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Principal Investigator: Valder R. Arruda
Organization: CHILDREN'S HOSP OF PHILADELPHIA
Fiscal Year: 2020
Award: $495,230
Funding agency: National Heart Lung and Blood Institute
Project 4 - Abstract
Novel approaches to enhance the biologic activity of FVIII and FIX are critical endeavors with potential for
improving protein- and gene-based therapy for hemophilia. We have identified novel variants and new
strategies that have a positive effect on the biological activity of FVIII and FIX. Promising preclinical studies on
efficacy and safety in small and large animal models provide the basis for translational studies using these
proteins; in particular the FIX-Padua (R338L) variant is already in early phase gene therapy clinical trials for
hemophilia B. The main goal of this application is to understand the biochemistry of the variants and in the
case of new variants, evaluate their potential in hemophilic models. We seek to use this approach to reveal
mechanistic aspects of intrinsic Xase function and thus provide evidence-based insights into the potential of
these variants in the treatment of human disease. The central tenets of our experimental strategies are based
on the fact that even modest enhancements of procoagulant function, as judged from a biochemical
perspective, can have a very significant impact on the success of therapeutic approaches. In aim 1 we
hypothesize that a FVIII derivative with a modified PACE/furin cleavage site results in its differential processing
by thrombin and/or FXa yielding a more stable/active cofactor. A second class of variant is based on a
molecule lacking the B-domain and acid region 3 with increased specific activity. These findings pose the
question whether vWF engagement, while an important determinant of FVIII circulating half-life, limits
activation by FXa, and if by relieving this constraint we can enhance available FVIIIa levels to promote clot
formation. In aim 2 we will characterize the FIX-Padua variant for which the molecular basis for its enhanced
function is uncertain. We will use biochemical and biophysical approaches to identify the mechanism of the
hyperfunctional molecule. Emerging structural information derived from FXa aptamers that prevent its binding
to FVa and the known structure of snake FVa-FX implicate a region of Xa not previously considered central in
Va binding. We hypothesize that the analogous interaction between FIXa and FVIIIa may be enhanced by
modifications in the heparin-binding exosite. If successful, our goal is to combine FIX-Padua with modifications
from the FIX-loop 90 to generate FIX variants with enhanced biologic properties. In aim 3 we will pursue
studies with variants of FIX that upon activation yield products with increasing zymogen-like character, are
long-lived because of their resistance to protease inhibition but may be functionally rescued upon their
assembly into the intrinsic Xase. Biochemical studies testing these ideas will be followed by approaches to
assess if such variants, and those combined on the FIX-Padua background, have therapeutic value for
hemophilia B. Together these approaches will provide new mechanistic insights into intrinsic Xase regulation
and activity as well as provide a platform for the development of new therapeutic agents for bleeding disorders.
Terms: <Acidic Region><Acids><Activated Blood Coagulation Factor IX><Activated Blood Coagulation Factor VIII><Activated Factor IX><Activated Factor VIII><Affect><Animal Model><Animal Models and Related Studies><Autoprothrombin III><Binding><Biochemical><Biochemistry><Biological><Biological Chemistry><Biological Function><Biological Process><Biology><Bleeding><Blood Coagulation Disorders><Blood Coagulation Factor><Blood Coagulation Factor X><Blood Plasma><Cell Body><Cells><Chemistry><Christmas Disease><Clinical><Clotting><Coagulation><Coagulation Disorder><Coagulation Factor IXa><Coagulation Factor VIII, Procoagulant Component><Coagulation Factor VIIIa><Coagulation Factor X><Coagulation Factors><Coagulation Process><Coagulopathy><DNA Therapy><DXS1253E><Data><Development><Disease><Disorder><Enzyme Gene><Enzyme Precursors><Enzymes><Esteroproteases><F8 gene><F8 protein><F8B><F8C><F9 gene><FVIII><Factor IX Deficiency><Factor IXa><Factor VIII Deficiency><Factor VIIIF8B><Factor VIIIa><Factor X><Gene Proteins><Gene Transfer Clinical><Gene therapy trial><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Goals><HEMA gene><Half-Life><Hemophilia><Hemophilia A><Hemophilia B><Hemorrhage><Hemostasis><Hemostatic Agents><Hemostatic function><Hemostatics><Heparin Binding><Hyperactive behavior><Hyperactivity><Hyperkinesia><Hyperkinesis><Hyperkinetic Movements><In Vitro><Kinetics><Laboratories><Lead><Link><Modeling><Modification><Molecular><Molecular Interaction><Motor Hyperactivity><Mutation><Pathway interactions><Pb element><Peptidases><Peptide Hydrolases><Phase><Physiologic><Physiological><Physiology><Plasma><Plasma Serum><Position><Positioning Attribute><Proenzymes><Property><Protease Gene><Proteases><Protein Gene Products><Proteinases><Proteins><Proteolytic Enzymes><Prower factor><Regulation><Reporting><Research><Research Resources><Resistance><Resources><Reticuloendothelial System, Serum, Plasma><Ristocetin Cofactor><Ristocetin-Willebrand Factor><Role><Safety><Series><Site><Snakes><Structure><Stuart Factor><Stuart-Prower Factor><Testing><Therapeutic><Therapeutic Agents><Thrombase><Thrombin><Thrombin-Activated Factor VIII><Thrombosis><Variant><Variation><Work><Xase><Xase complex><Zymogens><aptamer><base><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><bleeding disorder><blood loss><cancer procoagulant><cancer procoagulant A><clotting disorder><clotting factor><cofactor><cost><developmental><evidence base><fXase><factor X activator><factor X procoagulant><factor Xase><fibrinogenase><gain of function><gene therapy><gene therapy clinical trial><gene transfer trial><gene-based therapy><genetic therapy><genome mutation><genomic therapy><heavy metal Pb><heavy metal lead><heparin bound><human disease><improved><in vivo><in vivo Model><innovate><innovation><innovative><insight><interest><intrinsic fXase><male><model of animal><model organism><mouse model><murine model><neoplasm procoagulant A><neoplastic procoagulant><new approaches><new drug treatments><new drugs><new therapeutics><new therapy><next generation><next generation therapeutics><novel><novel approaches><novel drug treatments><novel drugs><novel strategies><novel strategy><novel therapeutics><novel therapy><pathway><patient population><pre-clinical study><preclinical study><prevent><preventing><procoagulant Blood-coagulation factor VIIIa><resistant><social role><success><thrombotic disease><thrombotic disorder><translational study><von Willebrand Factor><von Willebrand Protein>