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1
Rutledge, Elizabeth A., Christine L. Halbert, and David W. Russell. "Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2." Journal of Virology 72, no. 1 (January 1, 1998): 309–19. http://dx.doi.org/10.1128/jvi.72.1.309-319.1998.
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ABSTRACT Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.
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Xin, Ke-Qin, Hiroaki Mizukami, Masashi Urabe, Yoshihiko Toda, Kaori Shinoda, Atsushi Yoshida, Kenji Oomura, et al. "Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 forDendritic Cells." Journal of Virology 80, no. 24 (September 27, 2006): 11899–910. http://dx.doi.org/10.1128/jvi.00890-06.
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ABSTRACT The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.
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Chai, Zheng, Xintao Zhang, Amanda Lee Dobbins, Ellie Azure Frost, R. Jude Samulski, and Chengwen Li. "Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors." Viruses 11, no. 12 (December 9, 2019): 1138. http://dx.doi.org/10.3390/v11121138.
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Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better “communication” between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.
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Lin, Jianping, Yan Zhi, Lauren Mays, and James M. Wilson. "Vaccines Based on Novel Adeno-Associated Virus Vectors Elicit Aberrant CD8+ T-Cell Responses in Mice." Journal of Virology 81, no. 21 (August 22, 2007): 11840–49. http://dx.doi.org/10.1128/jvi.01253-07.
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ABSTRACT We recently discovered an expanded family of adeno-associated viruses (AAVs) that show promise as improved gene therapy vectors. In this study we evaluated the potential of vectors based on several of these novel AAVs as vaccine carriers for human immunodeficiency virus type 1 Gag. Studies with mice indicated that vectors based on AAV type 7 (AAV7), AAV8, and AAV9 demonstrate improved immunogenicity in terms of Gag CD8+ T-cell and Gag antibody responses. The quality of these antigen-specific responses was evaluated in detail for AAV2/8 vectors and compared to results with an adenovirus vector expressing Gag (AdC7). AAV2/8 produced a vibrant CD8+ T-cell effector response characterized by coexpression of gamma interferon and tumor necrosis factor alpha as well as in vivo cytolytic activity. No CD8+ T-cell response generated by any of the AAVs was effectively boosted with AdC7, a result consistent with the finding of a relative lack of cells expressing interleukin-2 (IL-2) or a central memory phenotype at 3 months after the prime. The primary response to an AdC7 vaccine differed from that generated by AAVs in that the peak effector response evolved into populations of Gag-specific T cells expressing high levels of cytokines, including IL-2, and with effector memory and central memory phenotypes. A number of mechanisms could be considered to explain the aberrant activation of CD8+ T cells by AAV, including insufficient inflammatory responses, CD4 help, and/or chronic antigen expression and T-cell exhaustion. Interestingly, the B-cell response to AAV-encoded Gag was quite vibrant and easily boosted with AdC7.
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Sabatino, Denise E., Amy M. Lange, Melinda Mucci, Rita Sarkar, Aaron M. Dillow, Timothy C. Nichols, Valder R. Arruda, and Haig H. Kazazian. "Long Term Dose-Dependent Correction of Hemophilia A Dogs Using AAV-8 and AAV-9-Mediated FVIII Gene Transfer." Blood 108, no. 11 (November 16, 2006): 999. http://dx.doi.org/10.1182/blood.v108.11.999.999.
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Abstract Hemophilia A (HA) is an X-linked bleeding disorder characterized by deficiency in clotting factor VIII (FVIII). Current treatment for hemophilia is protein replacement therapy while a gene-based therapy would provide continuous expression of even low levels of FVIII protein (>1% of normal) that is likely to improve the disease phenotype. It is challenging to utilize an AAV-mediated gene transfer approach for the FVIII cDNA (4.4kb) since the AAV vector can only efficiently accommodate a <5.3kb transgene cassette. The FVIII protein is composed of 2 chains -the heavy chain (HC) and the light chain (LC). FVIII undergoes proteolytic cleavage and processing of the 2 individual chains that form the active FVIII protein. In other studies in HA dogs (n=8), no dose-response and AAV serotype-dependent FVIII expression has been documented, which illustrates the difficulties in using a FVIII single-chain approach. We have utilized a 2-chain approach in which the 2.4kb LC cDNA is packaged in one AAV vector while the 2.5kb HC is packaged into a second AAV vector. Each construct contains a 695bp thyroxine-binding globulin gene promoter/enhancer fused to a 175bp intron along with a 263bp SV40 poly A signal. For this approach the LC and HC vectors packaged into either AAV8 or AAV9 were administered to HA dogs via the hepatic artery. Two male HA dogs received HC and LC in AAV8 and 2 male dogs received HC and LC in AAV9 at doses of 6x1012gc/vector/kg (low dose) or 1.25x1013gc/v/kg (high dose). At 150 days after vector infusion, the high dose group expressed FVIII at levels of 4.8% (AAV8) and 3% (AAV9) as detected by a functional assay (Coatest assay). FVIII remained stable for 797 days (AAV8) and >200 days (AAV9) (the longest time points to date) without any evidence of antibody formation to the transgene. In the low dose group at 150 days, FVIII levels were 1.5% (AAV8) and 0.5% (AAV9) cFVIII activity and were maintained in a follow up period of >150 days (AAV8) and >700 days (AAV9) without formation of antibodies to FVIII. Thus, no major differences between AAV8 and AAV9 vectors were observed. The transgene product is also functional based on shortening of whole blood clotting time (baseline values >50 min), in a dose-dependent manner, 10–15 min and 16–20 min for the high and low dose cohorts, respectively. Interestingly, high dose injection of AAV8 to 2 female HA dogs (1.25x1013 and 3x1013gc/v/kg) results in FVIII levels of 1–2%, which is consistent with data obtained in mice on the poor performance of AAV in mediating gene transfer to liver in female animals. Liver function tests and other blood chemistries were transiently elevated after the surgical procedure and were in normal limits within 4 days. Importantly, all dogs did not develop antibodies to FVIII. These findings suggest that FVIII chains efficiently assemble in vivo without increasing the protein immunogenicity. The 4 male dogs have remained asymptomatic with no spontaneous bleeds, whereas >20 bleeding episodes were expected for this group since untreated dogs require 5.5 plasma infusions/year. These data demonstrate for the first time, dose-dependent sustained expression of functional cFVIII in HA dogs by AAV8 and AAV9 vectors without formation of antibodies to cFVIII.
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Chen, Quan, Huan Luo, Chengcong Zhou, Huan Yu, Sai Yao, Fangda Fu, Rebecca Seeley, et al. "Comparative intra-articular gene transfer of seven adeno-associated virus serotypes reveals that AAV2 mediates the most efficient transduction to mouse arthritic chondrocytes." PLOS ONE 15, no. 12 (December 15, 2020): e0243359. http://dx.doi.org/10.1371/journal.pone.0243359.
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Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.
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Martini, Sabrina V., Adriana L. Silva, Debora Ferreira, Rafael Rabelo, Felipe M. Ornellas, Karina Gomes, Patricia R. M. Rocco, Hilda Petrs-Silva, and Marcelo M. Morales. "Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung." Cellular Physiology and Biochemistry 39, no. 2 (2016): 544–53. http://dx.doi.org/10.1159/000445646.
