Terminal antisense oligonucleotide modifications can enhance induced exon skipping.

Induction of specific exon skipping during the processing of the dystrophin gene transcript is being pursued as a potential therapy for Duchenne muscular dystrophy. Antisense oligonucleotides directed at motifs involved in pre-mRNA processing can manipulate dystrophin exon incorporation in the mature gene transcript. We have compared the exon skipping ability of oligodeoxyribonucleotides with compounds of the identical sequence incorporating 2'-O-methyl modified bases. Antisense oligonucleotides composed entirely of 2'-O-methyl modified bases on a phosphorothioate backbone were consistently more efficient at inducing exon skipping than comparable oligodeoxyribonucleotides. Chimeric antisense oligonucleotides, mixtures of unmodified and 2'-O-methyl modified bases, induced intermediate levels of exon skipping. In addition, we describe terminal modifications that may be incorporated into the 2'-O-methyl antisense oligonucleotides to further enhance efficiency of exon skipping. Our findings suggest that 2'-O-methyl antisense oligonucleotides should be considered for human clinical trials involving targeted exon skipping in dystrophin gene expression in preference to oligodeoxyribonucleotides.

Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons. METHODS: Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre-mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping. RESULTS: Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments. CONCLUSIONS: Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre-mRNA.