11. Reporter Gene-Based Analysis of rAAV Genome Circularization and Recombination

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Adeno-associated virus (AAV) vector DNA persists in non-dividing cells largely as circular and concatameric episomes. We have created a set of self-complementary AAV (scAAV) split-gene vectors with which to dissect the DNA recombination events that lead to stable episome formation. The scAAV vector provides two important advantages for these assays: 1) it forms double-strand DNA immediately upon uncoating, thus mimicking the structure of the conventional rAAV after second-strand synthesis has taken place; 2) it allows control of the specific orientation of the coding sequences relative to the open (O) and closed hairpin (C) terminal repeat ends of the genome. One such vector was constructed to report with GFP expression when the genome had circularized (intramolecular recombination between O and C terminal repeats). Additional sets of scAAV vectors were constructed to report when specific intermolecular recombination events had taken place. These included O to C end joining, as well as O-O, and C-C. The efficiency of each such event was measured in the presence and absence of drugs which inhibited specific cellular factors or processes. We found that circularization of rAAV genomes is extremely efficient in HeLa and HEK293 cells, with >97% of infecting genomes in the circular configuration before 24 hr. post-infection. Importantly however, circularization was not a prerequisite for gene expression. We have extended these studies to real-time reporting of rAAV genome circularization in mouse muscle tissue. For intermolecular events, we found that C-C recombination is the most efficient, followed by O-C and O-O events. Under conditions of DNA synthesis inhibition, recombination involving closed ends was most sensitive, with the result that the incidence of each type of event was reduced to approximately the same value. Inhibition of topoisomerase II with Etoposide resulted in strong, but non-specific, inhibition of each type of recombination. In contrast, inhibition of topoisomerase I with campothectin specifically reduced events involving the closed hairpin end, to the extent that the order of recombination preference was inverted relative to the untreated cells, with O-O most efficient, followed by O-C and C-C. Our results strongly suggest the involvement of a DNA synthesis step in rAAV genome circularization and concatemerization, as well as a requirement for topoisomerase I in processing the closed hairpin ends.