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Background/Aims: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.
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Domenger, Claire, and Dirk Grimm. "Next-generation AAV vectors—do not judge a virus (only) by its cover." Human Molecular Genetics 28, R1 (July 2, 2019): R3—R14. http://dx.doi.org/10.1093/hmg/ddz148.
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AbstractRecombinant adeno-associated viruses (AAV) are under intensive investigation in numerous clinical trials after they have emerged as a highly promising vector for human gene therapy. Best exemplifying their power and potential is the authorization of three gene therapy products based on wild-type AAV serotypes, comprising Glybera (AAV1), Luxturna (AAV2) and, most recently, Zolgensma (AAV9). Nonetheless, it has also become evident that the current AAV vector generation will require improvements in transduction potency, antibody evasion and cell/tissue specificity to allow the use of lower and safer vector doses. To this end, others and we devoted substantial previous research to the implementation and application of key technologies for engineering of next-generation viral capsids in a high-throughput ‘top-down’ or (semi-)rational ‘bottom-up’ approach. Here, we describe a set of recent complementary strategies to enhance features of AAV vectors that act on the level of the recombinant cargo. As examples that illustrate the innovative and synergistic concepts that have been reported lately, we highlight (i) novel synthetic enhancers/promoters that provide an unprecedented degree of AAV tissue specificity, (ii) pioneering genetic circuit designs that harness biological (microRNAs) or physical (light) triggers as regulators of AAV gene expression and (iii) new insights into the role of AAV DNA structures on vector genome stability, integrity and functionality. Combined with ongoing capsid engineering and selection efforts, these and other state-of-the-art innovations and investigations promise to accelerate the arrival of the next generation of AAV vectors and to solidify the unique role of this exciting virus in human gene therapy.
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Boye, Sanford L., Antonette Bennett, Miranda L. Scalabrino, K. Tyler McCullough, Kim Van Vliet, Shreyasi Choudhury, Qing Ruan, James Peterson, Mavis Agbandje-McKenna, and Shannon E. Boye. "Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors." Journal of Virology 90, no. 8 (February 10, 2016): 4215–31. http://dx.doi.org/10.1128/jvi.00200-16.
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ABSTRACTAdeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection.IMPORTANCEAAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.
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Lins-Austin, Bridget, Saajan Patel, Mario Mietzsch, Dewey Brooke, Antonette Bennett, Balasubramanian Venkatakrishnan, Kim Van Vliet, et al. "Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking." Viruses 12, no. 6 (June 20, 2020): 668. http://dx.doi.org/10.3390/v12060668.
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Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.
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Majowicz, Anna, Nolukholo Ncete, Floris van Waes, Nikki Timmer, Sander J. van Deventer, Johnny N. Mahlangu, and Valerie Ferreira. "Seroprevalence of Pre-Existing Nabs Against AAV1, 2, 5, 6 and 8 in South African Hemophilia B Patient Population." Blood 134, Supplement_1 (November 13, 2019): 3353. http://dx.doi.org/10.1182/blood-2019-128217.
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Introduction Several studies have shown that the induction of antibodies by natural exposure to various AAV serotypes can compromise the subsequent use of AAV as a gene therapy vector, limiting patient eligibility for AAV-delivered therapeutics. The implications of pre-existing antibodies to AAV serotypes are very different: Levels of anti-AAV2 or anti-AAV8 neutralizing antibodies (NABs) as low as 5 have been related to a decrease or even total impairment of AAV liver transduction after systemic delivery in humans. However, successful gene transfer has been reported in patients with anti-AAV5 NABs titers up to 340 and in non-human primates with titers up to 1030. Extensive surveys on the prevalence of anti-AAV antibodies in humans have been published. Results from these studies indicate that prevalence varies dependent on serotype, and that a significant proportion of individuals develop humoral immunity against various AAV serotypes early in life, starting around 2 years of age. Furthermore, the prevalence of antibodies to different AAV serotypes has been reported to vary according to geographical location. Study Objective We performed a NABs seroprevalence study in South African hemophilia B patient population (n=44) using a panel of AAV serotypes suitable for liver targeted therapy, to determine the AAV serotypes likely to be of greatest clinical applicability for the South African hemophilia B population. Methods Forty-four hemophilia B patient serum samples were obtained from Hemophilia Comprehensive Care Center in Johannesburg (South Africa). All the patient serum samples were analyzed for the presence of NABs against AAV serotypes 1, 2, 5, 6 and 8 with the use of highly sensitive luciferase-based bioassays. The assays entail incubation of the test serum samples dilution series with an AAV1, 2, 5, 6 or 8-based reporter vector that carries the luciferase gene. This incubation allows neutralizing antibodies in the test serum to bind to the reporter vector particles. These mixtures are subsequently transferred onto Hek293T cells, where reporter vector particles can transduce cells and mediate expression of luciferase. Anti-AAV1, 2, 5, 6 or 8 NABs titers were determined by calculation of the percentage of neutralization for each sample dilution and fitting the neutralization curve with a four-parameter method. Anti-AAV1, 2, 5, 6 or 8 NABs titer (IC50) is the dilution at which antibodies inhibit Hek293T cell transduction with AAV1, 2, 5, 6 or 8-LUC by 50%. The lowest patient serum dilution used in every assay was 8 and samples were considered positive when calculated anti-AAV NAB titer was ≥8. All analytical runs included proper negative and positive controls. Results and Discussion The presence of NABs against the AAV serotypes 1, 2, 5, 6 and 8 was determined in the serum of the hemophilia B patients (Fig. 1). The highest prevalence of NABs was found to be against the AAV2 serotype, 95% (n=42/44) followed by the AAV6 serotype, 82% (n=36/44), and the AAV1 serotype 77% (n=34/44). The prevalence of NABs against AAV5 and AAV8 was lower with 66% (n=29/44) for AAV5 and 64% (n=28/44) for AAV8. The serum samples positive for anti-AAV2 NABs had a high occurrence of titers above 1030 (39%) in comparison to anti-AAV1 NABs (20%), anti-AAV5 NABs (5%) or anti-AAV8 NABs (7%).The occurrence of samples with low titers (ranging from titer of 8 to titer of 50) was the highest for anti-AAV8 NABs (32%) and for anti-AAV5 NABs (27%), followed by anti-AAV2 (18%) and anti-AAV1 (5%) (Fig.1). Currently, an anti-AAV NABs titer of 5 is used as an exclusion criteria in most of the systemic AAV-based gene therapies. When applying a similar cut-off of 8, 23% of the analyzed patients could be treated with AAV1, 5% with AAV2, 34% with AAV5, 18% with AAV6 and 36% with AAV8-based therapeutics (Fig.2). However, we have previously reported that AAV5-neutralizing antibodies do not impair the efficacy of in vivo transduction of AAV5-based vector up to a measured titer of 340 in humans and 1030 in non-human primates. Therefore, applying the cut-off of 340 or 1030, either 84% or 95% of the South African Hemophilia B patients could benefit from treatment with AAV5-based gene therapy (Fig.2). Disclosures Majowicz: uniQure N.V.: Employment. van Waes:uniQure N.V.: Employment. Timmer:uniQure N.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Mahlangu:Takeda: Consultancy, Honoraria, Speakers Bureau; LFB: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Baxalta: Consultancy, Research Funding, Speakers Bureau; Freeline Therapeutics: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; Spark: Consultancy, Speakers Bureau; Chugai: Consultancy; Biomarin: Research Funding; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Novartis: Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; uniQure: Research Funding; World Federation of Haemophilia: Speakers Bureau. Ferreira:uniQure N.V.: Employment.
