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1
Wang, Xu-Shan, Benjawan Khuntirat, Keyun Qing, Selvarangan Ponnazhagan, Dagmar M. Kube, Shangzhen Zhou, Varavani J. Dwarki, and Arun Srivastava. "Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination." Journal of Virology 72, no. 7 (July 1, 1998): 5472–80. http://dx.doi.org/10.1128/jvi.72.7.5472-5480.1998.
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ABSTRACT The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.
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Tejero, Marcos, Ozgun F. Duzenli, Colin Caine, Hisae Kuoch, and George Aslanidi. "Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors." Genes 14, no. 10 (September 26, 2023): 1866. http://dx.doi.org/10.3390/genes14101866.
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The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.
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Favaro, Patricia, Harre D. Downey, Federico Mingozzi, Fraser Wright, Bernd Hauck, Katherine A. High, and Valder R. Arruda. "Safety of Recombinant Adeno-Associated Viral Vectors in a Large Animal Model." Blood 110, no. 11 (November 16, 2007): 2586. http://dx.doi.org/10.1182/blood.v110.11.2586.2586.
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Abstract Recombinant adeno-associated viral (AAV) vector is a promising gene-based strategy for the treatment of several inherited diseases. Using AAV serotype 2 (AAV-2), the most common tested vector in humans, we have determined that the risk of germline transmission and the immune responses to both transgene product and/or vector-capsid proteins are critical obstacles to the safety of this strategy (Nat Med12:342, 2006). Recently, novel and more potent serotypes have emerged such as AAV-8 that allows efficient liver transduction following peripheral intravenous injection (IV). The major determinant of vector safety relies on its tissue tropism. Here, we sought to compare the efficacy and safety profile of AAV-2 and AAV-8 in a large animal model. Male rabbits (∼3 kg) received IV injection of AAV-2 (n=11) or AAV-8 (n=11) encoding human F.IX (hF.IX) at doses ranging from 1×1012 to 1×1013vg/kg. Injections with AAV-2-hF.IX resulted in 6-fold lower expression of hF.IX than AAV-8-hF.IX for both low and high dose cohorts. Notably, no neutralizing antibody to hF.IX was detected with either serotypes. Eighteen weeks following the initial injections, animals were cross-administered with either AAV-2 or AAV-8. Whereas injection of AAV-8 led a 20% increase in transgene expression in animals initially injected with AAV-2-h.FIX, AAV-2 failed to boost hF.IX expression. Regarding germline safety, the presence of vector genomes in semen samples from the high-dose cohort (6 to 10 weeks after injection) was 3-5 fold higher for AAV-8 compared with AAV-2. There were no differences in the vector clearance in the semen among rabbits from the low-dose cohorts of AAV-2 and AAV-8. After 12 weeks, all semen samples from all cohorts tested negative. In another rabbit model, vasectomized prior to vector injection, we determined that semen samples lacking germ cells were also positive for vector-DNA sequences. The kinetics of vector clearance in these samples was dose- and time-dependent and serotype-independent. Because the presence of capsid in early-time points is critical for predicting possible immune responses against the viral vector, we determine the vector biodistribution one week after injection of 1×1013 vg/kg of AAV-2 or AAV-8. Rabbits were euthanized and their organs were harvested and analyzed for vector DNA presence through real-time quantitative PCR. Comparing to AAV-2, AAV-8 genomes were 2-5 fold times higher in all organs (spleen, lung, heart, and kidney), with the exception of liver where vector-DNA content was comparable (range from 25-69 copy number/cell). In addition, testes, accessory glands, and prostates were positive for the vector DNA, albeit in very low levels (the highest level of vector DNA found in those organs was 3copy number/cell in the testis of one rabbit injected with AAV-8). These differences may reflect the distributions of cellular receptors for AAV-2 and AAV-8, which may also explain the higher content of vector genome in the semen of high-dose AAV-8 cohort. Together our findings suggest that AAV-8 ensures higher transgene expression than AAV-2 and preexisting immunity to AAV-2, a naturally acquired virus in humans, may not limit AAV-8-mediated gene delivery. The overall kinetics of AAV vector clearance in the semen seems to be independent of the presence of germ cells and vector serotype. However, early biodistribution data of AAV-8 suggests a distinct safety profile from AAV-2.
