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

作　　者：何琼 王惠荟 程涛[3] 袁卫平[3] 马钰波 蒋永平[1] 任志华 

机构地区：[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 [5]石溪大学医学院病理部 [6]澳门科技大学澳门药物及健康应用研究院

出　　处：《Chinese Medical Sciences Journal》2017年第3期135-144,共10页中国医学科学杂志（英文版）

基　　金：Supported by the National Science and Technology Major Project(2011ZX09102-010-04)

摘　　要：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.

关 键 词：hemophilia B human induced pluripotent stem cells CRISPR/Cas9 genetic correction hepatic differentiation 

分 类 号：Q813[生物学—生物工程] Q987