral batches. To verify that all rAAV constructs utilized by us were viable and effective, we confirmed high infectivity rates in retinal pigmented epithelial cells (ARPE-19), which are known to be transduced by numerous rAAV serotypes. Based on the in vitro results, we next tested the cell type tropism of rAAV6 and rAAV2 in vivo, which were both injected in the barrel cortex at approximately equal doses. Three weeks later, the brains were sectioned and immunostained for viral GFP and the neuronal marker NeuN or the astrocytic marker GFAP. We found that rAAV6 strongly and preferentially transduced astrocytes (>90% of cells in the virus-infected areas), but not neurons (∼10% infection rate). On the contrary, rAAV2 preferentially infected neurons (∼65%), but not astrocytes (∼20%). Overall, our results suggest that rAAV6 can be used as a tool for manipulating gene expression (either delivery or knockdown) in rat astrocytes in vivo. PMID

  2. 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.

  3. Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

    PubMed

    Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter; Zhu, Yanqing; Yu, Hongwei; Wang, Ping; Bagel, Jessica; Vite, Charles H; Sikora, Tracey; Hinderer, Christian; Calcedo, Roberto; Yox, Alexander D; Steet, Richard A; Ruane, Therese; O'Donnell, Patricia; Gao, Guangping; Wilson, James M; Casal, Margret; Ponder, Katherine P; Haskins, Mark E

    2016-02-01

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

  4. Intravenous Administration of Self-complementary AAV9 Enables Transgene Delivery to Adult Motor Neurons

    PubMed Central

    Duque, Sandra; Joussemet, Béatrice; Riviere, Christel; Marais, Thibaut; Dubreil, Laurence; Douar, Anne-Marie; Fyfe, John; Moullier, Philippe; Colle, Marie-Anne; Barkats, Martine

    2009-01-01

    Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood–brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients. PMID:19367261

  5. AAV1.NT-3 gene therapy attenuates spontaneous autoimmune peripheral polyneuropathy.

    PubMed

    Yalvac, M E; Arnold, W D; Braganza, C; Chen, L; Mendell, J R; Sahenk, Z

    2016-01-01

    The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout non-obese diabetic mice shares clinical and histological features with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Secondary axonal loss is prominent in the progressive phase of this neuropathy. Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulates neurite outgrowth and myelination. The anti-inflammatory and immunomodulatory effects of NT-3 raised considerations of potential efficacy in the SAPP model that could be applicable to CIDP. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of 25-week-old SAPP mice. Measurable NT-3 levels were found in the serum at 7-week postgene delivery. The outcome measures included functional, electrophysiological and histological assessments. At week 32, NT-3-treated mice showed increased hind limb grip strength that correlated with improved compound muscle action potential amplitude. Myelinated fiber density was 1.9 times higher in the NT-3-treated group compared with controls and the number of demyelinated axons was significantly lower. The remyelinated nerve fiber population was significantly increased. These improved histopathological parameters from scAAV1.tMCK.NT-3 treatment occurred in the setting of reduced sciatic nerve inflammation. Collectively, these findings suggest a translational application to CIDP.

  6. Tropism-modified AAV vectors overcome barriers to successful cutaneous therapy.

    PubMed

    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

    2014-05-01

    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.

  7. Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

    PubMed

    Mack, David L; Poulard, Karine; Goddard, Melissa A; Latournerie, Virginie; Snyder, Jessica M; Grange, Robert W; Elverman, Matthew R; Denard, Jérôme; Veron, Philippe; Buscara, Laurine; Le Bec, Christine; Hogrel, Jean-Yves; Brezovec, Annie G; Meng, Hui; Yang, Lin; Liu, Fujun; O'Callaghan, Michael; Gopal, Nikhil; Kelly, Valerie E; Smith, Barbara K; Strande, Jennifer L; Mavilio, Fulvio; Beggs, Alan H; Mingozzi, Federico; Lawlor, Michael W; Buj-Bello, Ana; Childers, Martin K

    2017-04-05

    X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

  8. Major role of local immune responses in antibody formation to factor IX in AAV gene transfer.

    PubMed

    Wang, L; Cao, O; Swalm, B; Dobrzynski, E; Mingozzi, F; Herzog, R W

    2005-10-01

    The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.

  9. 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.