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Yang, Grace S., Michael Schmidt, Ziying Yan, Jonathan D. Lindbloom, Thomas C. Harding, Brian A. Donahue, John F. Engelhardt, Robert Kotin, and Beverly L. Davidson. "Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size." Journal of Virology 76, no. 15 (August 1, 2002): 7651–60. http://dx.doi.org/10.1128/jvi.76.15.7651-7660.2002.
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ABSTRACT Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.
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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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Jiang, Haiyan, David Lillicrap, Susannah Patarroyo-White, Tongyao Liu, Xiaobing Qian, Ciaran D. Scallan, Sandra Powell, et al. "Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs." Blood 108, no. 1 (July 1, 2006): 107–15. http://dx.doi.org/10.1182/blood-2005-12-5115.
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Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies. (Blood. 2006;108:107-115)
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Davidson, Cristin D., Alana L. Gibson, Tansy Gu, Laura L. Baxter, Benjamin E. Deverman, Keith Beadle, Arturo A. Incao, et al. "Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann–Pick disease type C1 mice." Life Science Alliance 4, no. 10 (August 18, 2021): e202101040. http://dx.doi.org/10.26508/lsa.202101040.
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Niemann–Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.
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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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Halbert, Christine L., Elizabeth A. Rutledge, James M. Allen, David W. Russell, and A. Dusty Miller. "Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes." Journal of Virology 74, no. 3 (February 1, 2000): 1524–32. http://dx.doi.org/10.1128/jvi.74.3.1524-1532.2000.
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ABSTRACT Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.
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Sabatino, Denise E., Ekaterina Altynova, Amy M. Lange, Shangzhen Zhou, Elizabeth P. Merricks, Aaron M. Dillow, Timothy C. Nichols, Valder R. Arruda, and Haig H. Kazazian. "Successful Long Term Therapeutic Expression of Factor VIII in Hemophilia A Dogs After Administration of AAV-cFVIII Using a Two-Chain or Single Chain Delivery Approach." Blood 114, no. 22 (November 20, 2009): 546. http://dx.doi.org/10.1182/blood.v114.22.546.546.
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Abstract Abstract 546 While adeno-associated virus (AAV) is a promising gene delivery vector, it has been challenging to deliver FVIII due to the large size of the FVIII cDNA and the high frequency of FVIII antibody formation in hemophilia A (HA) patients. We used two approaches to overcome the size limitation of AAV for FVIII: (1) two-chain delivery in which the canine FVIII (cFVIII) heavy chain (HC) is delivered in one AAV vector and the cFVIII light chain (LC) is delivered in a second AAV vector and (2) single chain delivery in which the B-domain deleted cFVIII cDNA with minimal regulatory elements is within one AAV vector. In the two-chain approach AAV-HC (4.0 Kb) and AAV-LC (3.9 Kb) with a liver specific promoter was co-injected at a dose of 6×1012 vector genomes/vector/kg or 1.25×1013vg/vector/kg using AAV8 or AAV9 via hepatic infusion. Five hemophilia A dogs treated with AAV-HC and AAV-LC expressed 0.5-11% cFVIII in a dose-dependent manner. The mean cFVIII activity based on Coatest assay for the low dose was 1.3% (>1220d)(Linus)(AAV8) and 0.6% (>1770d)(H19)(AAV9), while for the high dose it was 5.2% (800d)(F24)(AAV8) and 2.4% (>1270d)(Woodstock)(AAV9). One dog (J60) had a splenectomy due to a complication at the time of surgery and has maintained high levels of expression (mean 11.0%; >820d). The WBCT consistently remained at a mean of 17.6 min for low dose dogs and 13.7 min for high dose dogs compared to 8-12 min in normal dogs. Using novel reagents that we generated specific to cFVIII, we developed assays to detect cFVIII antigen levels and IgG antibodies. Despite receiving equal doses of each vector, at day 85 the cFVIII-LC antigen levels (71.7 ± 19.2 ng/ml) were >10-fold higher than would be predicted based on activity while the cFVIII-HC antigen levels (14.6 ± 9.2 ng/ml) were >3-fold higher than activity. Since functional FVIII synthesis relies on the co-transduction of AAV-HC and AAV-LC in the same cell, this suggests that only a portion of the vector co-transduces and expresses cFVIII in the same cell and that the light chain is secreted more efficiently than the HC. No IgG antibodies to cFVIII were detected at any time point in these dogs. Three dogs have maintained FVIII expression for >3.5 years and two dogs for >2 years with ongoing observation. No spontaneous bleeding episodes have been observed in these dogs for a cumulative observation of >16 years while >80 bleeding episodes would be expected during this time period. The second approach, the single chain delivery, overcomes the co-transduction requirement of the two-chain approach by ensuring that each transduced cell expresses functional FVIII. However, it is difficult to efficiently package the large 5.2 Kb single chain construct into an AAV vector. Since no significant differences were observed between AAV8 and AAV9 using the two-chain approach, we used AAV8 to deliver the single chain cFVIII by peripheral vein infusion at 2×1013vg/kg or 4×1013vg/kg. The mean cFVIII activity was 0.7% (>430d) for the low dose dog (L51) and 6.8% (>290d) and 2.2% (>110d) for the high dose dogs (M06, M50). cFVIII HC and LC ELISA showed that cFVIII antigen levels correlated with activity. WBCT was a mean of 19.1 min for L51, 15.3 min for M06 and 11.6 min for M50. No spontaneous bleeding episodes have been observed in these dogs. The high dose dogs had no IgG antibodies to FVIII. L51 had transient IgG antibodies to FVIII until d52 in the absence of a Bethesda titer. A rise in FVIII expression in L51 coincided with the disappearance of anti-cFVIII antibodies. Comparison of single chain and two-chain delivery of FVIII reveals that (1) long term therapeutic levels of cFVIII in a dose-dependent manner can be obtained with both delivery approaches; (2) circulating cFVIII antigen levels are >10-fold higher than activity in the two-chain delivery in contrast to single chain delivery in which antigen correlates with activity; and (3) high antigen levels may facilitate tolerance to FVIII in the setting of liver-directed gene transfer, since a transient non-inhibitory antibody was observed in only one dog with very low FVIII levels. Notably, no cellular toxicity due to continuous expression of various forms of FVIII was found in these animals based on long-term sustained FVIII expression levels and normal liver enzymes in all eight HA dogs. Further studies to characterize the immune responses to the transgene will define the optimal vector approach. These data will form the basis for clinical studies in humans with severe HA. Disclosures: No relevant conflicts of interest to declare.
