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

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,hemophiliaA can be considered an ideal target forgenemodification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mildrestorationofFVIIIlevels in plasma can prevent disease symptoms in patients with severehemophiliaA. In this study, we describe a universal genome correction strategy to restoreFVIIIexpressionin induced pluripotent stem cells (iPSCs) derived from a patient withhemophiliaA by the human elongation factor 1 alpha (EF1α)-mediated normalFVIIIgeneexpressionin theFVIIIlocusof the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from theFVIIIgenewith the human EF1α promoter. Aftergenetargeting, theFVIIIgenewas 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 thegene-correctediPSCscould generate functionally activeFVIIIprotein from the insertedFVIIIgene. This is the first demonstration that theFVIIIlocusis a suitable site for integration of the normalFVIIIgeneand can restoreFVIIIexpressionby the EF1α promoter in endothelial cells differentiated from thehemophiliaApatient-derivedgene-correctediPSCs.ope