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Zhang, Huang-Ge, Jinfu Xie, Igor Dmitriev, Elena Kashentseva, David T. Curiel, Hui-Chen Hsu, and John D. Mountz. "Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production." Journal of Virology 76, no. 23 (December 1, 2002): 12023–31. http://dx.doi.org/10.1128/jvi.76.23.12023-12031.2002.
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ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.
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Grimm, Dirk, Kusum Pandey, Hiroyuki Nakai, Theresa A. Storm, and Mark A. Kay. "Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype." Journal of Virology 80, no. 1 (January 1, 2006): 426–39. http://dx.doi.org/10.1128/jvi.80.1.426-439.2006.
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ABSTRACT We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 × 1010 to 1.8 × 1012 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.
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Wright, J. Fraser. "Coating of AAV Vectors with Human Albumin Blocks Antibody Binding and Enables Transduction of Human Hepatocytes in the Presence of Neutralizing Antibodies." Blood 112, no. 11 (November 16, 2008): 3542. http://dx.doi.org/10.1182/blood.v112.11.3542.3542.
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Abstract Neutralization of recombinant adeno-associated virus (AAV) gene transfer vectors by pre-existing antibodies is a significant barrier to clinical gene transfer using systemic administration. In a recent clinical trial for hemophilia B, while efficient gene transfer and therapeutic levels of hFIX were achieved in a human subject with low pre-existing antibodies to AAV2, no gene transfer was observed in a subject with a modest pre-existing AAV2 antibody titer of 1:17 who received the same dose (Manno et al. Nature Med.2006;12:342–347). With the objective to achieve consistent and efficient transduction in the presence of AAV antibodies, ‘humanized’ AAV vectors were generated by covalent attachment of human albumin (HSA) to the surface of purified AAV2 by cross linking the single free thiol (Cys34) of HSA to lysine residues on the vector surface using suflosuccinimidyl 4 [N-maleimidomethyl] cyclohexane-1-caboxylate (Sulfo-SMCC). Variable levels of HSA substitution up to 65 HSA molecules per virion were achieved, corresponding to a maximum of approximately one HSA molecule per virion capsid protein. Analysis by SDS-PAGE of the modified vector revealed the presence of a 130kDA polypeptide species in the conjugated vector, not observed in the unmodified vector, that was consistent with predicted molecular weight obtained by covalent attachment of HSA with AAV capsid protein VP3. Dynamic light scattering demonstrated an average radius of 16.6 nm for the HSA-conjugated vector compared with 11.6 nm for unconjugated vector, consistent with coating of the vector surface with HSA. Analysis by flow cytometry demonstrated that the binding of AAV2-specific monoclonal antibody A20 to unmodified vectors was progressively reduced to background levels in HSA-conjugated vectors with increasing HSA substitution. Quantitative analysis by Biacore indicated that the association rate and available sites per virion for antibody binding were reduced > 10- fold in HSA-conjugated vectors. The conjugated vectors were significantly more resistant than unmodified vectors to neutralization by AAV antibody, demonstrating comparable transduction in the presence of 16-fold higher concentration of MAb A20 following transduction of cultured human hepatocytes (HepG2). In conclusion, covalent coupling of HSA to the surface of recombinant AAV2 is demonstrated as a feasible method to modify AAV vectors to increase their resistance to neutralizing antibodies. ‘Humanized’ AAV vectors such as HSA-coated AAV that effectively mask viral epitopes recognized by antibodies and display human epitopes may enable more consistent transduction following systemic administration in vivo, and enable vector readministration.
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Fischer, Kyle B., Hannah K. Collins, and Edward M. Callaway. "Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors." Proceedings of the National Academy of Sciences 116, no. 52 (December 16, 2019): 27001–10. http://dx.doi.org/10.1073/pnas.1915974116.
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In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or “leak” expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant “cross-over insensitive” recognition sites combined with an “ATG-out” design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.
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Weger, Stefan. "High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector." Viruses 15, no. 1 (December 24, 2022): 64. http://dx.doi.org/10.3390/v15010064.
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The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV rep and cap genes for rAAV packaging. So far, however, such rAdV-rep/cap vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of cis and trans inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.
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Hewitt, F. Curtis, Chengwen Li, Steven J. Gray, Shelley Cockrell, Michael Washburn, and R. Jude Samulski. "Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors." Journal of Virology 83, no. 8 (February 11, 2009): 3919–29. http://dx.doi.org/10.1128/jvi.02466-08.
