Kinetics of recombinant adeno-associated virus-mediated gene transfer

Recombinant adeno-associated virus (rAAV) vectors have been shown to be use ful for efficient gene delivery to a variety of dividing and nondividing ce lls. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IS (hFIX) gene transfer into human p rimary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduct ion, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold highe r expression than that with a muscle-specific creatine kinase enhancer link ed to beta-actin promoter. Factor IX expression from transduced myoblasts a s well as myotubes reached levels as high as approximately 2 mu g of hFIX/1 0(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellu lar genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells sho wed that the conversion of single-stranded vector genomes to double-strande d DNA forms, but not the level of the integrated forms in HMW DNA, correlat ed with increasing expression of the transgene. Together, these results ind icate that rAAV can transduce both proliferating and terminally differentia ted muscle cells at about the same efficiency, that expression of transgene s increases linearly over their lifetime with no initial lag phase, and tha t increasing expression correlates with the appearance of double-stranded e pisomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.