Journal articles on the topic 'Adeno-Associated Viral (AAV) vectors'
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1
Bartlett, Jeffrey S., Rose Wilcher, and R. Jude Samulski. "Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors." Journal of Virology 74, no. 6 (March 15, 2000): 2777–85. http://dx.doi.org/10.1128/jvi.74.6.2777-2785.2000.
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ABSTRACT We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A1 prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.
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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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Xuan, Keh Min, Nur Ain Mohd Asri, Rafeah Suppian, Norazmi Mohd Nor, Maryam Azlan, and Frank Camacho. "The Use of Adeno-associated virus (AAV) in Vaccine Development." Asian Journal of Medicine and Biomedicine 6, S1 (November 10, 2022): 192–93. http://dx.doi.org/10.37231/ajmb.2022.6.s1.583.
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Adeno-associated virus (AAV) is a very tiny (20-26 nm) icosahedral and non-enveloped virus, and it belongs to the Parvoviridae family. AAV vectors are the most widely used option for gene therapy and delivery of therapeutic antibodies due to their relatively low immunogenicity, high safety profile, broad tropism, and their tendency to maintain long-term gene expression [1]. AVV vectors are developed by transfection of human embryonic kidney (HEK) 293 T cells with transgene, packaging and helper plasmids [2]. Several clinical studies have investigated the use of AAV vectors for gene therapy in treating of Parkinson’s disease, Alzheimer’s disease, heart disease, and prostate cancer [3]. AAV vectors have previously been used to treat muscular diseases, but in recent years, their usage as vaccine vectors to cure or prevent infectious diseases including HIV, HPV, and influenza has expanded [4].
Here, we discuss the advantages and disadvantages of the use of AAV in vaccine development, and future approaches in improving the drawbacks caused by AAV-based vaccines. Numerous animal investigations have been conducted to explore vaccine vectors against various illnesses, suggesting a possibility for AAV-based vaccinations. Clinical studies on humans are, however, uncommon because, in contrast to other viral vectors, AAV induces a poor humoral and cellular immune response. Additionally, infectious vaccinations often target a large group of healthy individuals across a variety of ages, including children and teenagers. Therefore, compared to AAV-based gene therapies, vaccinations based on AAV vectors need to be more cost-effective and need more robust safety control.
According to several research, AAV vector vaccines have been shown to induce a stronger or longer lasting antibody response in comparison to other vaccination approaches, such as DNA, recombinant proteins, inactivated viruses, or virus-like particles (VLPs) [5]. However, AAV vectors are thought to have a low immunogenic profile in comparison to other viral vectors. The main limitations of AAV vectors are their low transgenic capacity and widespread pre-existing immunity in humans [6]. Currently, strategies for improving AAV immunogenicity and circumventing pre-existing immunity are actively being investigated. The research undertaken so far have highlighted numerous significant benefits of AAV vectors for immunisation. Despite all the advantages, there are still a variety of challenges that limit the use of these vectors as a vaccine in humans. Thus, it is necessary to overcome these challenges in order to make AAV-based vaccines effective.
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Gonçalves, Manuel A. F. V., Ietje van der Velde, Josephine M. Janssen, Bram T. H. Maassen, Evert H. Heemskerk, Dirk-Jan E. Opstelten, Shoshan Knaän-Shanzer, Dinko Valerio, and Antoine A. F. de Vries. "Efficient Generation and Amplification of High-Capacity Adeno-Associated Virus/Adenovirus Hybrid Vectors." Journal of Virology 76, no. 21 (November 1, 2002): 10734–44. http://dx.doi.org/10.1128/jvi.76.21.10734-10744.2002.
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ABSTRACT Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.
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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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Yan, Ziying, Roman Zak, Yulong Zhang, and John F. Engelhardt. "Inverted Terminal Repeat Sequences Are Important for Intermolecular Recombination and Circularization of Adeno-Associated Virus Genomes." Journal of Virology 79, no. 1 (January 1, 2005): 364–79. http://dx.doi.org/10.1128/jvi.79.1.364-379.2005.
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ABSTRACT The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a β-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute β-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.
