Orthogonal Analysis of Dystrophin Protein and Mrna As A Surrogate Outcome for Drug Development

Abstract

Aim: 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. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: 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. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.

Safety, Tolerability, and Efficacy of Viltolarsen in Boys With Duchenne Muscular Dystrophy Amenable to Exon 53 Skipping

“…Dystrophin induction was assessed by Western blot, reverse transcription-polymerase chain reaction (RT-PCR), mass spectrometry (MS), and immunofluorescence (IF) staining using methods recently described (eMethods in Supplement 2). 2…”

“…uchenne muscular dystrophy (DMD) is an X-linked disorder affecting approximately 1 in 3500 to 5000 live male births. [1][2][3] Progressive weakness and skeletal muscle degeneration are caused by an absence of functional dystrophin protein secondary to loss-of-function variants in the DMD gene. 1,4 Patients with DMD typically exhibit dystrophin levels less than 3% of normal.…”

Exon-Skipping in Duchenne Muscular Dystrophy

“…After 4 weeks treatment period, the participants receiving placebo were randomized to viltolarsen for the remainder of the 24-week treatment period. The primary outcome was increase in dystrophin by immunoblot from baseline to 24-weeks, with secondary outcomes of dystrophin immunostaining, RT-PCR of dystrophin mRNA, and mass spectrometry quantitation of dystrophin (orthogonal approach [ 65 ]). All biopsy analyses were done blinded.…”

A Novel Method for Detecting Duchenne Muscular Dystrophy in Blood Serum of mdx Mice

“…This mouse model was chosen due to similar phenotypes experienced compared to humans with DMD. Specifically, within humans, an out-of-frame mutation in the DMD gene results in decreased production of the dystrophin protein in muscle fibers [ 9 , 10 ]; in the mdx mouse model, dystrophin is not expressed, thus making the mouse model a sufficient representation commonly used in the literature for exploring and studying DMD without the additional influence of confounding factors which may occur within humans [ 11 , 12 ].…”