Generation of a novel factor IX with augmented clotting activities in vitro and in vivo.
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.
Lin
C N
CN
Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Kao
C Y
CY
Miao
C H
CH
Hamaguchi
N
N
Wu
H L
HL
Shi
G Y
GY
Liu
Y L
YL
High
K A
KA
Lin
S W
SW
D000818:Animals; D001777:Blood Coagulation; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005164:Factor IX / Q000737:chemistry*; D015944:Factor VIIIa / Q000235:genetics; D005170:Factor X / Q000235:genetics; D014644:Genetic Variation; D002836:Hemophilia B / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D010314:Partial Thromboplastin Time; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance
D011994:Recombinant Proteins; D015944:Factor VIIIa; D005164:Factor IX; D005170:Factor X