Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVSI). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals withinAAVSl required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

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From the above, it appears that the sequence of the preintegration site is likely to be a determining factor in the site specificity of AAV integration. However, the question of whether the sequence of the preintegration site is sufficient, in terms of cellular parameters, to determine site specificity has not been answered. In this paper we describe experiments which directly address this issue by using an EpsteinBarr virus (EBV)-based shuttle vector which has been reported to be highly stable during propagation in mammalian cells (13). DNA containing the AAVSJ preintegration site was placed in the EBV-based shuttle vector and the ability ofthat DNA, when no longer in the normal context of the long (q) arm of chromosome 19, to direct AAV integration was assessed. We find that AAV integration is still directed by the preintegration DNA even when it is in an episome. The cellular DNA sequence directing integration was localized to a 510-nt region at the 5' end of the AAVSJ locus. (Fig. 1): p220.2/AAVS1(kb 0-0.51), p220.2/AAVS1(kb 0-1.6), p220.2/AAVS1(kb 0-3.5), and p220.2/AAVS1(kb 0-4.4) were derived by digestion of p220.2/AAVS1(kb 0-8.2) with, respectively, Xba I (site in the p220.2 polylinker)/Pvu II, BamHI, Xba I/Kpn I, and Xba I/Pvu II (partial digest) and religation of the digestion products. The 5'-end deletion mutants p220.2/ AAVS1(kb 0.51-1.6), p220.2/AAVS1(kb 0.51-3.5), and p220.2/AAVS1(kb 0.51-4.4) were derived, respectively, Abbreviations: AAV, adeno-associated virus; EBV, Epstein-Barr virus; EBNA-1, EBV-encoded nuclear antigen 1; SV40, simian virus 40.