Adeno-associated virus type 2-mediated gene transfer: Altered endocytic processing enhances transduction efficiency in murine fibroblasts

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, no npathogen ic human parvovirus that is currently in use as a vector for huma n gene therapy, However, the transduction efficiency of AAV vectors in diff erent cell and tissue types varies widely. In addition to the lack of expre ssion of the viral receptor and coreceptors and the rate-limiting viral sec ond-strand DNA synthesis,,which have been identified as obstacles to AAV-me diated transduction, we have recently demonstrated that impaired intracellu lar trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J, Hansen, It. Qing, H, J, Kwon, C, Mah, and A. Srivastava, J, Virol. 74:992-996, 2000). In this report, we document th at escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293, While virions were found in bot h early and late endosomes or lysosomes of infected 293 cells, they were lo calized predominantly to the early endosomes in NIH 3T3 cells. Moreover, tr eatment of cells with bafilomycin Al (Baf), an inhibitor of the vacuolar H-ATPase and therefore of endosomal-lysosomal acidification, decreased the t ransduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated tr ansduction in general, virions were detected in late endosomes and lysosome s, and these cells became sensitive to Baf-mediated inhibition of transduct ion. Thus, KCI treatment overcomes defective endocytic processing of AAV in murine fibroblasts, These studies provide insights into the underlying mec hanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy .