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

Duchenne肌营养不良症(DMD)是一种致命的肌肉萎缩疾病，患者通常在20多岁时死亡。许多研究，包括对基因替代疗法的研究，都是为了寻找治疗DMD的方法，到目前为止，最有希望的治疗方法是通过诱导外显子跳跃来挽救突变的dystrophin mRNA。根据对Dystrophin Kobe的分子分析结果，我们提出了一种治疗DMD的方法，即通过反义寡核苷酸诱导外显子跳跃，将Dstrophin的框外mRNA编辑成框内，从而将重度DMD转变为轻度DMD。在这里，我们开展了治疗DMD的RNA/ENA嵌合寡核苷酸的开发研究。首先，我们寻找能够诱导靶外显子跳跃的最佳RNA/ENA嵌合体。在重复了诱导培养的心肌细胞跳过外显子的实验后，我们成功地鉴定出了最有效的跳过外显子的RNA/ENA嵌合体。鉴定出的RNA/ENA嵌合体被转染到DMD患者的培养心肌细胞中，并被证明诱导了Dstrophin的表达。这种治疗策略被检验为应用于小鼠的治疗。我们获得了一只Dystrophin基因外显子52缺失的基因敲除小鼠。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跳过。

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.