Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity. Issue 5 (May 2015)

• Record Type: Journal Article
• Authors: Perot, Eloïse; Enjolras, Nathalie; Le Quellec, Sandra; Indalecio, Alice; Girard, Jonathan; Negrier, Claude; Dargaud, Yesim
• Is Part Of: Thrombosis research. Volume 135:Issue 5(2015)
• Page Start: 1017
• Page End: 1024
• Publication Date: 2015-05
• Subjects: Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2015.02.034
• Languages: English
• ISSNs: 0049-3848

Abstract

<abstract abstract-type="author" id="ab0005"> <title id="st0005">Abstract</title> <sec> <title id="st0010">Introduction</title> <p id="sp0005">Hemophilia B is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). The main treatment for hemophilia B is replacement therapy using FIX concentrates. Prophylactic treatment in severe hemophilia B is very effective but is limited by cost issues. Production of a recombinant FIX (rFIX) with enhanced clotting activity, offering the possibility of fewer infusions and fewer costs with similar efficacy, is one of the current challenges for hemophilia B treatment. The present study focused on an important amino acid sequence known to be involved in the interaction of activated FIX (FIXa) with its cofactor, activated factor VIII (FVIIIa).</p> </sec> <sec> <title id="st0015">Materials and Methods</title> <p id="sp0010">Using site-directed mutagenesis of glutamate E410 (c240, chymotrypsin numbering), four recombinant FIX-E410 (E410H, A, L and N) mutants were developed and produced by the human hepatoma cell line Huh-7.</p> </sec> <sec> <title id="st0020">Results</title> <p id="sp0015">The <italic>in-vitro</italic> clotting activity of mutant FIX molecules was 3 to 5-fold higher than wild-type recombinant FIX (FIX-WT). FIX-E410H compound showed the highest <italic>in-vitro</italic> procoagulant activity. Enhanced specific activity was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In hemophilia B mice, we observed significantly higher <italic>in-vivo</italic> clotting activity and thrombin generating capacity with FIX-E410H compared to FIX-WT. We demonstrated that increased procoagulant activity of FIX-E410H was mainly explained by 2.5- fold enhanced affinity of the mutant for human FVIIIa.</p> </sec> <sec> <title id="st5010">Conclusion</title> <p id="sp0020">We have engineered and characterized four improved FIX proteins with enhanced <italic>in-vitro</italic> and <italic>in-vivo</italic> activity. Future studies are required to evaluate the immunogenicity of FIX-E410.</p> </sec> </abstract>