Figure 1.

Responses of FVIII and FIX nonsense mutations to readthrough drugs. (A) Schematic representation of the BDD-FVIII, BDD-Gluc and FIX-Gluc fusion protein constructs. The mature Gaussia luciferase was joined with BDD-FVIII or FIX at the C terminus, separated by a glycine-rich linker. PTC were introduced into BDD-FVIII or FIX by site-directed mutagenesis. (B) Gluc fusion did not significantly affect the secretion and cofactor activity of BDD-FVIII. Recombinant FVIII (rFVIII) antigen and activity levels in the cultured media of HEK293 cells stably expressing the BDD-Gluc fusion were compared with HEK293 cells stably expressing the BDD-FVIII. Results are reported as mean ± standard deviation and are representative of at least three independent experiments. (C) (D) HEK293 cells stably expressing FVIII BDD-Gluc fusions (C) or FIX-Gluc fusions (D) of the indicated mutations were treated with candidate readthrough drugs at the indicated concentrations (μM). Secreted luciferase activities of drug-treated mutant fusion proteins were compared with activities of the wild-type (WT) fusion protein (relative luciferase activities: 12,008 ± 1,052 for FVIII BDD-Gluc and 626,610 ± 70,829 for FIX-Gluc) and plotted as percentages of WT levels. Background luciferase activity values of DMSO-treated cells (Online Supplementary Figure S2) were subtracted. Results are reported as mean ± standard deviation from three independent experiments.