FAH-Deficient Pigs

描述（由申请人提供）：肝病领域的转化研究受到大型动物模型短缺的限制，这反过来又限制了代谢性肝病、急性肝衰竭、慢性（肝硬化）肝病、肝细胞癌的新疗法的开发。虽然新的转基因和基因敲除啮齿动物模型促进了基础研究，但生物工程大型动物模型的开发一直存在问题。为了解决大型动物模型的短缺，我们利用了一种新的策略，结合基因打靶重组腺相关病毒DJ血清型（rAAVdj）和体细胞核移植（SCNT）的生物工程猪的生产。我们的原型模型是遗传性酪氨酸血症1型（HT 1）完成敲除基因编码延胡索酰乙酰乙酸水解酶（FAH）的猪。在人类中，FAH缺乏症与独特的表型相关，包括代谢紊乱（酪氨酸血症）、急性肝衰竭、肝硬化和肝细胞癌（HCC）。在小鼠中，FAH缺陷与免疫调节组合已用于产生具有人源化肝脏的小鼠（Azuma 2007 Nature Biotechnology; Bissig 2010 J Clinical Investigation）。这些FAH缺陷型小鼠用作正常人肝细胞稳健扩增的体内生物反应器。我们假设FAH缺陷平台扩大到猪是可能的，并且FAH缺陷猪也将作为正常人肝细胞更大规模体内扩增的替代宿主。需要大量供应人肝细胞用于包括新药的毒性测试、人肝细胞移植和基于细胞的肝支持装置（如生物人工肝）的应用。关于这项新技术的商业化， 我们已经通过rAAVdj和SCNT方法生产了Fah-无效杂合子母猪。该1期STTR（R41）申请将建立一个FAH缺陷猪群，并表征Fah缺失纯合子猪的表型。在未来的II期研究中，将评价FAH缺陷猪作为大规模生产人肝细胞的体内生物反应器。我们期望FAH缺陷猪将作为开发新的治疗方式的宝贵资源，并促进肝病领域的转化研究。此外，rAAVdj和SCNT方法可用于产生人类疾病的其他生物工程化大型动物模型。 公共卫生相关性：目前研究计划的目标是开发具有人类遗传性酪氨酸血症1型（HT 1）代谢紊乱的生物工程猪。1期研究旨在建立一个HT 1猪群，并表征这些猪的表型。II期研究将确定HT 1猪是否可用作大规模生产人肝细胞的体内生物反应器。正常人肝细胞有许多治疗应用，例如生物人工肝中的细胞来源或人类肝细胞移植，或安全性和毒性筛选的工业应用，或供制药公司在药物发现过程中使用。因此，对这些新猪的需求值得商业化。

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.