595. Enhanced Liver Transduction and Efficient Protection from Pre-Existing Neutralizing Antibodies with Exosome-Associated AAV8 Vectors
Authors: Amine Meliani, Zachary Fitzpatrick, Florence Boisgerault, Giuseppe Ronzitti, Fanny Collaud, Severine Charles, Casey A Maguire, Federico Mingozzi
Journal: Molecular Therapy (2016)

Adeno-associated virus (AAV) based vectors are ideal tools for in vivo gene transfer. The excellent safety profile and the highly efficient targeting of hepatocytes in vivo have been demonstrated in both preclinical studies and clinical trials. Although AAV vectors are not strongly immunogenic, they can give rise to both cellular and humoral immune responses. Additionally, as a result of exposure to wild-type AAV, an important fraction of humans harbor pre-existing neutralizing antibodies against the viral capsid, which can prevent successful transduction by AAV vectors. This pose a serious obstacle to the widespread use of AAV vectors in gene therapy. Recently, we have shown that AAV can associate with exosomes (exo-AAV) ultracentrifuged media samples from 293T producer cells. Exosome-associated AAV1, 2, and 9 serotype capsids were tested and found to have enhanced transduction and antibody evasion capabilities compared to conventional AAV vectors. With the goal of developing an enhanced vector for liver directed gene therapy, we sought to characterize the in vivo transduction and biodistribution profile of exo-AAV8 compared to conventional cell-lysated harvested, iodixanol-gradient purified AAV8 vectors purified from the same preparation. The efficiency of liver targeting of conventional AAV8 vs. exo-AAV8 expressing human factor IX (F.IX) under the control of liver specific promotor were tested in naïve and pre-immunized animals. C57BL/6 mice (n=5/group) were passively immunized with intravenous human immunoglobulin (IVIg) intraperitoneally, followed 24h later by the intravenous administration of either conventional AAV8 or exo-AAV8 vectors expressing coagulation factor IX (F.IX, 5×1010 vg/mouse). exo-AAV8 completely shielded the capsid vector from neutralizing antibodies at IVIg doses between 0.5mg and 2mg/mouse (NAb titer ~ 1:3.16), resulting in equivalent levels of F.IX transgene expression to naïve animals treated with conventional AAV8 vector. At the highest IVIg dose tested (8mg/mouse), residual levels of F.IX transgene expression were about 30% of naïve animals treated with conventional AAV8 vectors. We next evaluated the efficiency of exo-AAV8 vs. AAV8 vectors in naïve animals. In male mice, no statistically significant difference in F.IX transgene expression levels was observed between the two vector types; also indicated by the similar pattern of vector genome biodistribution. Interestingly, female mice (in which the efficiency of liver transduction with AAV is extremely low compared with male animals) treated with exo-AAV8 showed a dramatic increase in transgene expression, comparable to that of male mice receiving conventional AAV8 vectors. In conclusion, exo-AAV8 vectors present an enhanced liver transduction profile compared with conventionally purified AAV vectors, both in naïve female animals and in animals carrying anti-capsid neutralizing antibodies.