Research of antisense oligonucleotide therapy for muscular dystrophy using knockout mouse as a model

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Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease, and deletion mutations of the dystrophin gene that result in production of out-of-frame dystrophin mRNA have been identified in two-thirds of DMD cases. Transformation of an out-of-frame mRNA into an in-frame dystrophin message by inducing exon skipping and thereby enabling production of semi-functional internally deleted dystrophin is considered one of the approaches most likely to lead to success. We have reported that transfection of an antisense oligonucleotide that bind to a splicing enhancer sequence of exon 19 induced exon 19 skipping in cultured cells. Furthermore, intravenous infusion of the antisense oligonucleotide resulted in exon skipping in dystrophin mRNA and production of dystrophinmtein in muscle of DMD case with deletion of exon 20. Although an analysis of antisense oligonucleotide effect in DMD animal model is necessary for developing more effective strategy of this treatment, no DMD mod … More el mouse was not available. Unexpectedly, we can get the knockout mouse of dystrophin exon 52, then the effect of antisense oligonucleotide was analyzed using this model mouse.Because exon 52 of the dystrophin gene consists of 118 nucleotides, out-of-frame mRNA is produced in this model mouse. Induction of the skipping of exon 51 (233 bp) or exon 53 (212 bp) result in transformation of an out-of frame into an in-frame mRNA. Various antisense oligonucleotides against these exons were analyzed for activity inducing exon skipping and effective antisense oligonucleotides could be detected in each exon. And 4'-C-ethylene-bridged nucleic acids (ENA)/RNA chimera oligonucleotides which are more resistant against nucleases and more strongly bind to target sequences were used. In cultured myocyte of DMD model mouse transfection of the antisense oligonucleotide induced the skipping of each exon, and produced an in-frame dystrophin mRNA. Furthermore, these oligonucleotides could be administered intravenously and intraperitoneally to model mouse without any adverse events. Less