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Mingozzi, Federico, Jörg Schüttrumpf, Valder R. Arruda, Yuhong Liu, Yi-Lin Liu, Katherine A. High, Weidong Xiao, and Roland W. Herzog. "Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector." Journal of Virology 76, no. 20 (October 15, 2002): 10497–502. http://dx.doi.org/10.1128/jvi.76.20.10497-10502.2002.
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ABSTRACT Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes (∼15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.
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Hanazono, Yutaka, Kevin E. Brown, Atsushi Handa, Mark E. Metzger, Dominik Heim, Gary J. Kurtzman, Robert E. Donahue, and Cynthia E. Dunbar. "In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral Vectors: Direct Comparison With Retroviral Vectors." Blood 94, no. 7 (October 1, 1999): 2263–70. http://dx.doi.org/10.1182/blood.v94.7.2263.419k36_2263_2270.
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We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated β-galactosidase gene (β-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.
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Jewell, Andrew P., Melanie Cochrane, Jenny McIntosh, Reuben Benjamin, and Amit Nathwani. "Comparison of Viral Vectors for Gene Transfer into CLL Cells: Efficient Transduction with Adeno-Associated Virus-8 (AAV-8)." Blood 106, no. 11 (November 16, 2005): 2985. http://dx.doi.org/10.1182/blood.v106.11.2985.2985.
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Abstract Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.
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Monahan, Paul E., Claude Négrier, Michael Tarantino, Leonard A. Valentino, and Federico Mingozzi. "Emerging Immunogenicity and Genotoxicity Considerations of Adeno-Associated Virus Vector Gene Therapy for Hemophilia." Journal of Clinical Medicine 10, no. 11 (June 2, 2021): 2471. http://dx.doi.org/10.3390/jcm10112471.
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Adeno-associated viral (AAV) vector gene therapy has shown promise as a possible cure for hemophilia. However, immune responses directed against AAV vectors remain a hurdle to the broader use of this gene transfer platform. Both innate and adaptive immune responses can affect the safety and efficacy of AAV vector–mediated gene transfer in humans. These immune responses may be triggered by the viral capsid, the vector’s nucleic acid payload, or other vector contaminants or excipients, or by the transgene product encoded by the vector itself. Various preclinical and clinical strategies have been explored to overcome the issues of AAV vector immunogenicity and transgene-related immune responses. Although results of these strategies are encouraging, more efficient approaches are needed to deliver safe, predictable, and durable outcomes for people with hemophilia. In addition to durability, long-term follow-up of gene therapy trial participants will allow us to address potential safety concerns related to vector integration. Herein, we describe the challenges with current methodologies to deliver optimal outcomes for people with hemophilia who choose to undergo AAV vector gene therapy and the potential opportunities to improve on the results.
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Grimm, Dirk, Shangzhen Zhou, Hiroyuki Nakai, Clare E. Thomas, Theresa A. Storm, Sally Fuess, Takashi Matsushita, et al. "Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy." Blood 102, no. 7 (October 1, 2003): 2412–19. http://dx.doi.org/10.1182/blood-2003-02-0495.
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Abstract We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 × 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases. (Blood. 2003;102:2412-2419)
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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.
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Miller, Daniel G., Grant D. Trobridge, Lisa M. Petek, Michael A. Jacobs, Rajinder Kaul, and David W. Russell. "Large-Scale Analysis of Adeno-Associated Virus Vector Integration Sites in Normal Human Cells." Journal of Virology 79, no. 17 (September 1, 2005): 11434–42. http://dx.doi.org/10.1128/jvi.79.17.11434-11442.2005.
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ABSTRACT The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.
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Tickner, Zachary J., and Michael Farzan. "Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors." Pharmaceuticals 14, no. 6 (June 10, 2021): 554. http://dx.doi.org/10.3390/ph14060554.
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Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.
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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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Trapani. "Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina." Genes 10, no. 4 (April 9, 2019): 287. http://dx.doi.org/10.3390/genes10040287.
