Research for the exonic splicing enhancer sequences in dysttrophin gene

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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.