Adeno-associated virus (AAV) has been reported to integrate in a site-specific manner into chromosome 19 (a site designated AAVS1), a phenomenon that could be exploited for ex vivo targeted gene therapy. Recent studies employing LM-PCR to determine AAV integration loci; however, have, contrary to previous results with less reliable methods, concluded that the proclivity for AAV integration at AAVS1 is minimal. We tested this conclusion employing LM-PCR protocols designed to avoid bias. Hep G2 cells were infected with rAAV2-GFP and coinfected with wt AAV2 to supply Rep in trans. Sorted cells were cloned and cultured. In 26 clones that retained fluorescence, DNA was extracted and AAV-genomic junctions amplified by two LM-PCR methods. Sequencing was performed without bacterial cloning. Of these 26 clones it was possible to assign a genomic integration site to 14, of which 9 were in the AAVS1 region. In three additional clones, rAAV integration junction were to an integrated wt AAV genome while two were to an rAAV genome. We also show that integration of the AAV-GFP genome can be achieved without cointegration of the AAV genome. Based on the pattern of integrants we propose, for potential use in ex vivo targeted gene therapy, a simplified PCR method to identify clones that have rAAV genomes integrated into AAVS1.
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