  10. Aging, Terminal Decline, and Terminal Drop

    ERIC Educational Resources Information Center

    Palmore, Erdman; Cleveland, William

    1976-01-01

    Data from a 20-year longitudinal study of persons over 60 were analyzed by step-wise multiple regression to test for declines in function with age, for terminal decline (linear relationship to time before death), and for terminal drop (curvilinear relationship to time before death). There were no substantial terminal drop effects. (Author)

  11. Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

    PubMed

    Satkunanathan, Stifani; Wheeler, Jun; Thorpe, Robin; Zhao, Yuan

    2014-11-01

    Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat

  12. Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

    PubMed Central

    Satkunanathan, Stifani; Wheeler, Jun; Thorpe, Robin

    2014-01-01

    Abstract Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal

  13. Safety and Biodistribution Evaluation in Cynomolgus Macaques 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; Sharma, Alok K; Ver Hoeve, James N; Smith, Leia M; Arndt, Tara; Calcedo, Roberto; Gaskin, Chantelle; Robinson, Paulette M; 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 viral (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 the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10(11) or 4 × 10(12) vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. 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-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

  14. Safety and Biodistribution Evaluation in Cynomolgus Macaques 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; Sharma, Alok K; Ver Hoeve, James N; Smith, Leia; Arndt, Tara; Calcedo, Roberto; Gaskin, Chantelle; Robinson, Paulette; Knop, David R; Hauswirth, William W; Chulay, Jeffrey David

    2016-03-08

    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 the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n=2 males and 2 females per group) received a subretinal injection in one eye of 300 µL containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vg/mL) and were evaluated over a 3 month period prior to being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on Study Day 5 based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. 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-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

  15. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia

    PubMed Central

    Ye, Guo-jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T. Michael; Miller, Paul E.; Sharma, Alok K.; Ver Hoeve, James N.; Smith, Leia M.; Arndt, Tara; Calcedo, Roberto; Gaskin, Chantelle; Robinson, Paulette M.; Knop, David R.; Hauswirth, William W.; Chulay, Jeffrey D.

    2016-01-01

    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (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 the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. 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-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. PMID:27003753

  16. DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer

    PubMed Central

    Gurung, Neesha

    2016-01-01

    DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3′ of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified. PMID:28348864

  17. A delta configured auxiliary resonant snubber inverter

    SciTech Connect

    Lai, J.S.; Young, R.W.; Ott, G.W. Jr.; McKeever, J.W.; Peng, F.Z. |

    1995-09-01

    A delta ({Delta}) configured auxiliary resonant snubber inverter is developed to overcome the voltage floating problem in a wye (Y) configured resonant snubber inverter. The proposed inverter is to connect auxiliary resonant branches between phase outputs to avoid a floating point voltage which may cause over-voltage failure of the auxiliary switches. Each auxiliary branch consists of a resonant inductor and a reverse blocking auxiliary switch. Instead of using an anti-paralleled diode to allow resonant current to flow in the reverse direction, as in the Y-configured version, the resonant branch in the {Delta}-configured version must block the negative voltage, typically done by a series diode. This paper shows single-phase and three-phase versions of {Delta}-configured resonant snubber inverters and describes in detail the operating principle of a single-phase version. The extended three-phase version is proposed with non-adjacent state space vector modulation. For hardware implementation, a single-phase 1-kW unit and a three-phase 100-kW unit were built to prove the concept. Experimental results show the superiority of the proposed topology.

  18. Inverter Matrix for the Clementine Mission

    NASA Technical Reports Server (NTRS)

    Buehler, M. G.; Blaes, B. R.; Tardio, G.; Soli, G. A.

    1994-01-01

    An inverter matrix test circuit was designed for the Clementine space mission and is built into the RRELAX (Radiation and Reliability Assurance Experiment). The objective is to develop a circuit that will allow the evaluation of the CMOS FETs using a lean data set in the noisy spacecraft environment.

  19. Two-Stage Series-Resonant Inverter

    NASA Technical Reports Server (NTRS)

    Stuart, Thomas A.

    1994-01-01

    Two-stage inverter includes variable-frequency, voltage-regulating first stage and fixed-frequency second stage. Lightweight circuit provides regulated power and is invulnerable to output short circuits. Does not require large capacitor across ac bus, like parallel resonant designs. Particularly suitable for use in ac-power-distribution system of aircraft.

  20. "In Situ" Generation of Compressed Inverted Files.

    ERIC Educational Resources Information Center

    Moffat, Alistair; Bell, Timothy A. H.

    1995-01-01

    Discussion of index construction for large text collections highlights a new indexing algorithm designed to create large compressed inverted indexes "in situ." Topics include a computational model, inversion, index compression, merging, experimental test results, effect on retrieval performance, memory restrictions, and dynamic…

  1. 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.

  2. [Inverted papiloma and its rare forms].

    PubMed

    Bugová, G; Jeseňák, M; Wallenfels, P; Ondrušová, B; Hajtman, A

    2014-01-01

    Authors address the issue of a frequent benign tumour of the nasal cavity and paranasal sinuses -  inverted papilloma. They analyse the available diagnostic methods and treatment options. On the background of selected case reports of a rare malignant transformation they emphasize the need for longterm dispensarization as a part of management plan for patients with this oncological disease.

