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

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 (EF1?)-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 EF1? 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 EF1? promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.

SUBMITTER: Sung JJ

PROVIDER: S-EPMC6470126 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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

Sung Jin Jea JJ Park Chul-Yong CY Leem Joong Woo JW Cho Myung Soo MS Kim Dong-Wook DW

Experimental & molecular medicine 20190417 4

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 ...[more]

PMID: 30996250

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Project description:Essentials Platelet-specific FVIII gene therapy is effective in hemophilia A mice even with inhibitors. The impact of platelet adherence via VWF/GPIb? binding on platelet gene therapy was investigated. GPIb? does not significantly affect platelet gene therapy of hemophilia A with inhibitors. Platelet gene therapy induces immune tolerance in hemophilia A mice with pre-existing immunity. SUMMARY: Background We have previously demonstrated that von Willebrand factor (VWF) is essential in platelet-specific FVIII (2bF8) gene therapy of hemophilia A (HA) with inhibitory antibodies (inhibitors). At the site of injury, platelet adherence is initiated by VWF binding to the platelet GPIb complex. Objective To investigate the impact of GPIb? on platelet gene therapy of HA with inhibitors. Methods Platelet-FVIII expression was introduced by 2bF8 lentivirus (2bF8LV) transduction of hematopoietic stem cells (HSCs) from GPIb?null (Ibnull ) mice or rhF8-primed FVIIInull (F8null ) mice followed by transplantation into lethally irradiated rhF8-primed F8null recipients. Animals were analyzed by flow cytometry, FVIII assays and the tail bleeding test. Results After transplantation, 99% of platelets were derived from donors. The macrothrombocytopenia phenotype was maintained in F8null mice that received 2bF8LV-transduced Ibnull HSCs (2bF8-Ibnull /F8null ). The platelet-FVIII expression level in 2bF8-Ibnull /F8null recipients was similar to that obtained from F8null mice that received 2bF8LV-transduced F8null HSCs (2bF8-F8null /F8null ). The tail bleeding test showed that the remaining hemoglobin level in the 2bF8-Ibnull /F8null group was significantly higher than in the F8null control group, but there was no significant difference between the 2bF8-Ibnull /F8null and 2bF8-F8null /F8null groups. The half-life of inhibitor disappearance time was comparable between the 2bF8-Ibnull /F8null and 2bF8-F8null /F8null groups. The rhF8 re-challenge did not elicit a memory immune response once inhibitor titers dropped to undetectable levels after 2bF8 gene therapy. Conclusion GPIb? does not significantly impact platelet gene therapy of HA with inhibitors. 2bF8 gene therapy restores hemostasis and promotes immune tolerance in HA mice with pre-existing immunity.

Dataset Information

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

Publications

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

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