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ABSTRACT Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to “mobilization” of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.
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Lieber, André, Dirk S. Steinwaerder, Cheryl A. Carlson, and Mark A. Kay. "Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes." Journal of Virology 73, no. 11 (November 1, 1999): 9314–24. http://dx.doi.org/10.1128/jvi.73.11.9314-9324.1999.
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ABSTRACT Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.
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Hauck, Bernd, Wei Zhao, Katherine High, and Weidong Xiao. "Intracellular Viral Processing, Not Single-Stranded DNA Accumulation, Is Crucial for Recombinant Adeno-Associated Virus Transduction." Journal of Virology 78, no. 24 (December 15, 2004): 13678–86. http://dx.doi.org/10.1128/jvi.78.24.13678-13686.2004.
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ABSTRACT Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.
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Lebkowski, J. S., M. M. McNally, T. B. Okarma, and L. B. Lerch. "Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types." Molecular and Cellular Biology 8, no. 10 (October 1988): 3988–96. http://dx.doi.org/10.1128/mcb.8.10.3988-3996.1988.
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Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that is dependent on adenovirus or herpesvirus for reproductive functions. We describe the construction of recombinant AAV vectors containing the chloramphenicol acetyltransferase gene or the neomycin phosphotransferase gene. These vectors carried their respective genes into a wide variety of cell types, including primary skin fibroblasts and hematopoietic cells. Infection efficiencies varied with cell type and ranged up to 3.0%. Coinfection of two different recombinant viruses was also used to introduce two different sequences simultaneously into a given cell. Finally, methods for obtaining recombinant AAV vectors with minimal contamination of wild-type virus are described. These various attributes of AAV vectors make them a viable DNA transduction system.
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Lebkowski, J. S., M. M. McNally, T. B. Okarma, and L. B. Lerch. "Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types." Molecular and Cellular Biology 8, no. 10 (October 1988): 3988–96. http://dx.doi.org/10.1128/mcb.8.10.3988.
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Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that is dependent on adenovirus or herpesvirus for reproductive functions. We describe the construction of recombinant AAV vectors containing the chloramphenicol acetyltransferase gene or the neomycin phosphotransferase gene. These vectors carried their respective genes into a wide variety of cell types, including primary skin fibroblasts and hematopoietic cells. Infection efficiencies varied with cell type and ranged up to 3.0%. Coinfection of two different recombinant viruses was also used to introduce two different sequences simultaneously into a given cell. Finally, methods for obtaining recombinant AAV vectors with minimal contamination of wild-type virus are described. These various attributes of AAV vectors make them a viable DNA transduction system.
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Mendoza, Skyler D., Yasmine El-Shamayleh, and Gregory D. Horwitz. "AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses." Journal of Neurophysiology 117, no. 5 (May 1, 2017): 2004–13. http://dx.doi.org/10.1152/jn.00780.2016.
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Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously.NEW & NOTEWORTHY Adeno-associated viral vector (AAV)-mediated gene delivery is a valuable tool for neurophysiology, but variability in transduction efficiency remains a bottleneck for experimental success. Repeated vector injections can help overcome this limitation but affect humoral immune state and transgene expression in ways that are poorly understood. We show that AAV vector injections into the primate central nervous system trigger long-lasting and serotype-specific immune responses, raising the possibility that switching serotypes may promote successful vector readministration.
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Sollerbrant, Kerstin, Joacim Elmén, Claes Wahlestedt, Joel Acker, Hélene Leblois-Prehaud, Martine Latta-Mahieu, Patrice Yeh, and Michel Perricaudet. "A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors." Journal of General Virology 82, no. 9 (September 1, 2001): 2051–60. http://dx.doi.org/10.1099/0022-1317-82-9-2051.
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The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses – a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene – produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.
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Xiao, Xiao, Juan Li, Yeou-Ping Tsao, Devin Dressman, Eric P. Hoffman, and Jon F. Watchko. "Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy." Journal of Virology 74, no. 3 (February 1, 2000): 1436–42. http://dx.doi.org/10.1128/jvi.74.3.1436-1442.2000.
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ABSTRACT Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.
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Philpott, Nicola J., Catherine Giraud-Wali, Carolyn Dupuis, Janette Gomos, Henry Hamilton, Kenneth I. Berns, and Erik Falck-Pedersen. "Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-rep Sequence in cis." Journal of Virology 76, no. 11 (June 1, 2002): 5411–21. http://dx.doi.org/10.1128/jvi.76.11.5411-5421.2002.
