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. 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-SceI or multiple breaks created by treatment with etoposide or gamma-irradiation. Vector proviruses were found at I-SceI 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.