Research for the exonic splicing enhancer sequences in dysttrophin gene

我们提出了一种新的治疗Duchenne肌营养不良症(DMD)的分子疗法，通过使用反义寡核苷酸诱导外显子跳跃，将引起DMD的移码突变改变为轻度Becker肌营养不良症(BMD)的帧内缺失特征。我们已经阐明了dystrophin基因第19外显子中针对剪接增强子序列的反义寡核苷酸导致了第19外显子的跳过，而该外显子的跳过导致了DMD肌细胞中20外显子缺失的dystrophin蛋白的产生。为了应用这一策略来治疗频繁的缺失突变，我们检测了dystrophin基因缺失热点的外显子中的SES。由于外显子内的富嘌呤序列被认为是促进正确剪接的剪接增强剂，因此我们利用双倍果蝇的前-mRNA来检测来自3个外显子(外显子43、46和53)的富嘌呤序列的剪接增强活性。外显子43的序列对上游内含子剪接的激活作用最强。外显子53的序列显示相对较低的活性，而外显子46的序列几乎没有影响。为了进一步鉴定外显子53和46上的剪接增强子序列，将整个外显子分成30个核苷酸片段，分别检测它们的剪接增强子活性。在外显子53中，分别位于5‘端和3’端的两个片段具有很强的剪接增强活性，尽管它们不是外显子中最富嘌呤的区域。相反，所有来自外显子46的片段都几乎没有活性。这些结果表明，针对这些SESS的反义寡核苷酸是治疗DMD的有用工具。

We have proposed a novel molecular therapy for Duchenne muscular dystrophy (DMD), in which the frameshift mutation causing DMD is changed into the in-frame deletion characteristic of mild form Becker muscular dystrophy (BMD) by inducing exon skipping using an antisense oligonucleotide. We have already clarified that the antisense oligonucleotide against splicing enhancer sequence (SES) in exon 19 of dystrophin gene induced the skipping of exon 19, and the skipping of this exon resulted in the production of the dystrophin protein in DMD myocytes with the deletion of exon 20. To apply this strategy for the treatment of frequent deletion mutations, SES in the exons from deletion hot spot of dystrophin gene were examined.Because purine-rich sequences within exons are proposed to promote proper splicing as splicing enhancers, purine-rich sequences from three exons (exons 43,46 and 53) were examined for their splicing enhancer activity by using a Drosophila doublesex pre-mRNA. The most powerful activating effect on upstream intron splicing was seen with a sequence from exon 43. A sequence from exon 53 showed relatively low activity, whilst that from exon 46 had little effect. To characterize the splicing enhancer sequences in exons 53 and 46 further, entire exons were divided into 30nt fragments that were examined separately for their splicing enhancer activity. In exon 53, two fragments located at the 5' and 3' ends, respectively, had strong splicing enhancer activity, although they were not the most purine-rich regions of the exon. In contrast, all of the fragments derived from exon 46 had little activity. These results suggest that the antisense oligonucleotide aginst these SESs are useful tool for DMD treatment.