Deriving hepatocytes from disease specific iPS to treat metabolic liver disorders

描述（由申请人提供）：肝细胞移植是肝移植的一种微创替代方法，用于治疗肝功能衰竭或遗传性代谢紊乱患者。这种有前景的治疗方式广泛应用的主要障碍是高质量供体器官的稀缺和需要长时间的免疫抑制以防止同种异体移植排斥反应。本研究的目的是验证来自1型Crigler-Najjar综合征（CN1）患者皮肤的诱导多能干细胞（iPSC）的假设，这些患者由于尿苷二磷酸葡萄糖醛酸葡萄糖醛酸转移酶-1 （UGT1A1）缺乏而导致严重的终身黄疸，可以通过基因纠正，然后在培养中进行操作，产生表达活性UGT1A1的肝细胞。我们进一步假设，适当的宿主制备将允许CN1的Gunn大鼠模型的肝脏充分再生，以改善高胆红素血症。具体目标1是：(a)从正常受试者和CN1患者的皮肤成纤维细胞中生成和表征iPS细胞，(b)在基因组“安全港”通过同源重组纠正遗传缺陷，使用锌指核酸酶或tale核酸酶技术，以及(c)在培养中将遗传校正的iPSCs分化为肝细胞样细胞（iHep）。将所得肝细胞的基因表达谱和代谢功能与成人和胎儿肝细胞进行比较。具体目的2是(a)在适当的宿主制备后，将iHeps移植到Gunn大鼠的肝脏中，并评估移植的代谢效果。Gunn大鼠将接受以他克莫司为基础的免疫抑制方案治疗，该方案在我们的实验室中已被证明可以防止异种移植的人肝细胞的排斥反应。受者将接受肝脏预备照射，并通过单次注射表达肝细胞生长因子的腺载体刺激有丝分裂，以诱导移植的肝细胞在肝脏再生。随访血清胆红素水平，测定肝脏再生程度，用高压液相色谱法分析胆红素葡萄糖醛酸酯的胆汁排泄。(b)最后，将对一组受体大鼠和免疫缺陷小鼠进行终身观察，以评估移植的ipsc源性肝细胞的致瘤潜力。该项目的成功完成将提供自体肝细胞的可再生来源，在进一步修饰和扩大规模后，可将其转化为基于细胞移植的CN1和许多其他遗传性肝病的治疗。

DESCRIPTION (provided by applicant): Hepatocyte transplantation is being developed as a minimally invasive alternative to liver transplantation for patients with liver failure or inherite metabolic disorders. Major hurdles to the broad application of this promising therapeutic modality are the scarcity of high quality donor organs and the need for prolonged immunosuppression to prevent allograft rejection. This research proposal is to test the hypothesis that induced pluripotent stem cells (iPSC) derived from the skin of patients with Crigler-Najjar syndrome type 1 (CN1), who have severe lifelong jaundice due to uridinediphosphoglucuronate glucuronosltransferase-1 (UGT1A1) deficiency can be corrected genetically, and then manipulated in culture to generate hepatocytes expressing active UGT1A1. We further hypothesize that appropriate host preparation will permit sufficient repopulation of the livers of the Gunn rat model of CN1 to ameliorate hyperbilirubinemia. The specific aim 1 is (a) Generating and characterizing iPS cells from skin fibroblasts from normal subjects and CN1 patients, (b) correcting the genetic defect by homologous recombination at a genomic "safe harbor", using zinc-finger nuclease or TALE-nuclease technology, and (c) differentiating the genetically corrected iPSCs into hepatocyte-like cells (iHep) in culture. The gene expression profile and metabolic function of the derived hepatocytes will be compared with those of adult and fetal human hepatocytes. Specific aim 2 is (a) Transplanting the iHeps into the liver of Gunn rats following appropriate host preparation, and evaluating the metabolic effect of the transplantation. Gunn rats will be treated with a tacrolimus-based immunosuppressive regimen that has been shown to prevent the rejection of xenografted human hepatocytes in our laboratory. The recipients will be subjected to preparative hepatic irradiation and mitotic stimulation by a single injection of an adenovector, expressing hepatocyte growth factor to induce repopulation of the liver with the transplanted hepatocytes. Serum bilirubin levels will be followed, the extent of liver repopulation will be determined and biliary excretion of bilirubin glucuronides will be analyzed by high-pressure liquid chromatography. (b) Finally, a group of recipient rats and immunodeficient mice will be observed life-long to evaluate the tumorigenic potential of the transplanted iPSC-derived hepatocytes. Successful completion of this project will provide a renewable source of autologous hepatocytes that, after additional modification and scale up, could be translated into cell transplantation-based treatment of CN1 and many other inherited liver diseases.