The chaperone-like compound sodium phenylbutyrate improves intracellular trafficking, secretion and coagulant activity of factor IX impaired by the frequent p.R294Q mutation

Background: Amino acid substitutions caused by missense mutations, representing the most frequent cause of human disease, exert the most detrimental effect by impairing protein folding and intracellular processing, which can be improved by small molecules exhibiting chaperone-like activity. However, despite the potential of these compounds, only very few attempts have been made in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Methods: Cellular models mimicking severe HB were created to perform expression studies and evaluation of intracellular trafficking (immunofluorescence), protein (ELISA, Western blotting), and activity (coagulant assays) levels of recombinant (r)FIX variants before and after treatment of cells with chaperone-like compounds. Results: As model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations (p.Y115C, n=9; p.Y161C, n=5; p.Y305C, n=9) associated with severe/moderate type I HB. Mutations were characterized by expression of rFIX variants in HEK293 cells and investigations at the extracellular and intracellular level. In transient expression studies, all missense mutations resulted in impaired rFIX secretion (<1% of wild-type rFIX), in agreement with coagulation phenotypes in HB patients. At the intracellular level, immunofluorescence studies 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, in line with the observed defective secretion. The chaperone-like compound sodium phenylbutyrate (NaPBA) promoted secretion (from 0.26±0.06% to 1.5±0.3%) only of the rFIX-294Q variant in a dose-dependent manner, as also indicated by the appreciable and quantitatively improved trafficking to Golgi. Importantly, the shortening of coagulation times (from 80±0.1 to 62±3 seconds) corresponded to a concurrent increase in activity levels (from 0.5±0.04% to 3.0±0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Noticeably, the rFIX-294Q variant displayed a specific coagulant activity that was higher (~2.0) than that of wild-type rFIX at all treatment conditions, a feature that magnifies the functional impact of the NaPBA-mediated rescue. Conclusions: Altogether, our data detail for the first time the pathogenic mechanisms recurrent missense mutations associated with type I HB and candidate NaPBA, iin setting with poor access to standard substitutive therapy, as affordable “personalized” therapeutic tool for patients affected by the highly frequent p.R294Q mutation