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Variable nonsense suppression in Hemophilia A
chromogenic assay using not only the well established RTA geneticin and gentamicin, but also other RTA such as PTC, RTC13 and RTC14. RTA treatment resulted in a sig- nificant dose-dependent increase in FVIII:C in only five of the 12 variants (Table 2 and Figure 5). The strongest
readthrough response was that of p.W274X, in which a 3.7-fold increase in activity was obtained after treatment with 100 μg gentamicin/mL. Smaller increases in FVIII:C activity were measured at a lower dose of gentamicin (50 μg/mL) or geneticin (50 and 100 μg/mL). Increases in
A
B
C
Figure 5. Readthrough agents (RTA) increase FVIII coagulant activity in the supernatants of transfected Chinese hamster ovary (CHO) cells. (A) Fold increase of FVIII:C (relative to untreated controls) in the supernatants of cultured CHO cells transiently transfected with variants harboring a QX premature termination codons (PTC) (see legend to Figure 1). (B) Fold increase of FVIII:C in the supernatants of cultured CHO cells transiently transfected with variants harboring a WX PTC (see leg- end to Figure 1). (C) Fold increase of FVIII:C in the supernatants of cultured CHO cells transiently transfected with variants harboring a RX PTC (see legend to Figure 1). CT: untreated controls; GN50: 50 μg geneticin/mL; GN100: 100 μg geneticin/mL; GT50: 50 μg gentamicin/mL; GT100: 100 μg gentamicin/mL; PTC: PTC124 10 μM; RTC13 and RTC14: 10 μM. *P<0.05; **P<0.01; ***P<0.001. (N=3).
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