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. This includes 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 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. By these approaches it is hoped that the ultimate objective of gene therapy, the correction of human disease precisely at the tissue sites which manifest the disorder, will be achieved.
