Development of animal models for adeno-associated virus site-specific integration

The adeno-associated virus (AAV) is unique in its ability to target viral D NA integration to a defined region of human chromosome 19 (AAVS1). Since AA VS1 sequences are not conserved in a rodent's genome, no animal model is cu rrently available to study AAV-mediated site-specific integration. We descr ibe here the generation of transgenic rats and mice that carry the AAVS1 3. 5-kb DNA fragment. To test the response of the transgenic animals to Rep-me diated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, a nd fibroblasts were infected with wild-type wt AAV. PCR amplification of th e inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genom e integrated into the AAVS1 site in fibroblasts and hepatocytes. Integratio n in rat fibroblasts was also observed upon transfection of a plasmid conta ining the rep gene under the control of the p5 and p19 promoters and a dici stronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP- Neo sequence in the AAVS1 region was determined by Southern blot and FISH a nalysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo wa s assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are pro ficient for Rep-mediated targeting. These animals should allow further char acterization of the molecular aspects of site-specific integration and test ing of the efficacy of targeted integration of AAV recombinant vectors desi gned for human gene therapy.