CIRCULAR INTERMEDIATES OF RECOMBINANT ADENOASSOCIATED VIRUS HAVE DEFINED STRUCTURAL CHARACTERISTICS RESPONSIBLE FOR LONG-TERM EPISOMAL PERSISTENCE IN MUSCLE-TISSUE

Adeno-associated viral (AAV) vectors have demonstrated great utility f or long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue rema in unclear. Using a recomibinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rA AV genomes in muscle tissue. The majority of circular intermediates ha d a consistent head-to-tail configuration consisting of monomer genome s which slowly converted to large multimers of >12 kbp by 80 days post infection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular interme diates. Structural Features of these circular intermediates responsibl e for increased persistence included a DNA element encompassing two vi ral inverted terminal repeats (ITRs) in a head-to-tail orientation, wh ich confers a 10-fold increase in the stability of DNA following incor poration into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV c ircular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonvi ral gene delivery systems with increased efficiency.