Data Sheet 1_Non-viral gene therapy for hemophilia A: long-term outcomes of minicircle FVIII delivery in a mouse model.pdf

Hemophilia A, an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency, necessitates lifelong factor replacement therapy with high treatment burden. To explore a non-viral alternative, we evaluated minicircle DNA carrying the FVIII-E1984V mutation—engineered for improved stability and activity—as a gene therapy for sustained coagulation correction. Mini-circle DNA constructs (with the bacterial backbone excised) encoding either wild-type FVIII or E1984V-FVIII were delivered via hydrodynamic tail vein injection into FVIII-KO mice, a typical murine model of hemophilia A. Coagulation outcomes (aPTT, FVIII activity, tail-clip assay) and transgene persistence were monitored over 26 weeks. Minicircle DNA delivery demonstrated higher transfection efficiency in vitro and sustained coagulation improvement in vivo for at least 26 weeks, markedly exceeding the short durability of conventional FVIII infusion (days versus weeks). Furthermore, exogenous FVIII DNA and RNA persisted in hepatocytes without evidence of hepatotoxicity. These findings highlight minicircle DNA–based FVIII gene therapy as a promising strategy for hemophilia A. Future studies will focus on optimizing vector design for sustained expression, advancing toward clinical translation.