  3. 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.

  4. Inverted flat plate solar collector. Final report

    SciTech Connect

    Brown, M.A.

    1981-08-26

    Construction and testing of an inverted flat plate solar collector are described. Heat transfer and economic analysis were performed to optimize the collector design. The newly designed collector was tested against two other flat plate collectors and the results and comparison of efficiencies are presented. (BCS)

  5. 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.

  6. Inverting the Transition-to-Proof Classroom

    ERIC Educational Resources Information Center

    Talbert, Robert

    2015-01-01

    In this paper, we examine the benefits of employing an inverted or "flipped" class design in a Transition-to-Proof course for second-year mathematics majors. The issues concomitant with such courses, particularly student acquisition of "sociomathematical norms" and self-regulated learning strategies, are discussed along with…

  7. Expression of human alpha1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs.

    PubMed

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

    2010-06-01

    We evaluated the potential of lung-directed gene therapy for alpha1-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.

  8. 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

  9. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex-vivo

    PubMed Central

    Singh, Mandeep S.; Lipinski, Daniel M.; Barnea-Cramer, Alona O.; Walker, Nathan J.; Barnard, Alun R.; Hankins, Mark W.; MacLaren, Robert E.

    2016-01-01

    Gene therapy using adeno-associated viral vectors (AAV) for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4-/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4-/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex-vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treating retinal degenerations in which high levels of transgene expression are required. PMID:27416076

  10. 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.

  11. 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

  12. Operating temperatures of open-rack installed photovoltaic inverters

    SciTech Connect

    Zhang, Z.; Wang, L.; Kurtz, S.; Wu, J.; Quan, P.; Sorensen, R.; Liu, S.; Bai, J. B.; Zhu, Z. W.

    2016-11-01

    This paper presents a model for evaluating the heat-sink and component temperatures of open-rack installed photovoltaic inverters. These temperatures can be used for predicting inverter reliability. Inverter heat-sink temperatures were measured for inverters connected to three grid-connected PV (photovoltaic) test systems in Golden, Colorado, US. A model is proposed for calculating the inverter heat-sink temperature based on the ambient temperature, the ratio of the consumed power to the rated power of the inverter, and the measured wind speed. To verify and study this model, more than one year of inverter DC/AC power, irradiance, wind speed, and heat sink temperature rise data were collected and analyzed. The model is shown to be accurate in predicting average inverter temperatures, but will require further refinement for prediction of transient temperatures.

  13. Long-term correction of glycogen storage disease type II with a hybrid Ad-AAV vector.

    PubMed

    Sun, Bao-dong; Chen, Y-T; Bird, Andrew; Amalfitano, Andrea; Koeberl, Dwight D

    2003-02-01

    We administered an adenovirus-adeno-associated virus (Ad-AAV) vector encoding human acid alpha-glucosidase (hGAA) to acid alpha-glucosidase-knockout (GAA-KO) mice on day 3 of life by gastrocnemius injection. In contrast to previous results for muscle-targeted Ad vector in adult GAA-KO mice, the muscles of the hindlimb showed reduced glycogen content and persistent hGAA for as long as 6 months after neonatal Ad-AAV vector administration. Not only the injected gastrocnemius muscles, but also the hamstrings and quadriceps muscles produced therapeutic levels of hGAA as a result of widespread transduction with the Ad-AAV vector; moreover, hGAA activity was 50-fold elevated as compared to normal mice. Vector RNA was detected in the hindlimb muscles, the hearts, and the livers by northern blot analysis and/or by RT-PCR for as long as 6 months. The low levels of hGAA detected in the heart were attributable to transduction with the Ad-AAV vector, not to secretion of hGAA by the injected muscle and uptake by the heart. Finally, although an antibody response to hGAA was present, it did not prevent the correction of glycogen storage in the skeletal muscle of GAA-KO mice.

  14. AAV8-mediated Sirt1 gene transfer to the liver prevents high carbohydrate diet-induced nonalcoholic fatty liver disease

    PubMed Central

    Vilà, Laia; Elias, Ivet; Roca, Carles; Ribera, Albert; Ferré, Tura; Casellas, Alba; Lage, Ricardo; Franckhauser, Sylvie; Bosch, Fatima

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, and evidence suggests that it promotes insulin resistance and type 2 diabetes. Caloric restriction (CR) is the only available strategy for NAFLD treatment. The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD. Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet. Long-term hepatic SIRT1 overexpression led to upregulation of key hepatic genes involved in β-oxidation, prevented HC diet-induced lipid accumulation and reduced liver inflammation. AAV8-Sirt1–treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation. Moreover, HC feeding induced leptin resistance, which was also attenuated in AAV8-Sirt1–treated mice. Therefore, AAV-mediated gene transfer to overexpress SIRT1 specifically in the liver may represent a new gene therapy strategy to counteract NAFLD and related diseases such as type 2 diabetes. PMID:26015978

