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Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30–50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration. Keywords: AAV, hemophilia, neutralizing antibodies, NAbs, re-administration, human factor VIII, hFVIII, hFVIII inhibitor