Mutations in the human β-globin gene are the cause of β-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (β⁰/β⁺) and homozygous (β⁰/β⁰, β⁺/β⁺) β-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of β-hemoglobinopathies.

中文翻译：

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使用 CRISPR-Cas9 和腺相关病毒血清型 6 供体模板的 β-血红蛋白病通用基因校正方法

人β珠蛋白的突变β-血红蛋白病是世界上最常见的遗传性单基因血液病之一。新的治疗方法基于慢病毒载体 (LV) 或 CRISPR-Cas9 介导的基因破坏，以分别表达成人血红蛋白 (HbA) 或重新激活完全功能性的胎儿血红蛋白。尽管如此，LVs 存在插入突变的风险，而参与胎儿到成人血红蛋白转换的基因破坏转录因子（BCL11A、KLF1）可能会导致其他细胞过程的失调。因此，通过腺相关病毒提供 DNA 修复模板，结合 CRISPR-Cas9 和同源定向修复 (HDR) 的通用基因添加/校正方法可以减轻慢病毒基因转移和基因破坏策略的局限性，确保有针对性的整合和受控的转基因表达。在这项研究中，我们在健康供体的造血干细胞和祖细胞中获得了高基因添加率（高达 12%）和基因校正率（高达 38%），而无需任何细胞分选/富集或应用 HDR 增强剂。此外，这些方法在杂合子（β⁰ /β ⁺ ) 和纯合子 (β ⁰ /β ⁰ , β ⁺ /β ⁺ ) β-地中海贫血患者，HbA 显着增加并证明了该研究治疗β-血红蛋白病的普遍治疗潜力。