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Sample records for adeno-associated viruses aavs

  1. 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

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

    PubMed

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

    2016-10-01

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

  3. 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.

  4. 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2007-07-01

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

  8. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1999-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  12. Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro

    NASA Astrophysics Data System (ADS)

    Lange, Wienke; Jin, Lei; Maybeck, Vanessa; Meisenberg, Annika; Baumann, Arnd; Offenhäusser, Andreas

    2014-03-01

    Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

  13. 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

  14. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype

    PubMed Central

    2013-01-01

    Background The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Methods Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . Results We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. Conclusions In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect. PMID:23497173

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

    PubMed

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

    2002-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. 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

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

    PubMed

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

    2011-03-01

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

  20. Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors

    PubMed Central

    Bartlett, Jeffrey S.; Wilcher, Rose; Samulski, R. Jude

    2000-01-01

    We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A1 prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo. PMID:10684294

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

    PubMed

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

    1994-10-11

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

  2. 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

  3. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin

    PubMed Central

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Miller, Paul E.; Sharma, Alok K.; Ver Hoeve, James N.; Howard, Kellie; Knop, David R.; Neuringer, Martha; McGill, Trevor; Stoddard, Jonathan; Chulay, Jeffrey D.

    2015-01-01

    Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 1010 or 4 × 1011 vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS. PMID:26390090

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

    PubMed

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

    2016-01-01

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

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

    PubMed 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

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

    PubMed Central

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

    2009-01-01

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

  7. Identification and Characterization of Novel Adeno-Associated Virus Isolates in ATCC Virus Stocks

    PubMed Central

    Schmidt, Michael; Grot, Emmanuelle; Cervenka, Peter; Wainer, Sandra; Buck, Charles; Chiorini, John A.

    2006-01-01

    Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines. PMID:16641301

  8. Identification and characterization of novel adeno-associated virus isolates in ATCC virus stocks.

    PubMed

    Schmidt, Michael; Grot, Emmanuelle; Cervenka, Peter; Wainer, Sandra; Buck, Charles; Chiorini, John A

    2006-05-01

    Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.

  9. 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

  10. Structural Insights into Adeno-Associated Virus Serotype 5

    PubMed Central

    Govindasamy, Lakshmanan; DiMattia, Michael A.; Gurda, Brittney L.; Halder, Sujata; McKenna, Robert; Chiorini, John A.; Muzyczka, Nicholas; Zolotukhin, Sergei

    2013-01-01

    The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity. PMID:23926356

  11. Adeno-associated Virus as a Mammalian DNA Vector

    PubMed Central

    SALGANIK, MAX; HIRSCH, MATTHEW L.; SAMULSKI, RICHARD JUDE

    2015-01-01

    In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells. PMID:26350320

  12. Analysis of adeno-associated virus and HPV interaction.

    PubMed

    Hermonat, Paul L; You, Hong; Chiriva-Internati, C Maurizio; Liu, Yong

    2005-01-01

    It is slowly becoming accepted that adeno-associated virus (AAV) is another significant factor involved in cervical carcinogenesis. However, unlike human papillomavirus (HPV), which is positively associated with cervical cancer, AAV is negatively associated with this cancer. This negative association appears to be through a direct and complex bi-directional interaction between AAV and HPV. Essentially all assays used for studying HPV can be used for studying the AAV-HPV interaction. This is because both viruses are productive in the same tissue, the stratified squamous epithelium (skin). Their relationship can be studied on the level of the complete virus and their complete life cycle using the organotypic epithelial raft culture system, which generates a stratified squamous epithelium. Their relationship can be studied in various other tissue-culture models measuring oncogenic potential. Their interaction can also be studied on the component level, as both protein-protein and protein-DNA interactions are known. Their relationship has even been studied using transgenic animals. The AAV-HPV relationship can be broken down into two halves--AAV-encoded products, which affect HPV biology, and HPV-encoded products, which affect AAV biology. To date, the former are much better studied than the latter. The rep gene and its largest product, Rep78, are responsible for most of AAV's effects upon HPV. This chapter largely focuses on AAV's effect on the HPV life cycle.

  13. Engineering adeno-associated viruses for clinical gene therapy.

    PubMed

    Kotterman, Melissa A; Schaffer, David V

    2014-07-01

    Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

  14. 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

  15. Adeno-associated virus serotypes for gene therapeutics.

    PubMed

    Lisowski, Leszek; Tay, Szun Szun; Alexander, Ian Edward

    2015-10-01

    Gene transfer vectors based on adeno-associated virus (AAV) are showing exciting therapeutic promise in early phase clinical trials. The ability to cross-package the prototypic AAV2 vector genome into different capsids is a powerful way of conferring novel tropism and biology, with evolving capsid engineering technologies and directed evolution approaches further enhancing the utility and flexibility of these vectors. Novel properties of specific capsids show unpredictable species and cell-type specificity. Therefore, full realisation of the therapeutic potential of AAV vectors requires the development of more therapeutically predictive preclinical methods for evaluating capsid performance. This will strongly complement an iterative approach to the evaluation of capsid variants in the clinic and, should wherever possible, include the determination of gene transfer efficiencies.

  16. Adeno-associated virus vectors and neurological gene therapy.

    PubMed

    Ojala, David S; Amara, Dominic P; Schaffer, David V

    2015-02-01

    Gene therapy has strong potential for treating a variety of genetic disorders, as demonstrated in recent clinical trials. There is unfortunately no scarcity of disease targets, and the grand challenge in this field has instead been the development of safe and efficient gene delivery platforms. To date, approximately two thirds of the 1800 gene therapy clinical trials completed worldwide have used viral vectors. Among these, adeno-associated virus (AAV) has emerged as particularly promising because of its impressive safety profile and efficiency in transducing a wide range of cell types. Gene delivery to the CNS involves both considerable promise and unique challenges, and better AAV vectors are thus needed to translate CNS gene therapy approaches to the clinic. This review discusses strategies for vector design, potential routes of administration, immune responses, and clinical applications of AAV in the CNS.

  17. Detection of adeno-associated virus type 2 genome in cervical carcinoma

    PubMed Central

    Zheng, B Y; Li, X D; Wiklund, F; Chowdhry, S; Ångstrom, T; Hallmans, G; Dillner, J; Wallin, K L

    2006-01-01

    Adeno-associated virus (AAV) can impair the replication of other viruses. Adeno-associated virus seroprevalences have been reported to be lower among women with cervical cancer. In-vitro, AAV can interfere with the production of human papillomavirus virions. Adeno-associated virus-2 DNA has also been detected in cervical cancer tissue, although not consistently. To evaluate the role of AAV infection in relation to invasive cervical cancer, we performed a nested case–control study within a retrospectively followed population-based cohort. A total of 104 women who developed invasive cervical cancer on average 5.6 years of follow-up (range: 0.5 months–26.2 years) and 104 matched control-women who did not develop cervical cancer during the same follow-up time were tested for AAV and human papillomavirus by polymerase chain reaction. At baseline, two (2%) case-women and three (3%) control-women were positive for AAV-2 DNA. At the time of cancer diagnosis, 12 (12%) case-women and 3 (3%) matched control-women were positive for AAV-2 DNA. Persisting AAV infection was not evident. In conclusion, AAV-2 DNA was present in a low proportion of cervical cancers and we found no evidence that the presence of AAV in cervical smears of healthy women would be associated with reduced risk of cervical cancer. PMID:16736006

  18. Biosafety of recombinant adeno-associated virus vectors.

    PubMed

    Dismuke, David J; Tenenbaum, Liliane; Samulski, R Jude

    2013-12-01

    It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety. PMID:24195602

  19. Structure of Neurotropic Adeno-Associated Virus AAVrh.8

    PubMed Central

    Halder, Sujata; Van Vliet, Kim; Smith, J Kennon; Duong, Thao Thi Phuong; McKenna, Robert; Wilson, James M.; Agbandje-McKenna, Mavis

    2015-01-01

    Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5 Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ~84, ~91, and ~87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment. PMID:26334681

  20. Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

    PubMed

    Bell, Peter; Vandenberghe, Luk H; Wilson, James M

    2014-06-01

    Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-11-01

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

  3. 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.

  4. Heavy and Light Particles of Adeno-Associated Virus

    PubMed Central

    de la Maza, Luis M.; Carter, Barrie J.

    1980-01-01

    KB cells coinfected with adenovirus and adeno-associated virus (AAV) yielded two kinds of infectious AAV particles that banded in CsCl at densities of 1.45 and 1.41 g/cm2, respectively. The 1.45 band was found to be composed of a heterogeneous group of viral particles that could be subfractionated by velocity sedimentation. The main component from this band had a smaller S value (109) than the main component from the 1.41 band (111S), although both had the same DNA/protein ratio and the same density in metrizamide gradients. Continuous-label experiments showed that early after infection, both components (1.45 and 1.41) were generated in the same amounts, but this was followed by a relative increase in the proportion of the 1.41 component over the 1.45 particles. Pulse-chase analysis failed to demonstrate a precursor-product relationship between these two bands. The slower-sedimenting components from the 1.45 band were unstable in CsCl and were present in a greater proportion early after infection. These particles contained DNA that was enriched for the terminal sequences of the AAV genomes and was accessible to digestion with micrococcal nuclease. Images PMID:6245263

  5. Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

    SciTech Connect

    Malkinson, Mertyn . E-mail: malkins@agri.huji.ac.il; Winocour, Ernest . E-mail: ernest.winocour@weizmann.ac.il

    2005-06-05

    The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.

  6. Glycan binding avidity determines the systemic fate of adeno-associated virus type 9.

    PubMed

    Shen, Shen; Bryant, Kelli D; Sun, Junjiang; Brown, Sarah M; Troupes, Andrew; Pulicherla, Nagesh; Asokan, Aravind

    2012-10-01

    Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions. PMID:22787229

  7. Analysis of the production efficiency and titration of various recombinant adeno-associated viruses.

    PubMed

    Nam, Young Ran; Kim, Sung Jin; Lee, Boyoung; Chang, Jin Woo; Joo, Chul-Hyen; Kim, Yoo Kyum; Lee, Heuiran

    2004-10-01

    Recombinant adeno-associated virus type 2 (rAAV2) viral vector, a non-pathogenic human parvovirus, has recently emerged as a gene transfer vehicle for cancer gene therapy. To utilize rAAV2 properly and safely while carrying out preclinical and clinical studies, it is crucial to exactly titer the virus. We therefore compared biological infectious rAAV2 titers with physical titers of rAAV2 vectors encoding various transgenes with different sized viral genomes. Biological rAAV2 infectivity was assayed by measuring the number of virus particles able to transduce Hela cells using several detection methods, including X-gal staining and immunocytostaining. Physical titers of rAAV2 were determined using a commercially available rAAV2 particle-specific enzyme-linked immunosorbent assay. We found that total rAAV2 particle production was consistent within the limited size variations of the rAAV2 genome, regardless of the difference in transgenes. In contrast, the infectious titer of rAAV2 differed greatly, even for the same viruses, due to variation in the sensitivity of the relevant assays. Thus, the results suggest that both infectious virus titer and total virus particle should be precisely measured for rAAV2 vector utilized in each study.

  8. Adeno-Associated Virus Enhances Wild-Type and Oncolytic Adenovirus Spread

    PubMed Central

    Laborda, Eduardo; Puig-Saus, Cristina; Cascalló, Manel; Chillón, Miguel

    2013-01-01

    Abstract The contamination of adenovirus (Ad) stocks with adeno-associated viruses (AAV) is usually unnoticed, and it has been associated with lower Ad yields upon large-scale production. During Ad propagation, AAV contamination needs to be detected routinely by polymerase chain reaction without symptomatic suspicion. In this study, we describe that the coinfection of either Ad wild type 5 or oncolytic Ad with AAV results in a large-plaque phenotype associated with an accelerated release of Ad from coinfected cells. This accelerated release was accompanied with the expected decrease in Ad yields in two out of three cell lines tested. Despite this lower Ad yield, coinfection with AAV accelerated cell death and enhanced the cytotoxicity mediated by Ad propagation. Intratumoral coinjection of Ad and AAV in two xenograft tumor models improved antitumor activity and mouse survival. Therefore, we conclude that accidental or intentional AAV coinfection has important implications for Ad-mediated virotherapy. PMID:24020980

  9. Novel strategy for generation and titration of recombinant adeno-associated virus vectors.

    PubMed

    Shiau, Ai-Li; Liu, Pu-Ste; Wu, Chao-Liang

    2005-01-01

    Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

  10. Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.

    PubMed

    Seppen, Jurgen; Bakker, Conny; de Jong, Berry; Kunne, Cindy; van den Oever, Karin; Vandenberghe, Kristin; de Waart, Rudi; Twisk, Jaap; Bosma, Piter

    2006-06-01

    Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans. PMID:16581301

  11. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

    PubMed

    Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

    2016-02-01

    Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

  12. Adeno-associated viruses undergo substantial evolution in primates during natural infections.

    PubMed

    Gao, Guangping; Alvira, Mauricio R; Somanathan, Suryanarayan; Lu, You; Vandenberghe, Luk H; Rux, John J; Calcedo, Roberto; Sanmiguel, Julio; Abbas, Zahra; Wilson, James M

    2003-05-13

    Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.

  13. Fluorescent Calcium Indicator Protein Expression in the Mouse Brain Using Recombinant Adeno-Associated Viruses.

    PubMed

    Heindorf, Matthias; Hasan, Mazahir T

    2015-07-01

    One method for gene delivery and long-term fluorescent calcium indicator protein (FCIP) expression in mammalian neurons in vivo involves the introduction of FCIPs via recombinant adeno-associated virus (rAAV) vectors using constitutive and cell type-specific promoters. This protocol describes the use of rAAVs to express FCIPs in the brain for imaging. Human embryonic kidney 293 cells are first transfected using calcium phosphate. rAAV is then prepared using either an iodixanol gradient or a heparin column. After the virus is purified, its quality is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, estimation of genomic and functional virus titers by quantitative polymerase chain reaction, and expression in dissociated neurons. Mice are injected with rAAV using a stereotactic instrument and can be imaged ∼3 wk later. PMID:26134910

  14. In vivo model of adeno-associated virus vector persistence and rescue.

    PubMed Central

    Afione, S A; Conrad, C K; Kearns, W G; Chunduru, S; Adams, R; Reynolds, T C; Guggino, W B; Cutting, G R; Carter, B J; Flotte, T R

    1996-01-01

    Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue. PMID:8627804

  15. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    PubMed

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2014-10-30

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene.

  16. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    PubMed

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2015-03-01

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene. PMID:25359548

  17. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

    SciTech Connect

    Weger, Stefan . E-mail: stefan.weger@charite.de; Hammer, Eva; Goetz, Anne; Heilbronn, Regine

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

  18. Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

    PubMed Central

    Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

    2016-01-01

    Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

  19. Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification

    PubMed Central

    Arden, Erik; Metzger, Joseph M.

