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.

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

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.

Interactions of 4,4'-diaminoazobenzene derivatives with telomeric G-quadruplex DNA.

The development of small molecules to stabilize the G-quadruplex structure has garnered significant attention for anticancer drug discovery. Herein, we report the synthesis of several 4,4'-diaminoazobenzene derivatives containing different substituent groups and their ability to bind and stabilize telomeric G-quadruplex DNA. Circular dichroism (CD) spectroscopy was performed to characterize the quadruplex topologies, measure stabilization effects, and evaluate their capabilities for conformational photoregulation. 4,4'-Diaminoazobenzene derivatives were found to moderately stabilize quadruplex structures but not affect conformational photoregulation. This work further develops the design and general understanding of the stabilization effects of small molecules with telomeric G-quadruplex DNA.