A novel humanized FIX/FX hemophilia A mouse model for preclinical evaluation of FVIIIa-mimetic bispecific antibodies

Abstract The use of nonfactor products such as emicizumab, a bispecific antibody that mimics the cofactor function of activated factor VIII (FVIIIa) by bridging FIXa and FX, has significantly reduced bleeding episodes in people with hemophilia A (HA). However, emicizumab high species specificity in its FVIIIa-mimetic cofactor activity has limited in vivo functional evaluation to an acquired HA nonhuman primate model or congenital HA mouse model with infusion of human FIX (hFIX) and hFX proteins. To overcome the limitations of existing models, we generated hFIX/hFX double-knockin congenital HA model (humanized HA [HuHA]) mice. HuHA mice were established by the replacement of mouse FIX/FX genes with their human homologs and by the disruption of mouse FVIII. In HuHA mice, functional hFIX and hFX are constitutively expressed at levels equivalent to, or higher than, human plasma concentrations. Thrombin generation assays demonstrated dose dependently improved peak thrombin generation by both recombinant hFVIII (rhFVIII) and emicizumab. These HuHA mice were found to express sufficient levels of hFIX and hFX to support functional evaluation of rhFVIII and emicizumab activities. Furthermore, the tail-clip assay suggested potential hemostatic effects of both therapeutic agents in vivo, although the findings should be interpreted with caution given the limitations of the model. This novel mouse model, in which functional hFIX and hFX are constitutively expressed on an FVIII-deficient background, may provide a new platform for the preclinical evaluation of human-specific therapeutics for HA.