Analysis of site-specific transgene integration following cotransduction with recombinant adeno-associated virus and a Rep encoding plasmid

Recombinant adeno-associated virus (rAAV) has many advantages for gene therapeutic applications in comparison with other vector systems. One of the most promising features is the ability of wild-type (wt) AAV to integrate site-specifically into human chromosome 19. However, this feature is lost in rAAV vectors due to the removal of the rep-coding sequences. HeLa cells were transfected with a rep expression plasmid, infected by rAAV and grown with or without selection pressure. Single cell clones were generated and genomic DNA was analyzed for site-specific integration by Southern blotting analysis and fluorescence in situ hybridization (FISH). Transfection of HeLa cells with a rep expression plasmid followed by transduction with a rAAV vector resulted in site-specific integration of the transgene at AAVS1 on human chromosome 19 in 7 of 10 cell clones analyzed. In marked contrast, transduction of cells with rAAV alone did not result in any site-specific integration of the transgene. The high frequency with which the site-specific integration took place in the presence of Rep protein is comparable with the results observed with wtAAV. These results offer opportunities for the development of specifically integrating rAAV vectors.