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Updated: Jun 12, 2026
Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice
Published on: September 30, 2021
Generation of a novel factor IX with augmented clotting activities in vitro and in vivo
1Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Background:
Hemophilia B is an X-linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX.
Objective:
In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity.
Methods:
Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo.
Results:
One variant containing three substitutions (V86A/E277A/R338A, FIX-Triple) exhibited 13-fold higher specific clotting activity and a 10-fold increased affinity for human FVIIIa compared with FIX-wild-type (FIX-WT) and was thus investigated systematically in vivo. Liver-specific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific clotting activity compared with FIX-WT. Human FIX-Triple and FIX-WT knock-in mice were generated and it was confirmed that FIX-Triple has 7-fold higher specific clotting activity than FIX-WT under normal physiological conditions. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific clotting activity of FIX-Triple than FIX-WT.
Conclusions:
Our results indicate that the FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX-Triple is a good candidate for further evaluation in protein replacement therapy as well as gene-based therapeutic strategies.
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