Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro

Abstract

The human parvovirus adeno-associated virus (AAV) is unique in its ability 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 characterize 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 characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures 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 junction. 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 junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin 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 integration 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 should have an important impact on the use of rAAV vectors in human gene therapy.

Adeno-associated Virus as a Mammalian DNA Vector

“…In nondividing skeletal muscle fibers, ATM and DNA-PKcs were necessary for genome circularization wherease Nbs1 was not (137,151,152). Consistent with these in vivo data, elegant work performed in the mouse liver demonstrated that Artemis and DNA-PKcs process the AAV ITRs and promote the circularization/concatamerization of vector genomes, which is also supported by other reports (62,136,137,(153)(154)(155)(156). Collectively, with regards to the generation of persistent AAV vector episomes, it appears that the circularization event is probably mediated at the ITRs by different DNA repair pathways; homology directed repair in dividing cells and nonhomologous end joining (NHEJ) in terminally differentiated tissues (135,137,157).…”

“…Although the majority of AAV vector genomes persist as episomes, as with all exogenous DNA, a low percentage will undergo illegitimate host genome integration (62,(153)(154)(155)(156)(164)(165)(166). One major factor influencing this largely undesirable event is whether or not the cells are actively replicating/dividing (157,167).…”

Effect of poly(ADP‐ribose) polymerase 1 on integration of the adeno‐associated viral vector genome

“…The endonuclease activity of Rep mediates a nicking event at the terminal resolution site within the AAVS1 [20,21]. A poorly understood recombination event then occurs, resulting in partial duplication of the AAVS1 site and integration of the AAV genome [12,[20][21][22][23][24][25][26]. Although the role of AAV proteins in targeted integration has been extensively studied, the detailed mechanism and particularly the role of cellular factors in integration remains unclear.…”

Design and Packaging of Adeno-Associated Virus Gene Targeting Vectors

“…Typically, the foreign DNA encodes a transgene cassette with promoter and polyadenylation signals designed to express from an ectopic location. These vectors can transduce cells by a variety of mechanisms, including random chromosomal integration of the vector genome (29,38) or episomal transgene expression (2,7,18). If transduction is performed in the presence of the AAV rep gene, vectors can also be designed to integrate at the site-specific integration locus of wild-type AAV located on human chromosome 19 (3,24,25,33).…”