Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction

Abstract Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody–based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96–Padua as compared with AAV2/8-human FIX–Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.

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114. A Novel Adeno-Associated Virus Serotype (AAV)-8 Capsid Mutant Improves Human Coagulation Factor IX Expression In Vivo

Molecular Therapy ◽

10.1016/s1525-0016(16)34449-5 ◽

2013 ◽

Vol 21 ◽

pp. S47

Keyword(s):

Coagulation Factor ◽

Factor Ix ◽

Adeno Associated Virus ◽

Coagulation Factor Ix ◽

Virus Serotype

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Molecular coevolution of coagulation factor VIII and von Willebrand factor

Blood Advances ◽

10.1182/bloodadvances.2020002971 ◽

2021 ◽

Vol 5 (3) ◽

pp. 812-822

Author(s):

Philip M. Zakas ◽

Christopher W. Coyle ◽

Anja Brehm ◽

Marion Bayer ◽

Barbara Solecka-Witulska ◽

...

Keyword(s):

Molecular Evolution ◽

Factor Viii ◽

Von Willebrand Factor ◽

Coagulation Factor ◽

Ancestral Sequence ◽

Coagulation Factor Viii ◽

Ancestral Sequence Reconstruction ◽

Sequence Reconstruction ◽

Von Willebrand ◽

Willebrand Factor

Abstract Ancestral sequence reconstruction provides a unique platform for investigating the molecular evolution of single gene products and recently has shown success in engineering advanced biological therapeutics. To date, the coevolution of proteins within complexes and protein–protein interactions is mostly investigated in silico via proteomics and/or within single-celled systems. Herein, ancestral sequence reconstruction is used to investigate the molecular evolution of 2 proteins linked not only by stabilizing association in circulation but also by their independent roles within the primary and secondary hemostatic systems of mammals. Using sequence analysis and biochemical characterization of recombinant ancestral von Willebrand factor (VWF) and coagulation factor VIII (FVIII), we investigated the evolution of the essential macromolecular FVIII/VWF complex. Our data support the hypothesis that these coagulation proteins coevolved throughout mammalian diversification, maintaining strong binding affinities while modulating independent and distinct hemostatic activities in diverse lineages.

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Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽

10.1182/blood.v64.6.1220.bloodjournal6461220 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1220-1227

Author(s):

D Menache ◽

HE Behre ◽

CL Orthner ◽

H Nunez ◽

HD Anderson ◽

...

Keyword(s):

Animal Models ◽

Specific Activity ◽

Rabbit Model ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Clotting Factors ◽

Coagulation Factor Ix

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

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Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.11.5804 ◽

1997 ◽

Vol 94 (11) ◽

pp. 5804-5809 ◽

Cited By ~ 351

Author(s):

R. W. Herzog ◽

J. N. Hagstrom ◽

S.-H. Kung ◽

S. J. Tai ◽

J. M. Wilson ◽

...

Keyword(s):

Gene Transfer ◽

Blood Coagulation ◽

Human Blood ◽

Intramuscular Injection ◽

Coagulation Factor ◽

Factor Ix ◽

Stable Gene ◽

Adeno Associated Virus ◽

Coagulation Factor Ix ◽

Human Blood Coagulation

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Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽

10.1182/blood.v64.6.1220.1220 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1220-1227 ◽

Cited By ~ 47

Author(s):

D Menache ◽

HE Behre ◽

CL Orthner ◽

H Nunez ◽

HD Anderson ◽

...

Keyword(s):

Animal Models ◽

Specific Activity ◽

Rabbit Model ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Clotting Factors ◽

Coagulation Factor Ix

Abstract Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

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DEVELOPMENT OF A COAGULATION FACTOR X CONCENTRATE AS A BY-PRODUCT OF COAGULATION FACTOR IX PRODUCTION

10.1055/s-0038-1643919 ◽

1987 ◽

Author(s):

Shirley I Miekka ◽

David B Clark ◽

Doris Menache

Keyword(s):

Native Species ◽

Specific Activity ◽

Coagulation Factor ◽

Coagulation Factors ◽

Pilot Scale ◽

Factor Ix ◽

Red Cross ◽

Factor X ◽

Coagulation Factor Ix ◽

The Usa

The American Red Cross is developing a Coagu.lation Factor X (FX) concentrate to provide a safer alternative for replacement therapy in Factor X deficient patients, who can experience thromboembolic complications with current treatments. Based on a survey of hemophilia treatment centers, we estimate the frequency of the homozygous disorder to be approximately 1/150th that of hemophilia A, or about 65 patients in the USA. We have devised a method for producing FX as a by-product of our Coagulation Factor IX concentrate (FIX). The method starts with adsorption of cryoprecipitate supernatant plasma with DEAE-Sephadex resin followed by elution of Vitamin K-dependent coagulation factors. This material is adsorbed to sulfated dejctran resin and Factors II and X are eluted by increasing the salt concentration. At 0.45 M NaCl, FII elutes quickly while FX is retarded and can be recovered essentially free of FIX by collecting the slower eluting material. FIX is then recovered at still higher ionic strength. The pooled FX is concentrated, diafiltered and treated to inactivate viruses. Approximately 30% of plasma FX was recovered in pilot scale experiments (600 liters plasma). Specific activity was > 51 FX units / mg protein corresponding to a purity of around 50% and 3000-fold purification over plasma. The ratios of Factors "X : II : IX : Protein C were 1.0 : <0.03 : <0.03 : 0.2. The major contaminant, conprising nearly 50% of the protein, was found to be inter-alpha trypsin inhibitor (IaI), a serine protease inhibitor whose function in plasma has not yet been determined. This inhibitor is also present in the DEAE-Sephadex eluate and. in the FIX concentrate. However, Western blot and HPLC analyses have shown that IaI is present in two different forms.In FX it behaves as expected for the IaI monomer (Mr = 160 kDa), while in the DEAE-eluate and in FIX it exists also in r higher molecular weight form (≥400 kDa) corresponding either to aggregates, complexes or larger native species not previously described. The nature of the possible interaction of Ial w.vdi these coagulation factors is unknown and is currently boinn evaluated.

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