Genome editing of HBG1 and HBG2 to induce fetal hemoglobin
Genome editing of HBG1 and HBG2 to induce fetal hemoglobin
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DOI：
10.1182/bloodadvances.2019000820
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发表时间：
2019-11-12
期刊：
影响因子：
7.5
通讯作者：
Weiss, Mitchell J.
中科院分区：
文献类型：
--
作者：
Metais, Jean-Yves;Doerfler, Phillip A.;Weiss, Mitchell J.
通过聚集规则间隔的短回文重复序列/Cas9介导的DNA调控元件抑制伽马珠蛋白基因(HBG1和HBG2)的表达来诱导胎儿血红蛋白(HBF)是一种很有前途的治疗镰状细胞病(SCD)和β地中海贫血的策略，尽管最佳的技术方法和限制毒性尚未完全确定。我们破坏了一个HBG1/HBG2基因启动子基序，该基序与转录抑制因子BCL11A结合。将Cas9单引导RNA核糖核蛋白复合体电穿孔到正常和SCD供者CD34(+)造血干/祖细胞中，在体外和体内生成的红系后代中，HBF诱导到潜在的治疗水平。异种移植后16-17周，靶向编辑不影响CD34(+)细胞的再生或分化为红系、T、B或髓系细胞。通过环化确定的候选位置的定向测序没有检测到非靶点突变，以通过测序(Circle-Seq)体外报告切割效应(Circle-Seq)，这是一种体外基因组规模的检测Cas9活性的方法。含有3个核定位序列的工程化Cas9比含有2个核定位序列的常规Cas9更有效和一致地编辑人造血干/祖细胞。我们的研究提供了新的和重要的临床前证据，支持基于机制的方法来诱导HBF治疗血红蛋白疾病的安全性、可行性和有效性。
Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34(+) hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2r gamma(-/-)/Kit(W41/W41) immunodeficient mice. On-target editing did not impair CD34(+) cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and m essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.