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Gene therapy using adeno-associated viral (AAV) vectors currently represents the most promising approach for the treatment of many inherited retinal diseases (IRDs), given AAV’s ability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment epithelium, and their excellent safety and efficacy profiles in humans. However, one of the main obstacles to widespread AAV application is their limited packaging capacity, which precludes their use from the treatment of IRDs which are caused by mutations in genes whose coding sequence exceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to increase the transfer capacity of AAV vectors. This review will discuss these new developed strategies, highlighting the advancements as well as the limitations that the field has still to overcome to finally expand the applicability of AAV vectors to IRDs due to mutations in large genes.
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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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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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Shayakhmetov, Dmitry M., Cheryl A. Carlson, Hartmut Stecher, Qiliang Li, George Stamatoyannopoulos, and André Lieber. "A High-Capacity, Capsid-Modified Hybrid Adenovirus/Adeno-Associated Virus Vector for Stable Transduction of Human Hematopoietic Cells." Journal of Virology 76, no. 3 (February 1, 2002): 1135–43. http://dx.doi.org/10.1128/jvi.76.3.1135-1143.2002.
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ABSTRACT To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, ΔAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The ΔAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity ΔAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human γ-globin gene and the HS2 and HS3 elements of the β-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with ΔAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.
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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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Sellner, Leopold, Marlon R. Veldwijk, Jürgen A. Kleinschmidt, Stephanie Laufs, Julian Topaly, W. Jens Zeller, Frederik Wenz, and Stefan Fruehauf. "Gene Therapy of Chronic Myelogenous Leukemia Using Pseudotyped Recombinant Adeno-Associated Viral Vectors." Blood 114, no. 22 (November 20, 2009): 4506. http://dx.doi.org/10.1182/blood.v114.22.4506.4506.
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Abstract Abstract 4506 Introduction Gene transfer into malignant leukemia cells can be of relevance to overcome conventional therapy-resistance. Either suicide or immune stimulating gene therapy vectors may be a tool for second line treatment of imatinib-resistant chronic myelogenous leukemia (CML). Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency. Methods Pseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) were screened on a panel of human CML cell lines and primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations. Results On CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient (Fig. 1). For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells were observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p=0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p<0.001 vs rAAV2/6; Fig. 2). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors. Furthermore, donor-dependent differences in gene transfer efficiency were observed. Of note, data from experiments on CML cell lines seems to provide limited information about the transduction efficiency of rAAV vectors on primary CML cells. Conclusions Overall, pseudotyped rAAV and scAAV vectors offer efficient clinically relevant gene transfer into human CML cells with a potential for future clinical application. Disclosures: No relevant conflicts of interest to declare.
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Veldwijk, Marlon R., Leopold Sellner, Marius Stiefelhagen, Jürgen A. Kleinschmidt, Stephanie Laufs, Julian Topaly, Frederik Wenz, W. Jens Zeller, and Stefan Fruehauf. "Novel Efficient Primary Human Peripheral Blood Progenitor Cell-Targeted Recombinant Adeno-Associated Viral Vectors." Blood 110, no. 11 (November 16, 2007): 5144. http://dx.doi.org/10.1182/blood.v110.11.5144.5144.
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Abstract Introduction: G-CSF mobilized peripheral blood progenitor cells (PBPC) are an excellent target for the cure of acquired and inherited disorders of the hematopoietic system. AAV-based vectors have an advantageous safety profile, due to their low immunogenicity, the lack of any associated human disease and its episomal localization. Unfortunately, currently standard rAAV2-based vectors lack the required efficiency for gene transfer into human blood progenitor cells. To increase the gene transfer efficiency on primary hematopoietic cells we applied an AAV random peptide library on primary human CD34+ PBPC. Materials and Methods: The AAV random peptide library was applied on primary human CD34+ PBPC samples (3 selection rounds). Several mutant blood progenitor-targeted rAAV2 capsid clones were obtained and sequenced. Five new rAAV vectors with the most efficient clones were produced. Titers were verified using real time PCR-based titration. To determine efficiency and specificity, a panel of leukemia (CML and AML) and non-leukemia cell lines, as well as primary human CD34+ PBPC were transduced. GFP expression was determined against uninfected control cells. Results: On a panel of leukemia cell lines at least one capsid mutant was significantly more efficient (%GFP+; p<0.01) than standard rAAV2 vectors. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for hematopoietic cells. Significantly higher gene transfer efficiency (up to 16% GFP+ cells) could be obtained with the newly generated vectors compared to standard rAAV2 (up to 9-fold higher; p<0.01). Using a different approach, up to 25% gene transfer into CD34+ PBPC was observed. Conclusion: These novel vectors may finally enable efficient (>20%) gene transfer using rAAV-based vectors into primary human PBPC and may offer a safe alternative to other vector systems which is applicable in vivo.