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Huang, Lin-Ya, Ami Patel, Robert Ng, Edward Blake Miller, Sujata Halder, Robert McKenna, Aravind Asokan, and Mavis Agbandje-McKenna. "Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site." Journal of Virology 90, no. 11 (March 9, 2016): 5219–30. http://dx.doi.org/10.1128/jvi.00161-16.
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ABSTRACTThe adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering.IMPORTANCEThe adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.
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Hewitt, F. Curtis, Chengwen Li, Steven J. Gray, Shelley Cockrell, Michael Washburn, and R. Jude Samulski. "Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors." Journal of Virology 83, no. 8 (February 11, 2009): 3919–29. http://dx.doi.org/10.1128/jvi.02466-08.
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ABSTRACT Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to “mobilization” of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.
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Rajavel, Kavitha, Mila Ayash-Rashkovsky, Ying Tang, Bagirath Gangadharan, Maurus de la Rosa, and Bruce Ewenstein. "Co-Prevalence of Pre-Existing Immunity to Different Serotypes of Adeno-Associated Virus (AAV) in Adults with Hemophilia." Blood 134, Supplement_1 (November 13, 2019): 3349. http://dx.doi.org/10.1182/blood-2019-123666.
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Background: Recombinant and synthetic adeno-associated virus (AAV) vectors are in development for gene transfer in patients with hemophilia A (HA) or hemophilia B (HB). These include liver-directed recombinant AAV8 vectors BAX 888/SHP654/TAK-754 factor VIII (FVIII) gene therapy (GT) for severe HA, and SHP648/TAK-748 factor IX (FIX) GT for HB (Baxalta US Inc., a Takeda company, Lexington, MA, USA). However, environmental exposure to wild-type AAVs can result in individuals developing antibodies and cell-mediated immune responses to the naturally occurring AAV. While natural exposure to AAV does not result in any known disease in humans, presence of preexisting immunity can block delivery and prevent sustained expression of the transgene by an AAV-based vector in a gene therapy setting. Of the AAV serotypes, AAV2 is the most frequently encountered natural human infection and AAV5 and AAV8 have been the most commonly used vectors for hemophilia GT. Therefore, it is important to assess the prevalence and co-prevalence of antibody and T cell-mediated responses against each of these AAV serotypes and to better characterize the association between humoral and cellular immunity in people with hemophilia. Aims: To determine the prevalence of preexisting antibody-mediated immunity against AAV2, AAV5 and AAV8 and the association between AAV8-specific humoral and cell-mediated responses in adult patients with HA and HB in an international prospective, epidemiological study. Methods: This ongoing seroprevalence study involved adult male patients (18-75 years of age) with severe HA (<1% plasma FVIII activity) or severe/moderate HB (≤2% plasma FIX activity) recruited from hemophilia treatment centers in the United States and Europe (NCT03185897). Participants consented to collection of peripheral blood at either a single or multiple annual outpatient study visits, in order to explore fluctuations of the immune response over time. Local ethics committee approval was obtained. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 neutralizing antibodies (NAbs) were determined using a cell-based transduction inhibition assay, with seropositivity defined as a titer ≥1:5. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 binding antibodies (BAbs) were quantitated by indirect enzyme-linked immunosorbent assay (ELISA), with seropositivity defined as a titer ≥1:80. Cell-mediated immune responses to AAV8 peptide antigens were measured in peripheral blood mononuclear cells using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Samples with a signal ≥3 times background and >60 spots per million cells were defined as positive. Results: Here we present data from patients who completed at least a single visit at the time of the interim, one year data cut (November 9, 2018). Of 242 patients enrolled (mean ± SD age: 35.3 ± 11.4 years), 194 patients had HA and 48 patients had HB. The overall co-prevalence of NAbs and BAbs to AAV2, AAV5 and AAV8 was 39.7% (HA: 38.1%, 72/189; HB: 45.8%, 22/48) and 16.1% (HA: 16.5%, 31/188; HB: 14.6%, 7/48) respectively, with further details shown in Table 1. Overall, 38.3% of patients (82/214) exhibited a T cell-mediated immune response to AAV8 peptide antigens (HA: 35.9%, 61/170; HB: 47.7%, 21/44). Among patients with AAV-8-specific NAbs, 37.9% (39/103) demonstrated positive AAV8-specific ELISPOT results. (HA: 35.7%, 30/84; HB: 47.4%, 9/19). Conclusion: The findings from this ongoing study demonstrate that approximately 50% of patients with hemophilia have preexistent NAb responses to AAV2, AAV5 or AAV8 with 40% demonstrating co-prevalence to all 3 evaluated AAV serotypes. Similar percentages of patients exhibited a positive cellular response to AAV8 antigens. Further, patients with HB demonstrated a slightly higher co-prevalence and a higher cellular response than patients with HA. In the combined HA and HB cohorts, co-prevalence was almost 40% for AAV8-specific humoral and T-cell mediated immunity. These data will add to our appreciation of preexisting AAV immunity that prevent patient participation in gene therapy trials. Disclosures Rajavel: Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Ayash-Rashkovsky:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Tang:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. de la Rosa:Baxalta Innovations GmbH, a Takeda company: Employment. Ewenstein:Baxalta US Inc., a Takeda company: Employment, Equity Ownership, Other: a Takeda stock owner.
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Hauck, Bernd, and Weidong Xiao. "Characterization of Tissue Tropism Determinants of Adeno-Associated Virus Type 1." Journal of Virology 77, no. 4 (February 15, 2003): 2768–74. http://dx.doi.org/10.1128/jvi.77.4.2768-2774.2003.
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ABSTRACT Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.
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Medert, Rebekka, Andreas Jungmann, Staffan Hildebrand, Martin Busch, Dirk Grimm, Veit Flockerzi, Oliver J. Müller, Patrick Most, Dagmar Schumacher, and Marc Freichel. "Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart." Pflügers Archiv - European Journal of Physiology 473, no. 3 (February 13, 2021): 533–46. http://dx.doi.org/10.1007/s00424-021-02521-6.
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AbstractThe cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.
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Tseng, Yu-Shan, Brittney L. Gurda, Paul Chipman, Robert McKenna, Sandra Afione, John A. Chiorini, Nicholas Muzyczka, et al. "Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity." Journal of Virology 89, no. 3 (November 19, 2014): 1794–808. http://dx.doi.org/10.1128/jvi.02710-14.
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ABSTRACTThe clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions.IMPORTANCEThe adeno-associated viruses (AAVs) are promising vectors forin vivotherapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has preexisting AAV capsid antibodies and therefore must be excluded from clinical trials or vector readministration. This report represents our continuing efforts to understand the antigenic structure of the AAVs, specifically, to obtain a picture of “polyclonal” reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryo-electron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide information essential for generating antigenic escape vectors to overcome a critical challenge remaining in the optimization of this highly promising vector delivery system.
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Silveria, Mark A., Edward E. Large, Grant M. Zane, Tommi A. White, and Michael S. Chapman. "The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors." Viruses 12, no. 11 (November 18, 2020): 1326. http://dx.doi.org/10.3390/v12111326.
