Academic literature on the topic 'AAV recombinant vector'
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Journal articles on the topic "AAV recombinant vector"
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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.
Dissertations / Theses on the topic "AAV recombinant vector"
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Carty, Nikisha Christine. "Recombinant AAV Gene Therapy and Delivery." Scholar Commons, 2009. https://scholarcommons.usf.edu/etd/1890.
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Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution.
These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.
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Xu, Dan. "Cellular Immunity in Recombinant Adeno-Associated Virus Vector Mediated Gene Therapy." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1313504203.
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Jeanpierre, Lindsay. "Etude des réponses T CD8+ après transfert de gène par rAAV ciblant le muscle." Electronic Thesis or Diss., université Paris-Saclay, 2024. https://www.biblio.univ-evry.fr/theses/2024/interne/2024UPASL128.pdf.
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La thérapie génique par vecteurs dérivés des virus adéno-associés (rAAV), s'est imposée ces dernières décennies, comme une stratégie de traitement innovante, pour le traitement des maladies monogéniques rares. Elle est basée sur l'utilisation d'un vecteur d'origine virale, pour délivrer une séquence d'ADN fonctionnelle, dans les cellules présentant la mutation à l'origine de la maladie.Les avancées dans ce domaine ont conduit à la mise sur le marché de plusieurs produits, dont le ZolgenSMA®, utilisé pour le traitement de l'amyotrophie spinale, qui a permis de soigner plus de 4000 patients dans le monde à ce jour.Malgré ces résultats prometteurs, certains défis entravent encore l'efficacité et l'utilisation à plus large échelle de la thérapie génique par rAAV, notamment le développement de réponses immunitaires indésirables, pouvant compromettre l'efficacité du traitement, certaines fois conduisant à des complications sévères chez les patients. De tels effets délétères ont été recensés chez des patients traités avec le ZolgenSMA®, ou plus récemment dans des essais cliniques pour la dystrophie musculaire de Duchenne (DMD), soulignant la nécessité de mieux comprendre la mise en place de ces réponses immunitaires contre le rAAV.Les réponses innées et adaptatives activées par l'administration de rAAV, sont dirigées contre le vecteur lui-même, ainsi que contre le transgène codé par celui-ci. De nombreux travaux ont montré que de multiples facteurs sont impliqués dans la mise en place de ces réponses, dont la spécificité du vecteur pour le tissu cible, la dose injectée et la voie d'injection.Dans cette étude, nous nous sommes particulièrement intéressés à la caractérisation des lymphocytes T CD8+ dirigés contre le transgène, après transfert de gènes par rAAV ciblant les tissus musculaires. Nous avons pour cela, étudié la mise en place de ces réponses et leur fonctionnalité chez la souris, dans deux systèmes qui diffèrent par les vecteurs rAAV utilisés et leur voie d'injection.Nous avons premièrement démontré que la réponse CD8+ peut démarrer dès 24h, suite à l'administration intramusculaire de vecteur, en raison du drainage lymphatique rapide des particules d'AAV dans les ganglions drainants le site d'injection. De plus, l'induction précoce des réponses CD8+ dépend de la spécificité de l'expression du transgène dans le tissu cible et dans les cellules hématopoïétiques pouvant présenter l'antigène.Dans la deuxième partie de ce projet, nous avons pu démontrer que l'administration intraveineuse d'un vecteur ciblant spécifiquement le muscle, induit des réponses CD8+ anti-transgène dysfonctionnelles, notamment dans un modèle murin de dystrophie musculaire. Ces cellules dirigées présentent des marqueurs d'épuisement, semblent fonctionnelles, mais sont incapables de rejeter les cellules transduites par le vecteur, garantissant le maintien de la correction génique durant au moins 6 mois.Ces résultats enrichissent notre compréhension des réponses CD8+ dirigées contre le transgène, et contribuent à proposer des stratégies rAAV plus sûresGene therapy using adeno-associated virus-derived vectors (rAAV) has emerged in recent decades as an innovative treatment strategy for rare monogenic diseases. It is based on the use of a viral vector to deliver a functional DNA sequence into cells harboring the mutation responsible for the disease.Advancements in this field have led to the commercialization of several products, including Zolgensma®, which is used to treat spinal muscular atrophy and has helped over 4,000 patients worldwide to date.Despite these promising results, certain challenges still hinder the efficacy and broader application of rAAV gene therapy, including the development of undesirable immune responses that may compromise treatment efficacy, sometimes leading to severe complications in patients. Such adverse effects have been reported in patients treated with Zolgensma®, and more recently, in clinical trials for Duchenne muscular dystrophy (DMD), underscoring the need for a better understanding of these immune responses against rAAV.The innate and adaptive responses activated by rAAV administration are directed against both the vector itself and the encoded transgene. Numerous studies have shown that multiple factors are involved in triggering these responses, including the vector's specificity for the target tissue, the injected dose, and the route of administration.In this study, we focused specifically on characterizing CD8+ T lymphocytes directed against the transgene following rAAV-mediated gene