Background
AAV was originally discovered as a contaminating component, and 13 serotypes have been isolated so far: AAV1 to AAV13. AAV belongs to the genus Parvoviridae and is a type of tiny, non-enveloped linear single-stranded DNA virus. Its genome size is about 4700bp and its diameter is 20-26nm. AAV is a defective virus that requires helper viruses (adenovirus helper function genes include E1a, E1b, E2a, E4orf6 and VARNA, herpes virus helper function genes UL5, UL8, UL22, UL9 and regulatory proteins ICP0, ICP4, ICP22), etc. In order to infect host cells and produce new virus particles, when there is no helper virus, wild-type AAV can only integrate into the long arm of chromosome 19 to form a latent infection and replicate along with the host cell chromosome replication. The AAV genome is mainly composed of inverted repeats (ITRs) and 2 open reading frames (ORFs) between the 2 ITRs. The left ORF codes for Rep78, Rep68, Rep52 and Rep40, and the right ORF codes for the 3 capsid proteins VP1 (relative molecular mass 87000), VP2 (relative molecular mass 72000), VP3 (relative molecular mass 62000). The AAV capsid is composed of 60 capsid proteins arranged in an icosahedral structure. Different AAV serotypes have different spatial structures, sequences and tissue specificities of capsid proteins, so the cell surface receptors they recognize and bind to are also very different, which also leads to different tissue types and cell types transfected by different serotypes, and infection efficiency also vary.
Figure 1. Testing preexisting antibodies prior to AAV gene transfer therapy. (Source: Mendell JR, et al., 2022)
The liver is an organ that has been used for gene therapy earlier. It is now clear that many liver diseases are caused by single gene defects, so it is more suitable for clinical gene therapy than other complex diseases caused by multiple gene defects. The challenges facing liver-targeted gene therapy lie in the effective targeting of liver cells, the stability of the vector genome and sustained high-level expression. The emergence and use of rAAV vectors make it possible to solve this problem. rAAV vectors usually replace the rep and cap genes in wild-type AAV (Wt-AAV) with the target genes for research, leaving only the two ITRs at both ends of the AAV genome. rAAV vectors are currently the most popular vectors. Studies have shown that AAV2, AAV5, AAV8, AAV9 and AAV-DJ are the most commonly used serotypes in the liver. Studies have shown that AAV8, AAV9 and AAV-DJ all have strong affinity for the liver, among which AAV8 It is more hepatotropic and has low immunogenicity and high infection efficiency when injected into living animals. The in vivo hepatocyte transduction efficiency of AAV-DJ is similar to that of AAV8, and it can also show strong infection efficiency in in vitro experiments. Researchers can select appropriate serotypes based on experimental purposes and animal models. Among them, AAV7 and AAV8 have better effects on mouse liver infection; while in human livers, AAV2, AAV5 and AAV8 have stronger affinity.
AAV may cause adverse reactions when conducting clinical experiments on animals and humans. The first is the immune response. When rAAV infects antigen-presenting cells, a mild innate immune response can be induced due to the activation of Toll-like receptor 9 (TLR9). TLR9 recognizes unmethylated CpG in nucleic acids, thereby activating the classical pathway and non-methylated CpG. The classic NF-κB pathway promotes the expression of inflammatory cytokines and interferon response genes. Expression of these genes in turn generates CTL responses and stable neutralizing antibody titers. In order to avoid immune responses caused by AAV-mediated gene transfer, there are currently two commonly used methods: 1, Use immunosuppressive drugs to avoid CTL reactions, which was successful in human factor IX trials; 2, When designing rAAV vectors, DNA sequences that inhibit TLR9, such as multiple copies of TTAGGG from human telomeres, are integrated into the rAAV genome to evade the innate immune response.
Alternative Names
Adeno-associated virus serotype 8
References
- 1. Mendell JR, et al., Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations. Mol Ther Methods Clin Dev. 2022, 25:74-83.
References
Successful AAV8 readministration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease
J Gene Med
Authors: Choi SJ, Yi JS, Lim JA, Tedder TF, Koeberl DD, Jeck W, Desai AK, Rosenberg A, Sun B, Kishnani PS.
Abstract
Background: A major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice.
Methods: An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood.
Results: In n?ive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration.
Conclusions: Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naïve mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.
Split AAV8 Mediated ABCA4 Expression for Gene Therapy of Mouse Stargardt Disease (STGD1)
Hum Gene Ther
Authors: Li R, Jing Q, She K, Wang Q, Jin X, Zhao Q, Su J, Song L, Fu J, Wu X, Xu Q, Lu F, Wei Y, Yang Y
Abstract
Adeno-associated virus (AAV)-based gene therapy has been shown to be safe and effective in numerous animal models and clinical trials for various ophthalmic diseases. Stargardt disease (STGD1; MIM #248200) is the most common autosomal recessive macular dystrophy disease, and the most common form is caused by mutations in the ABCA4 gene, a gene with 6.8 kb coding sequence. Split intein approaches increase the capacity of dual AAV gene therapy, but at the cost of reduced protein expression, which may be insufficient to achieve a therapeutic effect. In this study, we designed various dual split intein ABCA4 vectors and showed that the efficiency of expression of full-length ABCA4 protein is dependent on combinations of types and split sites of the intein system. The most efficient vectors were identified through in vitro screening, and a novel dual AAV8-ABCA4 vector was constructed and subsequently proven to express full-length ABCA4 protein at a high level, reducing bisretinoid formation and correcting the visual function of ABCA4-knockout mice. Furthermore, we evaluated therapeutic effects of different dosages by subretinal injection in mice model. Both therapeutic effects and safety were guaranteed under the treatment of 1.00 × 109 GC/eye. These results support the optimized dual AAV8-ABCA4 approach in future clinical translation for treatment of Stargardt disease.
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