Background:
Adeno associated virus (AAV)–mediated microdystrophin delivery provides a targeted intervention to stabilize muscle fibers and slow the progression of Duchenne muscular dystrophy. Blood-accessible biomarkers offer promise as complementing exploratory endpoints to evaluate pharmaco-gene therapy efficacy for microdystrophin in preclinical and clinical trials.
Objective:
In this study, we report the identification of serum biomarkers in wild-type mice and in untreated and treated mdx mice using label-free mass spectrometry, and their relationship to microdystrophin restoration in treated cohorts.
Methods:
Serum and muscle extract samples were obtained from 9 mdx mice treated with AAV vector-μDys and 4 mdx mice treated with saline. As controls, samples from 3 age-matched wild-type mice were also included. Treated mdx mice were evaluated at two post-treatment time points (4 weeks and 16 weeks), to assess short and mid-term molecular responses. For serum protein quantification, a label-free workflow was employed where proteins were enzymatically digested into peptides and analyzed using the Evosep–TIMS-TOF system. For dystrophin quantification, samples were spiked with equal amounts of SILAC myotubes containing heavy 13C6-Arg and 13C6, 15N2-Lys prior to digestion, and the peptides were run on an Orbitrap mass spectrometer. Microdystrophin abundance was determined by Parallel Reaction Monitoring method, calculated as the ratio of light to heavy arginine peptides. Raw mass spectrometry data were processed using Proteome Discoverer v2.2 searched against the Mus musculus UniProt protein database using the Sequest HT algorithm.
Results and Conclusion:
LC-MS/MS analysis identified 46 proteins up-regulated and 50 down-regulated in serum from wild-type mice. In treated mdx mice, 44 proteins were up-regulated and 72 were down-regulated compared to untreated mdx mice. Several candidate biomarkers were found to show significant correlation with restored microdystrophin, including muscle-centric (Titin, Myomesin-3 , Creatine kinase M- type) , glycolytic Enzymes ( Fructose-bisphosphate Aldolase A and Beta-Enolase) as well as extracellular proteins (Gelsolin, Lumican and Adiponectin).If validated, these biomarkers, on integration with gene-therapy trials, could reduce reliance on repeated biopsies and enhance clinical monitoring, ultimately accelerating translation of therapeutic advances into meaningful patient outcomes.