264. Absence of CD8 T Cell Responses Against AAV Capsid Using Reduced Vector Dose or Library Selected Liver Targeted AAV2 Based Capsids
Authors: Brett Palaschak, Damien Marsic, David M. Markusic
Journal: Molecular Therapy (2015)

Predicting immune responses directed against the AAV capsid have proven to be a challenge in the translation of liver directed AAV gene therapy into the clinic. Previously we have described the first mouse model that allows us to evaluate the potential for CD8 T cell responses directed against AAV transduced hepatocytes and to define protocols and conditions to minimize this response (Martino et al. Blood 2013). Empirical data from both AAV2 and AAV8 clinical trials for hemophilia B have suggested that a threshold vector dose is needed for activation of capsid specific CD8 T cells and subsequent elimination of factor IX expressing hepatocytes. We sought to experimentally confirm this observation in our murine model by evaluating the impact of reducing AAV2 and AAV8 vector doses by one log. No reduction in FIX expression was observed for either AAV2 or AAV8 transduced mice receiving 1×1010 vg and capsid specific CD8 T cells, suggesting that that the reduced vector dose dropped the number of viral particles below a threshold for efficient MHC I presentation. In another set of studies we evaluated two recently described AAV2 capsid variants (AAV2-LiA and AAV2-LiC), which were selected for murine hepatocyte tropism from a highly complex rationally designed capsid library (Marsic et al. Mol Ther 2014). Both AAV2-LiA and AAV2-LiC vectors were not targeted by capsid specific CD8 T cells and resulted in stable FIX expression for the duration of the experiment. Our previous studies conducted on the AAV2-M3 (Y-F 444,500,730) capsid showed that these three mutations provided sustained FIX expression in the presence of capsid CD8 T cells. By design, AAV2-LiA contains two of the previously described (Y-F) mutations at positions 444 and 500 and AAV2-LiC contains one at position 500. These data now extend on this study and suggest that either a single Y-F mutation at position 500 or double at positions 444 and 500 may be sufficient in reducing capsid antigen presentation on hepatocytes. Although it is unclear if these single and double Y-F mutants are alone sufficient or if there are synergistic mutations in AAV2-LiA and AAV2-LiC that contribute to reduced capsid antigen presentation. Nonetheless, we were unable to detect the presence of infiltrating CD8 T cells both at the one week and four week time points where previous studies conducted on a similar AAV2 vector demonstrated loss of FIX expression and the presence of infiltrating CD8 T cells at the one week time point. Interestingly although AAV2-LiA was less efficient than AAV2-LiC in transgene expression in immune competent mice, our studies conducted in immune deficient mice showed that both vectors provided similar levels of FIX expression. These data suggest that AAV liver transduction efficiency is altered in immune deficient mice. Therefore, we would offer caution in the analysis and interpretation of AAV transgene expression data generated solely in immune deficient animals. Together, these data support a vector dose dependency threshold for capsid antigen presentation on hepatocyte MHC I molecules and demonstrates that the novel AAV2-LiA and LiC capsids avoid detection by capsid specific CD8 T cells.