514. Effect of DNA-PKcs on AAV Replication

Abstract

Top of pageAbstract DNA dependent protein kinase (DNA-PK) is a DNA repair enzyme with multiple functions. It has been shown that it plays an important role in determining the molecular fate of the rAAV genome in mouse muscle and liver. DNA-PKcs inhibits AAV integration in vitro and in vivo. In the present study, we sought to determine the effect of DNA-PKcs on recombinant adeno-associated virus (rAAV) replication. We co-infected 293 cells with rAAV vector (UF5) and recombinant herpes simplex virus (rHSV) helper virus. The replicated forms were detected by southern blot analysis. When cells were treated with a DNA-PKcs inhibitor, wortmannin (WM), rAAV replication is significantly reduced in a dose-dependent manner. Similar results were obtained from MO59K (DNA-PKcs positive) cells. In order to confirm this observation, we have employed small interference RNA (siRNA) to target DNA-PKcs. Transfection of this synthetic siRNA resulted in 70% to 90% reduction of the mRNA and the protein levels of DNA-PKcs. Remarkably, this treatment significantly decreased rAAV replication. In order to avoid the possible side effects by viral infection on DNA-PK activity, we co-transfected the siRNA treated 293 cells with vector (pUF5) and helper (pDG) plasmids. Results from these experiments again showed that targeting of DNA-PKcs decreased rAAV replication by at least two folds compared to the controls. Our results demonstrated that inhibition of DNA-PKcs by a DNA-PK inhibitor or siRNA decreased rAAV replication suggesting the important role of this cellular enzyme in AAV replication.