P.200 Automated quantification of dystrophin immunofluorescence in human and mouse muscle sections

Abstract

The large subsarcolemmal protein dystrophin plays a key role in maintaining sarcolemmal integrity in muscle cells. A large variety of mutations in the <i>DMD</i> gene can lead to markedly reduced dystrophin expression and functionality, resulting in dystrophinopathies that cause progressive muscle deterioration. Several gene therapies are in preclinical and clinical development with the goal of restoring dystrophin expression and function. Efforts to evaluate and optimize these potential therapies rely on sensitive, unbiased, and reliable methodologies for quantifying sarcolemmal dystrophin. To meet a growing need for dystrophin quantification methods that fit these criteria, we have developed and validated an automated approach for analyzing dystrophin expression by immunofluorescence. The analysis method provides detailed quantification of dystrophin signal intensity and the percent dystrophin-positive coverage at the sarcolemma of each muscle fiber in whole muscle sections stained for dystrophin and a sarcolemmal marker. Objectivity and high throughput are maintained by automatically calculating thresholds for dystrophin and the sarcolemmal marker based on appropriate negative controls and applying them consistently to all samples. Here we present validation 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 capsids carrying identical U7snRNAs for skipping the duplicated exon 2, both capsids showed robust dystrophin induction in mouse muscles (45-76% dystrophin-positive fibers), but this quantification approach was able to detect significant differences between them. 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.