Viral vectors such as rAAV and viral gene deleted adenoviruses show potential for liver directed gene therapy for genetic disorders such as hemophilia. In current proposed liver trial for rAAV-hFVIII or rAAV-hFIX, we plan to administer the vector into the hepatic artery. Even though after systemic delivery, a majority of the vector ends up in the liver, there is a substantial amount of promiscuous dissemination into other tissues. These can have unwanted side-effects. We propose to develop a clinically relevant approach to non-surgical isolation of the hepatic vasculature in a large animal model that would allow for asanguinous perfusion (AHP) of the liver in situ. We propose pre-clinical development to test the feasibility of this approach for rAAV-mediated gene transfer, and a new integrating plasmid based non-viral vector. We will compare the "standard" hepatic artery approach with new AHP methods. We plan to use these technologies to address scientific principles that are important for vector delivery into the liver. The specific questions to be addressed are: (1) is there a dose-response advantage to vascular isolation; (2) is there is difference in toxicity; (3) can we increase the number of hepatocytes that can be stably transduced with rAAV or non-viral vectors. We believe that the technology developed here will not only be useful for addressing these scientific principles but will be directly applicable to liver-directed gene therapy trials within the PEGT and other gene therapy investigators.