Relative human FVIII activity of FVIII constructs in vitro as determined by chromogenic assay. A total of 1 × 105 293T cells were transduced with 3 μL BDD FVIII, FVIII Fugu B, FVIII N6, SQ FVIII, SQ FVIII Fugu B, SQ FVIII N6, SQ FVIII (co), SQ FVIII Fugu B (co), or SQ FVIII N6 (co). At 48 hours, cell media was changed for 500 μL serum-free media. After a further 24 hours, incubation media was collected from all wells and assayed for factor VIII expression using (A) chromogenic-based assay, (B) a one-stage clotting assay to measure factor VIII cofactor activity, and (C) ELISA to measure FVIII antigen. Results were normalized on virus copy number per cell determined by quantitative PCR. Data are mean plus or minus SD; n = 5. *Statistical analyses were performed using general linear model based on 2-way analysis of variance with individual pairwise comparisons performed using Bonferroni simultaneous tests (Minitab 15 software). Results show a highly significant increase for SQ FVIII (co), SQ FVIII Fugu B (co), and SQ FVIII N6 (co) compared with their non–codon-optimized equivalents SQ FVIII, SQ FVIII Fugu B, and SQ FVIII N6, respectively (P < .0001). In addition, results for codon-optimized vectors also show a significant increase for SQ FVIII N6 (co) compared with SQ FVIII (co) (P < .0001) and a significant increase for SQ FVIII Fugu B (co) compared with both SQ FVIII (co) and SQ FVIII N6 (co) (P < .0001). (D) Transgene mRNA expression was quantified by quantitative RT-PCR, normalized to glyceraldehyde 3-phosphate dehydrogenase and compared with expression levels in cells transduced with BDD FVIII using the ΔΔ Ct method.
