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

Abstract

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 adenoassociated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction 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 demonstrated 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.

Boosting Anti‐idiotype Immune Response with Recombinant AAV Enhances Tumour Protection Induced by Gene Gun Vaccination

“…We found that repetitive Gene Gun administration is the most efficient strategy to induce anti-Id antibodies. Repetitive administration with rAAV was also performed; however, it failed to increase anti-Id titres, reasonable because of the induction of anti-AAV neutralizing antibodies, as previously reported [27][28][29][30][31].…”

Chronic Ethanol Increases Adeno-Associated Viral Transgene Expression in Rat Liver via Oxidant and NFκB-Dependent Mechanisms

“…1,2 Recombinant AAV (rAAV) is also a useful tool for liver-directed gene delivery because the virus can infect nondividing cells, integrate specifically into the host cell genome, and is relatively nonpathogenic compared with more commonly used adenoviral vectors. 1 Although rAAV provides many advantages over other vectors, its transduction efficiency in many tissues, including the liver, is limited either because of the requirement of synthesis of the second strand of DNA in the viral genome 3,4 or because of the lack of transgene expression caused by decreased promoter activity (i.e., inactivation of the cytomegalovirus [CMV] promoter). 5 Second-strand synthesis of the single-stranded AAV viral genome, which is required for the expression of the transgene, is hypothesized to be the limiting step in transduction; however, the cellular mechanisms for this process remain unclear.…”

“…Various genotoxic agents, such as etoposide and hydroxyurea, as well as coinfection with helper viruses such as adenovirus or herpes virus, have been shown to increase rAAV transduction. 3,4,6 Moreover, many agents shown to increase rAAV transduction in vitro also cause mild cellular damage and induce many cellular stress responses such as activation of nuclear transcription factor B (NFB). [7][8][9] An alternative hypothesis is that rAAV transduction is regulated through cellular proliferation or transcription factors rather than through an increase in cellular DNA synthesis.…”

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

“…In the present study, we found that Hoechst improves the efficiency of AAV-mediated transgene expression in human airway epithelia by activating the CMV promoter. Work by Walters et al [8] and Halbert et al [21] suggests that binding is a rate-limiting step for AAV2, whereas other studies have found that intracellular trafficking and second-strand synthesis are rate-limiting steps [12,13]. We have also previously found that binding and internalization are sequential rate-limiting steps, which an evolved virus (AAV2.5T) overcomes [10,11].…”