Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34 hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

中文翻译：

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一种新型 Cas9 基因组编辑的自体 CD34+ 细胞疗法的开发和 IND 支持研究，用于诱导胎儿血红蛋白治疗镰状细胞病


镰状细胞病（SCD）是一种常见的严重遗传性血液疾病。目前的药物疗法部分有效，同种异体造血干细胞移植与免疫毒性有关。对患者造血干细胞 (HSC) 进行基因组编辑以重新激活红系后代中的胎儿血红蛋白 (HbF)，为治疗 SCD 提供了另一种潜在的治疗方法。尽管 FDA 发布了评估基因组编辑风险的指南，但仍不清楚如何最好地对基因组编辑细胞产品进行临床前评估。在这里，我们描述了治疗性 γ-珠蛋白基因启动子编辑策略的严格临床前开发，该策略支持 FDA 批准的研究性新药申请。我们比较了 γ-珠蛋白启动子和增强子靶标，确定了一种有效的 HbF 诱导先导候选物，并在来自 SCD 患者的动员 CD34 造血干祖细胞 (HSPC) 中测试了我们的方法。我们观察到有效的编辑、HbF 诱导至预测的治疗水平以及镰状化的减少。通过单细胞分析，我们定义了 HbF 诱导和转录的异质性。通过 CHANGE-seq 进行灵敏且公正的脱靶发现，然后进行靶向测序，我们没有在编辑的 HSPC 中检测到脱靶活动。我们的研究为将新的 HSC 基因组编辑策略转化为治疗 SCD 和其他血液疾病的临床试验提供了蓝图。