Résumé :
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Duchenne muscular dystrophy (DMD) is the most common and fatal muscle wasting disease caused by a loss of dystrophin protein. Currently, no effective treatmentfor DMD is available. One of the major therapeutic approaches is to convert from severe to mild phenotype restoring the reading frame by inducing exon skipping withantisense oligonucleotides (AONs). However, to prepare large amount of AONs is still expensive, and it is troublesome for patients to get intramuscular or intravenousinjection of AONs regularly. Therefore, small chemicals that can modulate exon skipping are highly awaited for clinical use. Recently we found a dystrophinopathypatient who has a point mutation c.4303G>T in exon31 of the dystrophin gene that makes a stop codonp.Glu1435X. Unexpectedly, this patient expresses faint amount oftruncated, but functional dystrophin protein. The analysis of the patient mRNA revealed that the mutation promotes the exon31 skipping and restores the open readingframe of dystrophin, and it was shown that the mutation disrupts exonic splicing enhancer and creates exonic splicing silencer. This means that enough amount offunctional dystrophin protein could be obtained if we could induce more exon31 skipping in this patient. Therefore, we searched for small chemicals that enhance theexon31 skipping using the plasmid that contains mutant exon31 and flanking introns of dystorphin gene, and found that a CDC-like kinase (Clk)-specific inhibitor TG003promoted the exon31 skipping in HeLa cells in a dose-dependent manner. The effect on wild type exon31 plasmid was also investigated and TG003 did not cause exon31skipping. These results indicate that TG003 specifically promotes exon skipping with mutant exon31, but not with wild type exon31. Furthermore, TG003 promoted theexon31 skipping in the endogenous dystrophin gene in a dose-dependent manner and increased production of dystrophin protein in the patient muscle cells. Theseresults indicate the possibility of a novel therapy of DMD. Our findings may open the way to develop a new type of tailor-made medicine of the fatal genetic diseases withsmall chemicals.
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