CELLULAR RECOMBINATION PATHWAYS AND VIRAL TERMINAL REPEAT HAIRPIN STRUCTURES ARE SUFFICIENT FOR ADENOASSOCIATED VIRUS INTEGRATION IN-VIVO AND IN-VITRO

The human parvovirus adeno-associated virus (AAV) is unique in its abi lity to target viral integration to a specific site on chromosome 19 ( ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we c haracterize the terminal-repeat-mediated integration for wild-type (wt ), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterize d by a variety of techniques, including PCR, Southern hybridization, a nd fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to co mparison and analysis. In both immortalized and normal diploid human c ells, wt AAV targeted integration to ch-19. Integrated provirus struct ures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junct ion. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus j unctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell line s at the same locus on ch-2. In addition, provirus terminal repeat seq uences existed in both the flip and hop orientations, with microhomolo gy evident at the junctions. In all cases with the exception of the IT Rs, the vector integrated intact. rAAV junction sequence data were con sistent with the occurrence of genomic rearrangement by deletion and/o r rearrangement-translocation at the integration locus. Finally, junct ions formed in an in vitro system between several AAV substrates and t he ch-19 target site were isolated and characterized. Linear AAV subst rates typically utilized the end of the virus DNA substrate as the poi nt of integration, whereas products derived from AAV terminal repeat h airpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV , rAAV, and in vitro integration junctions suggest that the viral inte gration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch -19 in vivo requiring additional viral components. These studies shoul d have an important impact on the use of rAAV vectors in human gene th erapy.