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Author Notes:

Christopher B. Doering, Aflac Cancer and Blood Disorders Center, Emory University, 2015 Uppergate Drive NE Rm 450, Atlanta, GA 30322. Email: cdoerin@emory.edu

H.T.S., E.A.G., and C.B.D. conceived the project. P.L., H.T.S., and C.B.D. designed the experiments and analyzed the data; K.A.K., C.W.C., C.E.R., A.F., J.M.S., E.T.P., G.D., F.S., R.M.S., A.P., and M.C. performed experiments and analyzed the data; K.A.K. and C.B.D. drafted the manuscript; and K.A.K., P.L., E.A.G., H.T.S., and C.B.D. edited the manuscript.

This work was supported by grants HL137128 (H.T.S., C.B.D.) and U54 HL141981 (P.L.) from the National Heart, Lung and Blood Institute, National Institutes of Health (NIH); grant R01AR069137 (E.A.G.) from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH; and grant RGP0041 (E.A.G.) from the Human Frontier Science Program.

C.E.R., H.T.S., E.A.G., and C.B.D. are inventors on a patent application describing the ancestral FIX technology filed by Emory University, Children’s Healthcare of Atlanta, and Georgia Institute of Technology. H.T.S. and C.B.D. are inventors on a patent for liver-directed codon-optimization and promoter technology filed by Emory University and Children’s Healthcare of Atlanta. H.T.S. and C.B.D. are cofounders of Expression Therapeutics and own equity in the company. Expression Therapeutics has obtained licenses for the ancestral FIX, liver codon optimized FIX, and synthetic liver-directed promoter intellectual property. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies. The remaining authors declare no competing financial interests.

Subject:

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Hematology
  • HIGH-LEVEL EXPRESSION
  • FACTOR-VIII
  • GENE-THERAPY
  • GENERATION
  • VECTORS

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

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Journal Title:

BLOOD ADVANCES

Volume:

Volume 5, Number 17

Publisher:

, Pages 3333-3343

Type of Work:

Article | Final Publisher PDF

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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© 2021 by The American Society of Hematology

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