Therapeutic induction of dytrophin-positive "revertant" fibres in the mdx mouse

回复突变纤维是在DMD患者和动物模型的肌肉中发现的低丰度的肌营养不良蛋白阳性纤维。这些纤维似乎有一个选择性的优势，肌营养不良蛋白阴性纤维，因为它们随着年龄的增长而积累。肌营养不良蛋白mRNA的表征已经鉴定出缺失多个外显子的框内转录物，其排除无义突变或恢复缺失周围的阅读框。我们已经设计了反义寡核苷酸（AO）结合区域侧翼的外显子含有肌营养不良蛋白突变的mdx小鼠。AO干扰前mRNA的加工以排除突变并允许合成略微缩短的肌营养不良蛋白。使用AO修饰RNA加工允许基因在天然调控元件的控制下发挥功能。我们已经表明，AO可以诱导肌营养不良蛋白的表达，并提高营养不良（mdx）小鼠后肢肌肉的力量。我们的目标是改善这些结果，通过使用AO阻断剪接位点侧翼连续外显子，以诱导肌营养不良蛋白模仿的回复纤维。由于大多数回复突变体转录本缺失多个外显子，我们认为AO诱导的肌营养不良蛋白的功能能力可以通过去除多个外显子来改善。mdx小鼠骨骼肌细胞系用于评估AO。然而，为了确定诱导的肌营养不良蛋白在心肌和骨骼肌中的功效，必须在小鼠上进行实验。以前的工作，在体外和mdx小鼠的肌肉已经验证了这种方法。将通过a）血管内注射B）腹膜内注射在mdx小鼠中递送显示出前景的AO组合。将通过连续分析和终点分析评估治疗的疗效，包括生理学、临床、分子和组织学检测。将特别注意小鼠的健康和可能发生的任何不良副作用。

Revertant fibres are low-abundance, dystrophin-positive fibres found in muscle of DMD patients and animal models. These fibres appear to have a selective advantage over dystrophin negative fibres, as they accumulate with age. Characterisation of dystrophin mRNA has identified in-frame transcripts missing multiple exons, which either exclude a nonsense mutation or restore the reading frame around a deletion. We have designed antisense oligonucleotides (AOs) to bind regions flanking the exon containing the dystrophin mutation in the mdx mouse. The AOs interfere with processing of the pre-mRNA to exclude the mutation and allow a slightly shortened dystrophin to be synthesised. The use of AOs to modify RNA processing allows the gene to function under the control of natural regulatory elements. We have shown that AOs can induce dystrophin expression and improve strength in dystrophic (mdx) mouse hindlimb muscles. We aim to improve upon these results by using AOs to block splice sites flanking consecutive exons, in order to induce dystrophin which mimics that of revertant fibres. As most revertant transcripts are missing multiple exons, we believe that the functional capacity of AO-induced dystrophin can be improved upon by removing multiple exons. An mdx mouse skeletal muscle cell line is used for evaluation AOs. However, in order to determine the efficacy of the induced dystrophin in cardiac and skeletal muscle, experiments must be performed on mice. Previous work, in vitro and in muscles of mdx mice have validated this approach. Combinations of AOs which show promise will be delivered by a) intravascular injection b) intraperitoneal injection in mdx mice. The efficacy of the treatment will be assessed by both continual and end point analysis, which includes physiological, clinical, molecular and histological testing. Particular attention will be directed to the well-being of the mice and any adverse side effects which may occur.