transfer targeting muscle tissues. To this end, we examined the development and functionality of these responses in mice within two systems that differ in the rAAV vectors used and their route of injection.We first demonstrated that the CD8+ response can initiate as early as 24 hours after intramuscular vector administration, due to the rapid lymphatic drainage of AAV particles into the lymph nodes draining the injection site. Moreover, the early induction of CD8+ responses depends on the specificity of transgene expression in the target tissue and in hematopoietic cells capable of presenting the antigen.In the second part of this project, we showed that intravenous administration of a vector specifically targeting muscle induces dysfunctional anti-transgene CD8+ responses, particularly in a mouse model of muscular dystrophy. These transgene-specific cells exhibit exhaustion markers, appear functional, yet are unable to reject vector-transduced cells, ensuring sustained gene correction for at least six months.These findings enhance our understanding of CD8+ responses against the transgene and contribute to the development of safer rAAV-based strategies
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Ghenassia, Alexandre. "Induction de réponses mémoires lymphocytaires T CD8 et protection vaccinale après transfert de gènes par le vecteur AAV recombinant." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T032/document.
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La mémoire immunologique est le mécanisme biologique fondamental à la base du développement de la vaccination. La compréhension de ce mécanisme ainsi que de ses interactions avec les différents acteurs du système immunitaire a permis l’élaboration de vaccins qui sont aujourd’hui les garants d’une protection accrue face à l’émergence de maladies infectieuses potentiellement mortelles. La voie d’injection et le mode de transfert de ces vaccins sont des paramètres majeurs à prendre en considération car ils définissent une modulation des réponses immunitaires et de leurs spécificités d’action. De nos jours, seule la voie intramusculaire demeure la voie majoritaire d’administration de vaccins lors de la prophylaxie primaire en santé humaine. Au cours de notre étude, nous nous sommes intéressés à comparer l’injection d’un antigène (l’ovalbumine) selon deux voies d’administration : la voie intramusculaire et la voie intradermique. Nous nous sommes également appuyés sur une technologie du laboratoire qui consiste à transférer des gènes par des vecteurs AAV2/1 recombinants. Nous disposions de deux constructions de ces vecteurs ayant une spécificité pour cibler les cellules musculaires et permettant l’apport d’un effet auxiliaire par les lymphocytes T CD4+ lors d’injections dans des souris femelles. De plus, une de ces constructions nous permettait d’éviter la voie de présentation directe de l’antigène par les cellules dendritiques (DCs) aux lymphocytes T CD8+. Les capacités modulatrices de ces vecteurs nous permirent de montrer pour la première fois que le vecteur AAV2/1 recombinant était capable de faire exprimer un transgène au sein de la peau et d’y générer une réponse cellulaire forte. Nous avons également montré qu’il existait une synergie d’action entre l’effet auxiliaire et la voie intradermique qui améliorait considérablement les réponses cellulaires issues de la présentation croisée d’antigène. Enfin, nous avons pu démontrer que les lymphocytes T CD8+ générés suite à cette synergie d’action présentaient un profil phénotypique de cellules mémoires polyfonctionnelles et capables de protéger l’hôte face à un challenge pathogéniqueImmunological memory is the fundamental biological mechanism at the beginning of the development of vaccination. Understanding this mechanism and its interactions with the various players of the immune system has allowed the development of vaccines that are today the most effective barrier against the emergence of life-threatening infectious diseases. Route of injection and the nature of carriers of these vaccines are key parameters to be taken into consideration because they define a modulation of immune responses and their specific features. Nowadays, only the intramuscular injection route remains the major route of vaccines injection in the context of primary prophylaxis in human health. During our study, we were interested in comparing the injection of antigen (ovalbumin) following two routes of administration: intramuscular and intradermal routes. We also relied on a technology in the laboratory that involves the transfer of genes by rAAV2/1 vectors. We had two constructs of these vectors having specificity to target skeletal muscle cells and allowing us to provide a helper effect from CD4+ T cells during injections into female mice recipients. Moreover, one of these constructs enabled us to avoid the direct presentation of antigens by dendritic cells (DCs) to CD8+ T cells. The capacity of modulation of these vectors allowed us to show for the first time that the rAAV2/1 vector was able to trigger the expression of a transgene in the skin, and there to generate a strong cellular response. We have also shown that CD4+ T cell help and the intradermal route of immunization synergize to improve greatly cellular responses from the cross-presentation of antigens. Finally, we have demonstrated that CD8+ T cells generated following this synergism exhibited a phenotypic profile of polyfunctional memory cells and able to protect the host against a pathogenic challenge
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Ploquin, Aurélie. "Les vecteurs AAV recombinants : un nouvel outil de vaccination contre les Hénipavirus." Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2012. http://tel.archives-ouvertes.fr/tel-00756311.
