505. Analysis of AAV Genome Conversion and Stabilization

Abstract

Recombinant adeno-associated virus (rAAV) vectors have been widely used to deliver a variety of therapeutic genes in both in vitro and in vivo experiments. The administration of AAV vectors usually gives rise to persistent transgene expression. However, the doses required for meaningful gene expression are usually much higher than other viral vectors. Because AAV genomes are single-stranded DNA (ss), we examined the conversion and stability of AAV genomes in both tissue cultured cells and in vivo animal experiments. The special AAV vectors labeled with BrdU molecules were generated and an anti-BrdU antibody was used to specifically track free ss AAV DNA genomes. Although single stranded AAV genomes were detected by conventional Southern Blot anaylsis, the antibody tracking, which recognizes ssDNA labeled with BrdU but not double stranded (ds) DNA with BrdU, revealed no free ss DNA molecules after AAV transduction. We concluded that ss DNA accumulation is not an essential step for transduction. In addition, we observed significant AAV genome losses during AAV transduction. Overall, less than 0.1% of AAV genomes survive the degradation in the stages of intracellular trafficking, ss DNA to double stranded DNA conversion, and ds DNA to final stabilized forms. Through a quantitative analysis of newly synthesized ds AAV genomes, we discovered that more than 90% of these molecules did not persist although they were fully functional in expressing transgenes (such as lacZ or GFP) in both tissue culture cells and mouse liver. The long term expression of transgene products observed in vivo was therefore coming from a small amount of stabilized AAV genomes. Our results suggest that improving the AAV genome stability post transduction is critical for enhancing AAV transduction efficacy.