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Hansen, Jonathan, Keyun Qing, and Arun Srivastava. "Adeno-Associated Virus Type 2-Mediated Gene Transfer: Altered Endocytic Processing Enhances Transduction Efficiency in Murine Fibroblasts." Journal of Virology 75, no. 9 (May 1, 2001): 4080–90. http://dx.doi.org/10.1128/jvi.75.9.4080-4090.2001.
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ABSTRACT Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992–996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H+-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.
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Gao, Guangping, Luk H. Vandenberghe, Mauricio R. Alvira, You Lu, Roberto Calcedo, Xiangyang Zhou, and James M. Wilson. "Clades of Adeno-Associated Viruses Are Widely Disseminated in Human Tissues." Journal of Virology 78, no. 12 (June 15, 2004): 6381–88. http://dx.doi.org/10.1128/jvi.78.12.6381-6388.2004.
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ABSTRACT The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors.
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Mano, Miguel, Rudy Ippodrino, Lorena Zentilin, Serena Zacchigna, and Mauro Giacca. "Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction." Proceedings of the National Academy of Sciences 112, no. 36 (August 24, 2015): 11276–81. http://dx.doi.org/10.1073/pnas.1503607112.
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Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.
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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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Choi, Vivian W., Douglas M. McCarty, and R. Jude Samulski. "Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing." Journal of Virology 80, no. 21 (November 1, 2006): 10346–56. http://dx.doi.org/10.1128/jvi.00841-06.
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ABSTRACT Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.
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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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Davis, Adam S., Thais Federici, William C. Ray, Nicholas M. Boulis, Deirdre O'Connor, K. Reed Clark, and Jeffrey S. Bartlett. "Rational Design and Engineering of a Modified Adeno-Associated Virus (AAV1)-Based Vector System for Enhanced Retrograde Gene Delivery." Neurosurgery 76, no. 2 (December 29, 2014): 216–25. http://dx.doi.org/10.1227/neu.0000000000000589.
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ABSTRACT BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.
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Lunev, Evgenii, Anna Karan, Tatiana Egorova, and Maryana Bardina. "Adeno-Associated Viruses for Modeling Neurological Diseases in Animals: Achievements and Prospects." Biomedicines 10, no. 5 (May 15, 2022): 1140. http://dx.doi.org/10.3390/biomedicines10051140.
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Adeno-associated virus (AAV) vectors have become an attractive tool for efficient gene transfer into animal tissues. Extensively studied as the vehicles for therapeutic constructs in gene therapy, AAVs are also applied for creating animal models of human genetic disorders. Neurological disorders are challenging to model in laboratory animals by transgenesis or genome editing, at least partially due to the embryonic lethality and the timing of the disease onset. Therefore, gene transfer with AAV vectors provides a more flexible option for simulating genetic neurological disorders. Indeed, the design of the AAV expression construct allows the reproduction of various disease-causing mutations, and also drives neuron-specific expression. The natural and newly created AAV serotypes combined with various delivery routes enable differentially targeting neuronal cell types and brain areas in vivo. Moreover, the same viral vector can be used to reproduce the main features of the disorder in mice, rats, and large laboratory animals such as non-human primates. The current review demonstrates the general principles for the development and use of AAVs in modeling neurological diseases. The latest achievements in AAV-mediated modeling of the common (e.g., Alzheimer’s disease, Parkinson’s disease, ataxias, etc.) and ultra-rare disorders affecting the central nervous system are described. The use of AAVs to create multiple animal models of neurological disorders opens opportunities for studying their mechanisms, understanding the main pathological features, and testing therapeutic approaches.
