Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries.

BACKGROUND: Antisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. RESULTS: Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. CONCLUSION: The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.

Morpholino oligomer-mediated exon skipping averts 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 characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense 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 (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.

Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript.

Protein-truncating mutations in the dystrophin gene lead to the most common childhood form of muscle wasting, Duchenne muscular dystrophy. Becker muscular dystrophy, a condition that typically arises from dystrophin gene lesions that do not disrupt the reading frame, clearly indicates that substantial domains of the dystrophin protein are not essential. Potential therapeutic intervention exists during pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frame-shifting mutations from the mature mRNA. Appropriately designed antisense oligonucleotides (AOs), directed at amenable splicing motifs across the dystrophin gene transcript, block exon recognition and/or spliceosome assembly so that targeted exons are removed from the mature mRNA. We describe a panel of AOs designed to induce skipping of every exon within the human dystrophin gene transcript, with the exception of the first and last exons. Every exon targeted in vitro could be removed from the dystrophin mRNA, although some exons are more efficiently excluded than others. No single motif has emerged as a universal AO annealing site for redirection of dystrophin pre-mRNA processing, although the general trend is that the most efficient compounds are directed at motifs in the first half of the target exon.

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.