    2016-01-01

    Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV. These methods may also limit crude viral lysate processing volume resulting in a significant loss of viral titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across native serotype and bioengineered mosaic capsids. PMID:27294171

  20. Adeno-Associated Virus-Based Gene Therapy for CNS Diseases

    PubMed Central

    Hocquemiller, Michaël; Giersch, Laura; Audrain, Mickael; Parker, Samantha; Cartier, Nathalie

    2016-01-01

    Gene therapy is at the cusp of a revolution for treating a large spectrum of CNS disorders by providing a durable therapeutic protein via a single administration. Adeno-associated virus (AAV)-mediated gene transfer is of particular interest as a therapeutic tool because of its safety profile and efficiency in transducing a wide range of cell types. The purpose of this review is to describe the most notable advancements in preclinical and clinical research on AAV-based CNS gene therapy and to discuss prospects for future development based on a new generation of vectors and delivery. PMID:27267688

  1. Interaction of adeno-associated virus Rep78 with p53: implications in growth inhibition.

    PubMed

    Batchu, R B; Shammas, M A; Wang, J Y; Munshi, N C

    1999-08-01

    Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA virus belonging to the parvoviridae family. Onco-suppressive properties of AAV against adenovirus, a DNA tumor virus, have been well documented. Rep78, a major regulatory protein of AAV, is believed to be responsible for its antioncogenic properties. Most DNA tumor viruses disturb the cell cycle pathways by essentially abrogating the functions of p53. Here we present evidence that AAV acts as an antiproliferative agent against adenovirus by protecting the adenoviral-mediated degradation of p53 as confirmed by both Western blot analysis and immunoprecipitation analysis with anti-p53 antibody. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. Furthermore, the binding of purified p53 to the AAV Rep78 affinity column confirms their interaction. These results document for the first time that the antiproliferative effects of AAV against adenovirus are mediated, at least in part, by the interaction of AAV Rep78 with p53.

  2. The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

    PubMed

    Hölscher, Christina; Sonntag, Florian; Henrich, Katharina; Chen, Qingxin; Beneke, Jürgen; Matula, Petr; Rohr, Karl; Kaderali, Lars; Beil, Nina; Erfle, Holger; Kleinschmidt, Jürgen A; Müller, Martin

    2015-12-01

    Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo.

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

    PubMed

    Nash, Kevin R; Gordon, Marcia N

    2016-01-01

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

  4. Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4▿

    PubMed Central

    Govindasamy, Lakshmanan; Padron, Eric; McKenna, Robert; Muzyczka, Nicholas; Kaludov, Nikola; Chiorini, John A.; Agbandje-McKenna, Mavis

    2006-01-01

    The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core β strands (βB to βI) and helical (αA) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed “valleys” between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes. PMID:16971437

  5. Super-resolution imaging of nuclear import of adeno-associated virus in live cells

    PubMed Central

    Kelich, Joseph M; Ma, Jiong; Dong, Biao; Wang, Qizhao; Chin, Mario; Magura, Connor M; Xiao, Weidong; Yang, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9–12 nm and 2–20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy. PMID:26665132

  6. Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

    PubMed

    Kelich, Joseph M; Ma, Jiong; Dong, Biao; Wang, Qizhao; Chin, Mario; Magura, Connor M; Xiao, Weidong; Yang, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9-12 nm and 2-20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy. PMID:26665132

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

    PubMed

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

    2002-05-15

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

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

    PubMed

    Meng, Qinglin; Zhang, Binbin; Zhang, Chun

    2013-02-01

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

  9. Multiple human papillomavirus genes affect the adeno-associated virus life cycle.

    PubMed

    You, Hong; Liu, Yong; Prasad, C Krishna; Agrawal, Nalini; Zhang, Dazhi; Bandyopadhyay, Sarmistha; Liu, Hongmei; Kay, Helen H; Mehta, Jawahar L; Hermonat, Paul L

    2006-01-20

    The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.

  10. Recombinant adeno-associated virus vectors in the treatment of rare diseases

    PubMed Central

    Hastie, Eric; Samulski, R. Jude

    2016-01-01

    Introduction An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use. Areas covered In this review the biology of AAV and our ability to generate disease-specific variants is summarized. Limitations of current therapy are reviewed, with an emphasis on immune detection of virus, viral tropism and tissue targeting, and limitations of gene expression. Information for this review was found using PubMed and clinicaltrials.gov. Expert opinion Currently the scope of clinical trials of AAV gene therapy is concentrated in an array of phase I/II safety trials with less than two dozen rare diseases featured. Pre-clinical, translational studies are expanding in number as developments within the last decade have made generation of improved AAV vectors available to more researchers. Further, one bottleneck that is being overcome is the availability of disease models, which will allow for improved preclinical testing and advancement of AAV to more clinical applications.

  11. Recombinant adeno-associated virus vectors in the treatment of rare diseases

    PubMed Central

    Hastie, Eric; Samulski, R. Jude

    2016-01-01

    Introduction An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use. Areas covered In this review the biology of AAV and our ability to generate disease-specific variants is summarized. Limitations of current therapy are reviewed, with an emphasis on immune detection of virus, viral tropism and tissue targeting, and limitations of gene expression. Information for this review was found using PubMed and clinicaltrials.gov. Expert opinion Currently the scope of clinical trials of AAV gene therapy is concentrated in an array of phase I/II safety trials with less than two dozen rare diseases featured. Pre-clinical, translational studies are expanding in number as developments within the last decade have made generation of improved AAV vectors available to more researchers. Further, one bottleneck that is being overcome is the availability of disease models, which will allow for improved preclinical testing and advancement of AAV to more clinical applications. PMID:27668135

  12. Molecular detection of adeno-associated virus in cases of spontaneous and intentional human abortion.

    PubMed

    Pereira, Christiane Curi; de Freitas, Luciana Bueno; de Vargas, Paulo Roberto Merçon; de Azevedo, Maria Luiza Borges; do Nascimento, Jussara Pereira; Spano, Liliana Cruz

    2010-10-01

    Pregnancy failure is a common event and often of unknown cause. Some viruses are thought to cause abortions including the adeno-associated viruses (AAV), viruses which are regarded as being without any definitive association to any human disease. This study investigated AAV infection in 81 human abortions, both spontaneous and intentional that occurred up to the 23rd week of gestation. Nucleic acid of AAV-2, 3, and 5 types from 118 decidual and chorionic tissues, collected from the patients in this study, was amplified by nested-PCR. In situ hybridization (ISH) was developed with a digoxigenin-labeled AAV probe in paraffin embedded tissues from the AAV positive cases. AAV was observed in 28.4% (23/81) of the cases, of which, 78.3% (18/23) were in the decidua and 21.7% (5/23) in the extravillous trophoblast, the chorionic plate, or chorionic villi fragments. AAV-2, the only type detected, occurred in 32.3% (22/68) and in 7.7% (1/13) of the spontaneous and intentional abortions, respectively. ISH revealed AAV in the decidua, chorionic tissue or chorionic plate and extravillous trophoblast. The detection of only AAV-2 type indicates that it is the most frequent in the population studied and/or shows tissue tropism. The presence of AAV in decidual or trophoblastic cells in cases of abortion, as observed by ISH, implies that the virus could jeopardize the pregnancy. The significant predominance in spontaneous cases suggests possibly a causal association between AAV and abortion. PMID:20827766

  13. Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

    PubMed Central

    Rayaprolu, Vamseedhar; Kruse, Shannon; Kant, Ravi; Venkatakrishnan, Balasubramanian; Movahed, Navid; Brooke, Dewey; Lins, Bridget; Bennett, Antonette; Potter, Timothy; McKenna, Robert; Agbandje-McKenna, Mavis

    2013-01-01

    Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production. PMID:24067976

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

    PubMed

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

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

  15. Comparative analysis of adeno-associated virus capsid stability and dynamics.

    PubMed

    Rayaprolu, Vamseedhar; Kruse, Shannon; Kant, Ravi; Venkatakrishnan, Balasubramanian; Movahed, Navid; Brooke, Dewey; Lins, Bridget; Bennett, Antonette; Potter, Timothy; McKenna, Robert; Agbandje-McKenna, Mavis; Bothner, Brian

    2013-12-01

    Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production. PMID:24067976

  16. Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups.

    PubMed

    Rabinowitz, Joseph E; Bowles, Dawn E; Faust, Susan M; Ledford, Julie G; Cunningham, Scott E; Samulski, R Jude

    2004-05-01

    For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this

  17. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  18. Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

    PubMed Central

    Salganik, Maxim; Venkatakrishnan, Balasubramanian; Bennett, Antonette; Lins, Bridget; Yarbrough, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection. PMID:22915820

  19. Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

    NASA Astrophysics Data System (ADS)

    Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2007-05-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

    PubMed Central

    Koerber, James T; Jang, Jae-Hyung; Schaffer, David V

    2009-01-01

    Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (107), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4–6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology. PMID:18728640

  3. Adeno-associated virus-mediated gene transfer targeting normal and traumatized mouse utricle.

    PubMed

    Wang, G-P; Guo, J-Y; Peng, Z; Liu, Y-Y; Xie, J; Gong, S-S

    2014-11-01

    Balance dysfunction is closely associated with loss of vestibular hair cells (HCs). Gene therapy shows promise when used to protect or regenerate vestibular HCs to preserve or restore adequate vestibular function. Adeno-associated virus (AAV) vectors allow long-term gene expression in the absence of toxicity. To noninvasively define an AAV serotype exhibiting favorable tropism toward the vestibular sensory epithelium, we characterized the transgene expression potential of AAV vectors (serotypes 1, 2, 5, 6 and 8) inoculated into adult mouse utricle via canalostomy. We found that AAV8 was the most effective AAV vector in utricular gene transfer. Swim tests and measurements of auditory brainstem response revealed minimal loss of vestibular function and hearing after canalostomy. In the normal utricle after AAV8 infusion, transduction efficiency peaked at 7 days, and was maintained thereafter, in vestibular HCs, and at 3 days in supporting cells (SCs). In the streptomycin-lesioned utricle, the SC transduction efficiency peaked at 7 days and decreased at 30 days. In conclusion, AAV8-mediated gene transfer via canalostomy facilitates efficient and safe transduction in mouse vestibular sensory epithelium, and may in the future become clinically relevant for human vestibular gene therapy. PMID:25119376

  4. Improved transduction efficiencies of adeno-associated virus vectors by synthetic cell-permeable peptides.

    PubMed

    Tabata, Kitako; Sugano, Eriko; Murakami, Fumika; Yamashita, Tetsuro; Ozaki, Taku; Tomita, Hiroshi

    2016-09-30

    Various serotypes of adeno-associated virus (AAV) vectors have been used for gene therapy and as research tools. Among these serotypes, the AAV type 2 vector has been used successfully in human gene therapies. However, the transduction efficiency of AAV2 depends on the cell type, and this poses a problem in the efficacy of gene therapy. To improve the transduction efficiency of AAV2, we designed a small peptide consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor peptide and the HIV-Tat sequence Tat-Y1068. Pre- or co-treatment of CYNOM-K1 cells from cynomolgus monkey embryo skin with Tat-Y1068 increased the transduction efficiencies in a dose-dependent manner and caused p38 phosphorylation. The transduction efficiency of AAV2 into the rat fibroblast cell line RAT-1 highly expressing EGFR was less than the transduction efficiency of AAV2 into CYNOM-K1 cells. Tat-Y1068 increased the transduction efficiency in RAT-1 cells in the same manner as in CYNOM-K1 cells. In conclusion, cell-permeable peptides possessing the EGFR tyrosine kinase inhibitor function might serve as a useful ingredient of AAV2 vector solution for increasing the transduction efficiency of gene therapies.

  5. Improved transduction efficiencies of adeno-associated virus vectors by synthetic cell-permeable peptides.

    PubMed

    Tabata, Kitako; Sugano, Eriko; Murakami, Fumika; Yamashita, Tetsuro; Ozaki, Taku; Tomita, Hiroshi

    2016-09-30

    Various serotypes of adeno-associated virus (AAV) vectors have been used for gene therapy and as research tools. Among these serotypes, the AAV type 2 vector has been used successfully in human gene therapies. However, the transduction efficiency of AAV2 depends on the cell type, and this poses a problem in the efficacy of gene therapy. To improve the transduction efficiency of AAV2, we designed a small peptide consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor peptide and the HIV-Tat sequence Tat-Y1068. Pre- or co-treatment of CYNOM-K1 cells from cynomolgus monkey embryo skin with Tat-Y1068 increased the transduction efficiencies in a dose-dependent manner and caused p38 phosphorylation. The transduction efficiency of AAV2 into the rat fibroblast cell line RAT-1 highly expressing EGFR was less than the transduction efficiency of AAV2 into CYNOM-K1 cells. Tat-Y1068 increased the transduction efficiency in RAT-1 cells in the same manner as in CYNOM-K1 cells. In conclusion, cell-permeable peptides possessing the EGFR tyrosine kinase inhibitor function might serve as a useful ingredient of AAV2 vector solution for increasing the transduction efficiency of gene therapies. PMID:27614311

  6. Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

    PubMed Central

    Gruenert, Anja K.; Czugala, Marta; Mueller, Chris; Schmeer, Marco; Schleef, Martin; Kruse, Friedrich E.; Fuchsluger, Thomas A.

    2016-01-01

    Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic. PMID:27023329

  7. Systemic gene delivery to the central nervous system using Adeno-associated virus

    PubMed Central

    Bourdenx, Mathieu; Dutheil, Nathalie; Bezard, Erwan; Dehay, Benjamin

    2014-01-01

    Adeno-associated virus (AAV)-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood–brain barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery. PMID:24917785

  8. Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

    PubMed Central

    Park, Kyoung-Jin; Lee, Jongan; Park, June-Hee; Joh, Jae-Won; Kwon, Choon Hyuck David

    2016-01-01

    Background The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. Methods A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. Results The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). Conclusions AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients. PMID:27374713

  9. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

    PubMed Central

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su

    2016-01-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys. PMID:26924933

  10. Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors.

    PubMed

    Hirata, Roli K; Xu, Cong; Dong, Rong; Miller, Daniel G; Ferguson, Stacy; Russell, David W

    2004-01-01

    Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.

  11. A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus.

    PubMed

    Kelemen, Rachel E; Mukherjee, Raja; Cao, Xiaofu; Erickson, Sarah B; Zheng, Yunan; Chatterjee, Abhishek

    2016-08-26

    The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. PMID:27483453

  12. Adeno-associated virus type 2 binding study on model heparan sulfate surface

    NASA Astrophysics Data System (ADS)

    Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

    2003-11-01

    Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

  13. A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus.

    PubMed

    Kelemen, Rachel E; Mukherjee, Raja; Cao, Xiaofu; Erickson, Sarah B; Zheng, Yunan; Chatterjee, Abhishek

    2016-08-26

    The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity.

  14. Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

    PubMed

    Ohi, S

    2000-07-01

    With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

  15. Phenotypic correction of a mouse model for primary hyperoxaluria with adeno-associated virus gene transfer.

    PubMed

    Salido, Eduardo; Rodriguez-Pena, Marisol; Santana, Alfredo; Beattie, Stuart G; Petry, Harald; Torres, Armando

    2011-05-01

    Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors. PMID:21119625

  16. Adeno-associated virus activates an innate immune response in normal human cells but not in osteosarcoma cells.

    PubMed

    Laredj, Leila N; Beard, Peter

    2011-12-01

    Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

  17. Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface

    PubMed Central

    Bardelli, Martino; Zárate-Pérez, Francisco; Agúndez, Leticia; Linden, R. Michael

    2016-01-01

    ABSTRACT The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA+ domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a

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

    PubMed

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

    2005-02-01

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

  19. Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

    PubMed Central

    Nonnenmacher, Mathieu E.; Cintrat, Jean-Christophe; Gillet, Daniel

    2014-01-01

    ABSTRACT Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV

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

    SciTech Connect

    Zhao Weihong; Wu Jianqing ||; Zhong Li; Chen Linyuan; Weigel-Kelley, Kirsten A. |; Qing Keyun; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H. |; Srivastava, Arun |. E-mail: asrivastava@gtc.ufl.edu

    2006-09-30

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

  1. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.

