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Updated: Jul 9, 2025
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Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
Published on: June 3, 2014
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Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral
Christopher W Coyle1, Kristopher A Knight1, Harrison C Brown2
1Molecular and Systems Pharmacology Graduate Program, Graduate Division of Biological and Biomedical Sciences, Laney Graduate School, Emory University School of Medicine, Atlanta, Georgia, USA.
Journal of Thrombosis and Haemostasis : JTH
|November 28, 2023
Summary
Researchers resurrected ancient coagulation factor IX (FIX) variants, discovering An96 with enhanced activity. They identified key substitutions for a potent FIX gene therapy candidate (ET9) for hemophilia B.
Area of Science:
- Biochemistry and Molecular Biology
- Gene Therapy
- Protein Engineering
Background:
- Laboratory resurrection of ancient coagulation factor (F) IX variants identified An96, a variant with enhanced specific activity.
- This enhanced activity is independent of and additive to the FIX-Padua variant (p.Arg384Lys).
Purpose of the Study:
- Identify specific amino acid substitutions in An96 responsible for its enhanced activity.
- Develop a humanized An96 FIX transgene for potential gene therapy applications in hemophilia B.
Main Methods:
- Employed reductionist screening, including domain swapping and scanning residue substitution, guided by one-stage FIX activity assays.
- Conducted in vitro characterization of top candidates, including recombinant protein preparation, specific activity, and enzyme kinetic analysis.
- Packaged lead candidates into adeno-associated viral (AAV) vectors for delivery to hemophilia B mouse models.
Main Results:
- Five amino acid substitutions in An96 were found sufficient to retain enhanced activity in a human FIX background.
- An additional substitution (ET9) combined with the Padua variant yielded a 5-fold increase in specific activity, resulting in 51-fold greater activity than wild-type human FIX (hFIX).
- AAV2/8-ET9 treated hemophilia B mice showed plasma FIX activities 10-fold higher than AAV2/8-hFIX-Padua treated mice.
Conclusions:
- Novel amino acid substitutions conferring enhanced FIX activity were identified using ancestral sequence reconstruction and validated in an AAV-FIX gene therapy cassette.
- The developed gene therapy candidate (ET9) demonstrated high potency in a preclinical model.
- This approach of ancestral sequence reconstruction and refinement is a promising platform for optimizing protein drugs and gene therapy candidates.
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