Induction of revertant fibres in the mdx mouse using antisense oligonucleotides

Background Duchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein. Despite the primary dystrophin gene lesion, immunostaining studies have shown that at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or 'revertant' fibres. Fine epitope mapping has shown that the majority of transcripts responsible for revertant fibres exclude multiple exons, one of which includes the dystrophin mutation. Methods The mdx mouse model of muscular dystrophy has a nonsense mutation in exon 23 of the dystrophin gene. We have shown that antisense oligonucleotides (AOs) can induce the removal of this exon, resulting in an in-frame mRNA transcript encoding a shortened but functional dystrophin protein. To emulate one exonic combination associated with revertant fibres, we target multiple exons for removal by the application of a group of AOs combined as a "cocktail". Results Exons 19–25 were consistently excluded from the dystrophin gene transcript using a cocktail of AOs. This corresponds to an alternatively processed gene transcript that has been sporadically detected in untreated dystrophic mouse muscle, and is presumed to give rise to a revertant dystrophin isoform. The transcript and the resultant correctly localised smaller protein were confirmed by RT-PCR, immunohistochemistry and western blot analysis. Conclusion This work demonstrates the feasibility of AO cocktails to by-pass dystrophin mutation hotspots through multi-exon skipping. Multi-exon skipping could be important in expediting an exon skipping therapy to treat DMD, so that the same AO formulations may be applied to several different mutations within particular domains of the dystrophin gene

Induction of revertant fibres in the mdx Mouse using antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin mutations that preclude synthesis of a functional protein. The mdx mouse model has a nonsense mutation in exon 23. Removal of this single defective exon induces an in-frame mRNA transcript that encodes a shortened but still functional dystrophin protein. Despite the primary dystrophin gene lesion, at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or ‘revertant fibres’ which arise from some naturally occurring exon-skipping event. Immunostaining studies have shown that the majority of revertant fibres miss multiple exons flanking the DMD mutation. These revertant fibres could provide a template for more functional dystrophin design, rather than skipping of single exons. We aim to emulate these naturally occurring revertant fibres using either bi-functional or combination antisense oligonucleotides (AO) to induce multiple exon skipping. We have developed AO cocktails that consistently induce removal of exons 19–25 and 21–25, two revertant transcripts that have been detected in untreated dystrophic mouse muscle. We are investigating whether these ‘induced revertant’ transcripts generate a more functional dystrophin protein than the minimal exon 23 skip to by-pass the nonsense mutation

The influence of antisense oligonucleotide length on dystrophin exon skipping

Antisense oligonucleotides (AOs) can be used to redirect dystrophin pre-messenger RNA (mRNA) processing, to remove selected exons from the mature dystrophin mRNA, to overcome nonsense mutations, and/or restore the reading frame. Redundancy within the dystrophin protein allows some domains to be removed without seriously compromising function. One of the challenges for splicing blockade is to design AOs that efficiently remove targeted exons across the dystrophin pre-mRNA. AOs are initially designed to anneal to the more obvious motifs implicated in the splicing process, such as acceptor or donor splice sites and in silico predicted exonic splicing enhancers. The AOs are evaluated for their ability to induce targeted exon skipping after transfection into cultured myoblasts. Although no single motif has been implicated in the consistent induction of exon skipping, the length of the AO has emerged as an important parameter in designing compounds that redirect dystrophin pre-mRNA processing. We present data from in vitro studies in murine and human cells showing that appropriately designed AOs of 25–31 nucleotides are generally more effective at inducing exon skipping than shorter counterparts. However, there appears to be an upper limit in optimal length, which may have to be established on a case-by-case basis

Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide

Background Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy is usually caused by in-frame deletions allowing synthesis of some functional protein. Treatment options are limited, and we are investigating the potential of transcript manipulation to overcome disease-causing mutations. Antisense oligonucleotides have been used to induce specific exon removal during processing of the dystrophin primary transcript and thereby by-pass protein-truncating mutations. The antisense oligonucleotide chemistry most widely used to alter pre-mRNA processing is 2′-O-methyl-modified bases on a phosphorothioate backbone. Methods The present studies evaluate 2′-O-methylphosphorothioate, peptide nucleic acid and morpholino antisense oligonucleotides in the mdx mouse model of muscular dystrophy, which has a nonsense mutation in exon 23 of the dystrophin gene. Results We demonstrate dystrophin expression in mdx mouse tissues after localised and systemic delivery of a morpholino antisense oligonucleotide designed to target the dystrophin exon 23 donor splice site. Conclusions The stability of the morpholino structural type, and the fact that it can be delivered to muscle in the absence of a delivery reagent, render this compound eminently suitable for consideration for therapeutic exon skipping to address dystrophin mutations

Exon skipping prevents the onset of dystrophic pathology in the MDX mouse

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Antisense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, whilst maintaining or restoring the reading frame, and thereby overcome proteintruncating mutations. The mdx mouse has a nonsense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdx mouse, in vivo and in vitro. A cell-penetrating peptide-conjugated oligomer, targeted to the mouse dystrophin exon 23 donor splice site, was administered to mdx mice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The cell penetrating peptide greatly enhanced uptake of the phosphorodiamidate morpholino oligomer, resulting in widespread dystrophin expression. Treatment of neonatal mdx mice induced dystrophin expression and averted the onset of the dystrophic process that normally begins shortly before 3 weeks of age