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Les virus Hendra (HeV) et Nipah (NiV) sont des virus émergents appartenant à la famille des Paramyxovirus et au genre des Hénipavirus. Chaque année, ils sont responsables de nombreuses épidémies touchant plusieurs espèces animales dont les hommes, avec une forte morbidité et mortalité. À ce jour, aucun vaccin ni traitement ne sont commercialisés. Ce projet porte sur le développement d'un vaccin génétique pour lutter contre une infection par les Hénipavirus. La stratégie suivie, repose sur l'injection in vivo de vecteurs recombinants dérivés du virus Adéno-Associé (AAVr) codant pour la glycoprotéine d'enveloppe G du virus NiV. Une première expérience réalisée chez la souris, a montré qu'une seule injection de vecteurs AAVr par voie IM permet le développement d'une réponse humorale contre la protéine G, forte et stable dans le temps. Afin de tester le pouvoir protecteur de ce vaccin, des hamsters ont été infectés par les Hénipavirus, compte tenu de leur grande sensibilité à ces infections. L'injection de vecteurs AAVr chez ces animaux a permis de protéger 100 % des animaux infectés par le virus NiV et 50 % des animaux infectés par le virus HeV. Cette étude apporte une nouvelle approche de vaccination et de nouvelles perspectives concernant l'utilisation des vecteurs AAVr pour lutter contre des infections virales émergentes.
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Özgür, Günes Yasemin. "Preclinical gene therapy using recombinant AAV vectors in mouse models of two human diseases." Thesis, université Paris-Saclay, 2022. http://www.theses.fr/2022UPASL092.
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Nous avons obtenu une preuve de concept pour la thérapie génique de 2 maladies génétiques perte-de-fonction.L'acrodysostose est une maladie osseuse et rénale causée par des mutations de la sous-unité régulatrice de la protéine kinase A (PRKAR1A). Nous avons testé les effets d'un rAAV9-CAG-humanPRKR1A dans un modèle murin KI. hPRKAR1A a été exprimée dans les chondrocytes de la plaque de croissance et les cellules tubulaires rénales. L'architecture des chondrocytes et la longueur du squelette ont été améliorées.L'AMN est une axonopathie tardive de la moelle épinière causée par des mutations du gène ABCD1.Nous avons construit et développé un vecteur, rAAV9- MAG-humanABCD1-HA (hABCD1), et testé ses effets dans un modèle murin KO de la maladie.L'expression de hABCD1-HA a été observée dans les oligodendrocytes (OL) et les astrocytes (A). Nous avons développé un vecteur ciblant les oligodendrocytes (OL), rAAV9-MAG-humanABCD1-HA. Les déficits neurologiques ont été prévenus avec un suivi de 2 ans.C26:0-lysoPC (VLCFA) était diminué dans la moelle épinière des souris traitées.Chez le primate, l'injection intrathécale du vecteur rAAV9-MAG a induit un niveau élevé d'expression de hABCD1-HA dans les OL et les A de la moelle épinière et du cervelet. Le ciblage OL n'avait pas été obtenu auparavant chez les primates avec d'autres vecteurs ou promoteurs, ce qui ouvre la porte à l'application humaine de notre vecteur dans diverses maladies du système nerveux central (SNC)We have obtained proof-of concept for the gene therapy of two diseases.Acrodysostosis is a bone and kidney disease caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). We tested the effects of a rAAV9-CAG-humanPRKR1A in a knock-in mouse model. hPRKAR1A expression was found in growth plate chondrocytes, and kidney tubular cells. Chondrocyte architecture and skeleton length were improved.X-ALD AMN is a late-onset axonopathy of spinal cord caused by ABCD1 mutations. We made an original rAAV9-MAG-humanABCD1-HA (hABCD1) vector and tested its effects in a KO mouse model.hABCD1-HA expression was observed in numerous OL and astrocytes. Neurological deficits were prevented 24 months after injection. C26:0-lysoPC (VLCFA), was lower in spinal cord.In non-human primate, intrathecal injection of the rAAV9-MAG vector induced high hABCD1-HA expression in OL and astrocytes of spinal cord and cerebellum. OL targeting has not been obtained before in primates with other vectors or promoters. This opens the door to the human application of OL targeting in a number of CNS diseases
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Best, Victoria Maria. "Ongoing cellular responses to transgene products encoded by recombinant adeno-associated virus (rAAV) vectors." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1262213552.