  15. Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

    PubMed Central

    Gregorevic, Paul; Schultz, Brian R; Allen, James M; Halldorson, Jeffrey B; Blankinship, Michael J; Meznarich, Norman A; Kuhr, Christian S; Doremus, Caitlin; Finn, Eric; Liggitt, Denny; Chamberlain, Jeffrey S

    2009-01-01

    A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans. PMID:19471246

  16. Improved Intravitreal AAV-Mediated Inner Retinal Gene Transduction after Surgical Internal Limiting Membrane Peeling in Cynomolgus Monkeys.

    PubMed

    Takahashi, Kazuhisa; Igarashi, Tsutomu; Miyake, Koichi; Kobayashi, Maika; Yaguchi, Chiemi; Iijima, Osamu; Yamazaki, Yoshiyuki; Katakai, Yuko; Miyake, Noriko; Kameya, Shuhei; Shimada, Takashi; Takahashi, Hiroshi; Okada, Takashi

    2017-01-04

    The retina is an ideal target for gene therapy because of its easy accessibility and limited immunological response. We previously reported that intravitreally injected adeno-associated virus (AAV) vector transduced the inner retina with high efficiency in a rodent model. In large animals, however, the efficiency of retinal transduction was low, because the vitreous and internal limiting membrane (ILM) acted as barriers to transduction. To overcome these barriers in cynomolgus monkeys, we performed vitrectomy (VIT) and ILM peeling before AAV vector injection. Following intravitreal injection of 50 μL triple-mutated self-complementary AAV serotype 2 vector encoding EGFP, transduction efficiency was analyzed. Little expression of GFP was detected in the control and VIT groups, but in the VIT+ILM group, strong GFP expression was detected within the peeled ILM area. To detect potential adverse effects, we monitored the retinas using color fundus photography, optical coherence tomography, and electroretinography. No serious side effects associated with the pretreatment were observed. These results indicate that surgical ILM peeling before AAV vector administration would be safe and useful for efficient transduction of the nonhuman primate retina and provide therapeutic benefits for the treatment of retinal diseases.

  17. Tyrosine-mutated AAV2-mediated shRNA silencing of PTEN promotes axon regeneration of adult optic nerve

    PubMed Central

    Huang, ZhengRu; Hu, ZiZhong; Xie, Ping; Liu, QingHuai

    2017-01-01

    Activating PI3K/AKT/mTOR signaling pathway via deleting phosphatase and tensin homolog (PTEN) has been confirmed to enhance intrinsic growth capacity of neurons to facilitate the axons regeneration of central nervous system after injury. Considering conditional gene deletion is currently not available in clinical practice, we exploited capsid residue tyrosine 444 to phenylalanine mutated single-stranded adeno-associated virus serotype 2 (AAV2) as a vector delivering short hairpin RNA to silence PTEN to promote retinal ganglion cells (RGCs) survival and axons regeneration in adult rat optic nerve axotomy paradigm. We found that mutant AAV2 displayed higher infection efficiency to RGCs and Müller cells by intravitreal injection, mediated PTEN suppression, resulted in much more RGCs survival and more robust axons regeneration compared with wild type AAV2, due to the different extent of the mTOR complex-1 activation and glutamate aspartate transporter (GLAST) regulation. These results suggest that high efficiency AAV2-mediated PTEN knockdown represents a practicable therapeutic strategy for optic neuropathy. PMID:28323869

  18. Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver.

    PubMed

    Lu, Hui; Qu, Guang; Yang, Xiao; Xu, Ruian; Xiao, Weidong

    2011-05-01

    The capsid protein synthesis in targeted tissues resulting from residual contaminating replication-competent adeno-associated virus particles (rcAAV) remains a concern for hazardous immune responses that shut down the factor IX expression in the hemophilia B clinical trial. To systematically reduce/eliminate the effects of potential contaminating rcAAV particles, we designed a novel adeno-associated virus (AAV) helper (pH22mir) with a microRNA binding cassette containing multiple copies of liver-specific (hsa-mir-122) and hematopoietic-specific (has-mir-142-3p) sequences to specifically control cap gene expression. In 293 cells, the rep and cap gene from pH22mir functioned similarly to that of conventional helper pH22. The vector yields and compositions from pH22mir and pH22 were indistinguishable. The performance of vector produced in this new system was comparable to that of similar vectors produced by conventional methods. In the human hepatic cell line, the capsid expression was reduced significantly from cap-mir cassette driven by a cytomegalovirus promoter. In the liver, 99.9% of capsid expression could be suppressed and no cap expression could be detected by western blot. In summary, we demonstrated a new concept in reducing de novo capsid synthesis in the targeted tissue. This strategy may not only help AAV vectors in controlling undesirable capsid gene expression, but can also be adopted for lentiviral or adenoviral vector production.

  19. Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice.

    PubMed

    Heldermon, C D; Qin, E Y; Ohlemiller, K K; Herzog, E D; Brown, J R; Vogler, C; Hou, W; Orrock, J L; Crawford, B E; Sands, M S

    2013-09-01

    Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

  20. 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

  1. AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice.

    PubMed

    Hu, C; Lipshutz, G S

    2012-12-01

    Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors against the factor VIII (FVIII) protein; these 'inhibitors' more commonly affect 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. A serotype rh10 adeno-associated virus (AAV) was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant adeno-associated viral vector delivery. Virions of each FVIII chain were co-injected intravenously into mice on the second day of life. Mice express sustained levels of FVIII antigen ≥5% up to 22 months of life without development of antibodies against 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 against FVIII in this disease model where AAV is administered shortly after birth. These studies support the consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.

  2. Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy

    PubMed Central

    Cahoon, Judd M.; Rai, Ruju R.; Carroll, Lara S.; Uehara, Hironori; Zhang, Xiaohui; Medina, Reinhold J.; Das, Subtrata K.; Muddana, Santosh K.; Olson, Paul R.; Nielson, Spencer; Walker, Kortnie; Flood, Maggie M.; Messenger, Wyatt B.; Archer, Bonnie J.; Barabas, Peter; Krizaj, David; Gibson, Christopher C.; Li, Dean Y.; Koh, Gou Y.; Gao, Guangping; Stitt, Alan W.

    2015-01-01

    Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR. PMID:26340930

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

    PubMed

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

    2015-04-20

    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.

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

    PubMed

    Dinculescu, Astra; Stupay, Rachel M; 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.

  5. 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

  6. Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver.

    PubMed

    Mingozzi, Federico; Hasbrouck, Nicole C; Basner-Tschakarjan, Etiena; Edmonson, Shyrie A; Hui, Daniel J; Sabatino, Denise E; Zhou, Shangzhen; Wright, J Fraser; Jiang, Haiyan; Pierce, Glenn F; Arruda, Valder R; High, Katherine A

    2007-10-01

    Adeno-associated virus (AAV)-mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti-T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti-IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs.

  7. Inverter Anti-Islanding with Advanced Grid Support in Single- and Multi-Inverter Islands

    SciTech Connect

    Hoke, Andy

    2016-08-16

    As PV and other DER systems are connected to the grid at increased penetration levels, island detection may become more challenging for two reasons: 1. In islands containing many DERs, active inverter-based anti-islanding methods may have more difficulty detecting islands because each individual inverter's efforts to detect the island may be interfered with by the other inverters in the island. 2. The increasing numbers of DERs are leading to new requirements that DERs ride through grid disturbances and even actively try to regulate grid voltage and frequency back towards nominal operating conditions. These new grid support requirements may directly or indirectly interfere with anti-islanding controls. This report describes a series of tests designed to examine the impacts of both grid support functions and multi-inverter islands on anti-islanding effectiveness.

  8. 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.

  9. Local administration of AAV-DJ pseudoserotype expressing COX2 provided early onset of transgene expression and promoted bone fracture healing in mice.

    PubMed

    Lakhan, R; Baylink, D J; Lau, K-H W; Tang, X; Sheng, M H-C; Rundle, C H; Qin, X

    2015-09-01

    We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and μ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.

  10. Efficient transduction of vascular smooth muscle cells with a translational AAV2.5 vector: a new perspective for in-stent restenosis gene therapy.

    PubMed

    Lompré, A-M; Hadri, L; Merlet, E; Keuylian, Z; Mougenot, N; Karakikes, I; Chen, J; Atassi, F; Marchand, A; Blaise, R; Limon, I; McPhee, S W J; Samulski, R J; Hajjar, R J; Lipskaia, L

