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Differences in wild-type– and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies

Foley, JH; Shehu, E; Riddell, A; Gray, E; Goodale, A; Yu, IM; Verhoef, D; ... Nathwani, AC; + view all (2023) Differences in wild-type– and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies. Blood Advances , 7 (3) pp. 458-467. 10.1182/bloodadvances.2022007435. Green open access

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Abstract

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338LFIX activity in patients after gene therapy.

Type: Article
Title: Differences in wild-type– and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1182/bloodadvances.2022007435
Publisher version: https://doi.org/10.1182/bloodadvances.2022007435
Language: English
Additional information: © 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.
Keywords: Humans, Factor IX, Hemophilia B, Blood Coagulation Tests, Cysteine Endopeptidases
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology
URI: https://discovery.ucl.ac.uk/id/eprint/10166554
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