98. Evaluation of Proteasome Inhibitors on AAV-Mediated Transduction Efficiency in Retinal Bipolar Cells

Purpose: Recombinant adeno-associated virus (rAAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell-type specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in targeted gene expression in retinal bipolar cells, especially for optogenetic gene therapy for vision restoration. However, rAAV-mediated transduction efficiency in retinal bipolar cells is relatively low. The transduction efficiency could be affected by a number of factors, one of which is the proteasome-dependent virus degradation. In this study, we are examining the effect of proteasome inhibitors on the transduction efficiency of rAAV vectors in retinal bipolar cells. Methods: The expression of the transgene, mCherry, was used to evaluate the AAV transduction efficiency. Targeted expression of mCherry in retinal bipolar cells was achieved by rAAV2 vectors carrying an mGluR6 promoter. rAAV vectors at the concentration of 5 × 1012 vg/ml with or without containing proteasome inhibitors were intravitreally injected into the eyes of C57BL/6J mice at about one month of age. Animals were euthanized about one month after virus injection for assessing the expression of mCherry. Results: We tested the effects of three proteasome inhibitors, MG132, doxorubicin, and aclarubicin, on rAAV-mediated transduction efficiency in retinal bipolar cells. Retinas treated with doxorubicin from 200 µM to 800 µM exhibited a concentration-dependent increase in the transduction efficiency. Doxorubicin at the concentration of 2000 µM produced cytotoxicity as evidenced by the thinning of the retinas and decreased the number of mCherry-expressing bipolar cells. The optimal concentration of doxorubicin to enhance the AAV transduction efficiency was 500 µM. MG132 (100 µM, 200 µM, 500 µM) and aclarubicin (50 µM, 100 µM) were not found to enhance the transduction efficiency. Conclusions: Doxorubicin, a proteasome inhibitor, is effective in enhancing rAAV transduction in retinal bipolar cells in mice. Investigation of the mechanism of doxorubicin action may help to further improve the rAAV transduction efficiency in retinal bipolar cells in particular and rAAV-mediated gene delivery in general.