2ND-STRAND SYNTHESIS IS A RATE-LIMITING STEP FOR EFFICIENT TRANSDUCTION BY RECOMBINANT ADENOASSOCIATED VIRUS VECTORS

The ability of recombinant adeno-associated virus (AAV) to transduce c ells with a marker gene in vitro was found to be substantially increas ed by the presence of adenovirus. Transfection experiments with adenov irus genomic DNA suggest that this increase is not facilitated by aden ovirus-mediated viral uptake but is instead dependent on adenovirus ge ne expression. Using various adenovirus mutants, we were able to map t his function to early-region E4 open reading frame 6. Plasmid expressi on of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase i n transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV lif e cycle. Chemical and physical agents that have been shown to induce h elper-free replication of wild-type AAV were also able to stimulate re combinant AAV transduction, suggesting that the phenomenon might affec t AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis o f a second strand on the single-stranded genomic AAV DNA. These data s uggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have impo rtant implications for the use of AAV vectors in gene therapy protocol s.