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ABSTRACT The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.
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Cao, Lei, Yuhong Liu, Matthew J. During, and Weidong Xiao. "High-Titer, Wild-Type Free Recombinant Adeno-Associated Virus Vector Production Using Intron-Containing Helper Plasmids." Journal of Virology 74, no. 24 (December 15, 2000): 11456–63. http://dx.doi.org/10.1128/jvi.74.24.11456-11463.2000.
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ABSTRACT Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate therep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.
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Nakai, Hiroyuki, Roland W. Herzog, J. Nathan Hagstrom, Johannes Walter, Szu-Hao Kung, Edmund Y. Yang, Shing Jen Tai, et al. "Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver." Blood 91, no. 12 (June 15, 1998): 4600–4607. http://dx.doi.org/10.1182/blood.v91.12.4600.412k22_4600_4607.
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Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.
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Domenger, Claire, and Dirk Grimm. "Next-generation AAV vectors—do not judge a virus (only) by its cover." Human Molecular Genetics 28, R1 (July 2, 2019): R3—R14. http://dx.doi.org/10.1093/hmg/ddz148.
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AbstractRecombinant adeno-associated viruses (AAV) are under intensive investigation in numerous clinical trials after they have emerged as a highly promising vector for human gene therapy. Best exemplifying their power and potential is the authorization of three gene therapy products based on wild-type AAV serotypes, comprising Glybera (AAV1), Luxturna (AAV2) and, most recently, Zolgensma (AAV9). Nonetheless, it has also become evident that the current AAV vector generation will require improvements in transduction potency, antibody evasion and cell/tissue specificity to allow the use of lower and safer vector doses. To this end, others and we devoted substantial previous research to the implementation and application of key technologies for engineering of next-generation viral capsids in a high-throughput ‘top-down’ or (semi-)rational ‘bottom-up’ approach. Here, we describe a set of recent complementary strategies to enhance features of AAV vectors that act on the level of the recombinant cargo. As examples that illustrate the innovative and synergistic concepts that have been reported lately, we highlight (i) novel synthetic enhancers/promoters that provide an unprecedented degree of AAV tissue specificity, (ii) pioneering genetic circuit designs that harness biological (microRNAs) or physical (light) triggers as regulators of AAV gene expression and (iii) new insights into the role of AAV DNA structures on vector genome stability, integrity and functionality. Combined with ongoing capsid engineering and selection efforts, these and other state-of-the-art innovations and investigations promise to accelerate the arrival of the next generation of AAV vectors and to solidify the unique role of this exciting virus in human gene therapy.
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Ponnazhagan, Selvarangan, Gandham Mahendra, Sanjay Kumar, John A. Thompson, and Mark Castillas,. "Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands." Journal of Virology 76, no. 24 (December 15, 2002): 12900–12907. http://dx.doi.org/10.1128/jvi.76.24.12900-12907.2002.
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ABSTRACT The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1α receptor (FGFR1α), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1α-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.
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Chatterjee, Saswati, Wei Li, Christie Ann Wong, Grace Fisher-Adams, Di Lu, Mausumee Guha, James A. Macer, Stephen J. Forman, and K. K. Wong. "Transduction of Primitive Human Marrow and Cord Blood-Derived Hematopoietic Progenitor Cells With Adeno-Associated Virus Vectors." Blood 93, no. 6 (March 15, 1999): 1882–94. http://dx.doi.org/10.1182/blood.v93.6.1882.406k03_1882_1894.
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We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34+ and CD34+38− clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34+ and CD34+38− cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34+, CD34+38−LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.
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Booth, M. J., A. Mistry, X. Li, A. Thrasher, and R. S. Coffin. "Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids." Gene Therapy 11, no. 10 (February 26, 2004): 829–37. http://dx.doi.org/10.1038/sj.gt.3302226.
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Nakai, Hiroyuki, Roland W. Herzog, J. Nathan Hagstrom, Johannes Walter, Szu-Hao Kung, Edmund Y. Yang, Shing Jen Tai, et al. "Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver." Blood 91, no. 12 (June 15, 1998): 4600–4607. http://dx.doi.org/10.1182/blood.v91.12.4600.
Full textAbstract:
Abstract Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.
