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L. Martorell et al.
Figure 2. Readthrough agents (RTA) can increase FVIII antigen levels in the supernatants of some transfected Chinese hamster ovary (CHO) cells. Human FVIII:Ag levels analyzed by ELISA in the supernatants of CHO cells transiently transfected with the F8 variants, showing the increase in FVIII:Ag levels before and after RTA- treatment versus the F8 wild-type (WT) variant. BDD-FVIII: WT; CT: untreated controls; GN100: 100 μg geneticin/mL and GT100: 100 μg gentamicin/mL. *P<0.05; **P<0.01; ***P<0.001. (N=3).
FVIII immunodetection of F8BDD variants
Chinese hamster ovary lines transfected with the F8BDD vari-
ants were stained with several anti-FVIII antibodies for the immunodetection of FVIII in cell homogenates (Online Supplementary Table S1).
FVIII activity of F8BDD variants
The FVIII activity (FVIII:C) of the supernatants of cultured
fibroblasts or CHO cells transfected with F8BDD variants, treated or not with RTA, was assessed by the chromogenic method using the COAMATIC FVIII kit (Chromogenix, Werfen, Barcelona, Spain). The protocol was modified from Yatuv et al.20
Results
F8 mRNA levels after readthrough agent treatment
In the fibroblasts of HA-patients harboring nonsense mutations, F8 mRNA levels measured by quantitative real-time-polymerase chain reaction (qRT-PCR) were <60% (p.Q1636X: 46.23%±9.19; p.W1586X: 59.89%±5.55; p.R1960X: 57.09%±3,81) of those detected in control fibroblasts from healthy individuals or from the HA patients caused by the missense mutation. Treatment with the protein synthesis inhibitor cycloheximide, which also inhibits nonsense-mediated decay (NMD), restored the levels of PTC-containing transcripts to normal values, which suggested a role for NMD in our HA patients har- boring nonsense mutations (Figure 1B). We then analyzed the ability of RTA to suppress PTC and stabilize PTC-con-
taining mRNA, as reported in previous studies.21,22 Although some of the RTA increased F8 mRNA levels in the fibroblasts of HA patients, the differences were not statistically significant: p.W1586X: 59.89%±5.55 (CT) versus 86.01%±11.82 (100 μg geneticin/mL); p.Q1636X: 46.23%±9.19 (CT) versus 64.68%±12.41 (10 μM RTC13), and p.R1960X: 57.09%±3.82 (CT) versus 79.21%±5.66 (100 μg gentamicin/mL) (Figure 1B). A possible explana- tion for this could be that none of the nonsense mutations analyzed in patients' fibroblasts met the PTC rule.
In addition, we used CHO cells, which is the most com- monly used cell line for commercial production of FVIII,23 to analyze F8 mRNA levels to measure the effects of RTA. Time course qRT-PCR analysis using F8BDD-specific primers showed that the CHO cell model expressed high levels of F8 mRNA without any presence of NMD over time (Figure 1C).
Analysis of the human FVIII:Ag levels in the culture media
Although fibroblasts ectopically express F8 mRNA, they do not synthesize FVIII protein.24 For this reason, and because none of the nonsense mutations analyzed in patients' fibroblasts met the PTC rule, all protein studies were performed in the CHO cellular model. To determine the ability of the already established RTAs geneticin and gentamicin to increase FVIII production, FVIII:Ag levels in the culture supernatants of the treated CHO cells were analyzed by ELISA (Table 2 and Figure 2). Compared to
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