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突出了疾病特异性反应


通过 CRISPR-Cas9 表达胎儿血红蛋白 （HbF） 再激活是治疗镰状细胞病 （SCD） 的一种很有前途的策略。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。在健康供体 （HD） 和患者来源的 HSPC 中进行 CRISPR-Cas9 处理导致其红系后代中 LRF BS 破坏的频率很高，并且 HbF 合成有效。LRF BS 破坏不会损害 HSPC 植入和分化，但在 SCD 中比在 HD 细胞中更有效。然而，与 HD 细胞相比，SCD HSPCs 显示出植入减少和髓系偏倚。我们检测到脱靶活性和染色体重排，特别是在 SCD 样本中 （可能是因为整体编辑效率更高），但不影响靶基因表达和 HSPC 植入和分化。转录组学分析表明，编辑过程导致参与 DNA 损伤和炎症反应的基因上调，这在 SCD HSPCs 中更为明显。本研究为基于 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下进行安全性研究的必要性，即在患者来源的 HSPC 中。