115. Whole Genome Analysis Shows That AAV Integrates at Chromosome 19 AAV-S1 Sites and Other Chromosomal Sites That Frequently Share GGTC Micro-Homology with the AAV P5 Promoter

Adeno associated virus undergoes Rep-mediated integration into the AAV-S1 region of human chromosome 19 during latent infection but recombinant AAV (rAAV) vectors are missing essential viral components and lack such site-specificity. This was best shown recently by strategies that monitor rAAV integration events at the level of the whole eukaryotic genome. However, whole-genome analyses have not been used to analyse integration by wild-type AAV. We have used ligation-mediated PCR (LM-PCR) to monitor AAV integration under both latent and replication-permissive conditions of infection. Sequence analysis of fifty-two clones that met pre-determined criteria showed that, during replication, AAV integrated into many chromosomal sites via sequence microhomology that frequently involved a GGTC motif located within the AAV p5 element, with one site preferred. No clones containing specific AAV/AAV-S1 junctions were identified among authentic LM-PCR clones, although such clones were readily obtained using junction-specific PCR primers; suggesting that AAV-S1 integration events constituted only a small portion of the total integration product. During latent AAV infection, GGTC-mediated integration was significantly reduced, but such micro-homology-determined chromosomal junctions were still obtained in excess over AAV-S1 specific products. This new mechanism for the integration of AAV sequences into human chromosomes has a |[ldquo]|signature|[rdquo]| that suggests it may occur independently of Rep, although the detailed mechanism remains to be elucidated. Finally, since others have shown that the p5 element is necessary and sufficient for AAV-S1 chromosomal integration, in view of our new findings, the specificity of new rAAV vectors that include p5-region sequences for improved Repmediated integration should be closely monitored, especially by whole-genome approaches.