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Therapeutic base editing of human hematopoietic stem cells.

人类造血干细胞的治疗性碱基编辑。

  • 影响因子:19.14
  • DOI:10.1038/s41591-020-0790-y
  • 作者列表:"Zeng J","Wu Y","Ren C","Bonanno J","Shen AH","Shea D","Gehrke JM","Clement K","Luk K","Yao Q","Kim R","Wolfe SA","Manis JP","Pinello L","Joung JK","Bauer DE
  • 发表时间:2020-04-01
Abstract

:Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.

摘要

: 通过与可编程DNA结合蛋白连接的核苷酸脱氨酶进行碱基编辑代表了一种有前途的永久治疗血液疾病的方法,尽管其在移植造血干细胞 (hsc) 中的应用仍未被探索。在本研究中,我们纯化了A3A (N57Q)-BE3碱基编辑器,用于人外周血动员的CD34 + 造血干细胞和祖细胞 (HSPCs) 的核糖核蛋白 (RNP) 电穿孔。我们在 + 58的BCL11A红细胞增强子处观察到频繁的靶上胞嘧啶碱基编辑,几乎没有插入因子。在碱基编辑或核酸酶编辑后,红系子代中的胎儿血红蛋白 (HbF) 诱导是相似的。BCL11A增强子的单一治疗基础编辑分别防止镰状细胞病和 β-地中海贫血患者来源的hspc的红细胞后代的镰状细胞和改善珠蛋白链失衡。此外,通过联合破坏BCL11A红细胞增强子和校正HBB -28A>G启动子突变,可以实现有效的多重编辑。最后,如在初级和次级受体动物中测定的,可以在多谱系重新繁殖的自我更新的人hsc中以高频率产生碱基编辑,导致体内有效的HbF诱导。总之,这些结果证明了人hspc的RNP碱基编辑作为核酸酶编辑的可行替代方案用于HSC靶向治疗性基因组修饰的潜力。

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METHODS:BACKGROUND:Thalassemia is one of the most common monogenetic diseases in the south of China and Southeast Asia. Hemoglobin Bart's hydrops fetalis syndrome was caused by a homozygous Southeast Asian deletion (-/-) in the HBA gene. Few studies have proved the potential of screen for Bart's hydrops fetalis using fetal cell-free DNA. However, the number of cases is still relatively small. Clinical trials of large samples would be needed. OBJECTIVE:In this study, we aimed to develop a noninvasive method of target-captured sequencing and genotyping by the Bayesian method using cell-free fetal DNA to identify the fetal genotype in pregnant women who are at risk of having hemoglobin Bart hydrops fetalis in a large-scale study. STUDY DESIGN:In total, 192,173 couples from 30 hospitals were enrolled in our study and 878 couples were recruited, among whom both the pregnant women and their husbands were detected to be carriers of Southeast Asian type (-/αα) of α-thalassemia. Prenatal diagnosis was performed by chorionic villus sampling, amniocentesis, or cordocentesis using gap-polymerase chain reaction considered as the golden standard. RESULTS:As a result, we found that the sensitivity and specificity of our noninvasive method were 98.81% and 94.72%, respectively, in the training set as well as 100% and 99.31%, respectively, in the testing set. Moreover, our method could identify all of 885 maternal samples with the Southeast Asian carrier and 36 trisomy samples with 100% of sensitivity in T13, T18, and T21 and 99.89% (1 of 917) and 99.88% (1 of 888) of specificity in T18 and T21, respectively. CONCLUSION:Our method opens the possibility of early screening for maternal genotyping of α-thalassemia, fetal aneuploidies in chromosomes 13/18/21, and hemoglobin Bart hydrops fetalis detection in 1 tube of maternal plasma.

影响因子:1.74
发表时间:2020-02-01
DOI:10.1177/1049909119868657
作者列表:["Suarez ML","Schlaeger JM","Angulo V","Shuey DA","Carrasco J","Roach KL","Ezenwa MO","Yao Y","Wang ZJ","Molokie RE","Wilkie DJ"]

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血红蛋白病方向

由于血红蛋白分子结构异常(异常血红蛋白病),或珠蛋白肽链合成速率异常(珠蛋白生成障碍性贫血,又称海洋性贫血)所引起的一组遗传性血液病。

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