    PubMed Central

    Fisher, K J; Gao, G P; Weitzman, M D; DeMatteo, R; Burda, J F; Wilson, J M

    1996-01-01

    Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy. PMID:8523565

  2. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    SciTech Connect

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy; Kohlbrenner, Erik; Chiorini, John A.; McKenna, Robert; Muzyczka, Nicholas; Zolotukhin, Sergei; Agbandje-McKenna, Mavis

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  3. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    PubMed Central

    Liu, Yarong; Kim, Young Joo; Ji, Man; Fang, Jinxu; Siriwon, Natnaree; Zhang, Li I; Wang, Pin

    2014-01-01

    Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy. PMID:26015948

  4. Dual level inhibition of E2F-1 activity by adeno-associated virus Rep78.

    PubMed

    Batchu, R B; Shammas, M A; Wang, J Y; Munshi, N C

    2001-06-29

    E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have onco-suppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.

  5. Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal

    PubMed Central

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan

    2015-01-01

    We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3′ end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA. PMID:26297494

  6. Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.

    PubMed

    Merentie, M; Lottonen-Raikaslehto, L; Parviainen, V; Huusko, J; Pikkarainen, S; Mendel, M; Laham-Karam, N; Kärjä, V; Rissanen, R; Hedman, M; Ylä-Herttuala, S

    2016-03-01

    Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

  7. Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector

    PubMed Central

    Wollmann, Guido; Paglino, Justin C.; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N

    2015-01-01

    Vesicular stomatitis virus (VSV) shows promise as vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-D-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. PMID:25462341

  8. Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective

    PubMed Central

    Hastie, Eric

    2015-01-01

    Abstract Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications. PMID:25807962

  9. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

    PubMed

    Hastie, Eric; Samulski, R Jude

    2015-05-01

    Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

  10. Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

    PubMed

    Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

    2016-01-01

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications. PMID:27377618

  11. Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

    PubMed

    Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

    2016-07-05

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

  12. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17.

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

    Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells. Images PMID:1313913

  13. Germline viral “fossils” guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus

    PubMed Central

    Smith, Richard H.; Hallwirth, Claus V.; Westerman, Michael; Hetherington, Nicola A.; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B.; Koonin, Eugene V.; Agbandje-McKenna, Mavis; Kotin, Robert M.; Alexander, Ian E.

    2016-01-01

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV “fossils” provide novel capsid sequences for use in translational research and clinical applications. PMID:27377618

  14. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    SciTech Connect

    McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.; Stagg, Scott M.; Chapman, Michael S.

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  15. Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

    PubMed Central

    Whitford, Margaret H.; Arruda, Valder R.; Stedman, Hansell H.; Kay, Mark A.; High, Katherine A.

    2015-01-01

    Abstract Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

  16. Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus

    NASA Astrophysics Data System (ADS)

    Seisenberger, Georg; Ried, Martin U.; Endreß, Thomas; Büning, Hildegard; Hallek, Michael; Bräuchle, Christoph

    2001-11-01

    We describe a method, based on single-molecule imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye molecule. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resolution show various modes of motion of adeno-associated viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally observed so far.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-10-14

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

  20. Electron Microscopy Analysis of a Disaccharide Analog complex Reveals Receptor Interactions of Adeno-Associated Virus

    PubMed Central

    Xie, Qing; Spilman, Michael; Meyer, Nancy L.; Lerch, Thomas F.; Stagg, Scott M.; Chapman, Michael S.

    2013-01-01

    Mechanistic studies of macromolecular complexes often feature x-ray structures of complexes with bound ligands. The attachment of Adeno-Associated Virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8 Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5 Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable. PMID:24036405

  1. Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

    PubMed

    Wu, Hong-Xia; Wang, Hua-Lei; Guo, Xiao-Feng; Yang, Yu-Jiao; Ma, Jin-Zhu; Wang, Tie-Cheng; Gao, Yu-Wei; Zhao, Yong-Kun; Yang, Song-Tao; Xia, Xian-Zhu

    2013-10-01

    To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

  2. Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart.

    PubMed

    Prasad, Konkal-Matt R; Xu, Yaqin; Yang, Zequan; Toufektsian, Marie-Claire; Berr, Stuart S; French, Brent A

    2007-04-01

    Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P<0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Non-invasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.

  3. Retargeting transposon insertions by the adeno-associated virus Rep protein

    PubMed Central

    Ammar, Ismahen; Gogol-Döring, Andreas; Miskey, Csaba; Chen, Wei; Cathomen, Toni; Izsvák, Zsuzsanna; Ivics, Zoltán

    2012-01-01

    The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein–protein and protein–DNA interactions. PMID:22523082

  4. Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo.

    PubMed

    Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V; Nair, Sukesh C; Srinivasan, Narayanaswamy; Srivastava, Alok; Jayandharan, Giridhara R

    2013-04-01

    Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in

  5. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    PubMed Central

    Chung, Daniel C.; Fogelgren, Ben; Park, Kwon Moo; Heidenberg, Jessica; Zuo, Xiaofeng; Huang, Liwei; Bennett, Jean; Lipschutz, Joshua H.

    2011-01-01

    Background/Aims Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods Adeno-associated virus (AAV) offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber's congenital amaurosis, a disease of childhood blindness. Results Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury. PMID:22470395

  6. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    PubMed Central

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-01-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  7. TrkB gene therapy by adeno-associated virus enhances recovery after cervical spinal cord injury.

    PubMed

    Martínez-Gálvez, Gabriel; Zambrano, Juan M; Diaz Soto, Juan C; Zhan, Wen-Zhi; Gransee, Heather M; Sieck, Gary C; Mantilla, Carlos B

    2016-02-01

    Unilateral cervical spinal cord hemisection at C2 (C2SH) interrupts descending bulbospinal inputs to phrenic motoneurons, paralyzing the diaphragm muscle. Recovery after C2SH is enhanced by brain derived neurotrophic factor (BDNF) signaling via the tropomyosin-related kinase subtype B (TrkB) receptor in phrenic motoneurons. The role for gene therapy using adeno-associated virus (AAV)-mediated delivery of TrkB to phrenic motoneurons is not known. The present study determined the therapeutic efficacy of intrapleural delivery of AAV7 encoding for full-length TrkB (AAV-TrkB) to phrenic motoneurons 3 days post-C2SH. Diaphragm EMG was recorded chronically in male rats (n=26) up to 21 days post-C2SH. Absent ipsilateral diaphragm EMG activity was verified 3 days post-C2SH. A greater proportion of animals displayed recovery of ipsilateral diaphragm EMG activity during eupnea by 14 and 21 days post-SH after AAV-TrkB (10/15) compared to AAV-GFP treatment (2/11; p=0.031). Diaphragm EMG amplitude increased over time post-C2SH (p<0.001), and by 14 days post-C2SH, AAV-TrkB treated animals displaying recovery achieved 48% of the pre-injury values compared to 27% in AAV-GFP treated animals. Phrenic motoneuron mRNA expression of glutamatergic AMPA and NMDA receptors revealed a significant, positive correlation (r(2)=0.82), with increased motoneuron NMDA expression evident in animals treated with AAV-TrkB and that displayed recovery after C2SH. Overall, gene therapy using intrapleural delivery of AAV-TrkB to phrenic motoneurons is sufficient to promote recovery of diaphragm activity, adding a novel potential intervention that can be administered after upper cervical spinal cord injury to improve impaired respiratory function. PMID:26607912

  8. Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

    PubMed Central

    Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Kekilli, Demet; Samso, Monserrat

    2013-01-01

    The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle. PMID:23152528

  9. Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter

    PubMed Central

    Cronin, Therese; Vandenberghe, Luk H; Hantz, Péter; Juttner, Josephine; Reimann, Andreas; Kacsó, Ágota–Enikő; Huckfeldt, Rachel M; Busskamp, Volker; Kohler, Hubertus; Lagali, Pamela S; Roska, Botond; Bennett, Jean

    2014-01-01

    In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use. PMID:25092770

  10. The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

    PubMed Central

    Raupp, Christina; Naumer, Matthias; Müller, Oliver J.; Gurda, Brittney L.; Agbandje-McKenna, Mavis

    2012-01-01

    Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids 588QQNTA592 of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process. PMID:22718833

  11. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    SciTech Connect

    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  12. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  13. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    PubMed

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. PMID:26264580

  14. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    PubMed

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future.

  15. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    PubMed

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

  16. Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR.

    PubMed

    Rohr, Ulrich-Peter; Wulf, Marc-Andre; Stahn, Susanne; Steidl, Ulrich; Haas, Rainer; Kronenwett, Ralf

    2002-10-01

    In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.

  17. Adeno-associated virus-mediated expression of apolipoprotein (a) kringles suppresses hepatocellular carcinoma growth in mice.

    PubMed

    Lee, Kyuhyun; Yun, Sung-Tae; Kim, Young-Gun; Yoon, Yeup; Jo, Eui-Cheol

    2006-05-01

    Hepatocellular carcinoma (HCC) constitutes more than 90% of all primary liver cancers. HCC is a hypervascular tumor that develops from dedifferentiation of small avascular HCC and is therefore a good target for anti-angiogenic gene therapy. Recent studies have identified apolipoprotein(a) [apo(a)] kringles LK68 and LK8 (LKs) as having a potential antiangiogenic and anti-tumor activity, and the current study evaluates the therapeutic potential of gene therapy with recombinant adeno-associated virus carrying genes encoding LKs (rAAV-LK) in the treatment of hypervascular HCC. We generated rAAV-LK to obtain persistent transgene expression in vivo, which is essential for anti-angiogenic therapy. The rAAV-produced LKs substantially inhibited proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro, validating their anti-angiogenic potential. Intramuscular administration of rAAV-LK gave 60% to 84% suppression (P < .05) of tumor growth in mice bearing subcutaneously transplanted HCC derived from Huh-7 and Hep3B cells, respectively. Histological and immunohistochemical analyses of HCC tumor sections showed that a single administration of rAAV-LK gave rise to persistent expression of LKs that inhibited tumor angiogenesis and triggered tumor apoptosis, and, thus, significantly suppressed tumor growth. The administration of rAAV-LK provided a significant survival benefit (P < .05), and 3 of 10 rAAV-LK-treated mice were still alive without visible tumors and without clinical symptoms 188 days after treatment. In conclusion, rAAV-LK is a potential candidate for anti-angiogenic gene therapy in the treatment of HCC.

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

    PubMed Central

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

    2010-01-01

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

  19. Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

    SciTech Connect

    Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.; Murrin, L. Charles . E-mail: cmurrin@unmc.edu; Lockridge, Oksana . E-mail: olockrid@unmc.edu

    2006-07-15

    Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.

  20. Effects of adeno-associated virus serotype and tissue-specific expression on circulating biomarkers of propionic acidemia.

    PubMed

    Guenzel, Adam J; Hillestad, Matthew L; Matern, Dietrich; Barry, Michael A

    2014-09-01

    Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca(-/-)(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

  1. Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

    PubMed Central

    Zaiss, Anne K.; Foley, Erin M.; Lawrence, Roger; Schneider, Lina S.; Hoveida, Hamidreza; Secrest, Patrick; Catapang, Arthur B.; Yamaguchi, Yu; Alemany, Ramon; Shayakhmetov, Dmitry M.; Esko, Jeffrey D.

    2015-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1HEP mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1HEP mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1HEP mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These

  2. Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.

    PubMed

    Cordier, L; Hack, A A; Scott, M O; Barton-Davis, E R; Gao, G; Wilson, J M; McNally, E M; Sweeney, H L

    2000-02-01

    In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.

  3. Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo.

    PubMed

    Zhu, Aijun; Ren, Yu; Wang, Ning; Jin, Qiuyue; Zhang, Dongchang; Yang, Guangxiao; Wang, Quanying

    2015-11-01

    Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma.

  4. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  5. Long-Term Sex-Biased Correction of Circulating Propionic Acidemia Disease Markers by Adeno-Associated Virus Vectors

    PubMed Central

    Guenzel, Adam J.; Collard, Renata; Kraus, Jan P.; Matern, Dietrich

    2015-01-01

    Abstract Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined. PMID:25654275

  6. Long-term sex-biased correction of circulating propionic acidemia disease markers by adeno-associated virus vectors.

    PubMed

    Guenzel, Adam J; Collard, Renata; Kraus, Jan P; Matern, Dietrich; Barry, Michael A

    2015-03-01

    Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined. PMID:25654275

  7. Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo.

    PubMed

    Zhu, Aijun; Ren, Yu; Wang, Ning; Jin, Qiuyue; Zhang, Dongchang; Yang, Guangxiao; Wang, Quanying

    2015-11-01

    Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma. PMID:26143116

  8. Correction of Multiple Striated Muscles in Murine Pompe Disease Through Adeno-associated Virus-Mediated Gene Therapy

    PubMed Central

    Sun, Baodong; Young, Sarah P.; Li, Ping; Di, Chunhui; Brown, Talmage; Salva, Maia Z.; Li, Songtao; Bird, Andrew; Yan, Zhen; Auten, Richard; Hauschka, Stephen D.; Koeberl, Dwight D.

    2009-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) stems from the deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that systemic administration of an adeno-associated virus (AAV) vector containing a muscle specific regulatory cassette could drive efficacious transgene expression in GAA-knockout (GAA-KO) mice. AAV2/8 vectors containing the muscle creatine kinase (CK1) or hybrid α-myosin heavy chain enhancer-/muscle creatine kinase enhancer-promoter (MHCK7) cassettes were compared. The CK1 reduced glycogen content by approximately 50% in the heart and quadriceps, in comparison to untreated GAA-KO mice, whereas the MHCK7 containing vector reduced glycogen content even further: >95% in heart and >75% in the diaphragm and quadriceps. Administration of the MHCK7-containing vector significantly increased striated muscle function as assessed by increased Rotarod times at 18 weeks post-injection, whereas the CK1-containing vector did not increase Rotarod performance. Transduction efficiency was evaluated with an AAV2/8 vector in which MHCK7 drives alkaline-phosphatase, revealing that many more myofibers were transduced in the quadriceps than in the gastrocnemius. An AAV2/9 vector containing the MHCK7 cassette corrected GAA deficiency in the skeletal muscles of the distal limb, including the gastrocnemius, extensor digitalis longus, and soleus; furthermore, glycogen accumulations were substantially cleared by hGAA expression therein. Importantly, type IIb myofibers in the extensor digitalis longus were transduced, thereby correcting a myofiber type that is unresponsive to enzyme replacement therapy. In summary, AAV8 and AAV9-pseudotyped vectors containing the MHCK7 regulatory cassette achieved enhanced efficacy in Pompe disease mice. PMID:18560415

  9. Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice

    PubMed Central

    Hsiao, Hung-Bo; Chou, Ai-Hsiang; Lin, Su-I; Lien, Shu-Pei; Tao, Mi-Hua

    2014-01-01

    Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system. PMID:25243194

  10. Delivery of human EV71 receptors by adeno-associated virus increases EV71 infection-induced local inflammation in adult mice.

    PubMed

    Hsiao, Hung-Bo; Chou, Ai-Hsiang; Lin, Su-I; Lien, Shu-Pei; Liu, Chia-Chyi; Chong, Pele; Chen, Chih-Yeh; Tao, Mi-Hua; Liu, Shih-Jen

    2014-01-01

    Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

  11. Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles.

    PubMed

    McConnell, Kellie I; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E

    2014-11-28

    There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.

  12. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

    PubMed

    Li, Hua; Zhang, Feng-Lan; Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

    2015-01-01

    The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

  13. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants

    PubMed Central

    Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

    2015-01-01

    The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures. PMID:26035716

  14. My Life with Adeno-Associated Virus: A Long Time Spent Studying a Short Genome

    PubMed Central

    2013-01-01

    My 45 years of studying the molecular biology of adeno-associated virus are recounted. Additional activities as a mentor, department chair, and medical school administrator are described, as are my activities in the public sphere, which involved national issues related to science policy and medical education. PMID:23781880

  15. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

    PubMed Central

    Schreiber, Claire A.; Sakuma, Toshie; Izumiya, Yoshihiro; Holditch, Sara J.; Hickey, Raymond D.; Bressin, Robert K.; Basu, Upamanyu; Koide, Kazunori; Asokan, Aravind; Ikeda, Yasuhiro

    2015-01-01

    Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. PMID:26244496

  16. Recombinant adeno-associated virus-delivered anginex inhibits angiogenesis and growth of HUVECs by regulating the Akt, JNK and NF-κB signaling pathways.

