95. Comprehensive wtAAV Integration Site Analysis Reveals Integration Site Formation Throughout the Whole Viral Genome

To establish latency, wildtype adeno-associated virus (wtAAV) is able to integrate its DNA into a specific region on chromosome 19 of the human genome, named AAVS1. This targeted integration mechanism relies on the function of the two larger versions (Rep78/68) of the Rep protein family, expressed by wtAAV. Rep is able to bind a 12 bp long sequence, called Rep binding site (RBS). The RBS can be found both in the viral ITRs and in AAVS1. By the formation of a trimeric complex between wtAAV, Rep and the genomic locus, targeted integration can be achieved. However, the exact mechanisms of Rep-mediated integration still remain unknown. To analyze wtAAV IS distribution genome-wide, large-scale integration analyses using high-throughput next generation sequencing have been performed. These studies revealed that Rep-mediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with RBS can be found as well throughout the genome. Up to this point most integration sites (IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most currently available methods for the analysis of viral IS rely on amplification of the vector-genome junction with primers that bind in the vicinity of the viral ends. Therefore, knowledge about the nature and structure of the internal wtAAV genome remains limited. To address this question, we investigated ~3e6 wtAAV concatemeric structures generated by IS analyses using linear amplification-mediated (LAM) PCR to identify potential alternative breakpoints throughout the wtAAV genome. We discovered that breakpoints occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter, there are a number of other preferred breakpoints throughout the wtAAV genome. Using a novel approach involving targeted enrichment of AAV and AAVS1 sequences, we also were able to consider viral IS at any position of the AAV genome. Targeted HiSeq sequencing of wtAAV and AAVS1 determined several hundreds of exact IS and confirmed that wtAAV genome junctions form at any position of its genome. Those cryptic integration events, not picked up by the majority of methods currently available, shed new light onto wtAAV persistence and may further improve current AAV persistence analyses in gene therapy.