Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocy tes in experimental animals. Following portal-vein administration of rAAV v ectors in vivo, single-stranded (ss) rAAV genomes become double stranded (d s), circularized, and/or concatemerized concomitant with a slow rise and, e ventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DN A. The mechanism(s) of formation of stable ds rAAV genomes from input ss DN A molecules has not been delineated, although second-strand synthesis and g enome amplification by a rolling-circle model has been proposed. To begin t o delineate a mechanism, we produced rAAV vectors in the presence of bacter ial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mo use hepatocytes in vivo. A series of molecular analyses demonstrated that s econd-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Ra ther, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These f indings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsib le for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.