WT BDD-FVIII plasmid used for N to Q mutagenesis and the resulting FVIII variant constructs used for in vivo gene therapy. The WT BDD-FVIII plasmid construct is shown at the top. The 4 N-glycosylation sites, N41, N239, N1810, and N2118, in the A1, A3, and C1 domains are separately represented by red pins. N to Q mutagenesis was performed on the WT BDD-FVIII backbone to eliminate glycosylation on each of the 4 sites represented by the stop symbol. BDD, B domain deleted; bpA, bovine growth hormone polyadenylation site; hAAT, human α1-antitrypsin promoter; HCR, hepatic control region; hFIXintA, human factor IX intron 1; WT, wild type.