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

杜氏肌营养不良症（DMD）是最常见的遗传性肌肉疾病，并且在三分之二的DMD病例中已经鉴定出导致框外肌营养不良蛋白mRNA产生的肌营养不良蛋白基因的缺失突变。通过诱导外显子跳跃将框外mRNA转化为框内肌营养不良蛋白信息，从而能够产生半功能性内部缺失的肌营养不良蛋白，被认为是最有可能导致成功的方法之一。我们已经报道，转染的反义寡核苷酸，结合剪接增强子序列的外显子19诱导外显子19跳跃培养细胞。此外，静脉输注反义寡核苷酸导致dystrophin mRNA中的外显子跳跃，并在外显子20缺失的DMD病例的肌肉中产生dystrophin蛋白。尽管分析反义寡核苷酸在DMD动物模型中的作用对于开发更有效的治疗策略是必要的，但没有DMD模型， ...更多信息 el鼠标不可用。结果获得了dystrophin基因外显子52的敲除小鼠，并以此为模型分析了反义寡核苷酸的作用，由于dystrophin基因外显子52由118个核苷酸组成，因此在该模型小鼠中产生了框外mRNA。外显子51（233 bp）或外显子53（212 bp）跳跃的诱导导致框外mRNA转化为框内mRNA。分析针对这些外显子的各种反义寡核苷酸诱导外显子跳跃的活性，并且可以在每个外显子中检测到有效的反义寡核苷酸。并且使用了对核酸酶更具抗性并且更强地结合靶序列的4 ′-C-乙烯桥连核酸（ENA）/RNA嵌合体寡核苷酸。在培养的DMD模型小鼠心肌细胞中，反义寡核苷酸转染诱导了每个外显子的跳跃，并产生了符合读框的dystrophin mRNA。此外，这些寡核苷酸可以静脉内和腹膜内施用至模型小鼠而没有任何不良事件。少

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