Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene.

Steve D Wilton,Yoram Nevo,Kane Greer,Yakov Fellig,Susan Fletcher,Russell Johnsen

doi:10.3390/ijms21124511

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

Highlights

Duchenne muscular dystrophy (DMD), an X-linked recessive condition, is the most common and severe form of childhood muscle wasting
The differences in disease progression between DMD and Becker muscular dystrophy (BMD) form the basis of potential therapeutic interventions using antisense oligomers (AOs) that aim to induce exon skipping and generate a BMD-like dystrophin isoform from a DMD-mutated dystrophin gene [8,9,10]
Induced exon skipping is based on the observation that excising one or more exons from the DMD-mutated mature transcript can remove or by-pass mutations that cause premature termination of translation, allowing synthesis of an internally truncated semi-functional BMD-like dystrophin isoform

Summary

Introduction

Duchenne muscular dystrophy (DMD), an X-linked recessive condition, is the most common and severe form of childhood muscle wasting. Induced exon skipping is based on the observation that excising one or more exons from the DMD-mutated mature transcript can remove or by-pass mutations that cause premature termination of translation, allowing synthesis of an internally truncated semi-functional BMD-like dystrophin isoform. Those exon skipping studies in the clinic and in development describe restoration of the reading frame to address dystrophin genomic deletions, despite the fact that all early proof-of-concept and pre-clinical animal studies focussed on single exon excision to remove the mutated exon 23 in the mdx mouse dystrophinopathy model [11,12]. We compared this simple option to the conventional approach of restoring a semi-functional dystrophin by removing both copies of exon in addition to exon [14] and found the simplest to be the most efficient as shown by restoration of a near full-length protein

Results

AO Design and Synthesis

Cell Propagation

RNA Extraction and RT-PCR Assays

Gel Analysis and Imaging

Western Blotting