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Niemir, Natalia. "Gene transfer in the Sandhoff murine model using a specific recombinant AAV9 vector." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05S024/document.
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Koo, Taeyoung. "Studies on gene transfer in skeletal muscle cells and tissues using recombinant adeno-associated virus (AAV) vectors." Thesis, Royal Holloway, University of London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529039.
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Chtarto, Abdelwahed. "Contribution au développement de nouveaux vecteurs inductibles par la tétracycline et basés sur le parvovirus adéno-associé (AAV)." Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210983.
Full textAbstract:
Le parvovirus adéno-associé (AAV) possède un génome à ADN linéaire simple brin de 4,7kb encadré par deux séquences palindromiques inversées et identiques de 145 nucléotides appelées ITRs, requises en cis pour la réplication et l’encapsidation de l’ADN viral. Dans un AAV recombinant (rAAV), la totalité de la partie codante du génome viral est remplacée par une cassette d’expression et seuls les ITRs sont conservés.\
Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished
Book chapters on the topic "AAV recombinant vector"
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Hallek, M., C. M. Wendtner, R. Kotin, D. Michl, and E. L. Winnacker. "Recombinant Adeno-Associated Virus (r AAV) Vectors." In Gene Therapy, 73–91. Basel: Birkhäuser Basel, 1999. http://dx.doi.org/10.1007/978-3-0348-7011-5_6.
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Murlidharan, Giridhar, R. Jude Samulski, and Aravind Asokan. "Gene Therapy of CNS Disorders Using Recombinant AAV Vectors." In Translational Neuroscience, 9–32. Boston, MA: Springer US, 2016. http://dx.doi.org/10.1007/978-1-4899-7654-3_2.
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Lebkowski, J. S., T. B. Okarma, and R. Philip. "The Challenges of Recombinant Adeno-associated Virus Manufacturing: Alternative Use of Adeno-associated Virus Plasmid/Liposome Complexes for Gene Therapy Applications." In Adeno-Associated Virus (AAV) Vectors in Gene Therapy, 51–59. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-80207-2_4.
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Gupta, Megha. "Parvovirus Vectors: The Future of Gene Therapy." In Veterinary Medicine and Science. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.105085.
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The unique diversity of parvoviral vectors with innate antioncogenic properties, autonomous replication, ease of recombinant vector production and stable transgene expression in target cells makes them an attractive choice as viral vectors for gene therapy protocols. Amongst various parvoviruses that have been identified so far, recombinant vectors originating from adeno-associated virus, minute virus of mice (MVM), LuIII and parvovirus H1 have shown promising results in many preclinical models of human diseases including cancer. The adeno-associated virus (AAV), a non-pathogenic human parvovirus, has gained attention as a potentially useful vector. The improved understanding of the metabolism of vector genomes and the mechanism of transduction by AAV vectors is leading to advancement in the development of more sophisticated AAV vectors. The in-depth studies of AAV vector biology is opening avenues for more robust design of AAV vectors that have potentially increased transduction efficiency, increased specificity in cellular targeting, and an increased payload capacity. This chapter gives an overview of the application of autonomous parvoviral vectors and AAV vectors, based on our current understanding of viral biology and the state of the platform.
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Yan, Ziying, Teresa C. Ritchie, Dongsheng Duan, and John F. Engelhardt. "[20] Recombinant AAV-mediated gene delivery using dual vector heterodimerization." In Methods in Enzymology, 334–57. Elsevier, 2002. http://dx.doi.org/10.1016/s0076-6879(02)46065-x.
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R. Flotte, Terence. "Recombinant AAV vectors for gene transfer to the lung: a compartmental approach." In Adeno-Associated Viral Vectors for Gene Therapy, 83–101. Elsevier, 2005. http://dx.doi.org/10.1016/s0075-7535(05)31004-7.
Full textConference papers on the topic "AAV recombinant vector"
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Chulay, J., M. Brantly, L. Wang, C. Mueller, M. Humphries, L. Spencer, F. Rouhani, et al. "Clinical Evaluation of a Recombinant Adeno-Associated Virus (rAAV) Alpha-1 Antitrypsin (AAT) Gene Therapy Vector." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a2908.
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Flotte, Terence, Bruce Trapnell, Margaret Humphries, Brenna Carey, Roberto Calcedo, Martha Campbell-Thompson, Farshid Rouhani, et al. "Phase 2 Clinical Trial Of A Recombinant Adeno-Associated Virus (RAAV) Alpha-1 Antitrypsin (AAT) Gene Therapy Vector." In American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a2428.
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