Recombination activating genes ( RAGs ) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1 –/– mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

中文翻译：

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造血干细胞和祖细胞中的外显子敲除和敲入基因编辑可挽救 RAG1 免疫缺陷


重组激活基因（ RAG ）在淋巴分化过程中受到严格调节，它们的突变会导致一系列严重的免疫紊乱。造血干细胞和祖细胞（HSPC）移植是首选治疗方法，但受到供体可用性和毒性的限制。为了克服这些问题，我们开发了针对人类的纠正序列的基因编辑策略RAG1通过同源定向修复（HDR）对基因进行修复，并通过定制的二维、三维和体内异种移植平台对其进行验证，以评估表达和功能的拯救。而整合到内含子 1 中RAG1实现了次优校正，框内插入外显子 2 驱动了人类 RAG1 的生理表达和活性，从而破坏了未修复的低等位基因的显性负效应。增强型 HDR 介导的基因编辑能够纠正人类RAG1来自低效型患者的 HSPC RAG1克服 T 细胞和 B 细胞分化障碍的突变。基因校正效率超过了拯救免疫缺陷所需的功能性 HSPC 的最低比例拉格1 –/–小鼠，支持 HSPC 基因编辑用于治疗 RAG1 缺陷的临床转化。