Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

事实证明，实体器官（尤其是肝脏）中基于细胞的离体基因治疗在技术上具有挑战性。在这里，我们报告了肝细胞治疗临床应用的可行策略。我们首先通过大规模扩增患者来源的肝细胞来产生高质量的自体肝细胞。此外，增殖的患者源性肝细胞与通过筛选鉴定的AAV2.7m8变异体一起，能够实现CRISPR-Cas9介导的高效靶向整合，实现FAH或OTC致病突变的功能校正。重要的是，这些编辑过的肝细胞以高水平重新填充受损的小鼠肝脏并经历成熟，成功治疗了移植后患有酪氨酸血症的小鼠。我们的研究结合了体外大规模细胞扩增和患者来源的可移植肝细胞的基因编辑，这具有治疗人类肝脏疾病的潜力。