734. Association of DNA Polymerase μ with Recombinant Adeno-Associated Viral Vectors

Abstract

Recombinant adeno associated viral (rAAV) vectors have been successfully used for efficient delivery and stable transgene expression in animal models and clinical trials. Transduced vector genomes primarily persist as extra-chromosomal concatamers, with a low frequency of integration into host chromosomes. Investigations into possible mechanisms involved in vector genome integration and analyses of integration junctions reveal actively transcribed regions of chromosomes to be the hotspots for vector integration via a proposed nonhomologous end joining (NHEJ) mechanism of DNA repair (Nakai et.al 2003). Although the primary sensors of DNA damage, Ku, ATM and DNA-PK have been implicated in rAAV integration, there is limited information about the involvement of proteins that are directly involved in NHEJ. A recently discovered mammalian DNA polymerase |[mu]| (Pol|[mu]|) has been shown to be an important component of the NHEJ ligation complex and to have a functional interaction with Ku-DNA complex. In this study, we investigated the effect of rAAV on induction of Pol|[mu]| expression and the possible interaction with vector genomes. HEK293 cells transduced with rAAV2 at an MOI of 20,000 were incubated at 37|[deg]|C for 0, 0.5, 1, 3, 6, 12 and 24 hours. Total proteins were harvested at each time point and the lysates were analyzed by Western blotting using anti-Pol|[mu]| antibody. The results showed a nine-fold increase in Pol|[mu]| expression in cells incubated for 12h post transduction. A three fold increase in expression was observed at the 3h time point. The 0.5 and 1hr treatments did not show a significant increase over basal levels. Expression levels dropped down to 2 fold at the 24h time point.