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Cellular biological study on the treatment of Duchenne muscular dystrophy with nucleic acids

核酸治疗杜氏肌营养不良症的细胞生物学研究

基本信息

批准号：
16390301
负责人：
MATSUO Masafumi
金额：
$ 9.22万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390301/
关键词：
dystrophin exon skipping antisense Duchenne DMD

项目摘要

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, and its victims usually succumb in their twenties. Many studies, including investigations into gene-replacement therapy, have been conducted in a search for a treatment for DMD, and the most promising treatment to date is rescue of mutant dystrophin mRNA by induction of exon skipping. On the basis of results from the molecular analysis of dystrophin Kobe, we have proposed a treatment for DMD in which antisense oligonucleotides induce exon skipping to edit out-of frame dystrophin mRNA into in-frame, thereby converting severe DMD to a milder form. Here we conducted studies on development of RNA/ENA chimeric oligonucleotide for the treatment of DMD. At first we searched for the best RNA/ENA chimera that has ability to induce skipping of target exon. After repeating trials to induce exon skipping in cultured myocytes, we succeeded to identify the most powerful RNA/ENA chimera for exon skipping. The identified RNA/ENA chimera was transfected to a DMD patient's cultured myocytes and shown to induce dystrophin expression. This treatment strategy was examined for application to mouse treatment. We obtained a knockout mouse that has a deletion of exon 52 of the dystrophin gene. By identifying a proper RNA/ENA chimera, we are going to induce exon 51 skipping in the model mouse.

杜氏肌营养不良症（DMD）是一种致命的肌肉消耗性疾病，其受害者通常在二十多岁时死亡。已经进行了许多研究，包括对基因替代疗法的研究，以寻找DMD的治疗方法，迄今为止最有希望的治疗方法是通过诱导外显子跳跃来拯救突变型肌营养不良蛋白mRNA。根据肌营养不良蛋白科比的分子分析结果，我们提出了一种治疗DMD的方法，其中反义寡核苷酸诱导外显子跳跃，将框外肌营养不良蛋白mRNA编辑为框内，从而将严重的DMD转化为较温和的形式。本研究旨在开发RNA/ENA嵌合寡核苷酸治疗DMD。我们首先寻找能够诱导靶外显子跳读的最佳RNA/ENA嵌合体。在重复试验以诱导培养的肌细胞中的外显子跳跃后，我们成功地鉴定了用于外显子跳跃的最强大的RNA/ENA嵌合体。将鉴定的RNA/ENA嵌合体转染到DMD患者的培养的肌细胞中，并显示诱导肌营养不良蛋白表达。检查该治疗策略是否适用于小鼠治疗。我们获得了一个基因敲除小鼠，其具有肌营养不良蛋白基因的外显子52的缺失。通过鉴定合适的RNA/ENA嵌合体，我们将在模型小鼠中诱导外显子51跳跃。