Adeno-associated virus vectors integrate at chromosome breakage sites

Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at nonhomologous chromosomal locations by an unknown mechanism(1-5). We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this in cells containing a specific chromosomal double-strand break created by the endonuclease I-Scel or multiple breaks created by treatment with etoposide or gamma-irradiation. Vector proviruses were found at I-Scel cleavage sites, and sequencing of vector-chromosome junctions detected microhomologies, deletions and insertions that were similar when integration occurred spontaneously at random locations or at induced double-strand breaks. Infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration and suggest that AAV vectors can integrate at existing chromosome breaks rather than causing breaks themselves, which has implications for their clinical use.