The liver plays a pivotal role in numerous biochemical processes, including intermediary metabolism, synthesis and secretion of serum proteins, energy metabolism, and detoxification of drugs and other xenobiotics. Hepatocytes are, therefore, an important target for gene therapy, and important target for gene therapy in many genetic diseases that disrupt these processes. In the current application, our group is seeking to optimize the conditions for gene transfer into hepatocytes using recombinant adeno-associated virus (rAAV) vectors, which possess the inherent advantages of long-term stability, safety, and low immunogenicity. rAAV-mediated gene transfer is very efficient in myofibers and some other cell types, but previous reports have indicated that long-term rAAV transduction of the liver may be limited to maximum of approximately 5% of hepatocytes using current methods. This may not be sufficient for many genetic/metabolic disorders. The primary goal of the current application is to identify and circumvent the rate-limiting steps in rAAV-mediated transduction of hepatocytes and thereby increase the efficiency of rAAV-mediated transduction of hepatocytes to a point where it is sufficient for widespread clinical use. The optimization of hepatocyte gene transfer will be accomplished in the context of three gene therapy projects, each of which typifies a defect in one of the major hepatic functions: Project 1: gene therapy for phenylketonuria (a disorder of amino acid metabolism), Project 2: gene therapy for alpha 1-anti-trypsin deficiency (a defect in hepatic synthesis and secretion), and Project 3: gene therapy for glycogen storage (that affect a key process in energy metabolism, the release of free glucose from glycogen). In addition, a pilot study will investigate the feasibility of gene therapy in the murine mdr-2 knock-out mouse (a model defect in bile salt secretion analogous to progressive familial Intrahepatic cholestasis), in a paradigm whereby corrected cells could selectively repopulate the liver due to their survival advantage. In order to fully develop rAAV transduction of the liver, it may be necessary to enhance delivery to the hepatocyte, increase attachment to the cell membrane, improve internalization, nuclear entry and uncoating, maximize the transcriptional activity of vector genomes, and optimize conditions for therapeutic protein secretion. Since these disorders may ultimately require therapy in the newborn period or during child-bearing years, biological safety issues related to long-term genetic stability and biodistribution of rAAV genomes will also be addressed. These studies will be supported by the UF Vector Core Facility, for the production of high-titer, highly purified rAAV stocks, and the UF Immunology/Pathology Core. It is anticipated that by the end of this grant period, these basic improvements in rAAV-mediated delivery of genes to the liver will have improved the changes for successful gene therapy in one or more of these disorders.
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