Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

中文翻译：

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CRISPR-Cas9 治疗镰状细胞病的安全性和有效性研究强调了疾病特异性反应


通过 CRISPR-Cas9 重新激活胎儿血红蛋白 (HbF) 表达是治疗镰状细胞病 (SCD) 的一种有前景的策略。在这里，我们描述了一种基因组编辑策略，通过靶向 γ-珠蛋白启动子中淋巴瘤相关因子 (LRF) 阻遏物的结合位点 (BS) 来重新激活 HbF 表达。健康供体 (HD) 和患者来源的 HSPC 中的 CRISPR-Cas9 治疗导致其红系后代中出现高频率的 LRF BS 破坏和有效的 HbF 合成。 LRF BS 破坏不会损害 HSPC 植入和分化，但在 SCD 细胞中比在 HD 细胞中更有效。然而，与 HD 细胞相比，SCD HSPC 表现出植入减少和骨髓偏向性。我们检测到脱靶活性和染色体重排，特别是在 SCD 样本中（可能是因为总体编辑效率较高），但没有影响靶基因表达以及 HSPC 植入和分化。转录组分析表明，编辑过程导致参与 DNA 损伤和炎症反应的基因上调，这在 SCD HSPC 中更为明显。这项研究为基于 HbF 再激活的编辑策略提供了有效性和安全性的证据，并强调了在临床相关条件（即患者来源的 HSPC）中进行安全性研究的必要性。