Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral sequence reconstruction.

BACKGROUND

Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua.

OBJECTIVES

The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application.

METHODS

Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice.

RESULTS

Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua.

CONCLUSION

Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.

Informations de copyright

Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of competing interests H.C.B., G.D., S.N.G., C.B.D., H.T.S., K.A.K., and C.W.C. are inventors on patents and patent applications describing the ancestral FIX technology filed by Expression Therapeutics, Emory University, Children’s Healthcare of Atlanta, and Georgia Institute of Technology. C.B.D., H.T.S., and H.C.B. are inventors on 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, Inc, and own equity in the company. H.C.B., G.D., and S.N.G. are employees of Expression Therapeutics, Inc and own equity in the company. Expression Therapeutics, Inc has obtained licenses for FIX-An96, liver codon-optimized FIX, and synthetic liver-directed promoter intellectual property. K.C.C., P.C.S., and G.M.B. declare no conflicts of interest. The terms of these arrangements have been reviewed and approved by Emory University in accordance with its conflict of interest policies.