基于CRISPR/Cas9介导的LDLR基因定点敲入人源肝细胞移植治疗纯合型家族性高胆固醇血症的实验研究

纯合型家族性高胆固醇血症（HoFH）是一种严重的遗传性代谢病，儿童期即可发生危及生命的心血管并发症，低密度脂蛋白受体（LDLR）基因突变是主要致病原因，早期肝移植是目前唯一治愈手段。然而，供肝短缺、术后长期服用免疫抑制剂等问题严重限制其开展。肝细胞移植因创伤小、成本低等特点成为极具潜力的治疗手段，但肝细胞来源受限和移植后排异反应是目前面临的两大关键难题。为此，我们提出“自体肝细胞移植介导的基因治疗从根本上纠正脂质代谢紊乱”假说。我们前期建立了一套高效、稳定的人原代肝细胞分离和培养体系，开创性地实现了HoFH患者来源的肝细胞在体外大量扩增。进一步，我们将利用CRISPR/Cas9技术在体外实现野生型LDLR基因在该细胞中定点敲入并表达，将基因纠正后的人源肝细胞植入小鼠疾病模型验证其疗效及安全性。本研究拟同时解决肝细胞移植中细胞来源限制和移植后排斥两大难题，为HoFH治疗提供新的路径。

Homozygous familial hypercholesterolemia (HoFH) is a serious inherited metabolic disease. Life threatening cardiovascular complications can occur in childhood. Low density lipoprotein receptor (LDLR) gene mutation is the main cause of the disease. Early liver transplantation is the only curative method at present. However, the shortage of donor livers and the long-term use of immunosuppressants after transplantation seriously limit its implementation. Hepatocyte transplantation has become a potential treatment due to its advantages of minimal invasion and low costs. However, the limited cell source and immune rejection after transplantation are the two key obstacles. Therefore, we put forward the hypothesis that "gene therapy mediated by autologous hepatocyte transplantation can fundamentally correct the lipid metabolism disorder". We previously established an efficient and stable system for the isolation and culture of human primary hepatocytes, and achieved large-scale expansion of hepatocytes from HoFH patients in vitro. Furthermore, we will use the CRISPR/cas9 technique to have the wild-type LDLR gene knocked in and expressing in these cells in vitro, and implant the gene-corrected human hepatocytes into mouse disease models to verify its curative effect and safety. This study aims to solve the two obstacles of hepatocyte source restrictions and posttransplant rejections synchronously, and provide a new path for the treatment of HoFH.