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Ling, Chen, Yuan Wang, Yuan Lu, Lina Wang, Giridhara R. Jayandharan, George V. Aslanidi, Baozheng Li, et al. "Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell LinesIn Vitroand in Murine HepatocytesIn Vivo." Journal of Virology 89, no. 2 (October 29, 2014): 952–61. http://dx.doi.org/10.1128/jvi.02581-14.
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ABSTRACTWe have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell linesin vitroand in murine hepatocytesin vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy.IMPORTANCEThe results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.
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Hampson, David, Alexander Hooper, and Yosuke Niibori. "The Application of Adeno-Associated Viral Vector Gene Therapy to the Treatment of Fragile X Syndrome." Brain Sciences 9, no. 2 (February 2, 2019): 32. http://dx.doi.org/10.3390/brainsci9020032.
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Viral vector-mediated gene therapy has grown by leaps and bounds over the past several years. Although the reasons for this progress are varied, a deeper understanding of the basic biology of the viruses, the identification of new and improved versions of viral vectors, and simply the vast experience gained by extensive testing in both animal models of disease and in clinical trials, have been key factors. Several studies have investigated the efficacy of adeno-associated viral (AAV) vectors in the mouse model of fragile X syndrome where AAVs have been used to express fragile X mental retardation protein (FMRP), which is missing or highly reduced in the disorder. These studies have demonstrated a range of efficacies in different tests from full correction, to partial rescue, to no effect. Here we provide a backdrop of recent advances in AAV gene therapy as applied to central nervous system disorders, outline the salient features of the fragile X studies, and discuss several key issues for moving forward. Collectively, the findings to date from the mouse studies on fragile X syndrome, and data from clinical trials testing AAVs in other neurological conditions, indicate that AAV-mediated gene therapy could be a viable strategy for treating fragile X syndrome.
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Gorovits, Boris, Jean-Claude Marshall, Justin Smith, Laurence O. Whiteley, and Hendrik Neubert. "Bioanalysis of adeno-associated virus gene therapy therapeutics: regulatory expectations." Bioanalysis 11, no. 21 (November 2019): 2011–24. http://dx.doi.org/10.4155/bio-2019-0135.
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The number of gene therapy (GTx) modality therapies in development has grown significantly in the last few years. Adeno-associated virus (AAV)-based delivery approach has become most prevalent among other virus-based GTx vectors. Several regulatory guidelines provide the industry with general considerations related to AAV GTx development including discussion and recommendations related to highly diverse bioanalytical support of the AAV-based therapeutics. This includes assessment of pre- and post-treatment immunity, evaluation of post-treatment viral shedding and infectivity, as well as detection of transgene protein expression. An overview of the current regulatory recommendations as found in currently active and published draft US FDA and EMA guidance or guideline documents is presented herein.
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Assaf, Basel T., and Laurence O. Whiteley. "Considerations for Preclinical Safety Assessment of Adeno-Associated Virus Gene Therapy Products." Toxicologic Pathology 46, no. 8 (October 7, 2018): 1020–27. http://dx.doi.org/10.1177/0192623318803867.
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Progress in understanding the molecular bases of human health and disease in recent decades has flourished making it possible for the field of gene therapy (GT) to offer new possibilities for treating, and even curing, a plethora of medical conditions such as monogenic disorders and metabolic diseases. GT is a therapeutic intervention to genetically alter or modify living cells by means of gene delivery achieved using either viral vectors or nonviral vectors, with adeno-associated virus (AAV) vectors constituting market-share majority. Although GT is conceptually attractive, adverse and even fatal iatrogenic complications have marred the initial enthusiasm of clinical successes. The properties of investigational AAV-based GT may pose safety concerns unique from those of small molecule drugs and other macromolecular biologics, such as ectopic or unregulated expression of the transgene, long-term persistence, and off-target distribution. Herein, we discuss considerations in the design of a comprehensive preclinical safety program for AAV-based GT prior to administration in humans.
