酪氨酸血症Ⅰ型的体外疾病模型建立及基因修复治疗的实验研究

Hereditary tyrosinemia type 1 (HT1), which has been included in the first national list of rare diseases, is an inborn errors of metabolism in infancy. Defects in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism, causes accumulation of the toxic metabolites in hepatocytes. Acute onset of HT1 is characterized by severe liver involvement, most frequently leading to death, if untreated. Early treatment with NTBC will reduces the development of liver disease, however, there is no radical cure at present. Therefore, it is urgent to establish new research models and innovative treatments for HT1. Combination of iPSCs and gene editing technologies may be the effective way to cure this disease. The applicant has successfully derived HT1 patient-specific iPSCs cells with compound heterozygous mutations of FAH gene and established efficient differentiation methods of human pluripotent stem cells to induced hepatocyte in a stepwise manner. On the basis of this, patient-specific iPSCs will be induced to differentiate to hepatic progenitor cells and hepatocyte-like cells. Immunofluorescence, flow cytometry, quantitative RT-PCR and RNA-Seq will be carried out to explore the differences between HT1 and control groups in the phenotype, differentiation capacity, apoptosis, proliferation capacity, gene expression profile and so on. Furthermore, the mutated FAH gene will be repaired by ABEs base editing tools and the expanding and repopulating capacity of the gene corrected cells will be evaluated in FAH deficient mice. This study may provide an in vitro cell model and contribute to the pathogenesis and therapeutic targets study for HT1 disease.

原发性酪氨酸血症Ⅰ型（HT1）是新生儿罕见遗传代谢性疾病。患者FAH基因突变导致有毒代谢物在肝脏蓄积并危及生命，目前并无有效的根治药物。因此，亟需建立特异的疾病模型用于创新治疗技术开发，而iPSCs模型联合精准基因编辑手段可能从根本上治愈此疾病。申请人前期成功获得FAH复合突变的iPSCs模型，并建立了iPSCs定向分化成肝样细胞的分阶段诱导体系；拟应用免疫荧光染色、流式细胞分选、qRT-PCR和转录组测序等方法，系统研究病人来源的细胞其表型、分化能力、增殖、凋亡和基因表达谱等与正常对照的差异；进一步运用单碱基编辑修复突变基因，深入探讨基因修复-定向分化-肝祖细胞移植途径在FAH缺陷小鼠模型中再生功能性肝细胞并改善肝功能的作用。通过本研究可以建立HT1疾病细胞模型并有助于新药和治疗靶点筛选，也为罕见病HT1个体化精准治疗提供实验依据。

遗传性酪氨酸血症（HT1）是新生儿罕见代谢性疾病。FAH代谢酶功能缺陷导致有毒代谢物在肝脏蓄积并危及生命，尚无有效根治药物。本项目研究通过建立酪氨酸血症Ⅰ型疾病特异的HT1-iPSCs并定向肝脏祖细胞和肝样细胞诱导分化，成功建立了酪氨酸血症Ⅰ型细胞研究模型，为HT1疾病研究提供了一种新的研究思路。通过与正常对照细胞比较，发现HT1-iPSCs来源的细胞在肝脏祖细胞分化效率、增殖和肝样细胞分化能力并未见明显差异，但在高酪氨酸培养环境中肝样细胞分化能力减弱、凋亡细胞增加，与酪氨酸血症疾病检测结果相似，基因修复后能纠正疾病表型。我们进一步通过表达谱和免疫荧光检测发现疾病来源肝样细胞存在大量的异常DNA断裂，过表达FAH能逆转这个现象并促进细胞增殖，提示抑制异常DNA断裂可能为HT1疾病寻找治疗新靶点提供新线索。

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