Field Study and Correlative Studies of Factor IX Variant FIX-R338L in Participants Treated with Fidanacogene Elaparvovec

Abstract

Background: Fidanacogene elaparvovec, an adeno-associated virus-based gene therapy vector expressing the high-activity factor IX (FIX) variant FIX-R338L, is in development for hemophilia B. One-stage clotting (OS) assays and chromogenic substrate (CS) assays are commonly used to measure FIX-R338L variant activity. Data from ongoing trials suggest FIX activity varies between different OS and CS assays.

Material and methods: To better understand FIX-R338L activity in clinical samples, an international multisite field study was conducted across a central laboratory and 18 local laboratories, using standard protocols, reagents, and instrumentation, with individual participant samples from a phase 1/2a study of fidanacogene elaparvovec.

Results: Unlike the wild-type FIX control, FIX-R338L activity was higher with the OS silica-based assay versus OS ellagic acid-based and CS assays. Variation in FIX activity was greater at the lowest activity levels. Activated FIX (FIXa) in plasma could result in higher OS assay activity or increased thrombin generation, which could overestimate FIX activity. However, FIXa was not detected in the participant samples, indicating that it was not contributing to the OS assay differences. Since individuals on gene therapy may receive exogenous replacement FIX products, replacement products were spiked into patient plasma samples to target a therapeutic concentration. Exogenous FIX was additive to endogenous FIX-R338L, with no interference from FIX-R338L.

Conclusion: These results demonstrate FIX-R338L activity can be measured with OS and CS assays in clinical laboratories and provide insight into assay variability when measuring FIX with endogenously produced FIX-R338L. The findings may help establish best practices for measuring FIX-R338L activity (Clinicaltrials.gov identifier: NCT02484092).

Fig. 1

Measurement of FIX activity following addition of exogenous FIX to plasma samples from fidanacogene elaparvovec–treated patients. Exogenous FIX ( SHL, nonacog alfa; EHL, eftrenonacog alfa; plasma-derived) was spiked into FIX-deficient plasma, participant plasma (participants 1–3), and pooled normal wild-type plasma to reach a target therapeutic concentration of 0.50 IU/mL. Buffer was added to the participant plasmas or pooled normal plasma as a control to measure the baseline values. Plasma samples were analyzed in triplicate, and FIX activity values are shown as mean ± standard deviation IU/mL. CS, chromogenic substrate; EHL, extended half-life; FIX, factor IX; OS, one-stage; PD, plasma-derived; SHL, standard half-life.