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Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.
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Mietzsch, Mario, Ariana Jose, Paul Chipman, Nilakshee Bhattacharya, Nadia Daneshparvar, Robert McKenna, and Mavis Agbandje-McKenna. "Completion of the AAV Structural Atlas: Serotype Capsid Structures Reveals Clade-Specific Features." Viruses 13, no. 1 (January 13, 2021): 101. http://dx.doi.org/10.3390/v13010101.
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The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.
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Samaranch, Lluis, Azucena Pérez-Cañamás, Beatriz Soto-Huelin, Vivek Sudhakar, Jerónimo Jurado-Arjona, Piotr Hadaczek, Jesús Ávila, et al. "Adeno-associated viral vector serotype 9–based gene therapy for Niemann-Pick disease type A." Science Translational Medicine 11, no. 506 (August 21, 2019): eaat3738. http://dx.doi.org/10.1126/scitranslmed.aat3738.
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Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.
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Herzog, Roland W., Brad E. Hoffman, Irene Zolotukhin, Katherine A. High, Arun Srivastava, Ype P. de Jong, Koen Vercauteren, and Jing Xiao. "AAV3 Capsid Is Superior for In Vivo Gene Transfer to Human Hepatocytes Compared to Serotypes 5 and 8 in a Mouse/Human Chimeric Model." Blood 126, no. 23 (December 3, 2015): 4418. http://dx.doi.org/10.1182/blood.v126.23.4418.4418.
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Abstract Adeno-associated viral (AAV) gene transfer to the liver for the treatment of hemophilia B is currently being tested in multiple clinical trials and is also in the pipeline for hemophilia A. Current trials utilize AAV serotype 8, while other capsids including AAV5 will also be tested in hemophilia B patients in the near future. AAV8 shows superior transduction of murine liver and has directed long-term factor IX gene transfer in humans. However, efficacy was substantially lower in human than in murine liver. In order to better predict the performance of the various candidate capsids with liver tropism in humans, "humanized mice" harboring human hepatocytes have been adapted as a pre-clinical model of AAV liver gene transfer. Here, we utilized the liver chimeric FAH model. This model is based on the observation that adult immunodeficient fah-/- mice, which suffer from liver injury when cycled off the tyrosine catabolism blocker NTBC, support the proliferation of human hepatocytes. We used liver chimeric mice highly engrafted with fetal human hepatoblasts (human serum albumin levels 978-3737mcg/ml) or human pediatric hepatocytes (human serum albumin levels 388-3798mcg/ml). We produced AAV vectors expressing enhanced GFP reporter from a self-complementary genome. These included AAV5, AAV8, and also AAV3 (which in recent years has shown high transduction efficiency for human hepatocytes and human liver cancer cell lines in vitro and in murine xenograft models). Vectors were injected into the tail veins of humanized mice at a dose of 1x10^11 vector genomes/mouse. Livers were harvested 14 days later and analyzed for GFP expression in murine and human hepatocytes. Human cells were specifically identified by immunostain for FAH. We analyzed transduction efficiency by immunohistochemical stains of liver sections obtained from formalin fixed tissue (n=3 per vector). Transduced human FAH+GFP+ hepatocytes were visualized by confocal immunofluorescence microscopy followed by analysis with Velocity software. To further substantiate the results obtained by image analysis, we developed a flow cytometry-based method for detection of GFP-transduced FAH+ human hepatocytes and mCD81+ murine hepatocytes. Regardless of the method of analysis, AAV3 was the most efficient in transducing human hepatocytes, transducing 12-15% of human but only 3.5-5.5% of murine hepatocytes. AAV8 consistently transduced 5-6% of human hepatocytes while showing by far the highest efficiency for murine hepatocytes (on average 46%). Since the GFP signal in human hepatocytes transduced with AAV3 was higher than for AAV8 transduced cells, the difference in transduction efficiency of the human cells was likely greater in favor of AAV3 than the approximately 3-fold difference in percent GFP positivity. In contrast, AAV5 performed poorly, transducing only 0.1-0.3% of human hepatocytes and at best 0.8% of murine hepatocytes. This may in part also reflect the previously noted reduction in liver transduction when AAV5 is administered from a peripheral vein. Our model suggests that AAV5 is considerably less efficacious, in particular when given via a peripheral vein. In conclusion, AAV3-based vectors may improve liver-directed human gene therapy for hemophilia compared to the current prevailing AAV8 vector system. Disclosures Herzog: Novo Nordisk: Research Funding; Spark Therapeutics: Patents & Royalties: Patent licenses. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Srivastava:University of Florida: Patents & Royalties: AAV vector technology.
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Adam, Virginie S., Marco Crosariol, Sachin Kumar, Moyar Q. Ge, Sarah E. Czack, Soumitra Roy, Angela Haczku, Anna Tretiakova, James M. Wilson, and Maria P. Limberis. "Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus." Clinical and Vaccine Immunology 21, no. 11 (September 10, 2014): 1528–33. http://dx.doi.org/10.1128/cvi.00572-14.
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ABSTRACTInfluenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunodeficient (severe combined immunodeficient [SCID]) mice that were treated intranasally with AAV9 vector expressing a modified version of the broadly neutralizing anti-influenza antibody FI6 were protected and exhibited no signs of disease following an intranasal challenge with the mouse-adapted H1N1 influenza strain A/Puerto Rico/8/1934(H1N1) (PR8) (Mt. Sinai strain). Nonvaccinated mice succumbed to the PR8 challenge due to severe weight loss. We propose that airway-directed AAV9 passive immunization against airborne infectious agents may be beneficial in elderly and immunocompromised patients, for whom there still exists an unmet need for effective vaccination against influenza.
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Cheah, Pike-See, Shilpa Prabhakar, David Yellen, Roberta L. Beauchamp, Xuan Zhang, Shingo Kasamatsu, Roderick T. Bronson, et al. "Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin." Science Advances 7, no. 2 (January 2021): eabb1703. http://dx.doi.org/10.1126/sciadv.abb1703.
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Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a “condensed” form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.
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Bantel-Schaal, Ursula, Hajo Delius, Rainer Schmidt, and Harald zur Hausen. "Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses." Journal of Virology 73, no. 2 (February 1, 1999): 939–47. http://dx.doi.org/10.1128/jvi.73.2.939-947.1999.
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ABSTRACT We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.
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Passini, Marco A., Deborah J. Watson, Charles H. Vite, Daniel J. Landsburg, Alyson L. Feigenbaum, and John H. Wolfe. "Intraventricular Brain Injection of Adeno-Associated Virus Type 1 (AAV1) in Neonatal Mice Results in Complementary Patterns of Neuronal Transduction to AAV2 and Total Long-Term Correction of Storage Lesions in the Brains of β-Glucuronidase-Deficient Mice." Journal of Virology 77, no. 12 (June 15, 2003): 7034–40. http://dx.doi.org/10.1128/jvi.77.12.7034-7040.2003.
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ABSTRACT Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (β-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from β-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders.
