Functional consequences of Exon Skipping in LGMD 2C

We are requesting funds to support development and execution of the CK assay in LGMD 2C cells. Young et al. generated a gene-edited model of Duchenne Muscular Dystrophy using induced pluripotent stem cells. This goal of their study was to document that removing a large internal portion of the dystrophin gene could generate a stable mini-dystrophin useful for improving Duchenne Muscular Dystrophy. In doing this they established a method by which DMD cells released CK after exposure to osmotic shock conditions. This CK release is similar to what occurs in humans with muscular dystrophy. We propose to optimize the CK release assay for multiple different LGMD 2C mutations in order to demonstrate the efficacy of exon skipping. To this end, we will also generate a gene edited version of Mini-gamma by deleting exons 4, 5, and 7 from an individual with a frame disrupting deletion in exon 6. By permanently deleting exons 4, 5, and 7 using gene editing, we can generate a cell line that permanently expresses Mini-Gamma. This will be useful as a control for testing ASOs and providing proof-of-principle that gene editing can work for LGMD 2C.