319. Rationally Designed AAV Inverted Terminal Repeats Enhance Gene Targeting

Basic and clinical applications of mammalian genetic engineering rely on selective pressure and/or, ironically, DNA damage at or near the modification site to obtain relevant efficiencies. In regards to the later situation, continuously evolving endonuclease technologies remain focused on the generation of a site-specific DNA double strand break to stimulate homology directed repair (HDR). The stimulation of HDR is thus dictated by the ability of the endonuclease platform to specifically recognize its cognate site, with safety concerns dictated by its promiscuity influenced by its persistence. To eliminate the serious safety concerns associated with designer endonucleases while maintaining efficient HDR, it was hypothesized that rational modification of a viral DNA repair substrate would enhance gene editing without the requirement for induced chromosomal damage. Specifically, modifications of the adeno-associated virus (AAV) inverted terminal repeat sequence (ITR), in a viral vector context, were evaluated for stimulation of HDR in human cells. The results demonstrate an approximate 30-fold enhancement of AAV gene targeting using a particular rationally designed ITR sequence. Characterization of this event suggests that differences in the inherent ITR-initiated transcriptional activity and altered interactions with host DNA repair proteins contribute to the enhancement in HDR. The collective results demonstrate for the first time that alterations in the ITR sequence can enhance AAV gene targeting. Importantly, the refinement of such vectors may offer a safer alternative to site-specific endonuclease technologies as on- and off-target DNA cleavage concerns are eliminated.