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Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene
Author(s) -
Errington Stephen J.,
Mann Christopher J.,
Fletcher Sue,
Wilton Stephen D.
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.361
Subject(s) - exon skipping , exon , duchenne muscular dystrophy , rna splicing , oligonucleotide , dystrophin , microbiology and biotechnology , alternative splicing , biology , splice , antisense therapy , splice site mutation , genetics , gene , rna , locked nucleic acid
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. Copyright © 2003 John Wiley & Sons, Ltd.