Abstract
Adeno-associated virus (AAV)-based gene replacement therapies in Duchenne muscular dystrophy (DMD) aim to restore dystrophin function via the introduction of micro- or mini-dystrophins. We report dystrophin and mini-dystrophin concentrations generated by immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) in skeletal muscle biopsies from ambulatory participants with DMD in a phase 1b study of fordadistrogene movaparvovec, an AAV9-based gene replacement construct. The assay performed robustly for 26 months, as demonstrated by limited variability in calibration standards for peptides LLQV (dystrophin and mini-dystrophin) and LEMP (mini-dystrophin only), quality control samples consisting of spiked mini-dystrophin in DMD skeletal muscle lysate, as well as unspiked, pooled, non-dystrophic skeletal muscle lysate (normal pool). Average values for LLQV in the normal pool tested as part of clinical sample and long-term stability runs were similar to validated values. Biopsy samples showed minor or absent LLQV and absent LEMP signals pre-treatment with fordadistrogene movaparvovec infusion, but signals substantially increased at Days 60 and 360, on average. There was strong concordance in LEMP and LLQV expression change between Days 60 and 360 (R(2) = 0.91; p < 0.001). IA-LC-MS/MS enables reproducible, stable, and reliable quantification of dystrophin/mini-dystrophin following fordadistrogene movaparvovec infusion. ClinicalTrials.gov identifier: NCT03362502.