Accurate quantification of low-level dystrophin expression in muscle samples by a capillary western immunoassay


Translational Research

Poster Number: 158


Stefan Nicolau, MD, Nationwide Children's Hospital, Adrienne Bradley, Nationwide Children's Hospital, Tatyana Vetter, PhD, Nationwide Children's Hospital, Anthony Stephenson, Center for Gene Therapy, Nationwide Children's Hospital, Gabrielle Dufresne, Center for Gene Therapy, Nationwide Children's Hospital, Kevin Flanigan, MD, Nationwide Children's Hospital

The dystrophinopathies (Duchenne and Becker muscular dystrophies) encompass a spectrum of disorders caused by mutations in the DMD gene, resulting in reduced or absent dystrophin expression. The importance of accurate quantification of low-level dystrophin expression is highlighted by the observation that levels as little as 0.5% of normal may be associated with a meaningful attenuation of disease severity, as well as by the FDA approval of antisense oligonucleotides that restore approximately 1% of dystrophin expression.

We developed a capillary western immunoassay protocol for dystrophin quantification and validated its performance using mouse and healthy human donor muscle samples. Dystrophin quantification was performed on the ProteinSimple Jess instrument using 3µg of total protein and the C-terminal antibody ab154168. This assay was able to detect dystrophin levels of 0.1% of normal, and the quantification demonstrated good linearity at levels as low as 0.5% of normal. A coefficient of variation of 6.5% was observed between 11 technical replicates of a single sample processed concurrently, while the coefficient of variation was 11% when serial protein extractions and dystrophin quantifications were performed from the same muscle samples. This demonstrates the ability of the assay to reliably quantify low levels of dystrophin expression.

In subsequent studies, we employed this technique to assess dystrophin expression in a range of dystrophinopathy patient biopsies, to quantify the efficacy of gene editing in a mouse model of dystrophinopathy, and to demonstrate a response to a vectorized exon 2-skipping therapy in a patient-derived cell line carrying an exon 2 duplication. Compared to traditional western blotting, this capillary western method employs smaller amounts of tissue, requires less operator time, and offers high sensitivity for low levels of dystrophin expression.