State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Cell Prolif. 2023 May;56(5):e13467. doi: 10.1111/cpr.13467. Epub 2023 May 17.
Ex vivo gene manipulation in human hepatocytes is a promising therapeutic strategy in the treatment of inherited liver diseases. However, a major limitation is the lack of a highly efficient and safe genetic manipulation system for transplantable primary human hepatocytes (PHHs). Here, we reported that proliferating human hepatocytes (ProliHHs) cultured in vitro showed high susceptibility to lentivirus-mediated genetic modification and maintained cellular phenotypes after lentiviral infection. Human factor VIII expression was introduced through F8-Lentivirus-mediated transduction of ProliHHs followed by xenotransplantation into immunocompromised haemophilia A mice. We demonstrated that these F8-modified ProliHHs could effectively repopulate the mouse liver, resulting in therapeutic benefits in mouse models. Furthermore, no genotoxicity was detected in F8-modified ProliHHs using lentiviral integration site analysis. Thus, this study demonstrated, for the first time, the feasibility and safety of lentiviral modification in ProliHHs to induce the expression of coagulation factor VIII in the treatment of haemophilia A.
体外基因操作在人类肝细胞中是一种很有前途的治疗遗传性肝脏疾病的策略。然而，一个主要的限制是缺乏高效和安全的遗传操作系统，用于可移植的原发性人肝细胞（PHH）。在这里，我们报道了体外培养的增殖人肝细胞（ProliHHs）对慢病毒介导的基因修饰具有很高的易感性，并在慢病毒感染后保持细胞表型。通过 F8-慢病毒介导的 ProliHHs 的转导，引入人凝血因子 VIII 的表达，然后进行免疫缺陷血友病 A 小鼠的异种移植。我们证明，这些 F8 修饰的 ProliHHs 可以有效地重新填充小鼠肝脏，从而在小鼠模型中产生治疗效果。此外，使用慢病毒整合位点分析，在 F8 修饰的 ProliHHs 中未检测到遗传毒性。因此，这项研究首次证明了在 ProliHHs 中进行慢病毒修饰以诱导凝血因子 VIII 表达来治疗血友病 A 的可行性和安全性。
