Optimization of the Capsid of Recombinant Adeno-Associated Virus 2 (AAV2) Vectors: The Final Threshold?

Abstract

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

“…The rational design of the AAV capsid to avoid proteosomal degradation enhances transduction efficiencies (30,(35)(36)(37)(38). This is accomplished by the substitution of surface-exposed residues that otherwise would be phosphorylated and thereby target the capsid for ubiquitin-dependent proteasomal degradation.…”

Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses

“…15 We have also described that for the genome-modified AAV2 vectors, LC1 and LC2, in which one of the D-sequences in the viral inverted terminal repeats (ITRs) was deleted, the efficiency of transgene expression from the single-stranded genomes was significantly increased. 17 Thus, as depicted schematically in Figure 1, we generated the following set of QMssAAV2 vectors containing the firefly luciferase (Fluc) reporter gene flanked by either the unmodified ITRs (QM-ssAAV2-WT-Fluc), or with D-sequence-deleted ITRs (QM-ssAAV2-LC1-Fluc and QM-ssAAV2-LC2-Fluc).…”

“…By modifying the capsids, we have generated the nextgeneration AAV vectors in which specific surfaceexposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues were mutagenized, and various permutations and combinations of these mutations thereof, to significantly augment the transduction efficiency of these vectors, both in vitro and in vivo, by circumventing proteasome-mediated degradation. [14][15][16]30 At least one of these vectors has also shown to be less immunogenic 31 and, in a recent phase I clinical trial, shown efficacy for the potential gene therapy of Leber's hereditary optic neuropathy. 32 We have also described strategies involving genome modifications that lead to the generation of ssAAV vectors with which high efficiency of transgene expression can be achieved.…”

“…In such a pursuit, we developed the next-generation AAV serotype vectors, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues were mutagenized to achieve enhanced transduction efficiency at reduced vector doses. [14][15][16] In addition to the capsid-modified AAV vectors, we have also described the generation of genomemodified vectors, with which improved transgene expression from single-stranded AAV (ssAAV) vectors can be achieved. 17 In the present studies, we wished to examine whether combining the capsid modifications and genome modifications might lead to the generation of optimized AAV vectors capable of increased transduction at further reduced doses.…”

Adeno-Associated Virus: The Naturally Occurring Virus Versus the Recombinant Vector

“…6 A herculean effort by yet another long-term colleague, George Aslanidi, PhD, and colleagues, who mutagenized all 17 surface-exposed serines, all 15 surfaceexposed threonines, and various permutations and combinations thereof, led to the identification of the most efficient AAV2 vectors to date. 7 In addition, because ubiquitination occurs on lysines, all seven surface-exposed lysine residues on the AAV2 capsid were also mutagenized. 8 Remarkably, most, if not all, of the surfaceexposed tyrosine, serine, threonine, and lysine residues are highly conserved among all 10 commonly used AAV serotype vectors.…”