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

He Qiong; Wang Hui-hui; Cheng Tao; Yuan Wei-ping; Ma Yu-po; Jiang Yong-ping; Ren Zhi-hua

doi:10.24920/J1001-9294.2017.032

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

Chinese Medical Sciences Journal Vol. 32, Issue 3, Pages: 135-144(2017)

Orginal Article | Updated：2024-04-10

- Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells
- Affiliations：
  
  1. 1Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou 215126, China;
  2. 2Biopharmagen Corporation, Suzhou 215126, China
  3. 3State 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. 4iCell Gene Therapeutics LLC, Research & Development Division, Long Island High Technology Incubator, Stony Brook, NY 11794, USA;
  5. 5Department of Pathology, Stony Brook Medicine, Stony Brook, NY 11794, USA
  6. 6Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078, China
- Author bio：
  
  *Corresponding author Zhihua Ren, Tel: 86-512-62831269, Fax: 86-512-62831219, E-mail: renzh@biopharmagen.com; Yupo Ma, E-mail: yupo.ma@stonybrookmedicine.edu
- Funds：
- DOI：10.24920/J1001-9294.2017.032
  CLC：
- Received：16 March 2017，
  
  Published Online：26 September 2017，
  
  Published：2017-09
- Accepted：
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He Qiong, Wang Hui-hui, Cheng Tao, et al. Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells[J]. Chinese medical sciences journal, 2017, 32(3): 135-144.
DOI：

He Qiong, Wang Hui-hui, Cheng Tao, et al. Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells[J]. Chinese medical sciences journal, 2017, 32(3): 135-144. DOI： 10.24920/J1001-9294.2017.032.

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。

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 6

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.

关键词

Keywords

references

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