Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development.

Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.