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Duan, Dongsheng, Prerna Sharma, Jusan Yang, Yongping Yue, Lorita Dudus, Yulong Zhang, Krishna J. Fisher, and John F. Engelhardt. "Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue." Journal of Virology 72, no. 11 (November 1, 1998): 8568–77. http://dx.doi.org/10.1128/jvi.72.11.8568-8577.1998.
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ABSTRACT Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.
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Duan, Dongsheng, Qiang Li, Aimee W. Kao, Yongping Yue, Jeffrey E. Pessin, and John F. Engelhardt. "Dynamin Is Required for Recombinant Adeno-Associated Virus Type 2 Infection." Journal of Virology 73, no. 12 (December 1, 1999): 10371–76. http://dx.doi.org/10.1128/jvi.73.12.10371-10376.1999.
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ABSTRACT Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.
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Ponnazhagan, Selvarangan, Kirsten A. Weigel, Sudhanshu P. Raikwar, Pinku Mukherjee, Mervin C. Yoder, and Arun Srivastava. "Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes." Journal of Virology 72, no. 6 (June 1, 1998): 5224–30. http://dx.doi.org/10.1128/jvi.72.6.5224-5230.1998.
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ABSTRACT A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.
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Sarukhan, Adelaida, Sabine Camugli, Bernard Gjata, Harald von Boehmer, Olivier Danos, and Karin Jooss. "Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors." Journal of Virology 75, no. 1 (January 1, 2001): 269–77. http://dx.doi.org/10.1128/jvi.75.1.269-277.2001.
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ABSTRACT Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and to achieve safe and stable gene transfer in clinical trials.
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Bals, Robert, Weidong Xiao, Nianli Sang, Daniel J. Weiner, Rupalie L. Meegalla, and James M. Wilson. "Transduction of Well-Differentiated Airway Epithelium by Recombinant Adeno-Associated Virus Is Limited by Vector Entry." Journal of Virology 73, no. 7 (July 1, 1999): 6085–88. http://dx.doi.org/10.1128/jvi.73.7.6085-6088.1999.
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ABSTRACT The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.
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Galibert, Lionel, Aurélien Jacob, Adrien Savy, Yohann Dickx, Delphine Bonnin, Christophe Lecomte, Lise Rivollet, et al. "Monobac System–A Single Baculovirus for the Production of rAAV." Microorganisms 9, no. 9 (August 24, 2021): 1799. http://dx.doi.org/10.3390/microorganisms9091799.
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Large-scale manufacturing of rAAV is a bottleneck for the development of genetic disease treatments. The baculovirus/Sf9 cell system underpins the first rAAV treatment approved by EMA and remains one of the most advanced platforms for rAAV manufacturing. Despite early successes, rAAV is still a complex biomaterial to produce. Efficient production of the recombinant viral vector requires that AAV replicase and capsid genes be co-located with the recombinant AAV genome. Here, we present the Monobac system, a singular, modified baculovirus genome that contains all of these functions. To assess the relative yields between the dual baculovirus and Monobac systems, we prepared each system with a transgene encoding γSGC and evaluated vectors’ potency in vivo. Our results show that rAAV production using the Monobac system not only yields higher titers of rAAV vector but also a lower amount of DNA contamination from baculovirus.
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Stiefelhagen, Marius, Marlon R. Veldwijk, Anna Jauch, Volker Eckstein, Stephanie Laufs, Juergen A. Kleinschmidt, Frederik Wenz, Jens W. Zeller, Anthony D. Ho, and Stefan Fruehauf. "Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector." Blood 106, no. 11 (November 16, 2005): 4417. http://dx.doi.org/10.1182/blood.v106.11.4417.4417.
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Abstract Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells. Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH. Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH. Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.
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Huang, Jiayin. "The Mechanism of Adeno-associated Virus (AAV) Gene Therapy." Highlights in Science, Engineering and Technology 123 (December 24, 2024): 84–89. https://doi.org/10.54097/ma9x1289.
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Adeno-associated Virus (AAV) vector has become an important tool for vivo gene therapy due to its high specificity, low immunogenicity and long-term expressibility. There are multiple novel pharmaceuticals utilizing recombinant AAV vectors to treat genetic defect disease or disease related to gene expression disorder that are undergoing clinical trials. In this review, the basic biological features of AAV viruses are introduced, including their structure, classification, and interactions with hosts. This paper aims to elucidate the commonalities and differences among several AAV viruses used in clinical treatment by analyzing existing literature. The relevant genes for transduction of various types of AAV viruses are specifically analyzed in this article. By elaborating the process and mechanism of AAV virus transduction-related gene expression, this paper also attempts to summarize the current achievements of AAV therapy and explain the limitations still existing. It is hoped to contribute to the development and progress of AAV therapy in the future.
