Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4⁺ T-cell gene editing with Cas9 and a “one-size-fits-most” corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

中文翻译：

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在编辑 CD4+ T 细胞用于治疗 Hyper IgM1 时，对基因组完整性进行公正评估并消除目标位点的不良后果

Hyper IgM1 是一种由CD40LG突变引起的 X 连锁联合免疫缺陷病，可以通过使用 Cas9 的 CD4 ⁺ T 细胞基因编辑和“一刀切”的校正模板来治疗。与已建立的基因疗法相反，有关长程基因编辑后基因组改变的数据有限，并且对相关测定没有达成共识。我们开发了 drop-off 数字 PCR 检测方法，用于公正地检测大的目标缺失，并在编辑时以高频率发现它们。在编辑不同基因座和细胞类型以及使用替代 Cas9 和模板传递方法时，大的缺失也很常见。在CD40LG编辑的 T 细胞中，在培养物中对目标缺失进行反选择，并使用与基因校正耦合的选择器通过富集编辑细胞来进一步清除。然后，我们验证了光学基因组图谱对全基因组重排的无偏检测的敏感性，并发现在使用整合酶缺陷型慢病毒供体模板进行编辑后，对一个或多个载体副本进行目标捕获，这不会损害功能。没有检测到其他重复事件。编辑后的患者细胞显示出CD40LG调节表达和功能的忠实重建，并具有令人满意的安全性。在设计和测试类似的基因编辑策略时，应该预期并考虑大量缺失和供体模板整合。