In vivo efficacy of hFIX variants. (A) hFIX variant expression over time in groups of C57Bl/6 mice after hydrodynamic minicircle gene transfer to the liver at a dose of 7.5 μg/mouse for FIX-T (n = 5), FIX-ITV (n = 7), and FIX-WT (n = 5). (B) aPTT-based 1-stage assay in mouse plasma from groups of FVIII-KO mice treated with hFIX-WT or variant vectors at doses of 7.5 or 25 μg/mouse. (C) TAT complex levels in FVIII-KO mice after hFIX variant minicircle gene transfer at 25 μg/mouse. (D-E) Blood loss assay after tail dissection at 1.5-mm (D) or 3-mm (E) diameter in FVIII-KO mice receiving minicircle doses of 25 μg/mouse. Blood loss was measured by optic density measurement at 492 nm of hemoglobin lost over a period of 10 minutes. For all experiments, hFIX expression levels were determined by FIX ELISA. *P < .05 and **P < .01 according to ANOVA with Dunnett test for multiple comparisons with the corresponding FIX-WT group.