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

• Orginal Article •     Next Articles

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 Online:2017-09-26 Published:2017-09-27
  • Contact: Ma Yu-po,Ren Zhi-hua E-mail:yupo.ma@stonybrookmedicine.edu;renzh@biopharmagen.com
  • Supported by:
    △Supported by the National Science and Technology Major Project (2011ZX09102-010-04).

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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