Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs
Abstract Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal pro...
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| Format: | Article |
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Nature Publishing Group
2025-01-01
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| Series: | Experimental and Molecular Medicine |
| Online Access: | https://doi.org/10.1038/s12276-024-01375-z |
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| author | Do-Hun Kim Sang-Hwi Choi Jin Jea Sung Sieun Kim Hanui Yi Sanghyun Park Chan Wook Park Young Woo Oh Jungil Lee Dae-Sung Kim Jong-Hoon Kim Chul-Yong Park Dong-Wook Kim |
| author_facet | Do-Hun Kim Sang-Hwi Choi Jin Jea Sung Sieun Kim Hanui Yi Sanghyun Park Chan Wook Park Young Woo Oh Jungil Lee Dae-Sung Kim Jong-Hoon Kim Chul-Yong Park Dong-Wook Kim |
| author_sort | Do-Hun Kim |
| collection | DOAJ |
| description | Abstract Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA. |
| format | Article |
| id | doaj-art-05cf92cab96948a5bbc63e8ba01b0c0d |
| institution | Kabale University |
| issn | 2092-6413 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Publishing Group |
| record_format | Article |
| series | Experimental and Molecular Medicine |
| spelling | doaj-art-05cf92cab96948a5bbc63e8ba01b0c0d2025-02-09T12:14:14ZengNature Publishing GroupExperimental and Molecular Medicine2092-64132025-01-0157118419210.1038/s12276-024-01375-zLong-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCsDo-Hun Kim0Sang-Hwi Choi1Jin Jea Sung2Sieun Kim3Hanui Yi4Sanghyun Park5Chan Wook Park6Young Woo Oh7Jungil Lee8Dae-Sung Kim9Jong-Hoon Kim10Chul-Yong Park11Dong-Wook Kim12Department of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineDepartment of Biotechnology, College of Life Sciences and Biotechnology, Korea UniversityDepartment of Biotechnology, College of Life Sciences and Biotechnology, Korea UniversityDepartment of Physiology, Yonsei University College of MedicineDepartment of Physiology, Yonsei University College of MedicineAbstract Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.https://doi.org/10.1038/s12276-024-01375-z |
| spellingShingle | Do-Hun Kim Sang-Hwi Choi Jin Jea Sung Sieun Kim Hanui Yi Sanghyun Park Chan Wook Park Young Woo Oh Jungil Lee Dae-Sung Kim Jong-Hoon Kim Chul-Yong Park Dong-Wook Kim Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs Experimental and Molecular Medicine |
| title | Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs |
| title_full | Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs |
| title_fullStr | Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs |
| title_full_unstemmed | Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs |
| title_short | Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs |
| title_sort | long term correction of hemophilia a via integration of a functionally enhanced fviii gene into the aavs1 locus by nickase in patient derived ipscs |
| url | https://doi.org/10.1038/s12276-024-01375-z |
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