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Arruda, Valder R., Hansell H. Stedman, Virginia Haurigot, George Buchlis, Stefano Baila, Patricia Favaro, Yifeng Chen, et al. "Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B." Blood 115, no. 23 (June 10, 2010): 4678–88. http://dx.doi.org/10.1182/blood-2009-12-261156.
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Abstract Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.
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Akcan, Tugce, Sam Spyra, Hans W. Sollinger, and Tausif Alam. "LBSUN178 Teniposide Improves Adeno-associated Virus-mediated Insulin Gene Therapy In Diabetic Mice." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A282—A283. http://dx.doi.org/10.1210/jendso/bvac150.583.
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Abstract Type 1 Diabetes Mellitus (T1DM) results from the autoimmune destruction of insulin-producing pancreatic β cells. Despite advances in our understanding of the pathogenesis and management of diabetes, as well as treatment options, which primarily include injectable insulin and whole pancreas transplant, these advancements remain unsatisfactory, with only a small percentage of patients achieving their glycemic goals. Insulin gene therapy refers to the development of β cell substitutes by introducing an insulin producing gene into endogenous cell populations that do not normally produce insulin. This strategy has the promise to overcome the limitations of current treatments. Adeno-associated viral (AAV) vector-based gene therapy holds great promise for the treatment of inherited and many complex acquired disorders. The success of AAV vectors is determined by two major factors: 1) cell transduction efficiency, and 2) persistence of the introduced gene with minimal or no host immune response following vector administration. Previous research has suggested that certain small molecules can improve AAV vector transduction efficiency and lower the vector dose below the immunological response threshold. We explore the use of teniposide, a chemotherapeutic agent, in improving the efficacy of gene therapy using AAV. We co-administered teniposide at a final dosage of 20 mg/kg with an AAV vector encoding an insulin gene controlled by a liver-specific promotor into streptozotocin (STZ) induced diabetic Hsd: ICR mice. Teniposide co-treatment produced up to a 3-fold decrease in AAV dose compared to vector alone while reducing diabetic hyperglycemia to comparable degrees as AAV delivery alone. There was no additional systemic toxicity. While the mechanism of action of teniposide-enhanced viral delivery of gene therapy is unknown, our data suggest that teniposide co-administration could significantly enhance AAV transduction in vivo and provide a novel, useful strategy to improve the clinical efficacy of gene therapy. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.
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Schnepp, Bruce C., Ryan L. Jensen, Chun-Liang Chen, Philip R. Johnson, and K. Reed Clark. "Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues." Journal of Virology 79, no. 23 (December 15, 2005): 14793–803. http://dx.doi.org/10.1128/jvi.79.23.14793-14803.2005.
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ABSTRACT Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.
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Madrigal, Justin L., Shahin Shams, Roberta S. Stilhano, and Eduardo A. Silva. "Characterizing the encapsulation and release of lentivectors and adeno-associated vectors from degradable alginate hydrogels." Biomaterials Science 7, no. 2 (2019): 645–56. http://dx.doi.org/10.1039/c8bm01218k.
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In this study, we compare the ability of two different degradable alginate hydrogel formulations to efficiently deliver LV and AAV. We propose that release rates of viral vectors are dependent on the physical properties of both the hydrogels and vectors.
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Chen, Songfeng, Ming Luo, Hongwei Kou, Guowei Shang, Yanhui Ji, and Hongjian Liu. "A Review of Gene Therapy Delivery Systems for Intervertebral Disc Degeneration." Current Pharmaceutical Biotechnology 21, no. 3 (March 17, 2020): 194–205. http://dx.doi.org/10.2174/1389201020666191024171618.
