Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity

Thrombosis Research
Eloïse PérotYesim Dargaud

Abstract

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). 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. The in-vitro 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 in-vitro 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 ...Continue Reading

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Citations

Sep 19, 2015·Critical Reviews in Biotechnology·Jennifer DumontRashmi Kshirsagar
Nov 14, 2015·Biotechnology Letters·Robson Luis Ferraz do AmaralKamilla Swiech
Jun 21, 2019·JCI Insight·Benjamin J Samelson-JonesValder R Arruda
Nov 2, 2020·Biotechnology Letters·Aline Sousa BomfimElisa Maria Sousa Russo

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