Abstract
Hemophilia A (HA), an X-linked recessive congenital bleeding disorder, affects 80%-85% of patients with hemophilia. Nearly half of severe cases of hemophilia are caused by a 0.6-Mb genomic inversion (Inv22) that disrupts F8. Although viral-based gene therapy has shown therapeutic effects for hemophilia B (HB), this promising approach is not applicable for HA at the present stage; this limitation is mainly due to the large size of F8 cDNA, which far exceeds the adeno-associated virus (AAV) packaging capacity. We previously reported an in situ genetic correction of Inv22 in HA patient-specific induced pluripotent stem cells (HA-iPSCs) by using TALENs. We also investigated an alternative strategy for targeted gene addition, in which cDNA of the B-domain deleted F8 (BDDF8) was targeted at the rDNA locus of HA-iPSCs using TALENickases to restore FVIII function. Mesenchymal stem cells (MSCs) have low immunogenicity and can secrete FVIII under physiological conditions; in this study, MSCs were differentiated from F8-corrected iPSCs, BDDF8-iPSCs, and HA-iPSCs. Differentiated MSCs were characterized, and FVIII expression efficacy in MSCs was verified in vitro. The three types of MSCs were introduced into HA mice via intravenous injection. Long-term engraftment with restoration of FVIII function and phenotypic rescue was observed in HA mice transplanted with F8-corrected iMSCs and BDDF8-iMSCs. Our findings suggest that ex vivo gene therapy using iMSCs derived from F8-modified iPSCs can be feasible, effective, and promising for the clinical translation of therapeutic gene editing of HA and other genetic birth defects, particularly those that involve large sequence variants.