Chinese Medical Sciences Journal ›› 2017, Vol. 32 ›› Issue (3): 135-144.doi: 10.24920/J1001-9294.2017.032

• 论著 •    下一篇

CRISPR技术基因矫正乙型血友病来源iPS细胞的致病点突变并诱导肝系分化

何琼1, 王惠荟1,2, 程涛3, 袁卫平3, 马钰波4,5,6,*(), 蒋永平1, 任志华1,2,*()   

  1. 1 中国医学科学院 北京协和医学院,方舟生物医药研发中心,苏州 215126
    2 苏州方舟基因药业有限公司,苏州 215126
    3 中国医学科学院 北京协和医学院,干细胞医学中心,血液病医院,实验血液学国家重点实验室,天津 300020
    4 长岛高科技孵化器,iCell基因治疗有限责任公司研究开发部,石溪,纽约 11794,美国
    5 石溪大学医学院病理部,石溪,纽约 11794,美国
    6 澳门科技大学,澳门药物及健康应用研究院,澳门 999078,中国
  • 收稿日期:2017-03-16 出版日期:2017-09-27 发布日期:2017-09-27
  • 通讯作者: 马钰波,任志华 E-mail:yupo.ma@stonybrookmedicine.edu;renzh@biopharmagen.com

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

He Qiong1, Wang Hui-hui1,2, Cheng Tao3, Yuan Wei-ping3, Ma Yu-po4,5,6,*(), Jiang Yong-ping1, Ren Zhi-hua1,2,*()   

  1. 1Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou 215126, China;
    2 Biopharmagen Corporation, Suzhou 215126, China
    3 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;
    4 iCell Gene Therapeutics LLC, Research & Development Division, Long Island High Technology Incubator, Stony Brook, NY 11794, USA;
    5 Department of Pathology, Stony Brook Medicine, Stony Brook, NY 11794, USA
    6 Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078, China
  • Received:2017-03-16 Published:2017-09-27 Online:2017-09-27
  • Contact: Ma Yu-po,Ren Zhi-hua E-mail:yupo.ma@stonybrookmedicine.edu;renzh@biopharmagen.com

摘要:

目的 通过CRISPR/Cas9技术矫正乙型血友病病人诱导多能干细胞(iPSCs)的点突变基因缺陷,进一步证明CRISPR/Cas9技术用于遗传性疾病基因矫正的有效性,并为乙型血友病的基因细胞疗法提供参考依据。方法 首先,提取乙型血友病病人来源iPSCs基因组DNA,扩增凝血因子IX的8个外显子区域,并测序。其次,根据测序结果设计4条sgRNA,构建CRISPR/sgRNA质粒,并在293T细胞上进行剪切能力验证,挑选出一条具有较高剪切效率的sgRNA。同时根据突变位点设计129个核苷酸的含两个同义突变位点的单长链寡聚核苷酸 (ssODN),作为同源修复模板。再次,通过电击转染的方式将CRISPR/sgRNA质粒和ssODN导入iPS细胞内,低密度种板,嘌呤霉素筛选48 h,再培养5~7 d。然后,挑取55个单克隆,PCR扩增目标序列并直接测序筛选矫正克隆。通过在线软件,选择8个具有较高脱靶风险的位点,分别进行脱靶效应分析。最后,将矫正克隆在体外分化成肝脏细胞,用免疫荧光和酶联免疫法检测凝血因子IX的表达。结果 PCR测序结果显示该样本为6号外显子的点突变样本(c.676 C>T)。通过CRISPR/Cas9技术,成功测序45个克隆,筛选到了10个按照同源模板发生精确矫正的克隆,矫正效率达到了22% (10/45),并且未发现脱靶效应。最后我们对矫正克隆进行了为期21 d的肝细胞分化,ELISA结果显示,在分化末期凝血因子IX的表达可以达到6 ng/ml。结论 采用CRISPR/Cas9系统和长度为129个核苷酸的含两个同义突变的单链寡聚核苷酸,成功矫正了原乙型血友病iPS细胞株的致病点突变。这种人类乙型血友病的原位基因矫正方法为乙型血友病的基因细胞疗法提供了新的可能性。

关键词: 乙型血友病, 诱导多能干细胞, CRISPR/Cas9, 基因矫正, 肝系分化

Abstract: Objective

To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient.

Methods

First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.

Results

The cell line bore a missense mutation in the 6th coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure.

Conclusions

Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Key words: hemophilia B, human induced pluripotent stem cells, CRISPR/Cas9, genetic correction, hepatic differentiation

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