Production of an Oncolytic Adeno-Associated Virus Containing the Pro-Apoptotic TRAIL Gene Can Be Improved by shRNA Interference

Recombinant Adeno-associated virus (rAAV) is a popular vector for treating genetic diseases caused by absent or defective genes. rAAVs can be produced that contain a therapeutic transgene, i.e., a correct copy of the affected gene, which is then delivered into target cells. A further application of rAAV is to deliver pro-apoptotic genes such as TNF-related apoptosis-inducing ligand (<i>TRAIL</i>) into cancer cells, leading to tumor regression. However, rAAV production is expensive and insufficient yields may hinder wide-spread adoption especially in systemic conditions. During rAAV production, the therapeutic transgene may be expressed in the producer cell line, and in the case of an oncolytic gene, this would likely lead to cell death thus reducing rAAV yields. Here we demonstrate that expression of TRAIL during rAAV production in HEK293F cells negatively impacts rAAV yield. A shRNA-based strategy was developed to suppress the expression of TRAIL in rAAV-producing cells specifically during the production process. Incorporating a <i>TRAIL</i>-targeting shRNA expression cassette within the backbone of the rAAV genome-encoding plasmid during triple-transfection of HEK293F cells reduced transgene expression and led to a 60% increase in the yield of rAAV-<i>TRAIL</i> compared to controls.