Enhancing transduction efficiency of adeno-associated virus 9 by cell line engineering: implication for gene therapy potency assay

Abstract

Adeno-associated virus (AAV)-mediated gene therapy holds significant promises to treat or potentially cure various human diseases. Although AAV holds promise for their significant therapeutic potential, batch-to-batch differences can exist from manufacturing; and therefore, a potency assay is required for clinical development of AAV. Among different serotypes, due to its ability to cross blood–brain barrier and wide-spread transduction capability in vivo upon systemic administration, AAV9 has been widely utilized for the development of treatment of neurological disorders. However, as AAV9 is known to show poor transduction in vitro, establishing a robust in vitro potency assay have been difficult. To this end, we engineered HEK293T and Schwann-like cell lines to express previously identified common AAV receptor, AAVR or endogenous host factor involved in AAV endosomal escapes, GPR108 that can increase infectivity of AAVs in an attempt to increase transduction capability of AAV9. We found that AAVR overexpressed Schwann-like cell line showed significant increase in AAV9 transduction; whereas, GPR108 overexpression showed no effect on AAV9 transduction. On the other hand, GPR108 engineered HEK293T showed increase in AAV9 transduction; whereas, AAVR overexpressed HEK293T cell line showed modest increase in AAV9 transduction. Gene expression analysis showed that AAVR is highly expressed in HEK293T compared to Schwann-like cell line; whereas, GPR108 is highly expressed in Schwann-like cell line when compared to HEK293T. These results indicate that different cell lines may require different gene engineering to increase AAV9 infectivity and analysis of endogenous expression of AAV entry factors for cell line to be engineered can improve efficiency of cell line engineering for AAV transduction.