Secretion of wild-type factor IX upon readthrough over F9 pre-peptide nonsense mutations causing hemophilia B

Background: Nonsense mutations, which introduce premature termination codons (PTCs), are a relatively frequent cause of haemophilia. However, the pharmacological induction of ribosome readthrough over the PTC can lead to the synthesis of a full-length protein, with potential therapeutic implications. A relevant readthrough-mediated rescue of functional protein levels requires a complex interplay between favorable mRNA sequence features and the type of amino acid introduced at the PTC. In this view, nonsense mutations occurring in sequences encoding for protein regions that are poorly conserved, would represent ideal candidates for therapeutic readthrough. In particular, suppression of nonsense mutations affecting the pre-peptide regions of secreted proteins, that are intracellularly removed, would make negligible the impact of missense changes on secretion and function of the mature protein. Therefore in this study we selected three F9 nonsense mutations affecting the pre-peptide and pro-peptide regions of coagulation factor IX (FIX), which cause hemophilia B (HB): the p.G21X (TGA-T) in the variable hydrophobic core, the p.C28X (TGA-A) at the intracellular cleavage site and the p.K45X (TAG-A), affecting the pro-peptide cleavage site. Methods: Recombinant FIX (rFIX) nonsense and missense variants were transiently expressed in human embryonic kidney 293 cells. Protein levels and isoforms were evaluated by ELISA and Western Blotting, respectively. Pro-coagulant activity of rFIX was evaluated by aPTT-based assays. Results: Expression of the rFIX-28X and rFIX-45X nonsense variants did not result in appreciable secreted FIX levels. Conversely, the full-length FIX form was clearly detectable from rFIX-21X expressing cells, with estimated levels of 0.4±0.3% of rFIX-wt. The rFIX-21X also significantly responded (secretion, 4.1 ± 0.5% of wild-type; coagulant activity, 4.0 ± 0.3%) to the readthrough-inducer geneticin, while rescue of the p.C28X and p.K45X was prevented by constraints of adjacent cleavage sites. Strikingly, for the p.G21X mutation, the resulting specific coagulant activity (0.96 ± 0.11) was compatible with normal function, thus suggesting secretion of FIX with wild-type features upon readthrough and removal of pre-peptide. Expression of the predicted readthrough-deriving missense variants (Gly21Trp/Cys/Arg) revealed a preserved specific activity (ranging from 0.84 to 0.98), thus supporting our observation. Conclusion/summary: Overall, our data indicate that suppression of nonsense mutations in the pre-peptide core preserves mature protein features and specifically identified the p.G21X nonsense mutation as candidate for treatment with readthrough-inducing agents. More generally, this rational approach could be extended to nonsense mutations in protein regions removed during processing and moderately conserved (activation peptides, B-domain), in order to select responsive mutations, and thus patients, to be challenged with readthrough-inducing drugs.