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

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.

A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis.

Dystrophinopathies are caused by mutations in the DMD gene. Out-of-frame deletions represent most mutational events in severe Duchenne muscular dystrophy (DMD), while in-frame deletions typically lead to milder Becker muscular dystrophy (BMD). Antisense oligonucleotide-mediated exon skipping converts an out-of-frame transcript to an in-frame one, inducing a truncated but partially functional dystrophin protein. The reading frame rule, however, has many exceptions. We thus sought to simulate clinical outcomes of exon-skipping therapies for DMD exons from clinical data of exon skip-equivalent in-frame deletions, in which the expressed quasi-dystrophins are comparable to those resulting from exon-skipping therapies. We identified a total of 1298 unique patients with exon skip-equivalent mutations in patient registries and the existing literature. We classified them into skip-equivalent deletions of each exon and statistically compared the ratio of DMD/BMD and asymptomatic individuals across the DMD gene. Our analysis identified that five exons are associated with significantly milder phenotypes than all other exons when corresponding exon skip-equivalent in-frame deletion mutations occur. Most exon skip-equivalent in-frame deletions were associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. This study indicates the importance of genotype-phenotype correlation studies in the rational design of exon-skipping therapies.