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

Abstract

BackgroundDuchenne 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.MethodsAntisense 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.ResultsAntisense 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.ConclusionsAntisense 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.

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

“…Although amelioration of the dystrophic phenotype by exon skipping has only been reported in the mdx mouse [14,15], dystrophin exon skipping in human cells has been reported [16,25].…”

“…RT-PCR was performed across dystrophin exons 20-26 as previously described [12,25]. Amplification products were fractionated on 2% agarose gels, stained with ethidium bromide and the images captured by a Chemi-Smart 3000 gel documentation system (Vilber Lourmat, Marne La Vallee, France).…”

Antisense Modulation of Both Exonic and Intronic Splicing Motifs Induces Skipping of a DMD Pseudo-Exon Responsible for X-Linked Dilated Cardiomyopathy

“…In fact, to date only exonic sequences or splice site consensus sequences have been tested as AON targets, and in some cases with no effect, leading to the definition of poorly or even unskippable exons (Errington et al, 2003;Wilton et al, 2007). In those cases, searching for intronic splicing motifs as putative AON targets could represent an alternative solution.…”

The emperor's new dystrophin: finding sense in the noise

“…By contrast, another group found the mouse dystrophin acceptor splice site unresponsive to a 20mer AO, but specific, consistent, and reproducible skipping of Dmd exon 23 was achieved in primary mdx mouse myogenic cells after targeting the donor splice site [37]. Further studies refined AO design by exploring different annealing sites and AO length [38][39][40], evaluating different exonic targets, and studying the golden retriever muscular dystrophy dog model of DMD [41,42]. In addition, different chemistries were evaluated and this led to a division in exon skipping approaches, one group pursuing the use of 2 0 -OMemodified bases on a phosphorothioate backbone (2 0 -OMe AO) [43][44][45][46][47] and the others using phosphorodiamidate morpholino oligomers (PMOs) [22,[48][49][50][51][52][53][54][55][56].…”