Catalytic domain modification and viral gene delivery of activated factor VII confers hemostasis at reduced expression levels and vector doses in vivo

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVII...

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Bibliographic Details
Published in:Blood 2011-04, Vol.117 (15), p.3974-3982
Main Authors: Margaritis, Paris, Roy, Elise, Faella, Armida, Downey, Harre D., Ivanciu, Lacramioara, Pavani, Giulia, Zhou, Shangzhen, Bunte, Ralph M., High, Katherine A.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Catalytic Domain - genetics
Cell Line
Dependovirus - genetics
Disease Models, Animal
Factor VIIa - chemistry
Factor VIIa - genetics
Gene Expression Regulation
Gene Therapy
Gene Transfer Techniques
Genetic Therapy - methods
Genetic Therapy - mortality
Hematologic and hematopoietic diseases
Hemophilia A - blood
Hemophilia A - genetics
Hemophilia A - therapy
Hemostasis - physiology
Humans
Infectious diseases
Medical sciences
Mice
Mice, Inbred C57BL
Mice, Transgenic
Phenotype
Plasmids - genetics
Protein Structure, Tertiary
Thrombosis and Hemostasis
Viral diseases
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Summary:Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.
ISSN:0006-4971
1528-0020
DOI:10.1182/blood-2010-09-309732