FVIII constructs. (A) Single-chain FVIII construct. A 4.5-kb B domain-deleted canine FVIII cDNA was engineered such that residue S743 is in-frame with Q1630 while deleting most of the B domain. The cDNA is driven by synthetically derived 368-bp promoter/enhancer from insulin-like growth factor-binding protein, fused to 175 bp of a chimeric intron (IGBP/enh/intron, total size 543 bp). The FVIII sequence is followed by 263 bp of SV40 poly A signal and the entire cassette is flanked at both ends by the 145-bp ITRs. The total size of the expression cassette is 5.596 kb. (B) FVIII heavy-chain construct. The 2.5-kb cDNA contains the canine FVIII Kozak, initiator codon (bent arrow), signal peptide (57 bp), A1 and A2 domains followed by a stop codon after residue S743. The cDNA is driven by a 695-bp thyroxine-binding globulin gene promoter/enhancer fused to a 175-bp intron (TBG/enh/intron, total size 856 bp), has an SV40 poly A (263 bp) and 145-bp flanking ITRs. The size of the entire expression cassette is 4 kb. (C) FVIII light-chain construct. The 2.4-kb cDNA contains the canine FVIII Kozak sequence, initiator codon (bent arrow), and the signal peptide (57 bp) in-frame with the C-terminus of the B domain at residue G1555 through residue I1690, followed by A3, C1, and C2 domains. The light-chain construct is also driven by the 856 bp of TBG/enh/intron promoter, has the 263-bp SV40 poly A signal, and the 145-bp ITRs. The size of this expression cassette is 3.9 kb.