Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps
Mark A. Brimble, Pei-Hsin Cheng, Stephen M. Winston, Isaiah L. Reeves, Aisha Souquette, Yunyu Spence, Junfang Zhou, Yong‐Dong Wang, Christopher L. Morton, Marcus B. Valentine, Paul G. Thomas, Amit C. Nathwani, John T. Gray, Andrew M. Davidoff
Abstract
evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.