DESCRIPTION (provided by applicant): Translational research in the field of liver disease has been limited by a shortage of large animal models, which in turn has limited development of new therapies for metabolic liver disorders, acute liver failure, chronic (cirrhotic) liver disease, an hepatocellular carcinoma. While new transgenic and knockout rodent models of liver disease have facilitated basic research, the development of bioengineered large animal models of liver disease has been problematic. To address the shortage of large animal models, we have utilized a novel strategy combining gene targeting by recombinant adeno-associated virus DJ serotype (rAAVdj) and somatic cell nuclear transfer (SCNT) for the production of bioengineered pigs. Our prototypemodel is hereditary tyrosinemia type 1 (HT1) accomplished by knocking out the gene coding for fumarylacetoacetate hydrolase (FAH) in pigs. In humans, FAH deficiency is associated with a unique phenotype, which includes metabolic derangement (tyrosinemia),acute liver failure, cirrhosis, and hepatocellular carcinoma (HCC). In mice, FAH deficiency combined with immune modulation has been used to produce mice with humanized livers (Azuma 2007 Nature Biotechnology; Bissig 2010 J Clinical Investigation). TheseFAH deficient mice serve as in vivo bioreactors for the robust expansion of normal human hepatocytes. We postulate that scale up of the FAH deficient platform to pigs is possible, and that FAH deficient pigs will also serve as surrogate hosts for much larger scale in vivo expansion of normal human hepatocytes. An abundant supply of human hepatocytes is needed for applications including toxicity testing of new drugs, human hepatocyte transplantation, and cell- based liver support devices such as the bioartificial liver. With regard to commercialization of this novel technology, we have already produced Fah-null heterozygote female pigs by rAAVdj and SCNT methodology. This phase 1 STTR (R41) application will establish a herd of FAH-deficient pigs and characterize the phenotype of Fah-null homozygote pigs. In future phase 2 studies, FAH-deficient pigs will be evaluated as in vivo bioreactors for the large-scale production of human hepatocytes. We expect that the FAH-deficient pig will serve as a valuable resource for the development of novel therapeutic modalities and to facilitate translational research in the field o liver disease. Furthermore, the rAAVdj and SCNT methodology may be utilized to produce other bioengineered large animal models of human disease.PUBLIC HEALTH RELEVANCE: The goal of the current research program is to develop a bioengineered pig with the metabolic disorder of human hereditary tyrosinemia type 1 (HT1). Phase 1 studies are designed to establish a herd of HT1 pigs and characterize the phenotype of these pigs. Phase 2 studies will determine if HT1 pigs can serve as in vivo bioreactors for large scale production of human hepatocytes. There are many therapeutic applications for normal human hepatocytes such as a cell-source in a bioartificial liver or for hepatocyte transplantation in humans, or industrial applications for safety and toxicity screening, or for use by pharmaceutical companies in the drug discovery process. Thus the demand for these new pigs is worthy of commercialization.
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MAYO CLINIC ROCHESTER
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