Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination

Long-term recombinant AAV (rAAV) transgene expression in muscle has been as sociated with the molecular conversion of single-stranded rAAV genomes to h igh-molecular-weight head-to-tail circular concatamers. However, the mechan isms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermed iates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase ( Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular r ecombination of independent viral genomes. The GFP shuttle vector also enco ded ampicillin resistance and contained a bacterial origin of replication t o allow for bacterial rescue of circular intermediates from Hirt DNA of inf ected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which rea ched 33% of the total circular intermediates by 120 days postinfection. Fur thermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in ce ll lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering mul tiple DNA segments at a single locus, Such developments will expand the uti lity of rAAV fur splicing large gene inserts or large promoter-gene combina tions carried by two or more independent rAAV vectors.