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Castle, Michael J., Yuhsiang Cheng, Aravind Asokan, and Mark H. Tuszynski. "Physical positioning markedly enhances brain transduction after intrathecal AAV9 infusion." Science Advances 4, no. 11 (November 2018): eaau9859. http://dx.doi.org/10.1126/sciadv.aau9859.
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Several neurological disorders may benefit from gene therapy. However, even when using the lead vector candidate for intrathecal administration, adeno-associated virus serotype 9 (AAV9), the strength and distribution of gene transfer to the brain are inconsistent. On the basis of preliminary observations that standard intrathecal AAV9 infusions predominantly drive reporter gene expression in brain regions where gravity might cause cerebrospinal fluid to settle, we tested the hypothesis that counteracting vector “settling” through animal positioning would enhance vector delivery to the brain. When rats are either inverted in the Trendelenburg position or continuously rotated after intrathecal AAV9 infusion, we find (i) a significant 15-fold increase in the number of transduced neurons, (ii) a marked increase in gene delivery to cortical regions, and (iii) superior animal-to-animal consistency of gene expression. Entorhinal, prefrontal, frontal, parietal, hippocampal, limbic, and basal forebrain neurons are extensively transduced: 95% of transduced cells are neurons, and greater than 70% are excitatory. These findings provide a novel and simple method for broad gene delivery to the cortex and are of substantial relevance to translational programs for neurological disorders, including Alzheimer’s disease and related dementias, stroke, and traumatic brain injury.
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Thomas, Clare E., Theresa A. Storm, Zan Huang, and Mark A. Kay. "Rapid Uncoating of Vector Genomes Is the Key toEfficient Liver Transduction with Pseudotyped Adeno-Associated VirusVectors." Journal of Virology 78, no. 6 (March 15, 2004): 3110–22. http://dx.doi.org/10.1128/jvi.78.6.3110-3122.2004.
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ABSTRACT Transduction of the liver with single-stranded adeno-associated virus serotype 2 (AAV2) vectors is inefficient; less than 10% of hepatocytes are permissive for stable transduction, and transgene expression is characterized by a lag phase of up to 6 weeks. AAV2-based vector genomes packaged inside AAV6 or AAV8 capsids can transduce the liver with higher efficiency, but the molecular mechanisms underlying this phenomenon have not been determined. We now show that the primary barrier to transduction of the liver with vectors based on AAV2 capsids is uncoating of vector genomes in the nucleus. The majority of AAV2 genomes persist as encapsidated single-stranded molecules within the nucleus for as long as 6 weeks after vector administration. Double-stranded vector genomes packaged inside AAV2 capsids are at least 50-fold more active than single-stranded counterparts, but these vectors also exhibit a lag phase before maximal gene expression. Vector genomes packaged inside AAV6 or AAV8 capsids do not persist as encapsidated molecules and are more biologically active than vector genomes packaged inside AAV2 capsids. Our data suggest that the rate of uncoating of vector genomes determines the ability of complementary plus and minus single-stranded genomes to anneal together and convert to stable, biologically active double-stranded molecular forms.
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Liu, Shijie, Ke Li, Leonardo Wagner Florencio, Li Tang, Todd R. Heallen, John P. Leach, Yidan Wang, et al. "Gene therapy knockdown of Hippo signaling induces cardiomyocyte renewal in pigs after myocardial infarction." Science Translational Medicine 13, no. 600 (June 30, 2021): eabd6892. http://dx.doi.org/10.1126/scitranslmed.abd6892.
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Human heart failure, a leading cause of death worldwide, is a prominent example of a chronic disease that may result from poor cell renewal. The Hippo signaling pathway is an inhibitory kinase cascade that represses adult heart muscle cell (cardiomyocyte) proliferation and renewal after myocardial infarction in genetically modified mice. Here, we investigated an adeno-associated virus 9 (AAV9)–based gene therapy to locally knock down the Hippo pathway gene Salvador (Sav) in border zone cardiomyocytes in a pig model of ischemia/reperfusion-induced myocardial infarction. Two weeks after myocardial infarction, when pigs had left ventricular systolic dysfunction, we administered AAV9-Sav–short hairpin RNA (shRNA) or a control AAV9 viral vector carrying green fluorescent protein (GFP) directly into border zone cardiomyocytes via catheter-mediated subendocardial injection. Three months after injection, pig hearts treated with a high dose of AAV9-Sav-shRNA exhibited a 14.3% improvement in ejection fraction (a measure of left ventricular systolic function), evidence of cardiomyocyte division, and reduced scar sizes compared to pigs receiving AAV9-GFP. AAV9-Sav-shRNA–treated pig hearts also displayed increased capillary density and reduced cardiomyocyte ploidy. AAV9-Sav-shRNA gene therapy was well tolerated and did not induce mortality. In addition, liver and lung pathology revealed no tumor formation. Local delivery of AAV9-Sav-shRNA gene therapy to border zone cardiomyocytes in pig hearts after myocardial infarction resulted in tissue renewal and improved function and may have utility in treating heart failure.
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Gao, Guang-Ping, You Lu, Xun Sun, Julie Johnston, Roberto Calcedo, Rebecca Grant, and James M. Wilson. "High-Level Transgene Expression in Nonhuman Primate Liver with Novel Adeno-Associated Virus Serotypes Containing Self-Complementary Genomes." Journal of Virology 80, no. 12 (June 15, 2006): 6192–94. http://dx.doi.org/10.1128/jvi.00526-06.
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ABSTRACT Adeno-associated virus (AAV) vectors are being considered for in vivo applications of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second-generation vectors based on the novel serotypes AAV7 and AAV8 and containing self-complementary genomes in the nonhuman primate liver. Stable levels of transgene expression were achieved in cynomolgus macaques and suggest efficiencies at least 2 log higher than what could be achieved with AAV2 vectors using traditional single-stranded genomes. Analysis of DNAs from tissues revealed high levels of vector in the liver that appeared proportional to the relative amounts of transgene expression.
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Halbert, Christine L., James M. Allen, and A. Dusty Miller. "Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors." Journal of Virology 75, no. 14 (July 15, 2001): 6615–24. http://dx.doi.org/10.1128/jvi.75.14.6615-6624.2001.
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ABSTRACT Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations ofrep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.
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Legmann, R. "Transient transfection at large scale for clinical AAV9 vector manufacturing." Cytotherapy 22, no. 5 (May 2020): S151. http://dx.doi.org/10.1016/j.jcyt.2020.03.312.
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DeRosa, Samantha, Monica Salani, Sierra Smith, Madison Sangster, Victoria Miller-Browne, Sarah Wassmer, Ru Xiao, et al. "MCOLN1 gene therapy corrects neurologic dysfunction in the mouse model of mucolipidosis IV." Human Molecular Genetics 30, no. 10 (April 5, 2021): 908–22. http://dx.doi.org/10.1093/hmg/ddab093.
