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; 王惠荟; 程涛; 袁卫平; 马钰波; 蒋永平; 任志华

doi:10.24920/J1001-9294.2017.032

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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

中国医学科学杂志（英文版） 2017年32卷第3期页码：135-144

Orginal Article | Updated：2024-04-10

- 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
- 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：
  
  △Supported by the National Science and Technology Major Project (2011ZX09102-010-04).
- DOI：10.24920/J1001-9294.2017.032
  中图分类号：
- 收稿日期：2017-03-16，
  
  网络出版日期：2017-09-26，
  
  纸质出版日期：2017-09
- Accepted：
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何琼, 王惠荟, 程涛, 等. 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[J]. 中国医学科学杂志（英文版）, 2017,32(3):135-144.

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.
何琼, 王惠荟, 程涛, 等. 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[J]. 中国医学科学杂志（英文版）, 2017,32(3):135-144. DOI： 10.24920/J1001-9294.2017.032.

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

Chen Y , Schroeder JA , Kuether EL , Zhang G , Shi Q. Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(null) mice. Mol Ther 2014 ; 22 ( 1 ): 169 - 77 . doi: 10.1038/mt.2013.197 10.1038/mt.2013.197 .

Li T , Miller CH , Driggers J , Payne AB , Elliingsen D , Hooper WC. Mutation analysis of a cohort of US patients with hemophilia B. Am J Hematol 2014 ; 89 ( 4 ): 375 - 9 . doi: 10.1002/ajh.23645 10.1002/ajh.23645 .

Bowen DJ. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 2002 ; 55 ( 1 ): 1 - 18 . doi: 10.1136/mp. 55.1.1 10.1136/mp. 55.1.1 .

Seo JY , Jang MA , Kim HJ , Lee KO , Kim SH , Kim HJ. Sequence variation data of F8 and F9 genes in functionally validated control individuals: implications on the molecular diagnosis of hemophilia. Blood Res 2013 ; 48 ( 3 ): 206 - 10 . doi: 10.5045/br.2013.48.3.206 10.5045/br.2013.48.3.206 .

Takahashi K , Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006 ; 126 ( 4 ): 663 - 76 . doi: 10.1016/ j.cell.2006.07.024 10.1016/ j.cell.2006.07.024 .

Takahashi K , Okita K , Nakagawa M , Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2007 ; 2 ( 12 ): 3081 - 9 . doi: 10.1038/nprot.2007. 418 10.1038/nprot.2007. 418 .

Yamanaka S . Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors . Cell Prolif 2008 ; 41 Suppl 1 : 51 - 6 . doi: 10.1111/j.1365-2184.2008. 00493.x 10.1111/j.1365-2184.2008. 00493.x .

Iwamuro M , Komaki T , Kubota Y , Seita M , Kawamoto H , Yuasa T , et al. Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells. Cell Transplant 2010 ; 19 ( 6 ): 831 - 9 . doi: 10.3727/096368 910X508951 10.3727/096368 910X508951 .

Song Z , Cai J , Liu Y , Zhao D , Yong J , Duo S , et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 2009 ; 19 ( 11 ): 1233 - 42 . doi: 10.1038/cr.2009.107 10.1038/cr.2009.107 .

Paques F , Duchateau P. Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 2007 ; 7 ( 1 ): 49 - 66 . doi: 10.2174/156652307779940216 10.2174/156652307779940216 .

Carroll D. Genome engineering with zinc-finger nucleases. Genetics 2011 ; 188 ( 4 ): 773 - 82 . doi: 10.1534/genetics. 111.131433 10.1534/genetics. 111.131433 .

Wright DA , Li T , Yang B , Spalding MH. TALEN-mediated genome editing: prospects and perspectives. Biochem J 2014 ; 462 ( 1 ): 15 - 24 . doi: 10.1042/BJ20140295 10.1042/BJ20140295 .

Doudna JA , Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014 ; 346 ( 6213 ): 1258096 . doi: 10.1126/science. 1258096 10.1126/science. 1258096 .

Ran FA , Hsu PD , Wright J , Agarwala V , Scott DA , Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013 ; 8 ( 11 ): 2281 - 308 . doi: 10.1038/nprot.2013. 143 10.1038/nprot.2013. 143 .

Hsu PD , Lander ES , Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014 ; 157 ( 6 ): 1262 - 78 . doi: 10.1016/j.cell.2014.05.010 10.1016/j.cell.2014.05.010 .

Si-Tayeb K , Noto FK , Nagaoka M , Li J , Battle MA , Duris C , et al. Highly efficient generation of human hepatocyte- like cells from induced pluripotent stem cells. Hepatology 2010 ; 51 ( 1 ): 297 - 305 . doi: 10.1002/hep.23354 10.1002/hep.23354 .

Jinek M , East A , Cheng A , Lin S , Ma E , Doudna J. RNA-programmed genome editing in human cells. Elife 2013 ; 2 : e00471 . doi: 10.7554/eLife.00471 10.7554/eLife.00471 .

Jinek M , Chylinski K , Fonfara I , Hauer M , Doudna JA , Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012 ; 337 ( 6096 ): 816 - 21 . doi: 10.1126/science.1225829 10.1126/science.1225829 .

