TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction
TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction
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DOI：
10.1016/j.omtm.2018.12.008
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发表时间：
2019-03-15
影响因子：
4.7
通讯作者：
Rawlings, David J.
中科院分区：
文献类型：
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作者：
Lux, Christopher T.;Pattabhi, Sowmya;Rawlings, David J.
γ-血红蛋白启动子（HBG 1和HBG 2）内的元件的功能是结合转录复合物，介导胎儿血红蛋白表达的抑制。HBG 1启动子缺失13-bp的镰状细胞病（SCD）患者表现出临床上有利的胎儿血红蛋白（HPFH）表型遗传持久性。我们开发了靶向同源HBG启动子的TALEN以去抑制胎儿血红蛋白。用TALEN mRNA转染人CD 34（+）细胞导致HBG 1（43%）和HBG 2（74%）中的indel产生，包括13-bp HPFH缺失（类似于6%）。经编辑的细胞的红系分化揭示了如通过HPLC检测到的γ-血红蛋白表达的4.6倍增加。在人源化小鼠模型中对TALEN编辑的CD 34（+）细胞的体内评估证明了在造血细胞中indel的持续存在长达24周。二次移植后插入缺失率保持不变，这与长期再生干细胞（LT-HSC）的编辑一致。与模拟相比，在编辑的动物中通过流式细胞术检测到表达人γ-血红蛋白的F细胞的频率高约50%。总之，这些发现表明，在γ-血红蛋白启动子中TALEN介导的插入缺失产生导致体外和体内高水平的胎儿血红蛋白表达，表明这种方法可以在患有SCD或β-地中海贫血的患者中提供治疗益处。
Elements within the gamma-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34(+) cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (similar to 6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in gamma-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34(+) cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human gamma-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the gamma-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or beta-thalassemia.