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. 2025 Jul;31(4):799-806.
doi: 10.1111/hae.70053. Epub 2025 Jun 18.

Evaluation of One-Stage Assays for the Monitoring of Recombinant Human Factor IX Padua Activity After Etranacogene Dezaparvovec Gene Therapy

Jan Astermark  1 , Wolfgang Miesbach  2 , Michiel Coppens  3   4 , Sander Gielen  5 , Jaap Twisk  5 , Ricardo Dolmetsch  5 , Stephanie Verweij  5 , Paul E Monahan  6 , Bruce M Ewenstein  6 , Silpa Nuthalapati  6 , Nick Galante  6 , Guy Young  7 ; HOPE‐B study investigators
Affiliations

Evaluation of One-Stage Assays for the Monitoring of Recombinant Human Factor IX Padua Activity After Etranacogene Dezaparvovec Gene Therapy

Jan Astermark et al. Haemophilia. 2025 Jul.

Abstract

Introduction: Accurate and reproducible measures of factor activity are required to guide clinical decision-making following gene therapy for haemophilia B (HB). Highly significant discrepancies have been observed in measurements of various factor IX (FIX) concentrates that carry molecular modifications to extend their half-life, arguing for the need for careful analysis of new HB treatment modalities with respect to FIX assay performance.

Aim: To further characterise variability in FIX activity measured using different one-stage assays (OSAs) and chromogenic assays (CAs) in patients with HB receiving gene therapy utilising the FIX Padua variant and to assess whether assay differences were due to the FIX-Padua variant.

Methods: FIX activity was assessed centrally (OSA and CA) and locally (OSA only) using plasma samples collected from a phase 2b and phase 3 study of etranacogene dezaparvovec and in an in vitro study of wild-type (wt) recombinant human FIX (rhFIX) and rhFIX-Padua.

Results: Lower CA than OSA FIX activity for plasma samples from the phase 3 trial was observed (CA:OSA ratio: 0.408 [±0.049]-0.547 [±0.062]). Local OSA:central OSA FIX activity ratios were 0.789 (±0.314)-1.021 (±0.159). Local OSA:central OSA FIX activity ratios across methods and/or reagents were 0.81 (±0.02)-1.28 (±0.04) for rhFIX-wt-spiked samples and 0.67 (±0.02)-1.13 (±0.09) for rhFIX-Padua-spiked samples.

Conclusion: FIX activity differences between central and local OSAs were modest; similar differences were observed in vitro with rhFIX-wt versus rhFIX-Padua. Commonly available OSAs can be used to monitor patients post-etranacogene dezaparvovec administration; we recommend using the same assay platform throughout the post-treatment period.

Keywords: chromogenic assay; factor IX; gene therapy; haemophilia B; one‐stage assay.

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Conflict of interest statement

J.A.: Grant/research support from SOBI, Bayer, Takeda/Shire and CSL Behring; honorarium as member of advisory boards and speaker for BioMarin, Pfizer, Sparks, uniQure, CSL Behring, SOBI, Sanofi, Novo Nordisk, Bayer, Roche, Takeda/Shire and Octapharma. W.M.: Grant/research support from Bayer, Biotest, CSL Behring, LFB, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire; consultation/speaker fees from Bayer, Biomarin, Biotest, CSL Behring, Chugai, LFB, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Takeda/Shire; and consultation fees from Bayer, Biomarin, Biotest, CSL Behring, Chugai, Freeline, LFB, Novo Nordisk, Octapharma, Pfizer, Regeneron, Roche, Sanofi, Sobi, Takeda/Shire, and uniQure. M.C.: has received financial support for research from Anthos, Bayer, CSL Behring, Novo Nordisk, and Hoffmann‐La Roche; and honoraria for lecturing or consultancy from Alexion, CSL Behring, Daiichi Sankyo, Sanofi, Spark Therapeutics, Octapharma, Pfizer, Sobi, and Viatris. All funds were received by his institution. Non‐financial conflicts of interest: member of the gene therapy working group of the European Association for Haemophilia and Allied Disorders (EAHAD); and member of the European Reference Network (ERN) EuroBloodNet. S.G., J.T., R.D., S.V.: Employees of uniQure biopharma B.V. at the time of this research; RD is now an employee of Tempuro Bio. P.E.M., B.M.E., S.N., N.G.: Employees of CSL Behring. G.Y.: has received consulting fees from ASC Biotherapeutics, BioMarin, Centessa, CSL Behring, Genentech/Roche, Hema Biologics/LFB, Novo Nordisk, Octapharma, Pfizer, Sanofi Genzyme, Spark, and Takeda, and funds for research support from Sanofi.

Figures

FIGURE 1
FIGURE 1
Correlation of post‐treatment uncontaminated FIX activity levels measured by CA and OSA in the phase 3 HOPE‐B study central laboratory. CA, chromogenic assay; CI, confidence interval; FIX, factor IX; OSA, one‐stage assay.
FIGURE 2
FIGURE 2
Correlation of FIX activity levels measured by central and local laboratory OSAs in the phase 3 HOPE‐B study. aPTT, activated partial thromboplastin time; CI, confidence interval; FIX, factor IX; OSA, one‐stage assay.
FIGURE 3
FIGURE 3
Box/whisker plot of FIX activity ratios of local:central OSA ratios by reagent in the phase 3 HOPE‐B study. aPTT, activated partial thromboplastin time; FIX, factor IX; lab, laboratory.
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