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 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. Hemophilia A is a genetic disorder that causes bleeding due to a faulty gene on the X chromosome. Researchers　explored a new approach using stem cells and gene editing to improve treatment. They used induced pluripotent stem cells, which can become any cell type, from HA patients. By editing these cells with CRISPR/Cas9, they inserted a modified version of the faulty gene into a safe spot in the DNA. This study involved lab experiments and tests on mice to see if the edited cells could produce the necessary protein, Factor VIII, more effectively. The modified FVIII showed better activity and stability than the regular version. Mice treated with these cells had improved blood clotting and survival rates after injury. The researchers concluded that this method could be a promising step toward better treatments for HA. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.