Induction of revertant fibres in the mdx mouse using antisense oligonucleotides.
Induction of revertant fibres in the mdx mouse using antisense oligonucleotides.
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DOI：
10.1186/1479-0556-4-3
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发表时间：
2006-05-24
期刊：
影响因子：
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通讯作者：
Wilton, Stephen D
中科院分区：
文献类型：
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作者：
Fall, Abbie M;Johnsen, Russell;Wilton, Stephen D
背景技术背景：杜氏肌营养不良症是一种致命的遗传性疾病引起的肌营养不良蛋白基因突变，导致过早终止翻译和缺乏功能蛋白。尽管存在原发性肌营养不良蛋白基因损伤，但免疫染色研究表明，至少50%的DMD患者、mdx小鼠和DMD犬模型具有罕见的肌营养不良蛋白阳性或“回复突变”纤维。精细表位作图表明，大多数负责回复突变纤维的转录物排除多个外显子，其中之一包括肌营养不良蛋白mutation.METHODS：的mdx小鼠模型的肌营养不良蛋白基因的外显子23中有一个无义突变。我们已经表明，反义寡核苷酸（AO）可以诱导去除这个外显子，导致在框内的mRNA转录编码缩短，但功能性肌营养不良蛋白。为了模拟与回复突变纤维相关的一个外显子组合，我们通过应用一组AOs组合作为“cocktail”来去除多个外显子。结果：使用AOs的鸡尾酒，外显子19-25始终被从肌营养不良蛋白基因转录物中排除。这对应于在未治疗的营养不良小鼠肌肉中零星检测到的替代加工的基因转录物，并且推测其产生回复突变的肌营养不良蛋白同种型。RT-PCR、免疫组化和western blot analysis.CONCLUSION：这项工作证明了AO鸡尾酒通过多外显子跳跃绕过肌营养不良蛋白突变热点的可行性。多外显子跳跃在加速外显子跳跃疗法以治疗DMD中可能是重要的，使得相同的AO制剂可以应用于肌营养不良蛋白基因的特定结构域内的几种不同突变。
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.