315. Optimization of the Capsid of Recombinant Adeno-Associated Virus 6 (AAV6) Vectors for Liver-, and Muscle-Directed Gene Therapy

Abstract

Recombinant adeno-associated virus (AAV) vectors are currently in use in a number of Phase I/II clinical trials. However, large vector doses are needed to achieve therapeutic effects. The ubiquitinproteasome pathway plays a critical role in the intracellular trafficking of AAV vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. We have previously reported that substitution of critical amino acid residues dramatically increases the transduction efficiency of vectors based on AAV serotypes 2, 3, 5, and 8 by preventing capsid phosphorylation and subsequent proteasomal degradation.In the present studies, we document that the combination of mutations of specific tyrosine (Y), serine (S), threonine (T) and lysine (K) residues on the capsid of AAV6 vectors also significantly increases the transduction efficiency compared with the wild-type (WT) AAV6 vectors. AAV6 capsid contains 7 tyrosine, 17 serine, 19 threonine and 11 lysine residues, which are surface-exposed. Initially, 17 single-mutants were generated based on the AAV6 crystal structure and overlap of amino acid positions with previously studied serotypes: Y445F, Y705F, Y731F, S455V, S472V, S499V, S551V, S663V, S669V, T251V, T492V, T665V, T722V, K531E, K567E, K666E, K707E. Next, the best performing Y-, S-, T-, and K- mutations were combined onto a single AAV6 capsid to generate 9 double-mutants: Y705+731F, Y731F+T492V, T492V+S663V, T492V+S551V, T665V+S551V, S663V+S551V, T492V+K531E, S663V+K531E, T492V+K666E; 7 triple-mutants: Y445+705+731F, Y705+731F+S663V, Y705+731F+S551V, Y705+731F+T492V, Y705+731F+K531E, Y705+731F+K666E, T492V+S663V+K531E; and two quadruple-mutants: Y705+731F+T492V+S663V, and Y705+731F+T492V+K531E. We identified several AAV6-mutant vectors, such as S663+551V, Y705+731F+T492V and Y705+731F+K531E, which were particularly efficient. These vectors increased the transduction efficiency by approximately 5- to 8-fold in a murine hepatocyte cell line, H2.35, and approximately 3- to 5-fold in a murine myoblast cell line, C2C12, over the WT AAV6 vector in vitro. Similar results were obtained in murine hepatocytes and muscle in vivo after tail-vein or intra-muscular injections of these vectors, followed by whole-body bioluminescence imaging of C57BL/6 mice.These residues are localized towards the base of the protrusions surrounding the icosahedral 3-fold axes of the capsid (T492S, S551V and K531E), in the depression surrounding the 2-fold axes (Y705F and Y731Y), or the HI loop (Y663S). This study thus identifies these capsid surface regions as being critical for the trafficking and transduction properties of the AAV6 vectors.In summary, our studies have led to the generation of novel capsid-optimized AAV6 vectors for their potential use in liver-directed, and muscle-directed gene therapy in humans.