Chemical Modulation of AAV Trafficking

Adeno-associated virus is widely studied due to the promise it holds as a gene therapy vector. Gene therapy broadly describes strategies in which genetic material is introduced into a target cell in an effort to treat or cure disease. However, even with AAV being used as a gene delivery vector in over 100 clinical trials to date, there is still much unknown about the biology of the vector. Further understanding of the trafficking of the vector through the host cell will contribute to the safety and efficacy of the inevitable clinical trials and therapies that are to come. In this dissertation, we utilized small molecules to dissect and modulate the trafficking of AAV vectors. Firstly, we utilized numerous small molecules to dissect the potential role of several cellular degradation mechanisms in the AAV infectious pathway. We identified the ERAD inhibitor Eeyarestatin I (EerI) as a molecule that augments AAV transduction. EerI increased transduction by approximately 10-fold in a serotype, cell type, and genome type independent manner. Additionally, EerI and the proteasome inhibitor MG132 acted in distinct ways to augment AAV transduction. Further, EerI modulated the intracellular trafficking of AAV by redirecting AAV to enlarged Rab7/LAMP1 positive vesicles. This EerI-mediated redirection of AAV protected capsids from proteasomal degradation, thereby increasing the nuclear accumulation of AAV capsids. Next, we utilized ionomycin and BAPTA-AM to modulate the intracellular calcium environment and determined that intracellular calcium concentration influences AAV transduction. Ionomycin increases intracellular calcium concentration, and decreases transduction by approximately 10-fold. Ionomycin acts to block transduction at or before AAV nuclear entry. BAPTA-AM decreases intracellular calcium concentration, and increases transduction by approximately 10 to 100-fold in vitro and in vivo. BAPTA-AM likely acts at multiple steps in the AAV pathway to increase transduction. However, we identified that BAPTA-AM increased RNA transcription from the AAV vector genome, thereby increasing transgene protein levels. Taken together, we demonstrate multiple methods to modulate AAV trafficking using small molecules. Furthermore, we demonstrate that use of two of these molecules, EerI and BAPTA-AM, can augment AAV transduction, providing additional strategies for increasing AAV transduction in the clinic.Doctor of Philosoph