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Abstract Mucolipidosis IV (MLIV) is an orphan disease leading to debilitating psychomotor deficits and vision loss. It is caused by loss-of-function mutations in the MCOLN1 gene that encodes the lysosomal transient receptor potential channel mucolipin1, or TRPML1. With no existing therapy, the unmet need in this disease is very high. Here, we showed that AAV-mediated CNS-targeted gene transfer of the human MCOLN1 gene rescued motor function and alleviated brain pathology in the MLIV mouse model. Using the AAV-PHP.b vector in symptomatic mice, we showed long-term reversal of declined motor function and significant delay of paralysis. Next, using self-complementary AAV9 clinical candidate vector, we showed that its intracerebroventricular administration in post-natal day 1 mice significantly improved motor function, myelination and reduced lysosomal storage load in the MLIV mouse brain. Based on our data and general advancements in the gene therapy field, we propose scAAV9-mediated CSF-targeted MCOLN1 gene transfer as a therapeutic strategy in MLIV.
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Mingozzi, Federico, Xavier M. Anguela, Giulia Pavani, Yifeng Chen, Robert J. Davidson, Daniel J. Hui, Christian J. Hinderer, et al. "A Novel Strategy to Circumvent Pre-Existing Humoral Immunity to AAV." Blood 120, no. 21 (November 16, 2012): 2050. http://dx.doi.org/10.1182/blood.v120.21.2050.2050.
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Abstract Abstract 2050 Adeno-associated viral (AAV) vector-mediated gene transfer has shown great potential as a therapeutic platform for inherited and metabolic diseases. Systemic delivery of AAV vectors through the bloodstream is a safe, non-invasive, and potentially effective strategy to target a variety of organs, including liver, muscle, and brain. However, neutralizing antibodies (NAb) to AAV, highly prevalent in humans, constitute a major obstacle to successful gene transfer, particularly when a vector is delivered through the vasculature. Thus far, the liver was targeted to express the coagulation factor IX (F.IX) transgene in two clinical studies. In one study, a single-stranded AAV2 vector expressing the F.IX transgene was delivered through the hepatic artery to severe hemophilia B subjects at doses of 8×1010, 4×1011, and 2×1012 vector genomes (vg)/kg. Efficacy was observed in one subject from the high-dose cohort, who achieved peak F.IX transgene plasma levels of ∼10% of normal. The subjects infused at lower doses did not show any evidence of transgene expression, despite the fact that they did not have detectable NAb to AAV. In a second study, a self-complementary AAV8 vector expressing the F.IX transgene was delivered through peripheral vein infusion to severe hemophilia B subjects at doses similar to those administered in the AAV2 study, 2×1011, 6×1011, and 2×1012 vg/kg. All subjects enrolled in the AAV8 trial had evidence of transgene expression above baseline levels, despite the fact that some of the subjects had low-but-detectable anti-AAV8 NAb. Peak F.IX plasma levels at the high vector dose were 8–12% of normal, similar to the high dose of the AAV2 trial, suggesting that the vectors used in the two studies had comparable potency. Importantly, the vectors used in the two studies differed in empty capsid content, as the AAV2 vector preparation was essentially empty capsid-free and the AAV8 vector contained a 5–10 fold excess of empty capsids. The current study was undertaken to explore the role of empty capsids as a factor in the difference in outcome in the low- and mid- dose cohorts of the two trials. Our underlying hypothesis was that the presence of an excess of empty capsids effectively absorbs low-level neutralizing and non-neutralizing antibodies, and permits transduction even in their presence. Using a newly developed AAV antibody dot-blot assay, we demonstrate that adult human subjects with a low to undetectable NAb titer (1:1) as assessed by a commonly used assay do, in fact, carry significant amounts of anti-AAV antibodies. Conversely, children aged one year appear to be truly naïve for anti-AAV humoral immunity. Using C57BL/6 mice passively immunized with purified human IgG injected intraperitoneally 24 hours before vector administration, we further demonstrate that the same low levels of anti-AAV antibodies found in humans (NAb titer of 1:1–1:3) can block >90% of liver transduction after peripheral vein delivery of AAV8 vectors expressing F.IX at doses of 1×1012 vg/kg, comparable to those tested in the clinic. We next demonstrated that the inhibitory effect of low titer (1:1–1:3) anti-AAV antibodies can be overcome by adding a 5 to 10-fold excess of empty capsids to the final formulation of AAV8 vector, and that empty capsid content can be carefully titrated as a function of the animal's anti-AAV NAb in order to achieve efficient target organ transduction, even at titers >1:100. However, the beneficial effect of empty capsids on liver transduction is lost when a 1000-fold excess of AAV8 empty capsids are added to the formulation of AAV8 vectors, due to receptor binding competition. This inhibitory effect could be avoided by using AAV2 empty capsids, which efficiently protect AAV8 vectors from NAb without inhibiting transduction. These results were confirmed in non-human primates, a natural host for AAV8, in which a 5 to 6-fold increase in liver transduction was achieved by formulating vector in 5–10 fold excess AAV8 empty capsids, reaching levels of F.IX expression of 10 to 20% of normal. Application of these findings to the development of personalized formulations of vector product for intravascular delivery will facilitate safe, effective AAV-mediated gene transfer in settings in which vectors are delivered through the systemic circulation. Disclosures: Mingozzi: Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.
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Sarkar, Rita, Renee Tetreault, Guangping Gao, Lili Wang, Peter Bell, Randy Chandler, James M. Wilson, and Haig H. Kazazian. "Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype." Blood 103, no. 4 (February 15, 2004): 1253–60. http://dx.doi.org/10.1182/blood-2003-08-2954.
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Abstract Despite the popularity of adeno-associated virus 2 (AAV2) as a vehicle for gene transfer, its efficacy for liver-directed gene therapy in hemophilia A or B has been suboptimal. Here we evaluated AAV serotypes 2, 5, 7, and 8 in gene therapy of factor VIII (FVIII) deficiency in a hemophilia A mouse model and found that AAV8 was superior to the other 3 serotypes. We expressed canine B domain-deleted FVIII cDNA either in a single vector or in 2 separate AAV vectors containing the heavy- and light-chain cDNAs. We also evaluated AAV8 against AAV2 in intraportal and tail vein injections. AAV8 gave 100% correction of plasma FVIII activity irrespective of the vector type or route of administration.
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Wu, Zhijian, Aravind Asokan, Joshua C. Grieger, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, and R. Jude Samulski. "Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes." Journal of Virology 80, no. 22 (August 30, 2006): 11393–97. http://dx.doi.org/10.1128/jvi.01288-06.
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ABSTRACT Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.
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Ruppert, T., M. B. Heckmann, K. Rapti, D. Schultheis, A. Jungmann, H. A. Katus, L. Winter, et al. "AAV-mediated cardiac gene transfer of wild-type desmin in mouse models for recessive desminopathies." Gene Therapy 27, no. 10-11 (April 22, 2020): 516–24. http://dx.doi.org/10.1038/s41434-020-0147-7.
