FAH-Deficient Pigs

描述（由申请人提供）：由于缺乏大型动物模型，肝脏疾病领域的转化研究受到限制，这反过来又限制了代谢性肝脏疾病、急性肝衰竭、慢性（肝硬化）肝病和肝细胞癌新疗法的发展。虽然新的转基因和敲除啮齿动物肝脏疾病模型促进了基础研究，但生物工程大型肝脏疾病动物模型的发展一直存在问题。为了解决大型动物模型的不足，我们利用重组腺相关病毒DJ血清型（rAAVdj）基因靶向和体细胞核移植（SCNT）相结合的新策略来生产生物工程猪。我们的原型模型是遗传性酪氨酸血症1型（HT1），通过敲除猪富马酰乙酸水解酶（FAH）的基因编码来实现。在人类中，FAH缺乏与一种独特的表型相关，包括代谢紊乱（酪氨酸血症）、急性肝功能衰竭、肝硬化和肝细胞癌（HCC）。在小鼠中，FAH缺乏结合免疫调节已被用于产生人源化肝脏的小鼠（Azuma 2007 Nature Biotechnology; Bissig 2010 J Clinical Investigation）。这些FAH缺陷小鼠作为体内生物反应器，用于正常人肝细胞的强劲扩张。我们假设FAH缺陷平台在猪身上的规模是可能的，并且FAH缺陷猪也可以作为更大规模的正常人肝细胞体内扩增的替代宿主。大量的人肝细胞用于新药的毒性试验、人肝细胞移植和基于细胞的肝支持装置（如生物人工肝）。关于这项新技术的商业化，

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 prototype model 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). These FAH 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.