The ultimate objective of gene therapy is the correction of human disease precisely at the tissue sites which manifest the disorder. We are developing organ-specific gene therapies for hemophilia and other diseases using (1) viral and non-viral vectors that incorporate Adeno-Associated Virus (AAV) functions; and (2) novel methods for focal, organ-specific vector delivery in vivo, including catheter-mediated intraluminal delivery to ductular structures lined by polarized epithelial cells. Results: (1) In non-clonal Hepatoma G-2 cells, codelivery of the Rep protein by lipid-based transfection resulted in a long-term, Rep dose-dependent increase in the expression of a marker gene flanked by AAV ITRs. (2) In 293 cells the presence of both Rep (delivered by liposomes as either a protein or as a gene) and AAV ITRs increased the frequency of clonal cells with longterm transgene expression. (3) In order to develop an in vivo model for understanding AAV integration, a mouse transgenic for AAVS1 was created. (4) The manometric and histological effects of of catheter-mediated retrograde intrabiliary infusion were evaluated in mice and rabbits. (5) Viral and non-viral vector delivery was histologically modeled using fluorescent latex microspheres ranging in diameter from 20 nm - 200 nm. (6) We are presently evaluating the utility of non-toxic DC-Chol liposomes as vehicles for in vivo gene transfer to polarized epithelial cells by catheter-mediated intraluminal delivery.