    2013-09-01

    Coronary artery disease represents the leading cause of mortality in the developed world. Percutaneous coronary intervention involving stent placement remains disadvantaged by restenosis or thrombosis. Vascular gene therapy-based methods may be approached, but lack a vascular gene delivery vector. We report a safe and efficient long-term transduction of rat carotid vessels after balloon injury intervention with a translational optimized AAV2.5 vector. Compared with other known adeno-associated virus (AAV) serotypes, AAV2.5 demonstrated the highest transduction efficiency of human coronary artery vascular smooth muscle cells (VSMCs) in vitro. Local delivery of AAV2.5-driven transgenes in injured carotid arteries resulted in transduction as soon as day 2 after surgery and persisted for at least 30 days. In contrast to adenovirus 5 vector, inflammation was not detected in AAV2.5-transduced vessels. The functional effects of AAV2.5-mediated gene transfer on neointimal thickening were assessed using the sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2a (SERCA2a) human gene, known to inhibit VSMC proliferation. At 30 days, human SERCA2a messenger RNA was detected in transduced arteries. Morphometric analysis revealed a significant decrease in neointimal hyperplasia in AAV2.5-SERCA2a-transduced arteries: 28.36±11.30 (n=8) vs 77.96±24.60 (n=10) μm(2), in AAV2.5-green fluorescent protein-infected, P<0.05. In conclusion, AAV2.5 vector can be considered as a promising safe and effective vector for vascular gene therapy.

  11. DC-to-AC inverter ratio failure detector

    NASA Technical Reports Server (NTRS)

    Ebersole, T. J.; Andrews, R. E.

    1975-01-01

    Failure detection technique is based upon input-output ratios, which is independent of inverter loading. Since inverter has fixed relationship between V-in/V-out and I-in/I-out, failure detection criteria are based on this ratio, which is simply inverter transformer turns ratio, K, equal to primary turns divided by secondary turns.

  12. 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.

  13. 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…

  14. Neuromorphic System Based on CMOS Inverters and Si-Based Synaptic Device.

    PubMed

    Park, Jungjin; Kwon, Min-Woo; Kim, Hyungjin; Park, Byung-Gook

    2016-05-01

    We developed an analog neuron circuit that can work with Si-based synaptic devices. N-channel and p-channel synaptic devices connected to current mirrors constitute the synaptic connection and integration parts to implement the excitation and inhibition mechanisms of biological neurons. The normal inverter controlling delay time and the modified inverter making negative pulse constitute the action-potential generation part to generate output action-potential. Connecting output potential to the synaptic device, we implement the spike-timing-dependent-plasticity (STDP) mechanism, adjusting the conductance of synapse. As we have constituted the analog neuron circuit using 4-terminal synaptic device without additional switch and logic operation, we can emulate the operation of the neuron with minimum number of devices and power dissipation.

  15. AAV delivered artificial microRNA extends survival and delays paralysis in an Amyotrophic Lateral Sclerosis mouse model

    PubMed Central

    Stoica, Lorelei; Todeasa, Sophia H.; Cabrera, Gabriela Toro; Salameh, Johnny S.; ElMallah, Mai K.; Mueller, Christian; Brown, Robert H.; Miguel, Sena-Esteves

    2017-01-01

    Objectives Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis and death within five years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only FDA approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient AAV mediated RNAi gene therapy for ALS. Methods A single stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1G93A mice and impact on disease progression and survival assessed. Results This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9-treated SOD1G93A mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1G93A spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9-treated SOD1G93A mice displaying a phenotype reminiscent of patient pathophysiology. Interpretation These studies clearly demonstrate that an AAV9-delivered SOD1-specific artificial microRNA is an effective and translatable therapeutic approach for ALS. PMID:26891182

  16. Promise and problems associated with the use of recombinant AAV for the delivery of anti-HIV antibodies

    PubMed Central

    Fuchs, Sebastian P; Desrosiers, Ronald C

    2016-01-01

    Attempts to elicit antibodies with potent neutralizing activity against a broad range of human immunodeficiency virus (HIV) isolates have so far proven unsuccessful. Long-term delivery of monoclonal antibodies (mAbs) with such activity is a creative alternative that circumvents the need for an immune response and has the potential for creating a long-lasting sterilizing barrier against HIV. This approach is made possible by an incredible array of potent broadly neutralizing antibodies (bnAbs) that have been identified over the last several years. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for long-term delivery for a variety of reasons. The only products made from rAAV are derived from the transgenes that are put into it; as long as those products are not viewed as foreign, expression from muscle tissue may continue for decades. Thus, use of rAAV to achieve long-term delivery of anti-HIV mAbs with potent neutralizing activity against a broad range of HIV-1 isolates is emerging as a promising concept for the prevention or treatment of HIV-1 infection in humans. Experiments in mice and monkeys that have demonstrated protective efficacy against AIDS virus infection have raised hopes for the promise of this approach. However, all published experiments in monkeys have encountered unwanted immune responses to the AAV-delivered antibody, and these immune responses appear to limit the levels of delivered antibody that can be achieved. In this review, we highlight the promise of rAAV-mediated antibody delivery for the prevention or treatment of HIV infection in humans, but we also discuss the obstacles that will need to be understood and solved in order for the promise of this approach to be realized. PMID:28197421

