FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis.
Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.
Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chung-Yang
CY
Kao
Chia-Ni
CN
Lin
I-Shing
IS
Yu
Mi-Hua
MH
Tao
Hua-Lin
HL
Wu
Guey-Yueh
GY
Shi
Yung-Li
YL
Yang
Jau-Tsuen
JT
Kao
Shu-Wha
SW
Lin
D000818:Animals; D002712:Chlorides; D003029:Coagulants / Q000008:administration & dosage* / Q000378:metabolism / Q000633:toxicity; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005164:Factor IX / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D005290:Ferric Compounds; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D002836:Hemophilia B / Q000097:blood / Q000235:genetics / Q000628:therapy*; D006487:Hemostasis / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007262:Infusions, Intravenous; D017078:Laser-Doppler Flowmetry; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D009154:Mutation; D011994:Recombinant Proteins / Q000008:administration & dosage; D018570:Risk Assessment; D013916:Thrombelastography; D013997:Time Factors; D020246:Venous Thrombosis / Q000097:blood / Q000139:chemically induced / Q000235:genetics / Q000517:prevention & control*
D002712:Chlorides; D003029:Coagulants; D005290:Ferric Compounds; D011994:Recombinant Proteins; D005164:Factor IX; C024555:ferric chloride