A strategy with chaperone-like compounds to restore expression of factor IX variants affected by frequent missense mutations causing hemophilia B

Background Amino acid substitutions caused by missense mutations, representing the most frequent gene alteration associated with human disease including coagulation factor disorders, exert the most detrimental effect by impairing protein folding and intracellular trafficking. These effects at the protein level may be counteracted by small molecules with chaperone-like activity, with a concomitant improvement of the affected intracellular processes. Although some evidence exists for missense mutations associated with disorders involving intracellular proteins, only very few attempts have been made for secreted proteins, in particular in the coagulation field. Noticeably, a correction approach with chaperone-like compounds could be of relevance for coagulation factor disorders, where even modest increases of functional protein levels could have therapeutic implications. Aims The aims of our study were to i) detail the altered intracellular processing of a panel of factor IX (FIX) missense variants causing severe type I Hemophilia B (HB), and ii) to rescue their intracellular processing, secretion and activity through chaperone-like compounds. Methods Cellular models mimicking severe HB were created by transient and stable expression of recombinant FIX (rFIX) variants in Human Embryonic Kidney (HEK) 293 cells, followed by evaluation of secreted/intracellular protein levels (ELISA, Western Blotting), intracellular trafficking (immunofluorescence studies) and activity (coagulant assays) before and after treatment of rFIX-expressing cells with chaperone-like compounds. Results As model mutation we selected the F9 p.R294Q, representing the most frequent substitution (~100 patients) associated with severe/moderate type I HB. As comparison we chose other recurrent F9 missense mutations (p.Y115C, n=9; p.Y161C, n=5; p.Y305C, n=9; p.F424L, n=3). Transient expression studies indicated that the selected mutations severely impair rFIX secretion (0.2-0.8% of wild-type), thus recapitulating findings in HB patients (antigen levels <1%). Immunofluorescence studies on stably expressing cells revealed that, at variance from wild-type rFIX, missense variants mainly co-localized in the ER and scarcely with Golgi, thus indicating impaired intracellular trafficking. This pattern was comparable to that of the rFIX-450C variant, previously demonstrated by us to cause severe type I HB and here chosen as additional control. These observations were in accordance with the very low secreted levels observed in expression studies. Screening of a panel of chaperone-like compounds identified sodium phenylbutyrate (NaPBA) as active in improving trafficking to Golgi and appreciably promoting secretion (from ~0.3% to ~1.5%) of the rFIX-294Q variant in a dose-dependent manner. Noticeably, the rFIX-294Q variant revealed a remarkable specific coagulant activity that was higher (2.0±0.1) than that of wild-type rFIX in all treatment conditions (0.5, 1.0 and 2.0 mM NaPBA). Importantly, activity after treatment was improved in terms of shortening of coagulation times (from 80±0.1 sec to 62±3 at 2 mM NaPBA), with increased levels (~3%) that, if achieved in patients, would approach the therapeutic threshold. Summary/Conclusion Altogether our data contribute to detail molecular mechanisms underlying type I HB and candidate NaPBA as a potential “personalized” option for the high number of patients affected by the frequent p.R294Q mutation. In addition, our expression platform is proposed for other missense mutations leading to severe type I Hemophilia to select those being responsive to chaperone-like compounds.