    PubMed

    Ma, Ke; Wang, Chuying; Geng, Qianqian; Fan, Yangwei; Ning, Jing; Yang, Haixia; Dong, Xuyuan; Dong, Danfeng; Guo, Yuyan; Wei, Xin; Li, Enxiao; Wu, Yinying

    2016-06-01

    Anginex is an artificial synthetic small molecule β-sheet-forming peptide shown to have anti-angiogenesis and antitumor effects in various solid tumors. However, its molecular mechanism remains largely unclear and efficient delivery methods for anginex remains to be developed. We report on the development of recombinant adeno-associated virus (rAAV2)-delivered anginex and the underlying mechanism of anti-angiogenesis and antitumor effects of anginex. We have successfully developed the rAAV2 vector to efficiently express anginex (rAAV2‑anginex). Transduction of rAAV2-anginex significantly induced apoptosis, and inhibited the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells in vitro. Western blot analysis revealed that rAAV2‑anginex inhibited the phosphorylation of Akt, while inducing the phosphorylation of JNK and activation of the NF-κB signaling pathway. In an in vivo CAM assay and xenograft model of SKOV3, rAAV2-anginex significantly reduced microvessel density (MVD) and vascular endothelial growth factor 165 (VEGF165), as demonstrated by immunohistochemistry analysis. Importantly, rAAV2-anginex inhibited tumor growth in an ovarian cancer SKOV3 cell nude mouse xenograft model. Our results suggest that rAAV2-anginex may inhibit tumor angiogenesis and growth through regulating Akt, JNK and NF-κB signaling pathways. PMID:27035232

  17. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    PubMed Central

    Wang, Yuan-Yi; Zhu, Qing-San; Wang, Yi-Wei; Yin, Ruo-Feng

    2015-01-01

    Background: Thymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation. Methods: TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage. Results: NP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group. Conclusions: TB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases. PMID:26021512

  18. Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

    PubMed

    Dane, A P; Cunningham, S C; Kok, C Y; Logan, G J; Alexander, I E

    2015-11-01

    Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

  19. Treatment of congenital neurotransmitter deficiencies by intracerebral ventricular injection of an adeno-associated virus serotype 9 vector.

    PubMed

    Lee, Ni-Chung; Chien, Yin-Hsiu; Hu, Min-Hsiu; Liu, Wen-Shin; Chen, Pin-Wen; Wang, Wei-Hua; Tzen, Kai-Yuan; Byrne, Barry J; Hwu, Wuh-Liang

    2014-03-01

    Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice. PMID:24251946

  20. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    PubMed

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  1. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    SciTech Connect

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  2. Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

    PubMed

    Enomoto, Mitsuhiro; Hirai, Takashi; Kaburagi, Hidetoshi; Yokota, Takanori

    2016-01-01

    RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system. PMID:26472458

  3. Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

    PubMed

    Enomoto, Mitsuhiro; Hirai, Takashi; Kaburagi, Hidetoshi; Yokota, Takanori

    2016-01-01

    RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system.

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

    Balagúe, C; Kalla, M; Zhang, W W

    1997-01-01

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

  6. In vivo evaluation of adeno-associated virus gene transfer in airways of mice with acute or chronic respiratory infection.

    PubMed

    Myint, Melissa; Limberis, Maria P; Bell, Peter; Somanathan, Suryanarayan; Haczku, Angela; Wilson, James M; Diamond, Scott L

    2014-11-01

    Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼10(5) CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼10(5) CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer. PMID:25144316

  7. In Vivo Evaluation of Adeno-Associated Virus Gene Transfer in Airways of Mice with Acute or Chronic Respiratory Infection

    PubMed Central

    Myint, Melissa; Limberis, Maria P.; Bell, Peter; Somanathan, Suryanarayan; Haczku, Angela; Wilson, James M.

    2014-01-01

    Abstract Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼105 CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼105 CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer. PMID:25144316

  8. Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors.

    PubMed

    Watson, Deborah J; Passini, Marco A; Wolfe, John H

    2005-01-01

    Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors. PMID:15703488

  9. A retrograde adeno-associated virus for collecting ribosome-bound mRNA from anatomically defined projection neurons

    PubMed Central

    Cook-Snyder, Denise R.; Jones, Alexander; Reijmers, Leon G.

    2015-01-01

    The brain contains a large variety of projection neurons with different functional properties. The functional properties of projection neurons arise from their connectivity with other neurons and their molecular composition. We describe a novel tool for obtaining the gene expression profiles of projection neurons that are anatomically defined by the location of their soma and axon terminals. Our tool utilizes adeno-associated virus serotype 9 (AAV9), which we found to retrogradely transduce projection neurons after injection at the site of the axon terminals. We used AAV9 to express Enhanced Green Fluorescent Protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a), which enables the immunoprecipitation of EGFP-tagged ribosomes and associated mRNA with a method known as Translating Ribosome Affinity Purification (TRAP). To achieve high expression of the EGFP-L10a protein in projection neurons, we placed its expression under control of a 1.3 kb alpha-calcium/calmodulin-dependent protein kinase II (Camk2a) promoter. We injected the AAV9-Camk2a-TRAP virus in either the hippocampus or the bed nucleus of the stria terminalis (BNST) of the mouse brain. In both brain regions the 1.3 kb Camk2a promoter did not confer complete cell-type specificity around the site of injection, as EGFP-L10a expression was observed in Camk2a-expressing neurons as well as in neuronal and non-neuronal cells that did not express Camk2a. In contrast, cell-type specific expression was observed in Camk2a-positive projection neurons that were retrogradely transduced by AAV9-Camk2a-TRAP. Injection of AAV9-Camk2a-TRAP into the BNST enabled the use of TRAP to collect ribosome-bound mRNA from basal amygdala projection neurons that innervate the BNST. AAV9-Camk2a-TRAP provides a single-virus system that can be used for the molecular profiling of anatomically defined projection neurons in mice and other mammalian model organisms. In addition, AAV9-Camk2a-TRAP may enable the discovery of protein synthesis

  10. Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.

    PubMed

    Cordier, L; Gao, G P; Hack, A A; McNally, E M; Wilson, J M; Chirmule, N; Sweeney, H L

    2001-01-20

    Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

  11. A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors.

    PubMed

    Clark, K R; Voulgaropoulou, F; Johnson, P R

    1996-12-01

    Adeno-associated virus (AAV) vectors are being developed for in vivo and ex vivo gene transfer to human cells. At present, widespread usage of AAV vectors is limited primarily by difficulties in generating recombinant virions on a scale sufficient for in-depth preclinical and clinical trials. However, recent work in several laboratories suggests that this technical obstacle should be overcome in the near future. As a result, it can be anticipated that the interest in AAV vectors will expand, Thus, it becomes important to develop assay systems that will permit accurate quantification of the infectivity of AAV vectors derived from a variety of sources. We have developed an assay using a cell line that expresses AAV helper functions (rep and cap) upon induction by adenovirus infection. This assay system is based on the replication of input rAAV genomes rather than transgene expression (transduction). Thus, infectivity titrations in this system yield an estimation of rAAV infectious particles irrespective of the promoter or transgene present in the vector genome. Moreover, this assay method is more sensitive than conventional methods being used in other laboratories.

  12. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery.

    PubMed

    Weber, Michel; Rabinowitz, Joseph; Provost, Nathalie; Conrath, Hervé; Folliot, Sébastien; Briot, Delphine; Chérel, Yan; Chenuaud, Pierre; Samulski, Jude; Moullier, Philippe; Rolling, Fabienne

    2003-06-01

    We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

  13. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    SciTech Connect

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  14. Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

    PubMed Central

    Cecchini, Sylvain; Virag, Tamas

    2011-01-01

    Abstract The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 106 cells/ml, ≥1016 purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned. PMID:21381980

  15. Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

    SciTech Connect

    O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

    2009-03-15

    Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.

  16. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    SciTech Connect

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna . E-mail: anna.salvetti@univ-nantes.fr

    2005-05-10

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.

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

    PubMed

    Batchu, R B; Hermonat, P L

    1995-07-01

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

  18. A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells.

    PubMed

    Shayakhmetov, Dmitry M; Carlson, Cheryl A; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André

    2002-02-01

    To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.

  19. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity.

    PubMed

    Prasad, C Krishna; Meyers, Craig; Zhan, De-Jin; You, Hong; Chiriva-Internati, Maurizio; Mehta, Jawahar L; Liu, Yong; Hermonat, Paul L

    2003-09-15

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.

  20. Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters.

    PubMed Central

    Hörer, M; Weger, S; Butz, K; Hoppe-Seyler, F; Geisen, C; Kleinschmidt, J A

    1995-01-01

    The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterologous promoters. To gain insight into the molecular mechanism of Rep-mediated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papillomavirus type 18 upstream regulatory region HPV18 URR, the human immunodeficiency virus long terminal repeat, and the AAV-2 p5 and p19 promoters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C- and N-terminal sequences in which the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Rep52 was reduced and nearly completely abolished for Rep40. The contribution of the C terminus of Rep78 was reduced with respect to the inhibition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc binding in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains with the exception of the AAV-2 p5 promoter. Expression of the AAV-2 p5 promoter was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficiency virus long terminal repeat, and the HPV18 URR was not. Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition process. This finding suggests that transcriptional repression is mediated by protein-protein interactions of the Rep proteins either with multiple transcription factors or with target proteins of sequence-specific transcription factors of the basal

  1. Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system.

    PubMed

    Zacchigna, Serena; Zentilin, Lorena; Giacca, Mauro

    2014-05-23

    The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.

  2. Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 7

    SciTech Connect

    Quesada, Odayme; Gurda, Brittney; Govindasamy, Lakshmanan; McKenna, Robert; Kohlbrenner, Erik; Aslanidi, George; Zolotukhin, Sergei; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2007-12-01

    Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids have been produced which diffract X-rays to ∼3.0 Å resolution. Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids diffract X-rays to ∼3.0 Å resolution. The crystals belong to the rhombohedral space group R3, with unit-cell parameters a = 252.4, c = 591.2 Å in the hexagonal setting. The diffraction data were processed and reduced to an overall completeness of 79.0% and an R{sub merge} of 12.0%. There are three viral capsids in the unit cell. The icosahedral threefold axis is coincident with the crystallographic threefold axis, resulting in one third of a capsid (20 monomers) per crystallographic asymmetric unit. The orientation of the viral capsid has been determined by rotation-function searches and is positioned at (0, 0, 0) by packing considerations.

  3. Adeno-Associated Virus-2 and its Primary Cellular Receptor – Cryo-EM Structure of a Heparin Complex

    PubMed Central

    O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

    2009-01-01

    Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17kDa heparin fragment at 8.3Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R448/585, R451/588 and R350/487 from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R347/484, K395/532 and K390/527 that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multivalent attachment to symmetry-related binding sites. PMID:19144372

  4. Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle.

    PubMed

    Schnepp, Bruce C; Chulay, Jeffrey D; Ye, Guo-Jie; Flotte, Terence R; Trapnell, Bruce C; Johnson, Philip R

    2016-01-01

    Gene augmentation therapy as a strategy to treat alpha-1 antitrypsin (AAT) deficiency has reached phase 2 clinical testing in humans. Sustained serum levels of AAT have been observed beyond one year after intramuscular administration of a recombinant adeno-associated virus (rAAV) vector expressing the AAT gene. In this study, sequential muscle biopsies obtained at 3 and 12 months after vector injection were examined for the presence of rAAV vector genomes. Each biopsy sample contained readily detectable vector DNA, the majority of which existed as double-stranded supercoiled and open circular episomes. Episomes persisted through 12 months, although at slightly lower levels than observed at 3 months. There was a clear dose response when comparing the low- and mid-vector-dose groups to the high-dose group. The highest absolute copy numbers were found in a high-dose subject, and serum AAT levels at 12 months confirmed that the high-dose group also had the highest sustained serum AAT levels. Sequence analysis revealed that the vast majority of episomes contained double-D inverted terminal repeats ranging from fully intact to severely deleted. Molecular clones of vector genomes derived directly from the biopsies were transcriptionally active, potentially identifying them as the source of serum AAT in the trial subjects.

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

    PubMed Central

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

    2014-01-01

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

  6. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-04-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

  7. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed Central

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-01-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS. Images PMID:8139545

  8. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

    PubMed

    Hermens, W T; ter Brake, O; Dijkhuizen, P A; Sonnemans, M A; Grimm, D; Kleinschmidt, J A; Verhaagen, J

    1999-07-20

    Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

  9. Short-lived recombinant adeno-associated virus transgene expression in dystrophic muscle is associated with oxidative damage to transgene mRNA

    PubMed Central

    Dupont, Jean-Baptiste; Tournaire, Benoit; Georger, Christophe; Marolleau, Béatrice; Jeanson-Leh, Laurence; Ledevin, Mireille; Lindenbaum, Pierre; Lecomte, Emilie; Cogné, Benjamin; Dubreil, Laurence; Larcher, Thibaut; Gjata, Bernard; Van Wittenberghe, Laetitia; Le Guiner, Caroline; Penaud-Budloo, Magalie; Snyder, Richard O; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Preclinical gene therapy strategies using recombinant adeno-associated virus (AAV) vectors in animal models of Duchenne muscular dystrophy have shown dramatic phenotype improvements, but long-lasting efficacy remains questionable. It is believed that in dystrophic muscles, transgene persistence is hampered, notably by the progressive loss of therapeutic vector genomes resulting from muscle fibers degeneration. Intracellular metabolic perturbations resulting from dystrophin deficiency could also be additional factors impacting on rAAV genomes and transgene mRNA molecular fate. In this study, we showed that rAAV genome loss is not the only cause of reduced transgene mRNA level and we assessed the contribution of transcriptional and post-transcriptional factors. We ruled out the implication of transgene silencing by epigenetic mechanisms and demonstrated that rAAV inhibition occurred mostly at the post-transcriptional level. Since Duchenne muscular dystrophy (DMD) physiopathology involves an elevated oxidative stress, we hypothesized that in dystrophic muscles, transgene mRNA could be damaged by oxidative stress. In the mouse and dog dystrophic models, we found that rAAV-derived mRNA oxidation was increased. Interestingly, when a high expression level of a therapeutic transgene is achieved, oxidation is less pronounced. These findings provide new insights into rAAV transductions in dystrophic muscles, which ultimately may help in the design of more effective clinical trials. PMID:26029721

  10. Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

    NASA Astrophysics Data System (ADS)

    Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

    1997-12-01

    About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

  11. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  12. Triple trans-splicing adeno-associated virus vectors capable of transferring the coding sequence for full-length dystrophin protein into dystrophic mice.

    PubMed

    Koo, Taeyoung; Popplewell, Linda; Athanasopoulos, Takis; Dickson, George

    2014-02-01

    Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying "in tandem" sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.

  13. Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency.

    PubMed

    Gruntman, Alisha M; Flotte, Terence R

    2015-06-01

    The pathway to a clinical gene therapy product often involves many changes of course and strategy before obtaining successful results. Here we outline the methodologies, both clinical and preclinical, that went into developing a gene therapy approach to the treatment of alpha-1 antitrypsin deficiency lung disease using muscle-targeted recombinant adeno-associated virus. From initial gene construct development in mouse models through multiple rounds of safety and biodistribution studies in rodents, rabbits, and nonhuman primates to ultimate human trials, this review seeks to provide insight into what clinical translation entails and could thereby inform the process for future investigators.