  17. Cardiac AAV9-S100A1 gene therapy rescues postischemic heart failure in a preclinical large animal model

    PubMed Central

    Pleger, Sven T.; Shan, Changguang; Ksienzyk, Jan; Bekeredjian, Raffi; Boekstegers, Peter; Hinkel, Rabea; Schinkel, Stefanie; Leuchs, Barbara; Ludwig, Jochen; Qiu, Gang; Weber, Christophe; Kleinschmidt, Jürgen A.; Raake, Philip; Koch, Walter J.; Katus, Hugo A.; Müller, Oliver J.; Most, Patrick

    2014-01-01

    As a prerequisite to clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated viral (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 various animal models, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium Ca2+ 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 through retrograde coronary venous delivery, 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 this phenotype by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling and energy homeostasis. Transgene expression was restricted to cardiac tissue and extra-cardiac organ function was uncompromised indicating a favorable safety profile. This translational study shows the pre-clinical feasibility, long-term therapeutic effectiveness and a favorable safety profile of cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our study presents a strong rational for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure. PMID:21775667

  18. 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.

  19. Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

    PubMed Central

    Hammer, Eva; Heilbronn, Regine

    2014-01-01

    ABSTRACT The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3′ part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2–Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection. IMPORTANCE This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector

  20. A Plasma Display Terminal.

    ERIC Educational Resources Information Center

    Stifle, Jack

    A graphics terminal designed for use as a remote computer input/output terminal is described. Although the terminal is intended for use in teaching applications, it has several features which make it useful in many other computer terminal applications. These features include: a 10-inch square plasma display panel, permanent storage of information…

  1. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice.

    PubMed

    Yu, Wenhan; Mookherjee, Suddhasil; Chaitankar, Vijender; Hiriyanna, Suja; Kim, Jung-Woong; Brooks, Matthew; Ataeijannati, Yasaman; Sun, Xun; Dong, Lijin; Li, Tiansen; Swaroop, Anand; Wu, Zhijian

    2017-03-14

    In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.

  2. The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy.

    PubMed

    Youjin, Shen; Jun, Yin

    2009-03-01

    Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.

  3. 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.

  4. 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

  5. Mutagenic inverted repeat assisted genome engineering (MIRAGE).

    PubMed

    Nair, Nikhil U; Zhao, Huimin

    2009-01-01

    Here we describe a one-step method to create precise modifications in the genome of Saccharomyces cerevisiae as a tool for synthetic biology, metabolic engineering, systems biology and genetic studies. Through homologous recombination, a mutagenesis cassette containing an inverted repeat of selection marker(s) is integrated into the genome. Due to its inherent instability in genomic DNA, the inverted repeat catalyzes spontaneous self-excision, resulting in precise genome modification. Since this excision occurs at very high frequencies, selection for the integration event can be followed immediately by counterselection, without the need for growth in permissive conditions. This is the first time a truly one-step method has been described for genome modification in any organism.

  6. Micro-inverter solar panel mounting

    SciTech Connect

    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.

  7. 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

  8. 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.

  9. Inverter testing at Sandia National Laboratories

    SciTech Connect

    Ginn, J.W.; Bonn, R.H.; Sittler, G.

    1997-04-01

    Inverters are key building blocks of photovoltaic (PV) systems that produce ac power. The balance of systems (BOS) portion of a PV system can account for up to 50% of the system cost, and its reliable operation is essential for a successful PV system. As part of its BOS program, Sandia National Laboratories (SNL) maintains a laboratory wherein accurate electrical measurements of power systems can be made under a variety of conditions. This paper outlines the work that is done in that laboratory.

  10. 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.

  11. The Human Rhodopsin Kinase Promoter in an AAV5 Vector Confers Rod- and Cone-Specific Expression in the Primate Retina

    PubMed Central

    Alexander, John J.; Boye, Sanford L.; Witherspoon, Clark D.; Sandefer, Kristen J.; Conlon, Thomas J.; Erger, Kirsten; Sun, Jingfen; Ryals, Renee; Chiodo, Vince A.; Clark, Mark E.; Girkin, Christopher A.; Hauswirth, William W.; Gamlin, Paul D.

    2012-01-01

    Abstract Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting. PMID:22845794

  12. AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently

    PubMed Central

    Doria, Monica; Ferrara, Antonella

    2013-01-01

    Abstract 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. PMID:24116943

  13. The human rhodopsin kinase promoter in an AAV5 vector confers rod- and cone-specific expression in the primate retina.

