|
KMID : 0620920190510040045
|
|
Experimental & Molecular Medicine
2019 Volume.51 No. 4 p.45 ~ p.45
|
|
|
Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs
|
|
Sung Jin-Jea
Park Chul-Yong
Leem Joong-Woo
Cho Myung-Soo
Kim Dong-Wook
|
|
|
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 FVIII gene 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.
|
|
|
KEYWORD
|
|
|
Genetic engineering, Induced pluripotent stem cells
|
|
|
FullTexts / Linksout information
|
|
|
|
|
Listed journal information
|
|
    
|