  14. Enhancement of Recombinant Adeno-Associated Virus Type 2-Mediated Transgene Expression in a Lung Epithelial Cell Line by Inhibition of the Epidermal Growth Factor Receptor

    PubMed Central

    Smith, Andrew D.; Collaco, Roy F.; Trempe, James P.

    2003-01-01

    Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as gene delivery systems because they show long-term expression in vivo and transduce numerous cell types. Limitations to successful gene transduction from rAAVs have prompted investigations of a variety of treatments to enhance transgene expression from rAAV vectors. Tyrphostin-1, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, dramatically enhances rAAV transgene expression. Elegant studies have demonstrated that a single-strand D-sequence-binding protein (ssDBP) is phosphorylated by EGFR and binds to the D sequence element in the AAV terminal repeat (TR). Binding of the Tyr-phosphorylated ssDBP prevents conversion of single-stranded vector DNA to a double-strand conformation. We observed dramatic increases in transgene expression in lung epithelial cells (IB3) with tyrphostin treatment. Gel shift analysis of ssDBP revealed that its DNA binding characteristics were unchanged after tyrphostin treatment or adenovirus infection. Tyrphostin stimulated rAAV transgene expression to a greater extent than adenovirus coinfection. Southern hybridizations revealed that the vector DNA remained in the single-strand conformation in tyrphostin-treated cells but double-stranded replicative form monomer DNA was most abundant in adenovirus-infected cells. Northern analyses revealed that tyrphostin treatment enhanced mRNA accumulation more than in adenovirus-infected cultures even though replicative form DNA was undetectable. Analysis of the JNK, ERK, and p38K mitogen-activated protein kinase pathways revealed that tyrphostin treatment stimulated the activity of JNK and p38K. Our data suggest that tyrphostin-induced alteration of stress response pathways results in dramatic enhancement of transcription on linear vector DNA templates in the IB3 cell line. These results expand the downstream targets of the EGFR in regulating rAAV transduction. PMID:12743297

  15. Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

    PubMed Central

    Salganik, Maxim; Aydemir, Fikret; Nam, Hyun-Joo; McKenna, Robert; Agbandje-McKenna, Mavis

    2014-01-01

    A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>107-fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3- to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome. PMID:24198419

  16. Adeno-associated virus-like particles as new carriers for B-cell vaccines: testing immunogenicity and safety in BALB/c mice.

    PubMed

    Manzano-Szalai, Krisztina; Thell, Kathrin; Willensdorfer, Anna; Weghofer, Margit; Pfanzagl, Beatrix; Singer, Josef; Ritter, Mirko; Stremnitzer, Caroline; Flaschberger, Ingo; Michaelis, Uwe; Jensen-Jarolim, Erika

    2014-11-01

    Adeno-associated viruses (AAVs) are established vectors for gene therapy of different human diseases. AAVs are assembled of 60 capsomers, which can be genetically modified, allowing high-density display of short peptide sequences at their surface. The aim of our study was to evaluate the immunogenicity and safety of an adeno-associated virus-like particle (AAVLP)-displayed B-cell peptide epitope taking ovalbumin (OVA) as a model antigen or allergen from egg, respectively. An OVA-derived B-cell epitope was expressed as fusion protein with the AAV-2 capsid protein of VP3 (AAVLP-OVA) and for control, with the nonrelated peptide TP18 (AAVLP-TP18). Cellular internalization studies revealed an impaired uptake of AAVLP-OVA by mouse BMDC, macrophages, and human HeLa cells. Nevertheless, BALB/c mice immunized subcutaneously with AAVLP-OVA formed similarly high titers of OVA-specific IgG1 compared to mice immunized with the native OVA. The extent of the immune response was independent whether aluminum hydroxide or water in oil emulsion was used as adjuvant. Furthermore, in mice immunized with native OVA, high OVA-specific IgE levels were observed, which permitted OVA-specific mast-cell degranulation in a β-hexosaminidase release assay, whereas immunizations with AAVLP-OVA rendered background IgE levels only. Accordingly, OVA-immunized mice, but not AAVLP-OVA immunized mice, displayed an anaphylactic reaction with a significant drop of body temperature upon intravenous OVA challenge. From this mouse model, we conclude that AAVLPs that display B-cell epitope peptides on their surface are suitable vaccine candidates, especially in the field of allergy. PMID:25247267

  17. Effect of recombinant adeno-associated virus mediated transforming growth factor-beta1 on corneal allograft survival after high-risk penetrating keratoplasty.

    PubMed

    Zhou, Lianhong; Zhu, Xiangxiang; Tan, Jinquan; Wang, Jiong; Xing, Yiqiao

    2013-06-01

    Corneal transplantation is one of the most common and successful transplant surgeries performed around the world. However, the high-risk corneal transplantation remains a high level of corneal graft failure. Gene transfer of immunomodulatory molecules is considered as one potential strategy in preventing allograft rejection. It is worthy evaluating the effects of the immunemodulating agent on corneal allograft rejection. The purpose of this paper is to investigate the effects and mechanisms of recombinant adeno-associated virus mediated transforming growth factor-beta1 (rAAV-TGF-beta1) on corneal allograft survival using a high-risk rat model after penetrating keratoplasty (PKP). The mean survival time (MST) of corneal grafts was observed and immuno-histochemical staining of TGF-beta1 and Ox-62 was performed in the study. The MST showed significant prolongation in the rAAV-TGF-beta1 group compared to the allograft group. The rejection index (RI) at day 10 revealed was significantly greater in the allograft group than that of the other two groups. Besides the increase of TGF-beta1, the expression of Ox-62 decreasing in rAAV-TGF-beta1 transplanted recipients was detected after transplantation. In short, treatment with rAAV-TGF-beta1 prolongs corneal allograft survival and inhibits the Ox-62 expression in grafts after high-risk PKP.

  18. Cloning, expression and purification of full length Rep78 of adeno-associated virus as a fusion protein with maltose binding protein in Escherichia coli.

    PubMed

    Batchu, R B; Miles, D A; Rechtin, T M; Drake, R R; Hermonat, P L

    1995-03-17

    The adeno-associated virus (AAV) Rep78 protein is required for many aspects of AAV's life cycle including its DNA replication and the regulation of its gene expression. Because of increasing utilization of AAV as a gene therapy vector and its possible use as an anti-cancer/anti-viral agent, the complete characterization of its Rep78 regulatory protein is important. In order to study various functional aspects of Rep78, we have cloned and expressed the Rep78 gene in Escherichia coli using an inducible expression plasmid. The entire Rep78 open reading frame (nt 321 to 2185) was cloned into the LacZ inducible expression vector pMALc2. Upon induction of the Ptac promoter with isopropyl thio-beta-D-galactopyranoside (IPTG), Rep78 is produced as a fusion protein with maltose binding protein (MBP). This recombinant MBP-Rep78 protein displayed all the biochemical activities which are described for the wild type protein including binding to the AAV terminal repeats (TR), endonuclease activity, and helicase activity. Furthermore, for the first time, ATP binding by Rep78 is demonstrated.

  19. Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

    PubMed Central

    Venkatakrishnan, Balasubramanian; Yarbrough, Joseph; Domsic, John; Bennett, Antonette; Bothner, Brian; Kozyreva, Olga G.; Samulski, R. Jude; Muzyczka, Nicholas

    2013-01-01

    The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus. PMID:23427155

  20. Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking.

    PubMed

    Venkatakrishnan, Balasubramanian; Yarbrough, Joseph; Domsic, John; Bennett, Antonette; Bothner, Brian; Kozyreva, Olga G; Samulski, R Jude; Muzyczka, Nicholas; McKenna, Robert; Agbandje-McKenna, Mavis

    2013-05-01

    The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus. PMID:23427155

  1. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    PubMed

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  2. Suppressing tumor growth of nasopharyngeal carcinoma by hTERTC27 polypeptide delivered through adeno-associated virus plus adenovirus vector cocktail

    PubMed Central

    Liu, Xiong; Li, Xiang-Ping; Peng, Ying; Ng, Samuel S.; Yao, Hong; Wang, Zi-Feng; Wang, Xiao-Mei; Kung, Hsiang-Fu; Lin, Marie C.M.

    2012-01-01

    Nasopharyngeal carcinoma (NPC) is a metastatic carcinoma that is highly prevalent in Southeast Asia. Our laboratory has previously demonstrated that the C-terminal 27-kDa polypeptide of human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human glioblastoma and melanoma cells. In this study, we investigated the antitumor effect of hTERTC27 in human C666-1 NPC cells xenografted in a nude mouse model. A cocktail of vectors comprising recombinant adeno-associated virus (rAAV) and recombinant adenovirus (rAdv) that each carry hTERTC27 (rAAV-hTERTC27 and rAdv-hTERTC27; the cocktail was abbreviated to rAAV/rAdv-hTERTC27) was more effective than either rAAV-hTERTC27 or rAdv-hTERTC27 alone in inhibiting the growth of C666-1 NPC xenografts. Furthermore, we established three tumors on each mouse and injected rAAV/rAdv-hTERTC27 into one tumor per mouse. Although hTERTC27 expression could only be detected in the injected tumors, reduced tumor growth was observed in the injected tumor as well as the uninjected tumors, demonstrating that the vector cocktail could provoke an antitumor effect on distant, metastasized tumors. Further studies showed the observed antitumor effects included inducing necrosis and apoptosis and reducing microvessel density. Together, our data suggest that the rAAV/rAdv-hTERTC27 cocktail can potently inhibit NPC tumor growth in both local and metastasized tumors and should be further developed as a novel gene therapy strategy for NPC. PMID:23149313

  3. Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection.

    PubMed Central

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

    1995-01-01

    To further define the canonical binding site for the P5-promoted Rep proteins of the adeno-associated virus, a modified random oligonucleotide selection procedure was performed, using purified recombinant Rep protein. These results may explain the effects of Rep on cellular gene expression. PMID:7474165

  4. Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

    SciTech Connect

    Cassell, Geoffrey D.; Weitzman, Matthew D. . E-mail: weitzman@salk.edu

    2004-10-01

    Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha} interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.

  5. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    PubMed

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  6. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

    PubMed Central

    Bertran, J; Miller, J L; Yang, Y; Fenimore-Justman, A; Rueda, F; Vanin, E F; Nienhuis, A W

    1996-01-01

    Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. PMID:8794313

  7. ADENO-ASSOCIATED SATELLITE VIRUS INTERFERENCE WITH THE REPLICATION OF ITS HELPER ADENOVIRUS

    PubMed Central

    Parks, Wade P.; Casazza, Anna M.; Alcott, Judith; Melnick, Joseph L.

    1968-01-01

    Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80°C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This

  8. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

    PubMed

    Daily, Jennifer L; Nash, Kevin; Jinwal, Umesh; Golde, Todd; Rogers, Justin; Peters, Melinda M; Burdine, Rebecca D; Dickey, Chad; Banko, Jessica L; Weeber, Edwin J

    2011-01-01

    Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286) and Thr(305/306), resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  9. Adeno-Associated Virus Mediated Delivery of An Engineered Protein that Combines the Complement Inhibitory Properties of CD46, CD55 and CD59

    PubMed Central

    Leaderer, Derek; Cashman, Siobhan M.; Kumar-Singh, Rajendra

    2015-01-01

    Background A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. Herein we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. Methods Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC) was measured. Results SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59 respectively in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7% respectively. Conclusions When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach. PMID:25917932

  10. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

    PubMed Central

    SUN, Zhan; MA, Yi-Tong; CHEN, Bang-Dang; LIU, Fen

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the underlying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease. PMID:25593580

  11. A single injection of recombinant adeno-associated virus into the lumbar cistern delivers transgene expression throughout the whole spinal cord

    PubMed Central

    Guo, Yansu; Wang, Dan; Qiao, Tao; Yang, Chunxing; Su, Qin; Gao, Guangping; Xu, Zuoshang

    2015-01-01

    The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in sixty to ninety percent of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad CNS, the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ∼100 fold. Finally, the transduced areas were colocalized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases. PMID:26050084

  12. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

    PubMed Central

    Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

    1997-01-01

    We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

  13. A chimeric human APOBEC3A protein with a three amino acid insertion confers differential HIV-1 and adeno-associated virus restriction.

    PubMed

    Wang, Yaqiong; Wang, Zekun; Pramanik, Ankita; Santiago, Mario L; Qiu, Jianming; Stephens, Edward B

    2016-11-01

    Old World monkey (OWM) and hominid APOBEC3Aproteins exhibit differential restriction activities against lentiviruses and DNA viruses. Human APOBEC3A(hA3A)has weak restriction activity against HIV-1Δvifbut is efficiently restricted by an artificially generated chimeric from mandrills (mndA3A/G). We show that a chimeric hA3Acontaining the "WVS" insertion (hA3A[(27)WVS(29)]) conferred potent HIV-1restriction activity. Analysis of each amino acid of the "WVS" motif show that the length and not necessarily the charge or hydrophobicity of the amino acids accounted for restriction activity. Our results suggest that hA3A[(27)WVS(29)]restricts HIV-1at the level of reverse transcription in target cells. Finally, our results suggest that insertion of "WVS" into hA3Amodestly reduces restriction of adeno-associated virus 2(AAV-2)while insertion of the AC Loop1region of the mndA3A/G into hA3A abolished AAV-2 restriction, strengthening the role of this molecular interface in the functional evolution of primate A3A. PMID:27584592

  14. Evolutionary Relationships among Parvoviruses: Virus-Host Coevolution among Autonomous Primate Parvoviruses and Links between Adeno-Associated and Avian Parvoviruses

    PubMed Central

    Lukashov, Vladimir V.; Goudsmit, Jaap

    2001-01-01

    The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary groups of parvoviruses from vertebrates: (i) the human helper-dependent adeno-associated virus (AAV) serotypes 1 to 6 and the autonomous avian parvoviruses; (ii) the bovine, chipmunk, and autonomous primate parvoviruses, including human viruses B19 and V9; and (iii) the parvoviruses from rodents (except for chipmunks), carnivores, and pigs. Each of these three evolutionary groups could be further subdivided, reflecting both virus-host coevolution and multiple cross-species transmissions in the evolutionary history of parvoviruses. No parvoviruses from invertebrates clustered with vertebrate parvoviruses. Our analysis provided evidence for negative selection among parvoviruses, the independent evolution of their genes, and recombination among parvoviruses from rodents. The topology of the phylogenetic tree of autonomous human and simian parvoviruses matched exactly the topology of the primate family tree, as based on the analysis of primate mitochondrial DNA. Viruses belonging to the AAV group were not evolutionarily linked to other primate parvoviruses but were linked to the parvoviruses of birds. The two lineages of human parvoviruses may have resulted from independent ancient zoonotic infections. Our results provide an argument for reclassification of Parvovirinae based on evolutionary relationships among viruses. PMID:11222696

  15. Host Anti-antibody Responses Following Adeno-associated Virus-mediated Delivery of Antibodies Against HIV and SIV in Rhesus Monkeys.

    PubMed

    Martinez-Navio, José M; Fuchs, Sebastian P; Pedreño-López, Sònia; Rakasz, Eva G; Gao, Guangping; Desrosiers, Ronald C

    2016-02-01

    Long-term delivery of antibodies against the human immunodeficiency virus (HIV) using adeno-associated virus (AAV) vectors is a promising approach for the prevention or treatment of HIV infection. However, host antibody responses to the delivered antibody are a serious concern that could significantly limit the applicability of this approach. Here, we describe the dynamics and characteristics of the anti-antibody responses in monkeys that received either rhesus anti-simian immunodeficiency virus (SIV) antibodies (4L6 or 5L7) in prevention trials or a combination of rhesusized human anti-HIV antibodies (1NC9/8ANC195/3BNC117 or 10-1074/10E8/3BNC117) in therapy trials, all employing AAV1 delivery of IgG1. Eight out of eight monkeys that received the anti-HIV antibodies made persisting antibody responses to all three antibodies in the mix. Six out of six uninfected monkeys that received the anti-SIV antibody 4L6 and three out of six of those receiving anti-SIV antibody 5L7 also generated anti-antibodies. Both heavy and light chains were targeted, predominantly or exclusively to variable regions, and reactivity to complementarity-determining region (CDR)-H3 peptide could be demonstrated. There was a highly significant correlation of the magnitude of anti-antibody responses with the degree of sequence divergence of the delivered antibody from germline. Our results suggest the need for effective strategies to counteract the problem of antibody responses to AAV-delivered antibodies.

