Inhibitor development in mice treated with plasmids carrying WT-BDD-FVIII and mutated BDD-FVIII variants. Groups of mice were injected hydrodynamically with plasmids encoding mutated BDD-FVIII variants and WT BDD-FVIII, respectively. Second challenges were performed via hydrodynamic injection in all groups on day 86. (A) FVIII-specific IgG levels were analyzed by ELISA. (B) Anti-FVIII inhibitor titers were measured using Bethesda assay. Arrows indicate the time of FVIII plasmid challenges. The comparison of inhibitor titers between different treatment groups at days 100 and 107 are shown in the right panel of each figure. WT BDD-FVIII: N = 16; N41Q: N = 7; N239Q: N = 6; N1810Q: N = 8; N2118Q: N = 6; untreated: N = 4. Experiments for each group were repeated at least 3 times. Data are presented as averages from repeated experiments, with error bars indicating standard deviation. The P values were calculated using 2-way analysis of variance among all plasmid treated groups. Untreated mice were used as negative controls.