Dystrophin is a large cytoskeletal protein that plays a critical role in maintaining sarcolemmal integrity in muscle cells. Duchenne and Becker muscular dystrophies are caused by a variety of mutations in the dystrophin gene that markedly reduce dystrophin expression or produce less functional isoforms. Several gene therapy approaches are in preclinical development and clinical trials with the goal of restoring functional dystrophin expression in patients with dystrophinopathies. The ability to evaluate and compare the effects of various dystrophin mutations and gene therapies that aim to correct them depends on precise, sensitive, and reproducible approaches for quantifying dystrophin expression at the sarcolemma.
We have developed an automated and unbiased approach that meets the above criteria for quantifying dystrophin expression in immunofluorescence images of whole-tissue muscle sections stained for dystrophin along with a sarcolemmal marker. Dystrophin expression in each muscle fiber is quantified by the intensity of the dystrophin signal and the proportion of dystrophin-positive coverage at the sarcolemma. To ensure objectivity and efficiency, thresholds for dystrophin-positive and laminin-positive signal are derived empirically from appropriate controls and applied consistently to all samples.
Here we validate the sensitivity and reproducibility of this method in a set of 15 samples from patients with dystrophinopathies, and demonstrate its suitability for quantifying dystrophin in a mouse model of Dmd exon 2 duplication (Dup2) after treatment with two different AAV vectors delivering U7snRNAs developed for skipping exon 2 to restore dystrophin expression. Both vectors showed robust dystrophin induction in mouse muscle tissues, ranging between 45-76% dystrophin-positive fibers in skeletal muscle and 94-98% dystrophin-positive myocytes in the heart. These results confirm the viability and sensitivity of this dystrophin quantification approach in preclinical and clinical studies aiming to assess a wide range of dystrophin expression levels in both mouse and human muscle sections.