Lee Jeong Hyeon, Oh Hye-Kyung, Choi Beom Seok, Lee Ho Hyeon, Lee Kyu Jun, Kim Un Gi, Lee Jina, Lee Hyerim, Lee Geon Seong, Ahn Se Jun, Han Jeong Pil, Kim Seokjoong, Yeom Su Cheong, Song Dong Woo
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Gangwon 25354, Korea.
Research and Development Center, Toolgen Incorporated, Geumcheon-gu, Seoul 08501, Korea.
Mol Ther Nucleic Acids. 2022 Aug 4;29:551-562. doi: 10.1016/j.omtn.2022.08.002. eCollection 2022 Sep 13.
Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, , in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of editing using a small Cas9 protein derived from (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing or . Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient -editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.
最近，腺相关病毒介导的替代疗法的临床试验表明，其对包括血友病在内的几种肝脏遗传性疾病具有长期治疗效果。然而，当肝脏再生或发生生理性增殖时，治疗效果下降仍令人担忧。尽管使用成簇规律间隔短回文重复序列/Cas9系统进行基因组编辑为解决这一问题提供了契机，但低敲入效率可能会限制其在治疗相关表达中的应用。在此，我们鉴定出一个新基因，其中一个强启动子促进了敲入基因在肝细胞中的表达。我们还在血友病模型中研究了使用源自嗜热栖热菌的小型Cas9蛋白（CjCas9）进行编辑的效果。我们证明，腺相关病毒介导的CjCas9和供体的递送在载脂蛋白C3人源化小鼠中导致人因子9表达达到中等水平。此外，敲入驱动的表达在乙型血友病小鼠中诱导凝血功能大幅恢复。没有证据表明存在脱靶编辑或。总体而言，我们的研究结果证明了使用精确高效的编辑平台进行治疗相关表达，为开发针对多种单基因肝病的进一步长期疗法提供了见解。
