Immunodeficient FAH-/- Pigs

 描述(由申请人提供)：肝细胞移植已被提议作为遗传性肝脏代谢紊乱患者的一种细胞疗法。然而，肝细胞移植存在一些缺陷，限制了其临床应用。这些限制包括可移植肝细胞的短缺和供应不稳定，以及移植后对免疫抑制的终生依赖。我们假设，可筛查的先天性代谢错误，如遗传性酪氨酸血症1型(HT1)，可以通过体外基因治疗和体内细胞移植的个体化方法进行治疗。我们的方法是个体化的，因为患者自己的细胞可以在治疗中使用。在我们最初的概念验证研究中，将评估来自人类诱导多能干细胞(IPS)的成熟人类肝细胞和不太成熟的肝细胞样细胞。这些人类细胞将在一种新型的基因工程大型动物(猪)中扩增，该动物具有两种基因的纯合子突变：1)延胡索乙酸酯水解酶(FAH-/-)，催化酪氨酸代谢的最后一步；2)重组激活基因2(RAG2-/-)，产生严重的免疫缺陷。FAH缺乏导致受影响的肝细胞功能不稳定，并导致正常(FAH+)供体肝细胞的选择性扩增。免疫缺陷的FAH-/-RAG2-/-猪对FAH+人类细胞的排斥反应将通过胎儿耐受性进一步防止，即在免疫发育之前将供体细胞移植到子宫内。我们推测，宫内细胞移植也将为iPS来源的肝细胞样细胞的分化提供更自然的胎儿环境。一次或多次供体细胞注射(S)将在出生后进行，以促进植入。逐渐减少保护性药物2-硝基-4-三氟甲基苯甲酰基-1，3-环己二酮(NTBC)的使用，将允许FAH缺陷猪的移植细胞强劲扩张。这些在HT1大型动物模型中进行的概念验证研究将为肝细胞移植作为可筛查的先天性肝脏代谢疾病的个体化治疗提供基础。

 DESCRIPTION (provided by applicant): Hepatocyte transplantation has been proposed as a cellular therapy for patients with inherited metabolic disorders of the liver. However, hepatocyte transplantation has several shortcomings that limit its clinical application. These limitations include a shortage and inconsistent supply of transplantable hepatocytes and a life-long dependence on immunosuppression after transplantation. We hypothesize that screenable inborn errors of metabolism, such as Hereditary Tyrosinemia Type 1 (HT1), can be treated by an individualized approach involving ex vivo gene therapy and in vivo cell transplantation. Our approach is individualized since the patient's own cells may be used in the treatment. In our initial proof of concept studies, mature human hepatocytes and less mature hepatocyte-like cells derived from human induced pluripotent stem (iPS) cells will be evaluated. These human cells will be expanded in a novel, genetically engineered large animal (pig) possessing homozygote mutations of two genes: 1) fumarylacetoacetate hydrolase (FAH-/-), the enzyme that catalyzes the final step in tyrosine metabolism; 2) recombination activating gene 2 (RAG2-/-) to produce severe immunodeficiency. FAH deficiency causes functional instability in effected hepatocytes and results in a selective milieu for robust expansion of normal (FAH+) donor hepatocytes. Rejection of FAH+ human cells in immunodeficient FAH-/- RAG2-/- pigs will be further prevented by fetal tolerization, transplanting donor cells in utero prior to immune development. We postulate that in utero cell transplantation will also provide a more natural fetal environment for differentiation of iPS-derived hepatocyte-like cells. One or more injection(s) of donor cells wll be performed postnatally to enhance engraftment. Tapered use of the protective drug, 2-nitro-4-trifluoromethylbenzoyl-1,3- cyclohexanedione (NTBC), will allow robust expansion of transplanted cells in FAH- deficient pigs. These proof-of-concept studies performed in a large animal model of HT1 will provide a foundation for hepatocyte transplantation as an individualized treatment of screenable inborn errors of liver metabolism.