Evaluation of drug-induced readthrough over F9 nonsense mutations. (A) FIX activity levels in medium from cells expressing the rFIX nonsense variants upon treatment with G418, evaluated by chromogenic (red bars) and aPTT-based (blue bars) assays. Numbers above the bars report the activity/antigen ratio of the most responsive variants. Nonsense variants are indicated by amino acid numbering and grouped according to the 3 nonsense triplets (theoretical readthrough susceptibility, TGA≥TAG>TAA). The dotted line represents the selected threshold of 2%. The activity in medium from untreated cells was undetectable for all variants and the undetectable activity after treatment is indicated by asterisks. (B) Western blotting analysis on secreted (top) and intracellular (bottom) rFIX proteins transiently expressed from HEK293 cells untreated (−) or treated (+) with G418. The W240X(TGAC) and R384X stop codons, producing the highest rescue, are compared with PTCs displaying barely detectable (W240X and Y330X) or undetectable (L103X) readthrough. For the W240X PTCs, both nonsense triplets are indicated. The images are representative of at least 3 independent experiments. (C) Secreted full-length (red bars) and total (blue bars) rFIX levels after G418 treatment. Full-length rFIX levels were calculated by densitometric analysis of western blots shown in (B) and total antigen by ELISA. (D) Antigen (red bars), pro-coagulant activity (blue bars) levels, and activity/antigen ratio (specific activity, light green bars) of the most probable rFIX missense variants arising from misrecognition of TGA (R384W), TAG (W240Y), and TAA (L103Y/L103Q and Y330Q) stop codons. Readthrough over the W240X(TGAC) PTC is predicted to reintroduce the authentic amino acid (tryptophan, *W240W; Figure 1B). The dashed-line indicates the specific activity of WT rFIX. Results in (A,C-D) are reported as mean ± standard deviation from at least 3 independent experiments. Ag, antigen; M, molecular weight marker; n.d., not detectable.