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Variable nonsense suppression in Hemophilia A
Figure 3. Gentamicin increased FVIII biosynthesis in some transfected Chinese hamster ovary (CHO) cells. Representative images of the immunofluorescence detection of FVIII in transiently transfected CHO cells stained with antibodies against either FVIII heavy chain or FVIII light chain (both in green). Gentamicin treatment (GT50: 50 μg/mL) increased FVIII labeling using the anti-FVIII light chain antibody in CHO cells transiently transfected with variants harboring the p.W274X, p.Q462X, p.Q1764X, p.W2015X and p.W2131X premature termination codons.
the controls (CT), these two drugs, each at 100 μg/mL, sig- nificantly increased the human FVIII:Ag levels of the vari- ants p.W274X (CT=1.55 vs. 24.6% with gentamicin), p.Q462X (CT=3.65 vs. 18.75% with gentamicin), p.Q1764X (CT=1.6 vs. 13.45% with geneticin), p.W2015X (CT=0.5 vs. 15.9% with geneticin) and p.W2131X (CT=2.85 vs. 13% with geneticin). In the p.R1960X vari- ants, FVIII:Ag levels increased significantly only in cells treated with 100 μg geneticin/mL (CT=1.1 vs. 9.55%). Smaller, non-significant, increases were observed for the p.Q1715X, p.R1715X, p.W1726X, p.R18822x, p.R2071X and p.R2228X variants.
Immunodetection of FVIII after readthrough agent treatment
To establish the mechanism by which the previously used RTA increased FVIII production, protein levels were analyzed in transfected CHO cells by immunofluores- cence using antibodies generated against either heavy (N-terminal) or light (C-terminal) chains (Online Supplementary Table S1). Before RTA treatment, light chain staining was detected only in cells transfected with the
WT F8BDD variant (Figure 3). However, after treatment of the transfectants with 50 μg gentamicin/mL, staining for light chain was visible in cells transfected with variants p.W274X, p.Q462X, p.Q1764X, p.R1960X, p.W2015X, and p.W2131X (Figure 3). FVIII production in cell lysates was also analyzed, using an anti-FVIII antibody raised against FVIII heavy chain (Nt). Before RTA treatment, truncated FVIII accumulated intracellularly in cells trans- fected with the different constructs harboring PTC, which is, in addition to a faulty degradation, one of the possible consequences of the inability of FVIII to fold properly.23 In contrast, treatment with some of the RTA reduced the amount of intracellular accumulation (Figure 4 and Online Supplementary Figure S1). In the p.Q462X, the highest reduction in FVIII accumulation was observed with geneticin, at the two tested concentrations. The p.Q1764X and p.W2015X variants were also responsive to geneticin, but the highest reduction in intracellular accumulation of FVIII was achieved with RTC13. The p.R1822X showed a reduction in intracellular accumulation only with the high- er concentration of gentamicin tested. Other variants also showed a qualitative reduction (p.Q1705X for geneticin
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