  16. Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

    PubMed

    Gomez, Eric J; Gerhardt, Karl; Judd, Justin; Tabor, Jeffrey J; Suh, Junghae

    2016-01-26

    Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.

  17. Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

    PubMed

    Gomez, Eric J; Gerhardt, Karl; Judd, Justin; Tabor, Jeffrey J; Suh, Junghae

    2016-01-26

    Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts. PMID:26618393

  18. Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning

    PubMed Central

    Corti, Manuela; Cleaver, Brian; Clément, Nathalie; Conlon, Thomas J.; Faris, Kaitlyn J.; Wang, Gensheng; Benson, Janet; Tarantal, Alice F.; Fuller, Davis; Herzog, Roland W.; Byrne, Barry J.

    2015-01-01

    A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa−/− mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own

  19. CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

    PubMed

    Wu, Te-Lang; Li, Hua; Faust, Susan M; Chi, Emily; Zhou, Shangzhen; Wright, Fraser; High, Katherine A; Ertl, Hildegund C J

    2014-01-01

    Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

  20. A Novel Adeno-Associated Virus–Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine

    PubMed Central

    Zhu, Fengqin; Chen, Tian; Zhang, Yeqiong; Sun, Haixia; Cao, Hong; Lu, Jianxi; Zhao, Linshan; Li, Gang

    2015-01-01

    More than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV) vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine. PMID:26556235

  1. Adeno-associated virus serotype 8 gene therapy leads to significant lowering of plasma cholesterol levels in humanized mouse models of homozygous and heterozygous familial hypercholesterolemia.

    PubMed

    Kassim, Sadik H; Li, Hui; Bell, Peter; Somanathan, Suryanarayan; Lagor, William; Jacobs, Frank; Billheimer, Jeffrey; Wilson, James M; Rader, Daniel J

    2013-01-01

    Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a "humanized" mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5 × 10(11) genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly "humanized" mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5 × 10(11) GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH. PMID:22985273

  2. Adeno-Associated Virus Serotype 8 Gene Therapy Leads to Significant Lowering of Plasma Cholesterol Levels in Humanized Mouse Models of Homozygous and Heterozygous Familial Hypercholesterolemia

    PubMed Central

    Kassim, Sadik H.; Li, Hui; Bell, Peter; Somanathan, Suryanarayan; Lagor, William; Jacobs, Frank; Billheimer, Jeffrey; Rader, Daniel J.

    2013-01-01

    Abstract Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a “humanized” mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5×1011 genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly “humanized” mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5×1011 GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH. PMID:22985273

  3. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    SciTech Connect

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig Jameson, J. Larry

    2008-07-04

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the {beta}-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

  4. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice

    PubMed Central

    Heller, Kristin N.; Montgomery, Chrystal L.; Shontz, Kimberly M.; Clark, K. Reed; Mendell, Jerry R.; Rodino-Klapac, Louise R.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin–glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5–7 days of age to the mdx/utrn−/− mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn−/− mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

  5. Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

    PubMed Central

    Pasta, Saloni; Akhile, Omoye; Tabron, Dorothy; Ting, Flora; Shackleton, Cedric; Watson, Gordon

    2015-01-01

    Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies. PMID:26347274

  6. Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood

    PubMed Central

    1994-01-01

    Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK- Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU- granulocyte erythroid macrophage megakaryocyte colonies from mock- infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful

  7. Recombinant adeno-associated viral vector reference standards.

    PubMed

    Moullier, Philippe; Snyder, Richard O

    2012-01-01

    Reference standard materials (RSMs) exist for a variety of biologics including vaccines but are not readily available for gene therapy vectors. To date, a recombinant adeno-associated virus serotype 2 RSM (rAAV2 RSM) has been produced and characterized and was made available to the scientific community in 2010. In addition, a rAAV8 RSM has been produced and will be characterized in the coming months. The use of these reference materials by members of the gene therapy field facilitates the calibration of individual laboratory vector-specific internal standards and the eventual comparison of preclinical and clinical data based on common dosage units. Normalization of data to determine therapeutic dose ranges of rAAV vectors for each particular tissue target and disease indication is important information that can enhance the safety and protection of patients.

  8. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial.

    PubMed

    Ghazi, Nicola G; Abboud, Emad B; Nowilaty, Sawsan R; Alkuraya, Hisham; Alhommadi, Abdulrahman; Cai, Huimin; Hou, Rui; Deng, Wen-Tao; Boye, Sanford L; Almaghamsi, Abdulrahman; Al Saikhan, Fahad; Al-Dhibi, Hassan; Birch, David; Chung, Christopher; Colak, Dilek; LaVail, Matthew M; Vollrath, Douglas; Erger, Kirsten; Wang, Wenqiu; Conlon, Thomas; Zhang, Kang; Hauswirth, William; Alkuraya, Fowzan S

    2016-03-01

    MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed

  9. Effective inhibition of infectious bursal disease virus replication by recombinant avian adeno-associated virus-delivered microRNAs.

    PubMed

    Wang, Yongjuan; Sun, Huaichang; Shen, Pengpeng; Zhang, Xinyu; Xia, Xiaoli

    2009-06-01

    RNA interference (RNAi) is a novel antiviral strategy against a variety of virus infections. Infectious bursal disease virus (IBDV) causes an economically important disease in young chickens. This study demonstrated efficient inhibition of IBDV replication by recombinant avian adeno-associated virus (rAAAV)-delivered anti-VP1 and anti-VP2 microRNAs (miRNAs). In the viral vector-transduced cells, sequence-specific miRNA expression was detected by poly(A)-tailed RT-PCR. Reporter assays using a pVP2-EGFP vector showed significant and long-lasting inhibition of VP2-EGFP expression in cells transduced with anti-VP2 miRNA-expressing rAAAV-RFPmiVP2E, but not with the control miRNA-expressing rAAAV-RFPmiVP2con or anti-VP1 miRNA-expressing rAAAV-RFPmiVP1. Semi-quantitative RT-PCR and/or virus titration assays showed a significant inhibitory effect on homologous IBDV replication in cells transduced with rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E. For two heterologous IBDV isolates, transduction with rAAAV-RFPmiVP1 led to slightly weaker but similar inhibitory effects, whereas transduction with rAAAV-RFPmiVP2E resulted in significantly weaker and different inhibitory effects. These results suggest that rAAAV could act as an efficient vector for miRNA delivery into avian cells and that VP1 is the more suitable target for interfering with IBDV replication using RNAi technology.

  10. Adeno-associated virus mediated SOD gene therapy protects the retinal ganglion cells from chronic intraocular pressure elevation induced injury via attenuating oxidative stress and improving mitochondrial dysfunction in a rat model

    PubMed Central

    Jiang, Wenmin; Tang, Luosheng; Zeng, Jun; Chen, Baihua

    2016-01-01

    Purpose: This study aimed to determine whether chronic intraocular pressure (IOP) elevation induces retinal oxidative stress and alters mitochondrial morphology and function of retinal ganglion cells (RGC) and to explore the effects of AAV-SOD2 gene therapy on the RGC survival and mitochondrial dysfunction. Methods: Chronic experimental glaucoma was induced unilaterally in adult male Sprague-Dawley rats by laser burns at trabecular meshwork and episcleral veins 2 times with an interval of one week. One eye of each rat was intravitreally pretreated with recombinant adeno-associated virus expressing SOD2 (AAV-SOD2) or recombinant AAV expressing GFP (AAV-GFP) 21 days before glaucoma induction. RGCs counting, morphometric analysis of retina and optic nerve, and detection of activities of retinal SOD2 and catalase, MDA, mitochondrial morphology, mitochondrial dynamin protein OPA1 and DRP-1 expressions were conducted at 4, 8, 12 and 24 weeks. Results: Severe RGC loss, degeneration of optic nerve, reduced thickness of RGC layer and nerve fiber layer, significant decrease in total SOD and catalase activities, mitochondrial dysfunction and increased MDA were observed at 4, 8, 12 and 24 weeks after glaucoma. Pretreatment with AAV-SOD2 significantly reduced MDA and attenuated the damage to RGCs through a mitochondria-related pathway. Conclusion: AAV mediated pre-treatment with SOD2 is able to attenuate oxidative stress and improve mitochondrial dysfunction of RGC and optic nerve secondary to glaucoma. Thus, SOD2 may be used to prevent the retinal RGCs from glaucoma, which provides a promising strategy for glaucoma therapy. PMID:27158370

  11. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

    PubMed

    Monahan, Paul E; Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B; Wichlan, David G; Wu, Zhijian; Grieger, Joshua C; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W; Booth, Carmen J; Samulski, Jade J; Kafri, Tal; McPhee, Scott W J; Samulski, R Jude

    2015-02-01

    Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity

  12. Constitutive GABA expression via a recombinant adeno-associated virus consistently attenuates neuropathic pain.

    PubMed

    Lee, Boyoung; Kim, Jaehyung; Kim, Sung Jin; Lee, Heuiran; Chang, Jin Woo

    2007-06-15

    Peripheral neuropathic pain is a common clinical problem with few existing treatments. Previously, we constructed rAAV bearing GAD65 and demonstrated that GAD65 and GABA can be constitutively produced in the CNS. To investigate the beneficial effects of GAD65 produced by rAAV and resulting GABA release in peripheral neuropathic pain, we established a neuropathic pain rat model. The direct administration of rAAV-GAD65 to dorsal root ganglion induced constitutive GAD65 expression, which was readily detected by immunohistochemistry. Both allodynic and hyperalgeic behavior tests suggested that neuropathic pain was noticeably reduced, along with the transgenic GAD65 expression. Moreover, the magnitude of pain relief was maintained during the entire experimental period. Concomitantly, the significant enhancement in GABA release following transgenic GAD65 expression was identified in vivo. Taken all together, these results provide evidence that persistent GAD65 and subsequent GABA expression in DRGs via rAAV effectively attenuates peripheral neuropathic pain for long period of time.

  13. A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

    PubMed

    Gomes, Michelle M; Wang, Lingyan; Jiang, Han; Kahl, Christoph A; Brigande, John V

    2016-01-01

    There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders. PMID:27259920

  14. Manufacturing of recombinant adeno-associated viral vectors for clinical trials

    PubMed Central

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings. PMID:27014711

  15. Manufacturing of recombinant adeno-associated viral vectors for clinical trials.

    PubMed

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings.

  16. Construction and biological characterization of an interleukin-12 fusion protein (Flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus.

    PubMed

    Anderson, R; Macdonald, I; Corbett, T; Hacking, G; Lowdell, M W; Prentice, H G

    1997-06-10

    Interleukin-12 (IL-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. The combination of the immune costimulatory molecule B7.1 and IL-12 has been shown to be synergistic for T cell activation. By transfecting tumor cells with both IL-12 and B7.1 cDNAs, it may be possible to use these modified targets as vaccines. A major obstacle in designing a vector to deliver these genes results from the structure of IL-12. Functional IL-12 is a heterodimer composed of two distinct subunits that are encoded by separate genes on different chromosomes. Production of functional IL-12 requires the coordinated expression of both genes. This presents several problems in vectors, particularly those in which additional genes, either a co-stimulatory gene or a selectable marker, are inserted. Therefore, we have constructed a single cDNA that encodes a single-chain protein, called Flexi-12, which retains all of the biological characteristics of recombinant IL-12 (rIL-12). The monomeric polypeptide Flexi-12 is able to induce the proliferation of phytohemagglutinin (PHA) blasts, induce PHA blasts to secrete interferon-gamma (IFN-gamma) and additionally, by preincubation, enhance the killing of K562 targets by PBLs. These phenomena are in a dose-dependent manner comparable to that seen with rIL-12. We have also shown that tyrosine phosphorylation of the STAT 4 transcription factor, which has been shown to be unique to the IL-12 signaling pathway, occurs with Flexi-12 at levels similar to those seen with rIL-12. We have packaged Flexi-12 into a recombinant adeno-associated virus (AAV) and used this vector to infect acute myeloid leukemic (AML) blasts. Infected AML blasts produced between 2 and 6 ng of IL-12/10(6) cells per ml per 48 hr. These studies also confirm that AAV is an efficient delivery vehicle for cytokines to leukemic cells. Direct analysis of these modified cells acting

  17. Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection

    PubMed Central

    Lipinski, Daniel M.; Reid, Chris A.; Boye, Sanford L.; Peterson, James J.; Qi, Xiaoping; Boye, Shannon E.; Boulton, Michael E.; Hauswirth, William W.

    2015-01-01

    The ability to effectively deliver genetic material to vascular endothelial cells remains one of the greatest unmet challenges facing the development of gene therapies to prevent diseases with underlying vascular etiology, such as diabetes, atherosclerosis, and age-related macular degeneration. Herein, we assess the effectiveness of an rAAV2-based capsid mutant vector (Y272F, Y444F, Y500F, Y730F, T491V; termed QuadYF+TV) with strong endothelial cell tropism at transducing the vasculature after systemic administration. Intravenous injection of QuadYF+TV resulted in widespread transduction throughout the vasculature of several major organ systems, as assessed by in vivo bioluminescence imaging and postmortem histology. Robust transduction of lung tissue was observed in QuadYF+TV-injected mice, indicating a role for intravenous gene delivery in the treatment of chronic diseases presenting with pulmonary complications, such as α1-antitrypsin deficiency. The QuadYF+TV vector cross-reacted strongly with AAV2 neutralizing antibodies, however, indicating that a targeted delivery strategy may be required to maximize clinical translatability. PMID:26359319

  18. Mitochondria-Targeted Antiaging Gene Therapy with Adeno-associated Viral Vectors

    PubMed Central

    Li, Dejia; Duan, Dongsheng

    2015-01-01

    Transgenic expression of catalase in mitochondria using a transgenic strategy extends life span and prevents aging-related pathology in mice. However, transgenic overexpression is not suitable for a clinical application. Adeno-associated virus (AAV) is the most promising gene delivery vehicle. Here we outline strategies on the generation of an AAV vector expressing the mitochondria-targeted catalase gene (AV.RSV.MCAT). We also describe methods for evaluating physiological impact of AV.RSV.MCAT on muscle contractility and running performance in mice. PMID:23929105

  19. Adeno-associated virus-mediated knockdown of melanocortin-4 receptor in the paraventricular nucleus of the hypothalamus promotes high-fat diet-induced hyperphagia and obesity

    PubMed Central

    Garza, Jacob C; Kim, Chung Sub; Liu, Jing; Zhang, Wei; Lu, Xin-Yun

    2013-01-01

    Pharmacological and genetic studies have suggested that melanocortin-4 receptor (MC4R) signaling in the paraventricular nucleus of hypothalamus (PVN) regulates appetite and energy balance. However, the specific role of MC4R signaling in PVN neurons in these processes remains to be further elucidated in normally developed animals. In the present study, we employed RNA interference to determine whether MC4R knockdown in the PVN modulates food intake and body weight in adult rats. Adeno-associated viral (AAV) vectors encoding short hairpin RNAs targeting MC4R (AAV-shRNA-MC4R) were generated to induce MC4R knockdown in the PVN. By in situ hybridization, we detected a high-level expression of Dicer, a key enzyme required for shRNA-mediated gene silencing, along the entire rostrocaudal extent of the PVN. Bilateral injection of AAV-shRNA-MC4R vectors into the PVN of the adult rat resulted in significant and specific reduction of MC4R mRNA expression. Animals with MC4R knockdown exhibited an increase in food intake and excessive body weight gain when exposed to a high-fat diet. Our results provide evidence that AAV-mediated silencing of MC4R on PVN neurons promotes hyperphagia and obesity in response to the dietary challenge in the adult animal. PMID:18492813

  20. 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

  1. Avian Adeno-Associated Virus Vector Efficiently Transduces Neurons in the Embryonic and Post-Embryonic Chicken Brain

    PubMed Central

    Matsui, Ryosuke; Tanabe, Yasuto; Watanabe, Dai

    2012-01-01

    The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits. PMID:23144948

  2. Interference Between Two Adeno-associated Satellite Viruses: a Three-Component System

    PubMed Central

    Torikai, K.; Mayor, H. D.

