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Presentation Details
| Determination of REGV131-LNP1265–derived human factor IX activity using one-stage clotting and chromogenic substrate assays: Results from a comparative field study Karoline A.Meagher1, Robert J.Durso1, KehDih Lai1, Dan Chalothorn1, Natalie Niepoth1, Shane McCarthy1, Sara Hamon1, David E.Gutstein1, Meagan P.O’Brien1, Stefan Tiefenbacher2, William Pickering2, Mary Robinson3, Michael E.Burczynski1. 1Regeneron Pharmaceuticals, Inc., Tarrytown, NJ, USA.2Colorado Coagulation, Laboratory Corporation of America Holdings®, Englewood, CO, USA.3Colorado Coagulation, Laboratory Corporation of America Holdings® (at the time of the study), Englewood, CO, USA |
Abstract
Background: REGV131-LNP1265 is a CRISPR-based targeted gene insertion method using adeno-associated virus and lipid nanoparticles to deliver the F9 gene and CRISPR elements, respectively. REGV131-LNP1265 is being developed with the aim to permanently restore the body’s ability to independently produce wild-type human factor IX (hFIX) in people with hemophilia B. REGV131-LNP1265 demonstrated robust and durable hFIX expression in preclinical models. Monitoring hFIX activity after administration of REGV131-LNP1265 will be critical to assess the efficacy and durability of the F9 gene insertion. However, variability in measured hFIX activity between different assays has been reported and as such, the FDA recommends in vitro studies to compare the performance of one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs) (U.S. Department of Health and Human Services, FDA, 2020). Objectives: To evaluate the activity of REGV131-LNP1265 F9 gene insertion-derived wild-type hFIX across multiple assay formats, using different reagents and methods. Methods: hFIX activity was analyzed using commercially available hFIX (Enzyme Research Laboratories) diluted in hFIX-deficient immunodepleted human plasma (Precision BioLogic). Gene insertion samples were comprised of 10% mouse plasma from mice treated with preclinical grade REGV131-LNP1265, diluted with 90% hFIX-deficient immunodepleted human plasma to meet the minimum volume requirements needed by the laboratory. Dilution series of 12 concentrations (0–15 µg/mL) for hFIX and 10 concentrations for mouse-derived gene insertion samples (0–2 µg/mL) were evaluated in the present study. hFIX activity was assessed by OSA using Actin FSL, an ellagic acid-based trigger (Siemens Healthcare), and two FXIa-triggered CSAs, Rox FIX (Rossix AB) and BIOPHEN™ FIX (HYPHEN BioMed), all performed at Colorado Coagulation, a clinical laboratory. Correlation at each concentration was analyzed using the Spearman correlation coefficient. Results: Plasma-derived hFIX activity was very highly correlated between the OSA and CSAs (CSA [Rox FIX] vs OSA: ρ=0.9965, p<0.001; CSA vs CSA: ρ=1, p<0.001; CSA [BIOPHEN™ FIX] vs OSA: ρ=0.9965, p<0.001; Figure 1). Near-perfect correlation between the OSA and CSAs was demonstrated for mouse-derived gene insertion samples (CSA [Rox FIX] vs OSA: ρ=0.997, p<0.001; CSA vs CSA: ρ=0.997, p<0.001; CSA [BIOPHEN FIX™] vs OSA: ρ=1, p<0.001; Figure 2). Conclusions: Results showed that FIX activities measured in OSA and CSAs were correlated when used to analyze both hFIX and mouse-derived gene insertion samples, suggesting that OSAs and CSAs are suitable for analyzing hFIX activity after REGV131-LNP1265 administration. REGV131-LNP1265 holds promise as a novel treatment for people with hemophilia B and is being evaluated in an ongoing clinical trial (NCT06379789).
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.