Testing of hFIX variants in the presence of FVIII-inhibitory Abs. (A) Schematic illustration of the experiment. Anti-hFVIII Abs were induced by nonviral gene transfer of 50 μg/mouse of pSL1180-HCR/hAAT-FVIIIΔB vector by hydrodynamic liver-directed delivery into FVIII-KO mice. After confirming the absence of FVIII antigen and activity and the presence of high titers of anti-hFVIII Abs, mice received hFIX-T, hFIX-ITV, or hFIX-WT minicircle gene transfer (25 μg/mouse). (B) Blood loss assay after tail dissection at 1.5-mm diameter in FVIII-KO mice with inhibitory Abs. Blood loss was measured by optic density measurement at 492 nm of hemoglobin lost over a period of 10 minutes. hFIX expression levels were determined by FIX ELISA. **P < .01 according to ANOVA with Dunnett test for multiple comparisons with the corresponding FIX-WT group.