Increased gene therapy efficacy through the use of extended half-life clotting factors

ABSTRACT: The development of more effective gene therapy strategies for hemophilia B remains essential because results of multiple clinical trials show significant interpatient variability in clinical outcomes, with a fraction of patients (up to 13%) even returning to prophylaxis. Another major challenge in current adeno-associated viral (AAV)-based gene therapy for hemophilia B pertains to dose-dependent vector-related immune responses, requiring immune suppression. We investigated whether combining gene therapy with transgenes encoding extended half-life (EHL) coagulation factors could enhance therapeutic efficacy. AAV vectors encoding a hyperactive factor IX variant R338L (ie, FIX-Padua), genetically fused to a mutant human albumin (FIX-R338L-ALB[QMP]), were administered to both wild-type and hemophilia B mice. These chimeric transgenes were codon-optimized and driven by a hepatocyte-specific promoter. The AAV-FIX-R338L-ALB(QMP) vector demonstrated significantly improved efficacy compared with the control AAV-FIX-R338L vector, resulting in a sustained fourfold and threefold increase in FIX antigen levels and activity, respectively. These increases were consistent with durable correction of the bleeding phenotype. Importantly, immunogenicity or liver toxicity was not increased because no anti-FIX antibodies, alanine transaminase elevations, or liver immune infiltrations were detected after treatment. Furthermore, immune tolerance was achieved in most hemophilia B mice treated with AAV-FIX-R338L-ALB(QMP), even after active immunization with FIX protein and adjuvant, which was found to be mediated by regulatory T cells. In addition, no increase in thrombogenic risk was observed, indicated by stable D-dimer levels. These findings support the potential of FIX-R338L-ALB(QMP) fusion constructs encoding EHL FIX as a promising next-generation gene therapy for hemophilia B.