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Background: : Intervertebral Disc (IVD) degeneration is a major public health concern, and gene therapy seems a promising approach to delay or even reverse IVD degeneration. However, the delivery system used to transfer exogenous genes into intervertebral disc cells remains a challenge. Methods:: The MEDLINE, Web of Science, and Scopus databases were searched for English-language articles related to gene therapy for IVD degeneration articles from 1999 to May 2019. The keywords included “gene therapy” AND “intervertebral disc”. The history of the development of different delivery systems was analysed, and the latest developments in viral and non-viral vectors for IVD degeneration treatment were reviewed. Results: : Gene therapy delivery systems for IVD degeneration are divided into two broad categories: viral and non-viral vectors. The most commonly used viral vectors are adenovirus, adeno-associated virus (AAV), and lentivirus. Enthusiasm for the use of adenovirus vectors has gradually declined and has been replaced by a preference for lentivirus and AAV vectors. New technologies, such as RNAi and CRISPR, have further enhanced the advantage of viral vectors. Liposomes are the classic non-viral vector, and their successors, polyplex micelles and exosomes, have more potential for use in gene therapy for IVD degeneration. Conclusion:: Lentivirus and AAV are the conventional viral vectors used in gene therapy for IVD degeneration, and the new technologies RNAi and CRISPR have further enhanced their advantages. Nonviral vectors, such as polyplex micelles and exosomes, are promising gene therapy vectors for IVD degeneration.
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Pegoraro, Gianluca, Alessandro Marcello, Michael P. Myers, and Mauro Giacca. "Regulation of Adeno-Associated Virus DNA Replication by the Cellular TAF-I/Set Complex." Journal of Virology 80, no. 14 (July 15, 2006): 6855–64. http://dx.doi.org/10.1128/jvi.00383-06.
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ABSTRACT The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.
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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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Yan, Ziying, Roman Zak, G. W. Gant Luxton, Teresa C. Ritchie, Ursula Bantel-Schaal, and John F. Engelhardt. "Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors." Journal of Virology 76, no. 5 (March 1, 2002): 2043–53. http://dx.doi.org/10.1128/jvi.76.5.2043-2053.2002.
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ABSTRACT In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.
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Bharadwaj, Arpita S., Meagan Kelly, Dongsoo Kim, and Hengjun Chao. "Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells." Blood 115, no. 3 (January 21, 2010): 500–509. http://dx.doi.org/10.1182/blood-2009-08-239509.
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Abstract The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors, recognized as danger signals by the host immune system, activate dendritic cells (DCs), causing unwanted antivector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to coagulation factor IX (FIX) by direct intramuscular injection of adeno-associated virus (AAV)–FIX. AAV vectors are nonpathogenic and elicit minimal inflammatory response. We hypothesized that the nonpathogenic nature of AAV plays a critical role in induction of tolerance after AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX after intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs after administration of lipopolysaccharide and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and major histocompatibility complex backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced after intramuscular injection of AAV serotype 2 vector. Our results demonstrate that induction of FIX tolerance after AAV gene transfer is independent of DC activation status.
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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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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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Nam, Hyun-Joo, Michael Douglas Lane, Eric Padron, Brittney Gurda, Robert McKenna, Erik Kohlbrenner, George Aslanidi, et al. "Structure of Adeno-Associated Virus Serotype 8, a Gene Therapy Vector." Journal of Virology 81, no. 22 (August 29, 2007): 12260–71. http://dx.doi.org/10.1128/jvi.01304-07.
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ABSTRACT Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-Å resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded β-barrel and long loops between the β-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.
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Murlidharan, Giridhar, Travis Corriher, H. Troy Ghashghaei, and Aravind Asokan. "Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain." Journal of Virology 89, no. 7 (January 28, 2015): 3976–87. http://dx.doi.org/10.1128/jvi.02951-14.
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ABSTRACTAdeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA+ependymal lining to 2,8-linked PSA+migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain.IMPORTANCEViruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.
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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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Martino, Ashley T., Masataka Suzuki, David M. Markusic, Irene Zolotukhin, Renee C. Ryals, Babak Moghimi, Hildegund C. J. Ertl, Daniel A. Muruve, Brendan Lee, and Roland W. Herzog. "The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver." Blood 117, no. 24 (June 16, 2011): 6459–68. http://dx.doi.org/10.1182/blood-2010-10-314518.
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AbstractAlthough adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.
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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.