Development of circular AAV cargos for targeted seamless insertion with large serine integrases

Recent advancements in gene insertion have shifted from DNA-repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. Although I-PGI does not require DNA double-strand breaks (DSBs) for activity, linear cargo, like AAV, stimulates DNA end-joining activity after integration. To mitigate potential risks from DSBs, we developed two circular AAV cargos capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated seamless cargo integration in primary human hepatocytes (PHHs) and robust DSB-free insertion structures in vivo. We then investigated the delivery of a packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD exhibited functional seamless gene insertion in PHHs and showed cargo efficacy in vivo. Together, these findings provide evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.