Schematic representation of human FVIII variants designed and cloned into a SIN LV backbone. (A) Nine different human FVIII variants were designed and cloned into a LV backbone plasmid: BDD FVIII, B domain deleted human FVIII; FVIII Fugu B, BDD FVIII containing the Fugu B domain; FVIII N6, BDD FVIII containing the human N6 B domain; SQ FVIII, BDD FVIII containing a modified version of the SQ amino acid sequence SQ^m; SQ FVIII Fugu B, SQ FVIII containing the Fugu B domain between the SQ^m sequence to create the N terminal SQ^a and C terminal SQ^b sequences; and SQ FVIII N6, SQ FVIII containing the human N6 B domain. And constructs SQ FVIII (co), SQ FVIII Fugu B (co), and SQ FVIII N6 (co) are the same amino acid structure as constructs SQ FVIII, SQ FVIII Fugu B, and SQ FVIII N6, respectively, but are produced from a codon-optimized cDNA sequence. Dashes on constructs indicate asparagine (N)-linked glycosylation sites within the B domain only. (B) Schematics of SQ and modified SQ sequences; SQ^m, SQ^a, and SQ^b. The SQ sequence is a 14-amino acid bridge between the a2 and a3 domains of FVIII created by fusing Ser743 and Gln1638 in the B domain. The sequence promotes efficient intracellular cleavage by containing the 4 amino acid protease recognition site RHQR. A modified SQ sequence (SQ^m) was created containing a missense mutation from Lys1644 to Thr1644 caused by the creation of an MluI restriction enzyme site within the cDNA sequence for insertion of the Fugu and N6 B domains. SQ^a is the 11-amino acid sequence created at the N-terminal of the B domain after insertion of the N6 or Fugu B domain sequences into the SQ FVIII construct. SQ^b is the 5 amino acid sequence created at the C-terminal of the B domain after insertion of the N6 or Fugu B domain sequences into the SQ FVIII construct; this sequence retains the 4 amino acid protease recognition site. MluI restriction sites are shown underlined, and the K to T missense mutation is shown in red.