Characterization of adeno -associated virus 2 site -specific integration
Adeno-associated virus (AAV) is the only mammalian DNA virus known to be capable of establishing latency by integration into a specific site, called AAVS1, on Chromosome 19q13.4. The AAV cis and trans requirements for this process have been identified, yet, a complete picture has not emerged with regards to the precise mechanism. Specifically, the host protein requirements are not known, as well as how they could be interacting with the AAV genome.
This study has focused on two specific research objectives. The first objective was to determine if the AAV Rep protein, shown to be required for targeting site-specific integration, can minimize AAV random integration. The second objective was to study cellular repair proteins for an effect on AAV site specific integration.
My data has indicated that even in the presence of Rep, random integrations do take place, but, the limited number of identified AAV-cellular junctions precludes any definitive statements as to whether Rep can minimize random integration. In addition, I presented direct evidence that a protein called DNA-dependent protein kinase catalytic subunit (DNAPKcs) inhibits stable site-specific integration of single-stranded AAV Rep positive vectors. Moreover, the presence or absence of DNAPKcs did not affect the specific integration of self-complementary AAV Rep positive vectors, which also displayed more random integration. In addition, cellular repair proteins ligase I and ligase IV are not needed for AAV-AAVS1 junction formation, but the absence of ligase IV greatly reduced the frequency of AAV site-specific integration.
These findings contribute to a better understanding of the AAV site-specific integration process, suggesting that components of the non-homologous end joining pathway can modulate AAV site-specific integration. By systematically studying the different cellular repair proteins for an effect on specific integration, future work should be able to elucidate the mechanism. The outcome of such advancements would lead to an increased emphasis for the further development of AAV as a safe and efficient integrating vector for future gene therapy studies.