Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

造血干/祖细胞（HSPC）中的靶向基因组编辑是治疗免疫血液学疾病的一种有吸引力的策略。但是，原始HSPC中同源性指导编辑的效率有限，限制了校正细胞的产量，并可能影响临床翻译的可行性和安全性。这些问题需要在严格的临床前模型中解决，并通过开发更有效的编辑方法来克服。我们生成了人源化的X连锁严重联合免疫缺陷症（SCID-X1）小鼠模型，并从功能性HSPC的有限输入中评估了造血重建的功效和安全性，并根据不同类型的条件建立了完全校正的阈值。不料，移植少量祖细胞时，需要在HSPC输注之前进行调节，以保护小鼠免于淋巴瘤的发展。然后，我们设计了一种适合所有人的尺寸IL2RG（白介素2受体共同γ链）基因校正策略，并使用适用于校正人HSPC的相同试剂，在体内疾病模型中验证了已编辑的人基因，为在小鼠HSPC中靶向基因编辑提供了证据并证明了这一点。所述的功能性IL2RG -edited淋巴后代。最后，我们优化了人类HSPC的编辑试剂和方案，并达到了IL2RG的阈值使用临床相关的HSPC来源和高度特异性的锌指核酸酶或CRISPR（聚簇的，规则间隔的回文短重复序列）/ Cas9（CRISPR相关蛋白9）编辑长期预测的可安全挽救该疾病的细胞。总体而言，我们的工作为SCID-X1基因编辑的临床翻译建立了理论基础和指导原则，并为开发针对其他疾病的基因校正提供了框架。