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Wu, Ping, M. Ian Phillips, John Bui, and Ernest F. Terwilliger. "Adeno-Associated Virus Vector-Mediated Transgene Integration into Neurons and Other Nondividing Cell Targets." Journal of Virology 72, no. 7 (July 1, 1998): 5919–26. http://dx.doi.org/10.1128/jvi.72.7.5919-5926.1998.
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ABSTRACT The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.
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Davidoff, Andrew M., Catherine Y. C. Ng, Junfang Zhou, Yunyu Spence, and Amit C. Nathwani. "Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway." Blood 102, no. 2 (July 15, 2003): 480–88. http://dx.doi.org/10.1182/blood-2002-09-2889.
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AbstractA systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2– and AAV-5–based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5α dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.
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Li, Chengwen, and R. Jude Samulski. "Serotype-specific replicating AAV helper constructs increase recombinant AAV type 2 vector production." Virology 335, no. 1 (April 2005): 10–21. http://dx.doi.org/10.1016/j.virol.2005.02.008.
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Wendtner, Clemens-Martin, David M. Kofler, Hans D. Theiss, Christian Kurzeder, Raymund Buhmann, Carmen Schweighofer, Luca Perabo, et al. "Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors." Blood 100, no. 5 (September 1, 2002): 1655–61. http://dx.doi.org/10.1182/blood.v100.5.1655.h81702001655_1655_1661.
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B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 × 1011/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.
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Phillips, André W., Peisu Zhang, Mary Ellen Truckenmiller, Stuart D. Keir, Margaret Bouvier, Carlo Tornatore, and William J. Freed. "Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector." Restorative Neurology and Neuroscience 21, no. 1-2 (January 2003): 1–10. https://doi.org/10.3233/rnn-2003-00220.
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Purpose: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. Methods: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. Results: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). Conclusions: AAV may be a useful vector for the transduction of oncogenes to produce cell lines.
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Wang, Fei, Jiawen Sun, Wenyan Guo, and Yang Wu. "Application of the Insect Cell-Baculovirus Expression Vector System in Adeno-Associated Viral Production." Applied Sciences 14, no. 23 (November 25, 2024): 10948. http://dx.doi.org/10.3390/app142310948.
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Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is an efficient protein expression platform, which is famous for its high-level expression of complex protein in insect cells. The system is based on baculoviruses such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and the expression efficiency of the target proteins has been significantly improved by optimizing the viral vectors and cell lines. In recent years, IC-BEVS have shown great potential for Adeno-Associated Virus (AAV) production, particularly excelling in AAV structural protein expression and recombinant AAV production. The system not only improves the yield and purity of AAV, but also shortens the production cycle, providing an efficient and reliable tool for gene therapy. However, the system also has some challenges, including protein modification differences, limitations in expression levels, and production costs. This paper reviews the development of the insect baculovirus expression system, its application in AAV production, and its prospects in gene therapy, aiming to provide a systematic reference and outlook for research in related fields.
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Guilbaud, Mickaël, Gilliane Chadeuf, Fabio Avolio, Achille François, Philippe Moullier, Alessandra Recchia, and Anna Salvetti. "Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration." Journal of Virology 82, no. 5 (December 19, 2007): 2590–93. http://dx.doi.org/10.1128/jvi.01956-07.
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ABSTRACT The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.
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Xiao, Xiao, Juan Li, and Richard Jude Samulski. "Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus." Journal of Virology 72, no. 3 (March 1, 1998): 2224–32. http://dx.doi.org/10.1128/jvi.72.3.2224-2232.1998.
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ABSTRACT Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236–5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 105 viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.
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Akil, Omar, Frank Dyka, Charlotte Calvet, Alice Emptoz, Ghizlene Lahlou, Sylvie Nouaille, Jacques Boutet de Monvel, et al. "Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model." Proceedings of the National Academy of Sciences 116, no. 10 (February 19, 2019): 4496–501. http://dx.doi.org/10.1073/pnas.1817537116.