    1969-01-01

    Adenovirus-associated satellite viruses interfere with the replication of their helper adenoviruses. According to a previous report, this interference is not mediated by interferon. A three-component system comprising simian adenovirus SV15 and satellites types 1 and 4 was studied to determine whether satellite viruses also interfere with one another. Satellite type 1 interfered with the replication of type 4 and vice versa. The degree of interference was directly proportional to the dose of interfering satellite. The events leading to mutual satellite interference were operative during the first 12 hr of replication, the period associated with active synthesis of viral deoxyribonucleic acid. PMID:5786177

  3. Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation

    PubMed Central

    Saunders, Arpiar

    2015-01-01

    Recombinant adeno-associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre-On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre-Off) can be used in conjunction with Cre-On rAAVs or genomic Cre-reporter alleles to study brain circuits. Intracranial injection of Cre-On/Cre-Off rAAVs into spatially intermingled Cre+ and Cre- neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre+ neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre-Off rAAV systems, including caveats and methods of validation. PMID:26131660

  4. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers

    PubMed Central

    Yan, Ziying; Sun, Xingshen; Feng, Zehua; Li, Guiying; Fisher, John T.; Stewart, Zoe A.

    2015-01-01

    Abstract The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air–liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl– currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway. PMID:25763813

  5. 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

  6. Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

    PubMed Central

    Carlon, Marianne S.; Vidović, Dragana; Dooley, James; da Cunha, Marina Mori; Maris, Michael; Lampi, Youlia; Toelen, Jaan; Van den Haute, Chris; Baekelandt, Veerle; Deprest, Jan; Verbeken, Erik; Liston, Adrian; Gijsbers, Rik

    2014-01-01

    Abstract Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α1-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months. PMID:24548076

  7. Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

    PubMed

    Fleming, Jane; Spinoulas, Afroditi; Zheng, Maolin; Cunningham, Sharon C; Ginn, Samantha L; McQuilty, Robert C; Rowe, Peter B; Alexander, Ian E

    2005-08-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

  8. The potential of adeno-associated viral vectors for gene delivery to muscle tissue

    PubMed Central

    Nahid, M Abu; Gao, Guangping

    2014-01-01

    Introduction Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8+ T-cell response against AAV capsid in humans. Areas covered In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Expert opinion Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application. PMID:24386892

  9. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.

    PubMed

    Karumuthil-Melethil, Subha; Nagabhushan Kalburgi, Sahana; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D; Keimel, John G; Mark, Brian L; Mahuran, Don; Walia, Jagdeep S; Gray, Steven J

    2016-07-01

    GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α-β), "A" isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system.

  10. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.

    PubMed

    Karumuthil-Melethil, Subha; Nagabhushan Kalburgi, Sahana; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D; Keimel, John G; Mark, Brian L; Mahuran, Don; Walia, Jagdeep S; Gray, Steven J

    2016-07-01

    GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α-β), "A" isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system

  11. Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

    PubMed

    Strobel, Benjamin; Miller, Felix D; Rist, Wolfgang; Lamla, Thorsten

    2015-08-01

    Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking.

  12. Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

    PubMed

    Strobel, Benjamin; Miller, Felix D; Rist, Wolfgang; Lamla, Thorsten

    2015-08-01

    Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking. PMID:26222983

  13. Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.

    PubMed

    Piedra, Jose; Ontiveros, Maria; Miravet, Susana; Penalva, Cristina; Monfar, Mercè; Chillon, Miguel

    2015-02-01

    Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

  14. Novel Adeno-Associated Viral Vector Delivering the Utrophin Gene Regulator Jazz Counteracts Dystrophic Pathology in mdx Mice

    PubMed Central

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-01-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor “Jazz” that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. J. Cell. Physiol. 229: 1283–1291, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:24469912

  15. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    PubMed

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD.

  16. 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.

  17. Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery.

    PubMed

    Zhang, Chuanling; Yao, Tianzhuo; Zheng, Yongxiang; Li, Zhongjun; Zhang, Qiang; Zhang, Lihe; Zhou, Demin

    2016-02-01

    Virus-based nanoparticles have shown promise as vehicles for delivering therapeutic genes. However, the rational design of viral vectors that enable selective tropism towards particular types of cells and tissues remains challenging. Here, we explored structural-functional relationships of the adeno-associated virus 2 (AAV2) vector by expanding its genetic code during production. As a proof-of-principle, an azide moiety was strategically displayed on the vector capsid as a bioorthogonal chemical reporter. Upon bioorthogonal conjugation of AAV2 with fluorophores and cyclic arginyl-glycyl-aspartic acid ligands at certain modifiable sites, we characterized in vitro and in vivo AAV2 movement and enhanced tropism selectivity towards integrin-expressing tumor cells. Targeting AAV2 vectors resulted in selective killing of U87 glioblastoma cells and derived xenografts via the herpes simplex virus suicide gene thymidine kinase, with the potency of ganciclovir being increased by 25-fold. Our results demonstrated successful rational modification of AAV2 as a targeting delivery vehicle, establishing a facile platform for precision engineering of virus-based nanoparticles in basic research and therapeutic applications.

  18. 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.

  19. Viral and Cellular Components of AAV2 Replication Compartments

    PubMed Central

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

    2013-01-01

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

  20. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

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

    2013-01-01

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

  1. Adeno-associated virus Rep78/Rep68 promotes localized melting of the rep binding element in the absence of adenosine triphosphate.

    PubMed

    Lou, Hua Jane; Brister, J Rodney; Li, Jianwei Jeffery; Chen, Weijun; Muzyczka, Nicholas; Tan, Weihong

    2004-03-01

    We have applied fluorescence anisotropy and molecular beacon fluorescence methods to study the interactions between the Adeno-associated virus Rep78/Rep68 protein and the 23-bp Rep binding element (RBE). Rep78/Rep68 stably interacted with both the single- and double-stranded conformations of the RBE, but the interaction mechanisms of single- and double-stranded DNA appeared to be fundamentally different. The stoichiometry of Rep78 association with both the separate top and bottom strands of the RBE was 1:1, and the relative dissociation constant (K(D)) values of these associations were calculated to be 2.3x10(-8) and 3.2x10(-8) M, respectively. In contrast, the stoichiometry of Rep78 association with the double-stranded RBE was 2:1, and the dissociation constant was determined to be 4.2x10(-15) M(2). Moreover, Rep78/Rep68 interaction with the 23-bp duplex RBE appeared to cause localized melting of the double-stranded DNA substrate in the absence of adenosine triphosphate (ATP). This melting activity showed slower kinetics than binding and may contribute to the initiation of ATP-dependent Rep78 helicase activity.

  2. Tyrosine crosslinking reveals interfacial dynamics in adeno-associated viral capsids during infection

    PubMed Central

    Horowitz, Eric D.; Finn, M.G.; Asokan, Aravind

    2012-01-01

    Viral capsid dynamics are often observed during infectious events such as cell surface attachment, entry and genome release. Structural analysis of adeno-associated virus (AAV), a helper-dependent parvovirus, revealed a cluster of surface-exposed tyrosine residues at the icosahedral two-fold symmetry axis. We exploited the latter observation to carry out selective oxidation of Tyr residues, which yielded crosslinked viral protein (VP) subunit dimers, effectively “stitching” together the AAV capsid two-fold interface. Characterization of different Tyr-to-Phe mutants confirmed that the formation of crosslinked VP dimers is mediated by dityrosine adducts and requires the Tyr704 residue, which crosses over from one neighboring VP subunit to the other. When compared to unmodified capsids, Tyr-crosslinked AAV displayed decreased transduction efficiency in cell culture. Surprisingly, further biochemical and quantitative microscopy studies revealed that restraining the two-fold interface hinders externalization of buried VP N-termini, which contain a phospholipase A2 domain and nuclear localization sequences critical for infection. These adverse effects caused by tyrosine oxidation support the notion that interfacial dynamics at the AAV capsid two-fold symmetry axis play a role in externalization of VP N-termini during infection. PMID:22458529

  3. Adeno-associated virus transfer of a gene encoding SNAP-25 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism.

    PubMed

    Raghunath, Arvind; Perez-Branguli, Francesc; Smith, Leonard; Dolly, J Oliver

    2008-04-01

    Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

  4. Laser Photocoagulation Induces Transduction of Retinal Pigment Epithelial Cells by Intravitreally Administered Adeno-Associated Viral Vectors.

    PubMed

    Lee, Si Hyung; Kong, Yoon Jin; Lyu, Jungmook; Lee, Heuiran; Park, Keerang; Park, Tae Kwann

    2015-10-01

    Retinal transduction by intravitreally administered adeno-associated viral (AAV) vector is previously known to be extremely limited to the neural retina except AAV2 capsid type. Recently, we showed that prior laser photocoagulation enhances retinal transduction of intravitreally administered AAV vectors, including the outer retina and retinal pigment epithelium (RPE). Here, by performing short-pulse laser pretreatment on the mouse retina, we demonstrate RPE cells transduced by three different capsid types of AAV vectors, AAV2, AAV5, and AAV8, using RPE wholemounts. For all capsid types, laser pretreatment effectively induced the transduction of RPE cells in and around the laser site.

  5. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice.

    PubMed

    Joyeux, Luc; Danzer, Enrico; Limberis, Maria P; Zoltick, Philip W; Radu, Antoneta; Flake, Alan W; Davey, Marcus G

    2014-06-01

    Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 μl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken β-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target

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

    PubMed

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

    1999-07-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis

    PubMed Central

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  9. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

    PubMed

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  10. Copackaging of multiple adeno-associated viral vectors in a single production step.

    PubMed

    Doerfler, Phillip A; Byrne, Barry J; Clément, Nathalie

    2014-10-01

    Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step. Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.

  11. Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina.

    PubMed

    Boye, Shannon E; Alexander, John J; Witherspoon, C Douglas; Boye, Sanford L; Peterson, James J; Clark, Mark E; Sandefer, Kristen J; Girkin, Chris A; Hauswirth, William W; Gamlin, Paul D

    2016-08-01

    Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and

  12. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

    PubMed

    Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A; Balogh, Karla K; Tossi, Kerstin Pino; Roden, Richard B S; Christensen, Neil D

    2015-10-13

    Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.

  13. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

    PubMed

    Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A; Balogh, Karla K; Tossi, Kerstin Pino; Roden, Richard B S; Christensen, Neil D

    2015-10-13

    Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant. PMID:26382603

  14. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

    PubMed Central

    Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W.; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A.; Balogh, Karla K.; Tossi, Kerstin Pino; Roden, Richard B.S.; Christensen, Neil D.

    2016-01-01

    Vaccination with the minor capsid protein L2, notably the 17–36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17–36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum ± MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17–36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant. PMID:26382603

  15. 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.

  16. 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.

  17. Enhanced selective gene delivery to neural stem cells in vivo by an adeno-associated viral variant.

    PubMed

    Kotterman, Melissa A; Vazin, Tandis; Schaffer, David V

    2015-05-15

    Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into mature neuronal and glial cell types. NSCs are the subject of intense investigation, owing to their crucial roles in neural development and adult brain function and because they present potential targets for gene and cell replacement therapies following injury or disease. Approaches to specifically genetically perturb or modulate NSC function would be valuable for either motivation. Unfortunately, most gene delivery vectors are incapable of efficient or specific gene delivery to NSCs in vivo. Vectors based on adeno-associated virus (AAV) present a number of advantages and have proven increasingly successful in clinical trials. However, natural AAV variants are inefficient in transducing NSCs. We previously engineered a novel AAV variant (AAV r3.45) capable of efficient transduction of adult NSCs in vitro. Here, to build upon the initial promise of this variant, we investigated its in vitro and in vivo infectivity. AAV r3.45 was more selective for NSCs than mature neurons in a human embryonic stem cell-derived culture containing a mixture of cell types, including NSCs and neurons. It was capable of more efficient and selective transduction of rat and mouse NSCs in vivo than natural AAV serotypes following intracranial vector administration. Delivery of constitutively active β-catenin yielded insights into mechanisms by which this key regulator modulates NSC function, indicating that this engineered AAV variant can be harnessed for preferential modulation of adult NSCs in the hippocampus. The capacity to rapidly genetically modify these cells might greatly accelerate in vivo investigations of adult neurogenesis. PMID:25968319

  18. Enhanced selective gene delivery to neural stem cells in vivo by an adeno-associated viral variant

    PubMed Central

    Kotterman, Melissa A.; Vazin, Tandis; Schaffer, David V.

    2015-01-01

    Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into mature neuronal and glial cell types. NSCs are the subject of intense investigation, owing to their crucial roles in neural development and adult brain function and because they present potential targets for gene and cell replacement therapies following injury or disease. Approaches to specifically genetically perturb or modulate NSC function would be valuable for either motivation. Unfortunately, most gene delivery vectors are incapable of efficient or specific gene delivery to NSCs in vivo. Vectors based on adeno-associated virus (AAV) present a number of advantages and have proven increasingly successful in clinical trials. However, natural AAV variants are inefficient in transducing NSCs. We previously engineered a novel AAV variant (AAV r3.45) capable of efficient transduction of adult NSCs in vitro. Here, to build upon the initial promise of this variant, we investigated its in vitro and in vivo infectivity. AAV r3.45 was more selective for NSCs than mature neurons in a human embryonic stem cell-derived culture containing a mixture of cell types, including NSCs and neurons. It was capable of more efficient and selective transduction of rat and mouse NSCs in vivo than natural AAV serotypes following intracranial vector administration. Delivery of constitutively active β-catenin yielded insights into mechanisms by which this key regulator modulates NSC function, indicating that this engineered AAV variant can be harnessed for preferential modulation of adult NSCs in the hippocampus. The capacity to rapidly genetically modify these cells might greatly accelerate in vivo investigations of adult neurogenesis. PMID:25968319

  19. Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

    PubMed

    Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

    2015-08-01

    Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

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

    PubMed Central

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

    2016-01-01

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

  1. 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

  2. AAV vector integration sites in mouse hepatocellular carcinoma.

    PubMed

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

    2007-07-27

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

  3. Intracranial injection of adeno-associated viral vectors.

    PubMed

    Lowery, Rebecca L; Majewska, Ania K

    2010-01-01

    Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex. PMID:21113119

  4. A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

    PubMed Central

    Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

    2016-01-01

    Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

  5. Adeno-Associated Viral-Mediated LARGE Gene Therapy Rescues the Muscular Dystrophic Phenotype in Mouse Models of Dystroglycanopathy

    PubMed Central

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle

    2013-01-01

    Abstract Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Largemyd mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies. PMID:23379513

  6. Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

    PubMed Central

    Xia, Li; Yin, Shankai; Wang, Jian

    2012-01-01

    Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

  7. 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

  8. DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

    PubMed Central

    Schnödt, Maria; Schmeer, Marco; Kracher, Barbara; Krüsemann, Christa; Espinosa, Laura Escalona; Grünert, Anja; Fuchsluger, Thomas; Rischmüller, Anja; Schleef, Martin; Büning, Hildegard

    2016-01-01

    Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

  9. 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.

