Reducing encapsidated impurity DNA derived from plasmid backbone by modifying the p5 terminal resolution site in rAAV vector production

Abstract Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5Δloop variant. Furthermore, the p5Δloop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy. Competing Interest Statement This study was conducted as part of the authors' professional duties at Synplogen Co. Ltd. SS is a Director of Synplogen Co. Ltd., while the other authors are employees. YN, NM, and SS are inventors on a patent application filed by Synplogen Co., Ltd. based on the findings reported in this paper. Footnotes Electronic address: y_nishimura{at}synplogen.com, s_hataya{at}synplogen.com, s_saito{at}synplogen.com All figures updated to improve resolution.