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Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5′ and the other the 3′ portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea ofOtof−/−mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5′ and 3′ cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.
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Grimm, Dirk, Joyce S. Lee, Lora Wang, Tushar Desai, Bassel Akache, Theresa A. Storm, and Mark A. Kay. "In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses." Journal of Virology 82, no. 12 (April 9, 2008): 5887–911. http://dx.doi.org/10.1128/jvi.00254-08.
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ABSTRACT Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.
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Dong, Biao, Hiroyuki Nakai, and Weidong Xiao. "Characterization of Genome Integrity for Oversized Recombinant AAV Vector." Molecular Therapy 18, no. 1 (January 2010): 87–92. http://dx.doi.org/10.1038/mt.2009.258.
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Deodato, B., N. Arsic, L. Zentilin, M. Galeano, D. Santoro, V. Torre, D. Altavilla, et al. "Recombinant AAV vector encoding human VEGF165 enhances wound healing." Gene Therapy 9, no. 12 (June 2002): 777–85. http://dx.doi.org/10.1038/sj.gt.3301697.
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Cao, L., M. During, and W. Xiao. "Replication competent helper functions for recombinant AAV vector generation." Gene Therapy 9, no. 18 (September 2002): 1199–206. http://dx.doi.org/10.1038/sj.gt.3301710.
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Ogasawara, Yoji, Hiroaki Mizukami, Masashi Urabe, Akihiro Kume, Yumi Kanegae, Izumu Saito, John Monahan, and Keiya Ozawa. "Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP switching system." Journal of General Virology 80, no. 9 (September 1, 1999): 2477–80. http://dx.doi.org/10.1099/0022-1317-80-9-2477.
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Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5·5×108 vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.
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Hüser, Daniela, Stefan Weger, and Regine Heilbronn. "Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production." Journal of Virology 77, no. 8 (April 15, 2003): 4881–87. http://dx.doi.org/10.1128/jvi.77.8.4881-4887.2003.
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ABSTRACT Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.
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George, Lindsey A. "Hemophilia gene therapy: ushering in a new treatment paradigm?" Hematology 2021, no. 1 (December 10, 2021): 226–33. http://dx.doi.org/10.1182/hematology.2021000254.
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Abstract After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.
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Zhang, Yi, Narendra Chirmule, Guang-ping Gao, and James Wilson. "CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells." Journal of Virology 74, no. 17 (September 1, 2000): 8003–10. http://dx.doi.org/10.1128/jvi.74.17.8003-8010.2000.
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ABSTRACT Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding β-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ 10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L−/−) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a β-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZ transduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.
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RICHTER, MATTHIAS, AKIKO IWATA, JOHN NYHUIS, YOSHIO NITTA, A. DUSTY MILLER, CHRISTINE L. HALBERT, and MARGARET D. ALLEN. "Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo." Physiological Genomics 2, no. 3 (April 27, 2000): 117–27. http://dx.doi.org/10.1152/physiolgenomics.2000.2.3.117.
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Richter, Matthias, Akiko Iwata, John Nyhuis, Yoshio Nitta, A. Dusty Miller, Christine L. Halbert, and Margaret D. Allen. Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo. Physiol Genomics 2: 117–127, 2000.—Adeno-associated virus (AAV) vectors might offer solutions for restenosis and angiogenesis by transducing nondividing cells and providing long-term gene expression. We investigated the feasibility of vascular cell transduction by AAV vectors in an in vivo rabbit carotid artery model. Time course of gene expression, inflammatory reaction to the vector, and effects of varying viral titer, exposure time, and intraluminal pressures on gene expression were examined. Recombinant AAV vectors with an Rous sarcoma virus promoter and alkaline phosphatase reporter gene were injected intraluminally into transiently isolated carotid segments. Following transduction, gene expression increased significantly over 14 days and then remained stable to 28 days, the last time point examined. Medial vascular smooth muscle cells were the main cell type transduced even with an intact endothelial layer. Increasing the viral titer and intraluminal pressure both enhanced transduction efficiency to achieve a mean of 34 ± 7% of the subintimal layer of smooth muscle cells expressing gene product. A mild inflammatory reaction, composed of T cells with only rare macrophages, with minimal intimal thickening was demonstrated in 40% of transduced vessels; inflammatory cells were not detected in sham-operated control arteries. These findings demonstrate that AAV is a promising vector for intravascular applications in coronary and peripheral vascular diseases.