  10. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery.

    PubMed

    Wang, Qizhao; Dong, Biao; Firrman, Jenni; Roberts, Sean; Moore, Andrea Rossi; Cao, Wenjing; Diao, Yong; Kapranov, Philipp; Xu, Ruian; Xiao, Weidong

    2014-08-01

    Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (∼5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. 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

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

    PubMed Central

    Pacak, Christina A; Byrne, Barry J

    2011-01-01

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

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

    PubMed

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

    2016-10-01

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

  16. Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector.

    PubMed

    Huang, Wei; McMurphy, Travis; Liu, Xianglan; Wang, Chuansong; Cao, Lei

    2016-06-01

    Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications. PMID:26857843

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  19. 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

  20. 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

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

    PubMed

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

    2015-01-01

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

  2. 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

  3. Comparative Efficacy and Safety of Multiple Routes of Direct CNS Administration of Adeno-Associated Virus Gene Transfer Vector Serotype rh.10 Expressing the Human Arylsulfatase A cDNA to Nonhuman Primates

    PubMed Central

    Rosenberg, Jonathan B.; Sondhi, Dolan; Rubin, David G.; Monette, Sébastien; Chen, Alvin; Cram, Sara; De, Bishnu P.; Kaminsky, Stephen M.; Sevin, Caroline; Aubourg, Patrick

    2014-01-01

    Abstract Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5×1012 genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS

  4. Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

    PubMed

    Burnham, Brenda; Nass, Shelley; Kong, Elton; Mattingly, MaryEllen; Woodcock, Denise; Song, Antonius; Wadsworth, Samuel; Cheng, Seng H; Scaria, Abraham; O'Riordan, Catherine R

    2015-12-01

    Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

  5. 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

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

    PubMed

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

    2012-02-01

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

  7. 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.

  8. 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.

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

    PubMed

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

    2011-01-01

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

  10. AAV-mediated gene targeting methods for human cells

    PubMed Central

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

    2013-01-01

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

  11. Retinal gene delivery by rAAV and DNA electroporation

    PubMed Central

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

    2013-01-01

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

  12. Partial Correction of the CNS Lysosomal Storage Defect in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis by Neonatal CNS Administration of an Adeno-Associated Virus Serotype rh.10 Vector Expressing the Human CLN3 Gene

    PubMed Central

    Sondhi, Dolan; Scott, Emma C.; Chen, Alvin; Hackett, Neil R.; Wong, Andrew M.S.; Kubiak, Agnieszka; Nelvagal, Hemanth R.; Pearse, Yewande; Cotman, Susan L.; Cooper, Jonathan D.

    2014-01-01

    Abstract Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3Δex7/8 knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3Δex7/8 mice were administered 3×1010 genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3Δex7/8 mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3Δex7/8 mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3Δex7/8 mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

  13. 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

  14. AAV Gene Therapy for MPS1-associated Corneal Blindness

    PubMed Central

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

    2016-01-01

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

  15. Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

    PubMed Central

    Lock, Martin; Alvira, Mauricio; Vandenberghe, Luk H.; Samanta, Arabinda; Toelen, Jaan; Debyser, Zeger

    2010-01-01

    Abstract Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV–adenovirus, AAV–herpesvirus, and AAV–baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 × 1014 genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies. PMID:20497038

  16. Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials.

    PubMed

    Minella, A L; Mowat, F M; Willett, K L; Sledge, D; Bartoe, J T; Bennett, J; Petersen-Jones, S M

    2014-10-01

    The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

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

    PubMed

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

    2015-06-01

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

  18. Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice

    PubMed Central

    Penrod, Rachel D.; Wells, Audrey M.; Carlezon, William A.; Cowan, Christopher W.

    2015-01-01

    Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery. PMID:26426386

  19. Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice.

    PubMed

    Penrod, Rachel D; Wells, Audrey M; Carlezon, William A; Cowan, Christopher W

    2015-01-01

    Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery.

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

    PubMed Central

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

    2012-01-01

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

  1. Lentiviral and adeno-associated vector-based therapy for motor neuron disease through RNAi.

    PubMed

    Towne, Chris; Aebischer, Patrick

    2009-01-01

    RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. Lentivirus and adeno-associated virus have been used to knockdown levels of mutated superoxide dismutase 1 (SOD1) in the G93A SOD1 mouse model of familial amyotrophic lateral sclerosis (fALS) to result in beneficial therapeutic outcomes. This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection.

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

    PubMed

    Asokan, Aravind

    2010-05-01

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

  3. 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

  4. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.

    PubMed

    Allay, James A; Sleep, Susan; Long, Scott; Tillman, David M; Clark, Rob; Carney, Gael; Fagone, Paolo; McIntosh, Jenny H; Nienhuis, Arthur W; Davidoff, Andrew M; Nathwani, Amit C; Gray, John T

    2011-05-01

    To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2 × 10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

  5. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    PubMed

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV. PMID:27181212

  6. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    PubMed

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.

  7. Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing.

    PubMed

    Kapranov, Philipp; Chen, Lingxia; Dederich, Debra; Dong, Biao; He, Jie; Steinmann, Kathleen E; Moore, Andrea R; Thompson, John F; Milos, Patrice M; Xiao, Weidong

    2012-01-01

    The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology. PMID:21875357

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

    PubMed

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

    2008-05-01

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

  9. 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 ...

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2010-06-20

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

  16. 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

  17. Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors.

    PubMed

    Trepel, M; Körbelin, J; Spies, E; Heckmann, M B; Hunger, A; Fehse, B; Katus, H A; Kleinschmidt, J A; Müller, O J; Michelfelder, S

    2015-10-01

    Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2013-07-01

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

  3. 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

  4. 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.

  5. 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

  6. 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

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

    PubMed

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

    2016-01-01

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

  8. Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model.

    PubMed

    Sugano, E; Tabata, K; Takahashi, M; Nishiyama, F; Shimizu, H; Sato, M; Tamai, M; Tomita, H

    2016-02-01

    We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa. PMID:26440056

  9. Phase I/II trial of adeno-associated virus-mediated alpha-glucosidase gene therapy to the diaphragm for chronic respiratory failure in Pompe disease: initial safety and ventilatory outcomes.

    PubMed

    Smith, Barbara K; Collins, Shelley W; Conlon, Thomas J; Mah, Cathryn S; Lawson, Lee Ann; Martin, Anatole D; Fuller, David D; Cleaver, Brian D; Clément, Nathalie; Phillips, Dawn; Islam, Saleem; Dobjia, Nicole; Byrne, Barry J

    2013-06-01

    Pompe disease is an inherited neuromuscular disease caused by deficiency of lysosomal acid alpha-glucosidase (GAA) leading to glycogen accumulation in muscle and motoneurons. Cardiopulmonary failure in infancy leads to early mortality, and GAA enzyme replacement therapy (ERT) results in improved survival, reduction of cardiac hypertrophy, and developmental gains. However, many children have progressive ventilatory insufficiency and need additional support. Preclinical work shows that gene transfer restores phrenic neural activity and corrects ventilatory deficits. Here we present 180-day safety and ventilatory outcomes for five ventilator-dependent children in a phase I/II clinical trial of AAV-mediated GAA gene therapy (rAAV1-hGAA) following intradiaphragmatic delivery. We assessed whether rAAV1-hGAA results in acceptable safety outcomes and detectable functional changes, using general safety measures, immunological studies, and pulmonary functional testing. All subjects required chronic, full-time mechanical ventilation because of respiratory failure that was unresponsive to both ERT and preoperative muscle-conditioning exercises. After receiving a dose of either 1×10(12) vg (n=3) or 5×10(12) vg (n=2) of rAAV1-hGAA, the subjects' unassisted tidal volume was significantly larger (median [interquartile range] 28.8% increase [15.2-35.2], p<0.05). Further, most patients tolerated appreciably longer periods of unassisted breathing (425% increase [103-851], p=0.08). Gene transfer did not improve maximal inspiratory pressure. Expected levels of circulating antibodies and no T-cell-mediated immune responses to the vector (capsids) were observed. One subject demonstrated a slight increase in anti-GAA antibody that was not considered clinically significant. These results indicate that rAAV1-hGAA was safe and may lead to modest improvements in volitional ventilatory performance measures. Evaluation of the next five patients will determine whether earlier intervention can

  10. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

    Okada, Takashi; Takeda, Shin'ichi

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  18. 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

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

    PubMed

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

    2016-01-01

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

  20. 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.

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

    PubMed

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

    2016-01-01

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

  2. 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

  3. 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.

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

    PubMed Central

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

    2016-01-01

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

  6. 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...

  7. Production of CFTR-null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer

    PubMed Central

    Rogers, Christopher S.; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A.; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W.; Kabel, Amanda C.; Yan, Ziying; Spate, Lee; Wax, David; Murphy, Clifton N.; Rieke, August; Whitworth, Kristin; Linville, Michael L.; Korte, Scott W.; Engelhardt, John F.; Welsh, Michael J.; Prather, Randall S.

    2008-01-01

    Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (ΔF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice. PMID:18324337

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

    Samulski, R. Jude

    2014-01-01

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

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

    PubMed

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

    2016-01-01

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

  11. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    PubMed Central

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

    2016-01-01

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

  12. Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.

    PubMed

    Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

    2016-09-01

    The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

  13. Artificial evolution with adeno-associated viral libraries.

    PubMed

    Perabo, Luca; Huber, Anke; Märsch, Stephan; Hallek, Michael; Büning, Hildegard

    2008-02-01

    After attracting the attention of the scientific community due to a number of favourable characteristics that make it an attractive vector for human gene therapy [1,2], AAV has been thoroughly investigated in the past two decades. Standard technologies for the manipulation of the viral genome and for efficient packaging and purification protocols have paved the road for trial and error manipulation by educated guesses to study viral infectious biology by reverse genetics and to generate improved vectors for human gene transfer. However, despite remarkable progress, our limited knowledge of molecular mechanisms implicated in virus-cell interactions has been a limiting factor. Combinatorial engineering and high-throughput selection techniques hold the potential to boost technological improvement by offering the possibility to screen large numbers of randomly generated clones by appropriate selection protocols. These approaches not only require lesser knowledge of viral biology, but can also be employed as valuable tools to investigate molecular mechanisms that drive the infection process. In this review we recapitulate the rationale for employment of combinatorial methods in AAV vector development and the accomplishments achieved so far, discussing current limitations and interesting developments that are in sight.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2015-10-29

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2012-11-01

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

  1. 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

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

    PubMed

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

    2016-01-01

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

  3. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    PubMed

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes. PMID:24043756

  4. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    PubMed

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

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

    PubMed Central

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

    2015-01-01

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

  6. 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

  7. 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.

  8. AAV2/8-humanFOXP3 gene therapy shows robust anti-atherosclerosis efficacy in LDLR-KO mice on high cholesterol diet.

    PubMed

    Cao, M; Theus, S A; Straub, K D; Figueroa, J A; Mirandola, L; Chiriva-Internati, M; Hermonat, P L

    2015-07-18

    Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.

  9. 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

  10. Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

    PubMed

    Tshilenge, Kizito-Tshitoko; Ameline, Baptiste; Weber, Michel; Mendes-Madeira, Alexandra; Nedellec, Steven; Biget, Marine; Provost, Nathalie; Libeau, Lyse; Blouin, Véronique; Deschamps, Jack-Yves; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Pichard, Virginie; Rolling, Fabienne

    2016-06-01

    Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons. PMID:27229628

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

    PubMed Central

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

    2011-01-01

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

  12. Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

    PubMed

    Mizutani, Takeomi; Li, Rui; Haga, Hisashi; Kawabata, Kazushige

    2015-09-18

    The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

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

    PubMed

    Boisgerault, Florence; Mingozzi, Federico

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. 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

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

    PubMed

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

    2016-06-01

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

  18. 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.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-02-19

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. 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

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

    PubMed

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

    2014-01-01

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

  5. 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

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

    PubMed

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

    2016-01-01

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

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-01-01

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

  13. The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

    PubMed Central

    Kyostio-Moore, Sirkka; Berthelette, Patricia; Piraino, Susan; Sookdeo, Cathleen; Nambiar, Bindu; Jackson, Robert; Burnham, Brenda; O’Riordan, Catherine R; Cheng, Seng H; Armentano, Donna

    2016-01-01

    Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency. PMID:26958574

  14. PEO-PPO-PEO Carriers for rAAV-Mediated Transduction of Human Articular Chondrocytes in Vitro and in a Human Osteochondral Defect Model.

    PubMed

    Rey-Rico, Ana; Frisch, Janina; Venkatesan, Jagadesh Kumar; Schmitt, Gertrud; Rial-Hermida, Isabel; Taboada, Pablo; Concheiro, Angel; Madry, Henning; Alvarez-Lorenzo, Carmen; Cucchiarini, Magali

    2016-08-17

    Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA.

  15. Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model.

    PubMed

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

    2011-07-20

    As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

  16. Distinct Expression Patterns of AAV8 Vectors with Broadly Active Promoters from Subretinal Injections of Neonatal Mouse Eyes at Two Different Ages.

    PubMed

    Xiong, Wenjun; Cepko, Constance

    2016-01-01

    The retinal expression patterns were analyzed following the injection of serotype 8 adeno-associated virus (AAV8) vectors that utilize two broadly active and commonly used sets of transcription regulatory sequences. These include the human cytomegalovirus (CMV) immediate early (IE) enhancer/promoter and the hybrid CAG element (also known as CAGGS or CBA) composed of a partial human CMV IE enhancer and the chicken β-actin promoter and intron. Subretinal delivery to postnatal day 0 (P0) or 6 (P6) mouse eyes resulted in efficient labeling of retinal cells, but with very distinct patterns. With P0 delivery, AAV8-CMV-GFP selectively labelled photoreceptors, while AAV8-CAG-GFP efficiently labeled both outer and inner retinal neurons, including photoreceptors, horizontal cells, amacrine cells and retinal ganglion cells. With P6 delivery, both vectors led to efficient labeling of photoreceptors and Müller glia cells, but not of inner retinal neurons. Our results suggest that the cell types that express the genes encoded by subretinally delivered AAV8 vectors are determined by both the timing of the injection and the regulatory sequences.

  17. AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease

    PubMed Central

    Katz, Martin L.; Tecedor, Luis; Chen, Yonghong; Williamson, Baye G.; Lysenko, Elena; Wininger, Fred A.; Young, Whitney M.; Johnson, Gayle C.; Whiting, Rebecca E. H.; Coates, Joan R.; Davidson, Beverly L.

    2016-01-01

    The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit. PMID:26560358

  18. Targeting Channelrhodopsin-2 to ON-bipolar Cells With Vitreally Administered AAV Restores ON and OFF Visual Responses in Blind Mice

    PubMed Central

    Macé, Emilie; Caplette, Romain; Marre, Olivier; Sengupta, Abhishek; Chaffiol, Antoine; Barbe, Peggy; Desrosiers, Mélissa; Bamberg, Ernst; Sahel, Jose-Alain; Picaud, Serge; Duebel, Jens; Dalkara, Deniz

    2015-01-01

    Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell–specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration. PMID:25095892

  19. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

    PubMed Central

    Cereso, Nicolas; Pequignot, Marie O; Robert, Lorenne; Becker, Fabienne; De Luca, Valerie; Nabholz, Nicolas; Rigau, Valerie; De Vos, John; Hamel, Christian P; Kalatzis, Vasiliki

    2014-01-01

    Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC–derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model. PMID:26015956

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  10. Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol

    PubMed Central

    Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

    2016-01-01

    Background Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. Objective The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. Methods We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate thei