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Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs

Authors
 Jin Jea Sung  ;  Chul-Yong Park  ;  Joong Woo Leem  ;  Myung Soo Cho  ;  Dong-Wook Kim 
Citation
 EXPERIMENTAL AND MOLECULAR MEDICINE, Vol.51(4) : 45, 2019 
Journal Title
 EXPERIMENTAL AND MOLECULAR MEDICINE 
ISSN
 1226-3613 
Issue Date
2019
Abstract
Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIIIgene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.
Files in This Item:
T201902186.pdf Download
DOI
10.1038/s12276-019-0243-1
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Physiology (생리학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
Yonsei Authors
Kim, Dong Wook(김동욱) ORCID logo https://orcid.org/0000-0002-5025-1532
Park, Chul Yong(박철용) ORCID logo https://orcid.org/0000-0002-4467-9268
Leem, Joong Woo(임중우) ORCID logo https://orcid.org/0000-0002-1605-2230
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/170375
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