Quantitative Evaluation of Exon Skipping in Immortalized Muscle Cells In Vitro

Abstract

Exon skipping through the use of antisense oligonucleotides (AOs) is currently one of the most promising approaches for treating Duchenne muscular dystrophy (DMD). While we now have a number of AO drug candidates in clinical trials, we are still faced with issues of poor or controversial efficacy in some of these drugs. This is the case with eteplirsen, an exon 51-skipping AO that is the first and only FDA-approved drug for DMD to date. Effective procedures must, therefore, be set up for the in vitro screening of potential AOs for DMD treatment. Here, we describe one such procedure using immortalized DMD patient-derived muscle cells. Aside from allowing for the quantitative evaluation of candidate AOs based on their exon skipping efficiency and dystrophin protein rescue levels, these immortalized cells are stable, pure, easy to grow, and not subject to confounding by senescence-related issues. This procedure enables a more reliable screening of AOs prior to their entry in clinical trials and greatly facilitates the search for more efficacious candidate exon skipping AOs for DMD treatment.