Cone L , Ran FA , Cox D , Lin S , Barretto R , Habib N , et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013 ; 339 ( 6121 ): 819 - 23 . doi: 10.1126/ science.1231143 10.1126/ science.1231143 .

Mali P , Yang L , Esvelt KM , Aach J , Guell M , DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013 ; 339 ( 6121 ): 823 - 6 . doi: 10.1126/science. 1232033 10.1126/science. 1232033 .

Tabebordbar M , Zhu K , Cheng JK , Chew WL , Widrick JJ , Yan WX , et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2016 ; 351 ( 6271 ): 407 - 11 . doi: 10.1126/science.aad5177 10.1126/science.aad5177 .

Yin H , Xue W , Chen S , Bogorad RL , Benedetti E , Grompe M , et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 2014 ; 32 ( 6 ): 551 - 3 . doi: 10.1038/nbt.2884 10.1038/nbt.2884 .

Ding Q , Strong A , Patel KM , Ng SL , Gosis BS , Regan SN , et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 2014 ; 115 ( 5 ): 488 - 92 . doi: 10.1161/CIRCRESAHA.115.304351 10.1161/CIRCRESAHA.115.304351 .

Lin SR , Yang HC , Kuo YT , Liu CJ , Yang TY , Sung KC , et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo . Mol Ther Nucleic Acids 2014 ; 3 : e186 . doi: 10.1038/mtna.2014.38 10.1038/mtna.2014.38 .

Guan Y , Ma Y , Li Q , Sun Z , Ma L , Wu L , et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 2016 ; 8 ( 5 ): 477 - 88 . doi: 10.15252/emmm.201506039 10.15252/emmm.201506039 .

Xie F , Ye L , Chang JC , Beyer AI , Wang J , Muench MO , et al. Seamless gene correction of b-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res 2014 ; 24 ( 9 ): 1526 - 33 . doi: 10.1101/gr. 173427.114 10.1101/gr. 173427.114 .

Schwank G , Koo BK , Sasselli V , Dekkers JF , Heo I , Demircan T , et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cysticfibrosis patients. Cell Stem Cell 2013 ; 13 ( 6 ): 653 - 8 . doi: 10.1016/j.stem.2013.11.002 10.1016/j.stem.2013.11.002 .

Li HL , Fujimoto N , Sasakawa N , Shirai S , Ohkame T , Sakuma T , et al. Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep 2015 ; 4 ( 1 ): 143 - 54 . doi: 10.1016/j.stemcr.2014. 10.013 10.1016/j.stemcr.2014. 10.013 .

Ousterout DG , Kabadi AM , Thakore PI , Majoros WH , Reddy TE , Gersbach CA. Multiplex CRISPR/Cas9-based genome editing forcorrectionofdystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun 2015 ; 6 : 6244 . doi: 10.1038/ncomms7244 10.1038/ncomms7244 .

Fu Y , Foden JA , Khayter C , Maeder ML , Reyon D , Joung JK , et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013 ; 31 ( 9 ): 822 - 6 . doi: 10.1038/nbt.2623 10.1038/nbt.2623 .

Hsu PD , Scott DA , Weinstein JA , Ran FA , Konermann S , Agarwala V , et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013 ; 31 ( 9 ): 827 - 32 . doi: 10.1038/nbt.2647 10.1038/nbt.2647 .

Wu XB , Andrea JK , Philip AS. Target specificity of the CRISPR-Cas9 system. Quant Biol 2014 , 2 ( 2 ): 59 - 70 . doi: 10.1007/s40484-014-0030-x 10.1007/s40484-014-0030-x .

Ashrani AA , Reding MT , Shet A , Osip J , Humar A , Lake JR , et al. Successful liver transplantation in a patient with severe haemophilia A and a high-titre factor Ⅷ inhibitor. Haemophilia 2004 ; 10 ( 6 ): 735 - 7 . doi: 10.1111/j.1365-2516. 2004.01030.x 10.1111/j.1365-2516. 2004.01030.x .

Wilde J , Teixeira P , Bramhall SR , Gunson B , Mutimer D , Mirza DF. Liver transplantation in haemophilia. Br J Haematol 2002 ; 117 ( 4 ): 952 - 6 . doi: 10.1046/j.1365-2141. 2002.03528.x 10.1046/j.1365-2141. 2002.03528.x .

Gordon FH , Mistry PK , Sabin CA , Lee CA. Outcome of orthotopic liver transplantation in patients with haemophilia. Gut 1998 ; 42 ( 5 ): 744 - 9 . doi: 10.1136/gut.42.5.744 10.1136/gut.42.5.744 .

Nathwani AC , Reiss UM , Tuddenham EG , Rosales C , Chowdary P , McIntosh J, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014 ; 371 ( 21 ): 1994 - 2004 . doi: 10.1056/NEJMoa 1407309 10.1056/NEJMoa 1407309 .

Sharma A , Easow MM , Sriganesh V , Neely JA , Kalipatnapu S. Gene therapy for haemophilia . Cochrane Database Syst Rev 2014 ; 11:CD010822. doi: 10.1002/ 14651858.CD010822.pub2 10.1002/ 14651858.CD010822.pub2 .

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