Hemophilia A mice were injected hydrodynamically with plasmids encoding mutated FVIII variants and WT BDD-FVIII, respectively. Second challenges were performed via hydrodynamic injection in all groups on Day 86. (A) From left to right, the wild-type (WT)-BDD-FVIII plasmids used for N to Q mutagenesis, the resulting FVIII variant constructs used for in vivo gene therapy, and relative FVIII antibody response. (B) FVIII-specific IgG levels were analyzed by ELISA following first and second plasmid challenges. Anti-FVIII immune responses were significantly reduced in mice treated with the plasmid carrying FVIII N2118Q variant.