Exons 45–55 Skipping Using Antisense Oligonucleotides in Immortalized Human DMD Muscle Cells

Abstract

Antisense oligonucleotides (AOs) have demonstrated high potential as a therapy for treating genetic diseases like Duchene muscular dystrophy (DMD). As a synthetic nucleic acid, AOs can bind to a targeted messenger RNA (mRNA) and regulate splicing. AO-mediated exon skipping transforms out-of-frame mutations as seen in DMD into in-frame transcripts. This exon skipping approach results in the production of a shortened but still functional protein product as seen in the milder counterpart, Becker muscular dystrophy (BMD). Many potential AO drugs have advanced from laboratory experimentation to clinical trials with an increasing interest in this area. An accurate and efficient method for testing AO drug candidates in vitro, before implementation in clinical trials, is crucial to ensure proper assessment of efficacy. The type of cell model used to examine AO drugs in vitro establishes the foundation of the screening process and can significantly impact the results. Previous cell models used to screen for potential AO drug candidates, such as primary muscle cell lines, have limited proliferative and differentiation capacity, and express insufficient amounts of dystrophin. Recently developed immortalized DMD muscle cell lines effectively addressed this challenge allowing for the accurate measurement of exon-skipping efficacy and dystrophin protein production. This chapter presents a procedure used to assess DMD exons 45–55 skipping efficiency and dystrophin protein production in immortalized DMD patient-derived muscle cells. Exons 45–55 skipping in the DMD gene is potentially applicable to 47% of patients. In addition, naturally occurring exons 45–55 in-frame deletion mutation is associated with an asymptomatic or remarkably mild phenotype as compared to shorter in-frame deletions within this region. As such, exons 45–55 skipping is a promising therapeutic approach to treat a wider group of DMD patients. The method presented here allows for improved examination of potential AO drugs before implementation in clinical trials for DMD.