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AbstractMutations in the human desmin gene cause autosomal-dominant and recessive cardiomyopathies and myopathies with marked phenotypic variability. Here, we investigated the effects of adeno-associated virus (AAV)-mediated cardiac wild-type desmin expression in homozygous desmin knockout (DKO) and homozygous R349P desmin knockin (DKI) mice. These mice serve as disease models for two subforms of autosomal-recessive desminopathies, the former for the one with a complete lack of desmin protein and the latter for the one with solely mutant desmin protein expression in conjunction with protein aggregation pathology in striated muscle. Two-month-old mice were injected with either a single dose of 5 × 1012 AAV9-hTNT2-mDes (AAV-Des) vector genomes or NaCl as control. One week after injection, mice were subjected to a forced swimming exercise protocol for 4 weeks. Cardiac function was monitored over a period of 15 month after injection and before the mice were sacrificed for biochemical and morphological analysis. AAV-mediated cardiac expression of wild-type desmin in both the homozygous DKO and DKI backgrounds reached levels seen in wild-type mice. Notably, AAV-Des treated DKO mice showed a regular subcellular distribution of desmin as well as a normalization of functional and morphological cardiac parameters. Treated DKI mice, however, showed an aberrant subcellular localization of desmin, unchanged functional cardiac parameters, and a trend toward an increased cardiac fibrosis. In conclusion, the effect of a high-dose AAV9-based desmin gene therapy is highly beneficial for the heart in DKO animals, but not in DKI mice.
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Manso, Ana Maria, Sherin I. Hashem, Bradley C. Nelson, Emily Gault, Angel Soto-Hermida, Elizza Villarruel, Michela Brambatti, et al. "Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease." Science Translational Medicine 12, no. 535 (March 18, 2020): eaax1744. http://dx.doi.org/10.1126/scitranslmed.aax1744.
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Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.
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Zabner, Joseph, Michael Seiler, Robert Walters, Robert M. Kotin, Wendy Fulgeras, Beverly L. Davidson, and John A. Chiorini. "Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer." Journal of Virology 74, no. 8 (April 15, 2000): 3852–58. http://dx.doi.org/10.1128/jvi.74.8.3852-3858.2000.
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ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.
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Pattali, Rithu, Yongchao Mou, and Xue-Jun Li. "AAV9 Vector: a Novel modality in gene therapy for spinal muscular atrophy." Gene Therapy 26, no. 7-8 (June 26, 2019): 287–95. http://dx.doi.org/10.1038/s41434-019-0085-4.
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Woodard, Kenton T., Katharine J. Liang, William C. Bennett, and R. Jude Samulski. "Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism." Journal of Virology 90, no. 21 (August 24, 2016): 9878–88. http://dx.doi.org/10.1128/jvi.01568-16.
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ABSTRACTMany adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescencein situhybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on humanex vivoretinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery.IMPORTANCEAdeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.
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Shi, Yimin, Rustom Falahati, and Karin ML Gaensler. "Tolerance Induction by Neonatal Gene Delivery." Blood 112, no. 11 (November 16, 2008): 4628. http://dx.doi.org/10.1182/blood.v112.11.4628.4628.
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Abstract Advances in the design and efficiency of gene delivery vectors have enabled the initiation of clinical trials in gene therapy for genetic and other disorders. However, the development of inhibitory immune responses to vector antigens and to therapeutic proteins remains an obstacle. Efforts to limit these immune responses by immunosuppressive and immuno-modulatory approaches have met with limited success. Our approach is to deliver and express viral vectors early in immune ontogeny and thereby induce immune tolerance to both vectors and therapeutic proteins. We have previously shown that in utero delivery of AAV-2 vectors produces lifelong gene expression without immune responses, and that augmented levels of gene expression are achieved with re-administration of AAV vectors. Because fetal injections are limited by technical issues, our current focus is to use a neonatal model for defining the critical period when tolerance to vector and transgene may be achieved by primary injection. We are also exploring mechanisms of tolerance induction to neo-antigens. We have delivered AAV serotypes 1 and 8 with higher transduction efficiencies than AAV-2, to assess the expression levels, duration, and tissue distribution of luciferase by semi-quantitative longitudinal in vivo bioluminescence assays. In both C57BL/6 and BALB/c strains, neonatal injection of AAV1-Luc or AAV8-Luc by either IP, IV or IT routes produces lifelong gene expression. After IP injection at day 1–2 of life, gene expression increases 10–20 fold over the next several months. Highest levels of expression were achieved by IP injection, with lowest levels observed after IV injection. Injection of AAV1-Luc achieved higher levels of luciferase expression than did injection of AAV8- Luc. In contrast to the localized distribution of AAV1 mediated luciferase expression in the injected area, widespread, systemic expression of luciferase mediated by AAV8 after neonatal delivery is observed, regardless of the route of delivery. The effect of this altered tropism on gene expression levels and tolerance induction is being examined. In both C57BL/6 and BALB/c mice, IP injection of AAV1-Luc or AAV8-Luc at 1–2 days, 1 week, 2 weeks, or 3 weeks of age produced lifelong expression of luciferase and resulted in increasing levels of antibody responses against AAV1 or AAV8 with increasing age at primary injection. Antibody titers to AAV1 or AAV8 in animals injected at day 1–2 of life were comparable to background levels in uninjected animals. In C57BL/6 mice receiving a primary injection of AAV8-Luc, secondary injection of AAV8-Luc boosted the antibody response to AAV8 in the animals first injected at 1 week, 2 weeks or 3 weeks, but not in the animals injected at 1–2 day of life. We are currently exploring whether augmented expression with re-administration of AAV vectors in adult animals is due to an active process such as tolerance, partial tolerance or anergy. Developing strategies for the induction of tolerance to gene delivery vectors and therapeutic gene products will be an important advance for gene therapy for genetic and other disorders.
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Wang, Lili, Roberto Calcedo, Timothy C. Nichols, Dwight A. Bellinger, Aaron Dillow, Inder M. Verma, and James M. Wilson. "Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy." Blood 105, no. 8 (April 15, 2005): 3079–86. http://dx.doi.org/10.1182/blood-2004-10-3867.
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AbstractAdeno-associated virus 8 (AAV8), a new member of the AAV family isolated from nonhuman primates, is an attractive candidate for hepatic gene transfer applications because of 10- to 100-fold improved transduction efficiency in mouse liver models. Additionally, AAV8 has lesser frequency of pre-existing immunity in humans. These properties could solve some of the problems associated with AAV2 vectors. The benefits of AAV8 demonstrated in mouse models, however, have not been confirmed in larger animals. In this study, we evaluate the efficacy and safety of AAV2/8 vector in both naive and AAV2-pretreated hemophilia B dogs. Two naive hemophilia B dogs that received a single intraportal administration of AAV2/8 vector have achieved sustained expression of 10% and 26% of normal levels of canine factor IX (cFIX) for more than a year. In an AAV2-pretreated hemophilia B dog, cFIX expression increased from less than 1% to 16% of normal levels when treated with an AAV2/8 vector, and a high level of expression has lasted for more than 2 years. No significant liver toxicity or cFIX-specific antibodies have been detected in these animals. Studies here have demonstrated the safety and improved efficacy of AAV2/8 vector in large-animal models for liver-directed gene therapy.
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Wu, Zhijian, Edward Miller, Mavis Agbandje-McKenna, and Richard Jude Samulski. "α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6." Journal of Virology 80, no. 18 (September 15, 2006): 9093–103. http://dx.doi.org/10.1128/jvi.00895-06.
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ABSTRACT Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection.