镰状突变的高水平校正会在红细胞分化过程中被放大。
High-level correction of the sickle mutation is amplified during erythroid differentiation.
作者信息
Magis Wendy, DeWitt Mark A, Wyman Stacia K, Vu Jonathan T, Heo Seok-Jin, Shao Shirley J, Hennig Finn, Romero Zulema G, Campo-Fernandez Beatriz, Said Suzanne, McNeill Matthew S, Rettig Garrett R, Sun Yongming, Wang Yu, Behlke Mark A, Kohn Donald B, Boffelli Dario, Walters Mark C, Corn Jacob E, Martin David I K
机构信息
Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, Oakland, CA 94609, USA.
Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA.
出版信息
iScience. 2022 May 10;25(6):104374. doi: 10.1016/j.isci.2022.104374. eCollection 2022 Jun 17.
BACKGROUND
A point mutation in sickle cell disease (SCD) alters one amino acid in the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction.
RESULTS
An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one β-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. erythroid differentiation markedly enriches for corrected β-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage.
SIGNIFICANCE
These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.
背景
镰状细胞病（SCD）中的一个点突变改变了血红蛋白β-珠蛋白亚基中的一个氨基酸，导致贫血和多器官损伤，通常会使寿命缩短数十年。由于SCD是由单个突变引起的，并且造血干细胞（HSC）可以采集、操作并回输给个体，因此它是基因校正的一个有吸引力的靶点。
结果
优化后的Cas9核糖核蛋白（RNP）与单链DNA寡核苷酸供体共同作用，在超过30%的长期植入的人类HSC中至少校正了一个β-珠蛋白等位基因。采用高保真Cas9变体后，也观察到了高效校正且脱靶事件极少的情况。红细胞分化显著富集了校正后的β-珠蛋白等位基因，表明携带一个或多个校正等位基因的成红细胞具有生存优势。
意义
这些发现表明，通过适合临床转化的优化方案，可以在自体HSC中校正镰状突变。
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