    PubMed

    Boye, Shannon E; Alexander, John J; Boye, Sanford L; Witherspoon, Clark D; Sandefer, Kristen J; Conlon, Thomas J; Erger, Kirsten; Sun, Jingfen; Ryals, Renee; Chiodo, Vince A; Clark, Mark E; Girkin, Christopher A; Hauswirth, William W; Gamlin, Paul D

    2012-10-01

    Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

  14. Long-term suppression of experimental arthritis following intramuscular administration of a pseudotyped AAV2/1-TNFR:Fc Vector.

    PubMed

    Sandalon, Ziv; Bruckheimer, Elizabeth M; Lustig, Kurt H; Burstein, Haim

    2007-02-01

    We previously reported that administration of an adeno-associated virus 2 (AAV2) vector encoding a rat tumor necrosis factor (TNF) receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats with streptococcal cell wall-induced arthritis resulted in suppression of joint inflammation and cartilage and bone destruction, as well as expression of joint proinflammatory cytokines. In this study, we used an alternate rat model of arthritis to compare the serum levels and duration of TNFR:Fc protein expression following intramuscular administration of pseudotyped AAV-TNFR:Fc vectors based on serotypes 1, 2, and 5. All three pseudotyped AAV-TNFR:Fc vectors led to sustained expression of serum TNFR:Fc protein for at least one year. Serum TNFR:Fc protein levels in rats administered intramuscularly with AAV2/1-TNFR:Fc vector were up to 100- and 10-fold higher than in rats administered the AAV2-TNFR:Fc or AAV2/5-TNFR:Fc vectors, respectively. A single intramuscular administration of AAV2/1-TNFR:Fc vector at vector doses ranging from 10(10) to 10(12) DNase-resistant particles (DRP) per animal, resulted in complete and long-term suppression of recurrent joint inflammation for at least 150 days. Our results establish a proof of concept for administration of an AAV2/1-TNFR:Fc vector to the muscle to achieve long-term, sustained and therapeutically relevant levels of TNFR:Fc protein to treat chronic systemic inflammatory joint diseases.

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

    PubMed

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

    2014-09-01

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

  16. Self-complementary AAV2.5-BMP2-coated Femoral Allografts Mediated Superior Bone Healing Versus Live Autografts in Mice With Equivalent Biomechanics to Unfractured Femur

    PubMed Central

    Yazici, Cemal; Takahata, Masahiko; Reynolds, David G; Xie, Chao; Samulski, R Jude; Samulski, Jade; Beecham, E Jeffrey; Gertzman, Arthur A; Spilker, Mark; Zhang, Xinping; O'Keefe, Regis J; Awad, Hani A; Schwarz, Edward M

    2011-01-01

    Structural allografts used for critical bone defects have limited osteogenic properties for biointegration. Although ex vivo tissue-engineered constructs expressing bone morphogenetic protein-2 (BMP2) have demonstrated efficacy in critical defect models, similar success has not been achieved with off-the-shelf acellular approaches, including allografts coated with freeze-dried single-stranded adeno-associated virus (ssAAV-BMP2). To see whether the self-complementary AAV serotype 2.5 vector (scAAV2.5-BMP2) could overcome this, we performed side-by-side comparisons in vitro and in the murine femoral allograft model. Although ssAAV-BMP2 was unable to induce BMP2 expression and differentiation of C3H10T1/2 cells in culture, scAAV2.5-BMP2 transduction led to dose-dependent BMP2 expression and alkaline phosphatase activity, and displayed a 25-fold increased transduction efficiency in vivo. After 6 weeks, the ssAAV-BMP2 coating failed to demonstrate any significant effects. However, all allografts coated with 1010 scAAV2.5-BMP2 formed a new cortical shell that was indistinguishable to that formed by live autografts. Additionally, coated allografts experienced reduced resorption resulting in a threefold increase in graft bone volume versus autograft. This led to biomechanical superiority versus both allografts and autografts, and equivalent torsional rigidity to unfractured femur. Collectively, these results demonstrate that scAAV2.5-BMP2 coating overcomes the major limitations of structural allografts, which can be used to heal critical defects of any size. PMID:21206485

  17. 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.

  18. 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.

  19. 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

  20. Dynamic performance of a STATCON at an HVDC inverter feeding a very weak AC system

    SciTech Connect

    Zhuang, Y.; Menzies, R.W.; Nayak, O.B.; Turanli, H.M.

    1996-04-01

    This paper investigates the dynamic performance of the advanced static var compensator or STATCON at a high voltage direct current (HVDC) converter terminal where the ac system has a very low short circuit ratio (SCR). The STATCON is based on a nine-level GTO thyristor inverter. The studies include operating characteristics of the STATCON under various ac and dc disturbances. The simulation results are compared with other types of reactive power compensation options available for such applications. It is shown that the STATCON has clear advantages over the other compensators, in areas such as; fault response time, voltage support ability, and dc recovery, while operating with very weak ac systems.