Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis

Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV trans-duction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demon-strated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy. Adeno-associated virus (AAV) is a human parvovirus being considered as a gene delivery vehicle for human gene therapy. This small, nonenveloped virus contains a 4.7-kb single-stranded DNA genome that encodes regulatory and structural genes called rep and cap, respectively (13). Recombinant form