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

• Published in: Experimental & molecular medicine 2019, 51(0), , pp.1-9
• Main Authors: Sung, Jin Jea, Park, Chul-Yong, Leem, Joong Woo, Cho, Myung Soo, Kim, Dong-Wook
• Format: Article
• Language: English
• Subjects: 42/100; 42/41; 631/1647/1511; 631/532/2064/2158; Biomedical and Life Sciences; Biomedicine; Cell lines; Cells, Cultured; Coagulation factor VIII; Coagulation factors; CRISPR; Disease; Endothelial cells; Exons - genetics; Factor VIII - genetics; Factor VIII - metabolism; Factor VIII deficiency; Gene Editing; Gene expression; Gene targeting; Genetic Engineering - methods; Genome editing; Genomes; HEK293 Cells; Hemophilia; Hemophilia A - genetics; Hemophilia A - metabolism; Homology; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Medical Biochemistry; Molecular Medicine; Mutation; Neomycin; Nuclease; Pluripotency; Promoter Regions, Genetic - genetics; Stem Cells; Streptococcus pyogenes - genetics; Streptococcus pyogenes - metabolism; Transcription; Transcription activator-like effector nucleases; Zinc finger proteins; 생화학
• ISSN: 1226-3613; 2092-6413
• DOI: 10.1038/s12276-019-0243-1

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. Hemophilia: One gene therapy fits all A strategy to restore the expression of the gene encoding blood clotting factor VIII (FVIII) offers new hope to patients with hemophilia A. Hemophilia A is a rare bleeding disorder caused by a variety of mutations in the FVIII gene which affect the function of FVIII protein. At present, the main treatment option relies on the injection of expensive clotting-factor concentrates to restore functional levels of the FVIII. Dong-Wook Kim and colleagues at Yonsei University in Seoul, South Korea, have used genome editing techniques to insert a corrected version of the FVIII gene into stem cells derived from a patient with severe hemophilia A. When these cells differentiated into the cells lining blood vess