Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver

Recombinant adeno-associated virus (rAAV) vectors allow for sustained expre ssion of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.I X (hF.IX), AAV-EF1 alpha-F.IX (hF.IX expression was controlled by the human elongation factor 1 alpha [EF1 alpha] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchy ma, and the forms of rAAV vector DNA extracted from the liver were analyzed . Southern blot analyses suggested that rAAV vector integrated into the hos t genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences, To f urther confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, inc luding ITR deletions and amplifications of the vector and cellular DNA sequ ences. The breakpoints of the vector were mostly located within the ITRs, a nd cellular DNA sequences were recombined with the vector genome in a nonho mologous manner. Two rAAV-targeted DNA sequences were identified as the mou se rRNA gene and the alpha 1 collagen gene. These observations serve as-dir ect evidence of rAAV integration into the host genome of mouse liver and al low us to begin to elucidate the mechanisms involved in rAAV integration in to tissues in vivo.