91. Development of Optimized [“Opt”] AAV Vectors by Combining Capsid-Modified NextGen and Genome-Modified GenX AAV Vectors for High-Efficiency Transduction at Further Reduced Doses
Authors: Chen Ling, Baozheng Li, Wenqin Ma, George Aslanidi, Arun Srivastava
Journal: Molecular Therapy (2016)

There is little doubt that AAV did not evolve for the purposes of delivery of therapeutic genes. The use of first generation AAV vectors, albeit successful, is unlikely to reach its full potential. We have described the development of capsid-modified next generation [NextGen] AAV vectors for both AAV2 (Proc Natl Acad Sci USA, 105: 7827-7832, 2008; Mol Ther., 18: 2048-2056, 2010; Vaccine, 30: 3908-3917, 2012; PLoS One, 8: e59142, 2013) and AAV3 (Gene Ther., 19: 375-384, 2012; Hum Gene Ther., 25: 1023-1034, 2014) serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses Fig. 1A). We have also described the development of genome-modified generation X [GenX] AAV vectors (J Virol., 89: 952-961, 2015), in which the transcriptionally-inactive, single-stranded AAV genome was modified to achieve improved transgene expression (Fig. 1B). Thus, we reasoned that encapsidation of GenX AAV genomes into NextGen AAV capsid might lead to further increased transduction at further reduced vector doses. To this end, the following sets of ssAAV2 as well as ssAAV3 serotype vectors containing the firefly luciferase (Fluc) reporter gene were generated: (i) wild-type (WT) genome and Y444+500+730F+T492V AAV2 quadruple-mutant (QM) capsid (WT-Fluc-AAV2/QM); (ii) two GenX genomes and AAV2 QM capsid (LC1-Fluc-AAV2/QM and LC2-Fluc-AAV2/QM); (iii) WT genome and S663V+T492V AAV3 double-mutant (DM) capsid (WT-Fluc-AAV3/DM); and (iv) two GenX genomes and AAV3 DM capsid (LC1-Fluc-AAV3/DM and LC2-Fluc-AAV3/DM). The combination of the modified-genomes with the capsid-mutants led to ~5-6-fold increase with both AAV2 and AAV3 serotype vectors following transduction of a human hepatocellular carcinoma (HCC) cell line, Huh7, at an MOI of 1,000 vgs/cell under identical conditions in vitro. When male C57BL/6 mice were injected via tail-vein with WT-Fluc-AAV2/QM, LC1-Fluc-AAV2/QM, and LC2-Fluc-AAV2/QM vectors at a relatively low dose of 5×109 vgs/mouse, the AAV2 QM capsid-mutant vectors led to ~6-10-fold increase in transgene expression in the liver. Similarly, when WT-Fluc-AAV3/DM, LC1-Fluc-AAV3/DM, and LC2-Fluc-AAV3/DM vectors were injected via tail-vein in NSG mice xenografted with human liver tumors at relatively low dose of 3×109 vgs/mouse, led to ~5-8-fold increase with the combination of modified-genomes with the AAV3 DM capsid-mutant vectors, which was restricted to human liver tumors. Taken together, these data document that the combination of NextGen capsids and GenX genomes leads to the generation of optimized [Opt] AAV serotype vectors (Fig. 1C), which transduce cells and tissues more efficiently, both in vitro and in vivo, at significantly reduced doses. These studies have significant implications in the potential use of the Opt AAV serotype vectors in human gene therapy.