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 delivery to the murine hepatobiliary tree. Results: (1) The manometric and histological effects of catheter-mediated retrograde biliary infusion were evaluated in mice. (2) The effect of retrograde infusion parameters upon distribution of adenoviral and adeno-associated viral vectors was histologically modeled using fluorescent latex spheres 20 and 100 nm in diameter. (3) Low pressure retrograde infusion resulted in a canalicular pattern of sphere distribution, while high pressure infusion led to sinusoidal and subsequent systemic sphere distribution. (4) Selective use of different infusion parameters altered the histological pattern of gene transfer immunohistochemically detected followed retrograde infusion of a recombinant LacZ adenovirus. High pressure infusion led to a mixed pattern of cholangiocyte and hepatocyte gene transfer, while low pressure infusion resulted in cholangiocyte much greater than hepatocyte gene transfer. These findings suggest that high pressure retrograde biliary infusion leads to a disruption of canalicular tight junctions, resulting in sinusoidal passage of infusate. (5) A major problem observed in several laboratories has been the unintended generation of wildtype AAV during recombinant virus production. We found that production in Cos cells of recombinant Bgal and Factor IX AAV virus using a modified cotransfection system resulted in persistently high (5-25 %) levels of wildtype AAV as determined by genomic dot blot. Generation of wildtype AAV appears to have resulted from a combination of both homologous and non-homologous recombination. These findings suggest that cell-line based production systems may be necessary in order to generate virus with the purity required for appropriate preclinical in vivo studies of recombinant AAV virus safety, expression, and integration.
