Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy

Figures

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  Figure 1. Intracellular fate of the first generation of AAV vectors (A) Schematic representation of the first generation of AAV2 vectors that contain 7 surface-exposed tyrosine (Y) residues, undergo ① phosphorylation, ubiquitination, and proteasome-mediated degradation in the cytoplasm, and the broken down peptides trigger the MHC class I-mediated CD8+ cytotoxic T cell response. Site-directed mutagenesis ② of each of the Y residues to phenylalanine (F) residues leads to the next generation (“NextGen”) of AAV vectors, and the transduction efficiency of each Y-F mutant vector is increased, ranging between ∼2- and 11-fold. The combination of the three most efficient mutations ③ into one capsid results in the triple-mutant vector, the transduction efficiency of which is up to ∼30-fold higher than the first generation of AAV2 vectors. (B) Schematic representation of the life cycle of the first generation of AAV2 vectors that in addition to 7 Y residues, also contain 15 serine (S), 17 threonine (T), and 10 lysine (K) residues that are surface exposed and undergo phosphorylation and ubiquitination, followed by proteasome-mediated degradation in the cytoplasm. Site-directed mutagenesis each of…

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  Figure 2. Rate-limiting step of viral second-strand DNAsynthesis of the first generation of ssAAV vectors (A) Schematic model of inhibition of AAV second-strand DNA synthesis by a cellular chaperone protein, FKBP52, phosphorylated forms of which bind to the single-stranded D-sequence at the 3′ end of the AAV-ITR, which is also the “packaging signal” of the AAV genome and is indispensable. (B) The D-sequence at the 5′ end of the AAV-ITR, on the other hand, is dispensable, deletion of which leads to generation X (“GenX”) AAV vectors, transgene expression from which is significantly higher than that from conventional ssAAV vectors.

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  Figure 3. Strategies for the development of capsid-, genome-, and capsid+genome-modified AAV serotype vectors (A) Schematic structures of first generation, NextGen, ssAAV, GenX, and optimized (Opt) AAV2 vectors. (B) Schematic structures of Opt AAV2, Opt AAV3, and Opt AAV6 serotype vectors.

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  Figure 4. Strategies for dampening the host humoral immune response to AAV vectors ① Transient depletion of B lymphocytes using CD20 antibody, ② AAV D-sequence-mediated suppression of MHC class II gene expression, and ③ transient degradation of pre-existing IgG antibodies using IgG-cleaving IdeS and IdeZ bacterial endopeptidases. Modified image from.121

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  Figure 5. Schematic structures of the 10 most commonly used recombinant AAV serotype vectors AAV3 and AAV6 vectors have been documented to be selectively tropic for high-efficiency transduction of primary human hepatocytes and primary human hematopoietic stem cells, respectively.