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

反向纤维是在DMD患者和动物模型的肌肉中发现的低丰度、肌营养不良蛋白阳性纤维。随着年龄的增长，这些纤维似乎比肌营养不良蛋白阴性纤维具有选择性优势。对肌营养不良蛋白mRNA的表征已经确定了框内转录本缺失多个外显子，这要么排除了无义突变，要么恢复了缺失周围的阅读框。我们设计了反义寡核苷酸（AOs）来结合mdx小鼠含有肌营养不良蛋白突变的外显子两侧的区域。AOs干扰前mrna的加工，以排除突变，并允许合成稍微缩短的肌营养不良蛋白。使用AOs修饰RNA加工允许基因在自然调控元件的控制下发挥作用。我们已经证明，AOs可以诱导肌营养不良（mdx）小鼠后肢肌肉的肌营养不良蛋白表达并提高肌力。我们的目标是通过使用AOs阻断连续外显子两侧的剪接位点来改善这些结果，以诱导模仿反向纤维的肌营养不良蛋白。由于大多数逆转转录本缺失多个外显子，我们认为通过去除多个外显子可以改善ao诱导的肌营养不良蛋白的功能。用mdx小鼠骨骼肌细胞系评价AOs。然而，为了确定诱导的肌营养不良蛋白对心脏和骨骼肌的功效，必须在小鼠身上进行实验。先前的工作，在体外和mdx小鼠的肌肉中证实了这种方法。有希望的AOs组合将通过a)血管内注射b)腹腔注射给mdx小鼠。治疗的效果将通过持续和终点分析来评估，包括生理、临床、分子和组织学检测。将特别关注小鼠的健康状况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.