Protein engineering and pharmacological approaches to develop novel treatment strategies for coagulation disorders

Haemophilia A (HA) and B (HB) as well as factor VII (FVII) or X (FX) deficiencies are well-characterized haemorragic genetic disorders, whose etiology, even at the molecular level, has been extensively described. Treatment options for patients affected by these diseases are currently available and mainly consist of replacement therapy with the missing factor. However, there are still important limitations and research in the field is boosted towards the implementation of current therapeutic options or the discovery of innovative procedures. In the present work we aimed at providing consistent examples of novel treatment strategies for bleeding disorders, via protein engineering or pharmacological approaches. The different systems here proposed intend to (i) provide selection criteria for an individually-targeted therapy of patients carrying specific mutations (personalized medicine) and to (ii) ameliorate pharmacokinetic features of therapeutic proteins in order to reduce the burden of treatment. In the first part of the thesis, we studied the molecular event of ribosome readthrough in the framework of nonsense mutations causing HB or FVII deficiency. We took advantage of an in vitro platform for the expression of factor IX nonsense variants. We were able to show that specific mRNA and protein constraints limit the number of mutations that could be productively suppressed by readthrough induction. In particular, the potential re-insertion of the original residue in place of the premature stop codon strongly favors a productive functional output. Moreover, this correction approach is predicted to be efficient for those mutations that fall in a protein domain removed during biosynthesis (i.e. pre-peptide). We also studied ribosome readthrough in order to elucidate the molecular mechanisms underlying phenotypes’ severity in FVII deficiency, by two paradigmatic homozygous nonsense mutations. The data obtained suggest that the appreciable rescue for one mutation was driven by reinsertion of the wild-type residue, whereas the minimal function seen for the other one was explained by missense changes permitting FVII secretion and function. Together, these data may lay the foundation for the rational selection of patients who could benefit from treatment with readthrough-inducing drugs. In the second part of the thesis, we exploited protein engineering to extend coagulation factors half-life. Among the several known strategies, we choose the fusion with human serum albumin (HSA), a protein with an extremely long half-life due to a neonatal Fc receptor (FcRn)-mediated recycling mechanism. In the context of by-passing therapy for haemophilias, we aimed at providing next-generation recombinant activated FVII-albumin (rFVIIa-HSA) fusion proteins by taking advantage of albumin engineered variants with improved pharmacokinetic features. The rFVIIa-HSA variants showed a by-passing activity in hemophilic plasma comparable to that of rFVIIa alone, both in vitro and in vivo. Furthermore, the fusion proteins comprising engineered albumin bind to FcRn with increased affinity compared to wild-type albumin. So, these molecules would have the potential to last remarkably longer in circulation; if translated into patients, this would eventually mean needing less frequent injections, which represents a great achievement in terms of patients’ quality of life. Finally, we proposed a fusion protein between FX and albumin, in order to fulfill the unmet need of a recombinant product for FX deficiency therapy. We were able to show for the first time that fusion with albumin is compatible with a FX-HSA chimaera endorsed of robust procoagulant activity; we have also preliminarily demonstrated that albumin actually confers to FX prolonged persistence in circulation in a mouse model. Nevertheless, the molecule here proposed could be the platform for albumin engineering aimed at further improving pharmacokinetic properties of the therapeutic protein(s).