Delayed expression of adeno-associated virus vector DNA

Two previous reports indicated that recombinant adenoassociated virus (rAAV) vectors were dependent on helper adenovirus (Ad) for efficient conversion of single-stranded (ss) rAAV DNA to the double-stranded (ds) form. This finding is somewhat paradoxical, however, since during a latent infection wild-type (wt)-AAV is rapidly converted to a ds form in the absence of Ad, Our hypothesis was that the effect observed in the previous studies was due to kinetic factors, i.e. to a relative delay in conversion to ds-DNA rather than to an absolute requirement for Ad, To test this, Hela cells were infected with a rAAV-CMV-green fluorescent protein (GFP) vector either in the presence or absence of Ad. Within the first 2 days, Ad infection resulted in a 4-fold increase in AAV vector expression and an augmentation of conversion to a ds-AAV DNA, By 6 days, however, the total number of GFP-expressing cells in the Ad-free culture had exceeded the original number in the Ad co-infected cells, and the conversion to ds-DNA episomes was substantial and ongoing.