Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction

Numerous environmental influences have been demonstrated to enhance recombi nant adeno-associated virus (rAAV) transduction. Such findings are the foun dation of developing new and innovative strategies to improve the efficienc y of rAAV as a gene therapy vector. Several of these environmental factors included genotoxic stresses such as UV and gamma irradiation as well as cer tain adenoviral gene products such as E4orf6. The mechanisms by which these environmental stimuli increase rAAV transduction are only partially unders tood but have been suggested to involve both endocytosis and uptake of viru s to the nucleus, as well as conversion of single-stranded DNA viral genome s to double-stranded expressible forms. Two molecular intermediates of rAAV genomes, which have been demonstrated to correlate with transgene expressi on and/or the persistence of rAAV, include both replication form (Rf) monom ers and dimers as well as circular intermediates. In the present study, we demonstrate that augmentation of rAAV transduction by UV irradiation and th e adenoviral protein E4orf6 correlates with distinct increases in either ci rcular or replication form intermediates, respectively. UV irradiation of p rimary fibroblasts at 15 J/m(2) resulted in a 15-fold induction of head-to- tail circular intermediates, with minimal induction of replication form rAA V genomes. In contrast, E40rf6-augmented rAAV transduction was correlated w ith the formation of replication form intermediates, with no alteration in the abundance of circular intermediates. These findings demonstrate that rA AV transduction can occur through two independent molecular pathways that c onvert single-stranded AAV genomes to expressible forms of DNA.