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L. Martorell et al.
FVIII:C after exposure to 100 μg geneticin/mL were also observed in the p.W2015X and p.W2131X variants (2.9- and 2.7-fold, respectively) and in the p.Q462X and p.Q1764X variants (up to 2.6- and 3.3-fold, respectively). These variants were also responsive, although to a lesser extent, to treatment with 100 μg gentamicin/mL but the increases in FVIII:C were smaller. Variants responsive to either gentamicin or geneticin showed a slight response to other RTA, particularly to PTC124 and RTC13, but these increases were not statistically significant.
In silico analysis of the premature termination codons Variable responses to RTA-induced nonsense suppres- sion related to the sequences adjacent to the PTC have been reported. We therefore analyzed the PTC type and the context effect in both the patients' fibroblasts and the 12 F8BDD variants (Tables 1 and 2, and Online Supplementary Table S2). In patients' fibroblasts respon- sive to RTA, we would expect at least a partial restoration of the F8 mRNA levels; however, none of the PTC sequence context filled the PTC rule, so we cannot expect any mRNA stabilization. In contrast, in the CHO model, we could evaluate the readthrough effect on the produc- tion of full-length protein and on the restoration of FVIII:C activity. The results suggested a TGA≥TAG hier- archy of PTC suppression sensitivity and a positive RTA readthrough effect on both FVIII:Ag levels and FVIII:C activity for sequences in which a C was located at posi- tion +4 and a T at position −1 as present in variants pW274X, pQ462X, pQ1764X and pW2015X. Furthermore, the pW2131X variant also showed respon- siveness even when a G is present at position -1 instead of a T. In contrast, variants pR1715X, pR2071X and pR2228X, even containing a TGA and a T at position –1 and a C at position +4 were unable to significantly increase FVIII:C activity. Similarly, the pR1822X variant was also unresponsive to RTA, despite the fact that it
contains a TGA and a T at position –1.
Although positions −5 (A, C>G) and +8 (G>C) were also
reported to influence readthrough, our data show that T, GandAatposition−5aswellasTorCatposition+8 promoted PTC suppression, as measured by FVIII:C lev- els; other tested combinations may have positive effects measured by FVIII:Ag levels. Whether positive effects are associated with yet other nucleotide combinations could not be determined due to the limited number of F8 vari- ants tested (Online Supplementary Table S2).
Discussion
The present study represents a comprehensive analysis of factors influencing the responsiveness to RTA of a series of nonsense mutations that are representative of those causing HA. Readthrough therapy is not expected to restore the synthesis and activity of the recoded pro- teins to curative levels in most patients harboring non- sense mutations. However, in the case of hemophilia, even a relatively small percentage of restoration may be very valuable. Thus, in patients with severe HA (<1% of FVIII:C), a slight increase in activity to 2-5% could signif- icantly improve the bleeding phenotype as reported for FIX production in severe HB patients treated with gen- tamicin.9
Our study uses a CHO-based HA model in which
NMD is absent, as cells were transfected with plasmid containing the F8 cDNA, but lacking the exon-exon junc- tion complexes (EJC) which are necessary for PTC-con- taining mRNA to be targeted by NMD, thus providing a clean tool to amplify and detect eventual readthrough activity of RTA. While cDNA models do not mimic the in vivo situation (in which NMD is present), they can be used for assessing potential readthrough effects and to identify new RTA.10,11,17,25 In practice, to show a readthrough effect in vivo or in primary cells, RTA will probably have to be combined with NMD inhibition. Nonetheless, the CHO-based HA model allowed us to demonstrate that the effectiveness of readthough therapy is mutation-specific and that in vitro assays may be useful for classifying patients that can likely benefit from RTA.
It has recently become clear that different types of endothelial cells, including liver sinusoidal endothelial cells (LSEC), constitute the main source of FVIII produc- tion in humans,24,26-29 perhaps to a greater extent than hepatocytes. This may explain why, although ectopically expressed at the mRNA level, none of the fibroblasts from HA patients analyzed showed a significant stabi- lization of F8 mRNA levels. However, as the PTC sequence context does not fill the PTC rule, we cannot draw any conclusions as to the effectiveness of the RTA used. Furthermore, since fibroblasts do not produce FVIII,24 we could not expect to detect FVIII protein.
Despite the limitations of this study, which include the relatively low number of mutations analyzed, the intrin- sic variability in the transfection efficiencies, and the low levels of FVIII:C achieved (in the low range of detection by FVIII:C assays, i.e. 0.5-3%), the observed increases in the FVIII:C activity in some mutations suggest a potential phenotypic improvement, from severe to moderate/mild HA. Thus, RTA treatment significantly rescued FVIII pro- duction in some of the F8BDD variants, as measured by FVIII:Ag levels. This increase was associated with a qual- itative reduction in intracellular accumulation of FVIII observed in the western blots, and a concomitant increase in light-chain FVIII detection by immunofluorescence. Moreover, data obtained in the FVIII:C analyses high- lighted the importance of the amino acid incorporated upon RTA treatment in the partial restoration of FVIII:C activity. In our study, after drug treatment, only variants that natively encoded tryptophan (Trp=W) or glutamine (Gln=Q) and had a cytosine at position +4, regardless of the PTC type, were able to significantly increase FVIII:C in the culture supernatants, while this activity was not increased in variants natively encoding arginine (Arg=R), even when a cytosine at position +4 was present, likely because the RTA may promote the incorporation of amino acids that do not necessarily restore FVIII function- ality.
Nonsense suppression was observed in only five of the 12 transfected F8BDD variants (p.Q462X, p.Q1764X, p.W274X, p.W2015X and p.W2131X). This apparent dis- crepancy between protein levels and restored activity can be explained by the context-dependent ability of RTA to restore the full-length recoded protein13 and by the pre- ferred aminoacyl tRNA that will recognize each PTC in RTA-treated cells.15 Accordingly, upon secretion, only full- length protein that has incorporated the native amino acid (or another one with similar chemical properties) is expected to have a greater chance of restoring maximum or partial levels of FVIII:C activity. Additionally, the phy-
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haematologica | 2020; 105(2)