Duchenne muscular dystrophy is a fatal muscle disease, caused by mutations in DMD, leading to loss of dystrophin expression. Phosphorodiamidate morpholino splice-switching oligonucleotides (PMO-SSOs) have been used to elicit the restoration of a partially functional truncated dystrophin by excluding disruptive exons from the DMD messenger. The 30-mer PMO eteplirsen (EXONDYS51) developed for exon 51 skipping is the first dystrophin-restoring, conditionally FDA-approved drug in history. Clinical trials had shown a dose-dependent variable and patchy dystrophin restoration. The main obstacle for efficient dystrophin restoration is the inadequate uptake of PMOs into skeletal muscle fibers at low doses. The excessive cost of longer PMOs has limited the utilization of higher dosing. We designed shorter 25-mer PMOs directed to the same eteplirsen-targeted region of exon 51 and compared their efficacies in vitro and in vivo in the mdx52 murine model. Our results showed that skipped-dystrophin induction was comparable between the 30-mer PMO sequence of eteplirsen and one of the shorter PMOs, while the other 25-mer PMOs showed lower exon-skipping efficacies. Shorter PMOs would make higher doses economically feasible, and high dosing would result in better drug uptake into muscle, induce higher levels of dystrophin restoration in DMD muscle, and, ultimately, increase the clinical efficacy.

Duchenne肌营养不良症是一种致命的肌肉疾病，由DMD突变引起，导致肌营养不良蛋白表达丧失。磷酸二酰胺基吗啉代剪接切换寡核苷酸（PMO-SSOs）已用于通过从DMD中排除破坏性外显子来引发部分功能性截短的肌营养不良蛋白的恢复。信使。为跳过第51外显子而开发的30聚体PMO teteplirsen（EXONDYS51）是历史上第一种恢复抗肌萎缩蛋白，有条件的FDA批准的药物。临床试验显示剂量依赖性可变和肌营养不良蛋白的修复。有效肌营养不良蛋白恢复的主要障碍是低剂量下PMO无法充分吸收到骨骼肌纤维中。更长的PMO成本过高，限制了更高剂量的使用。我们设计了针对25外显子的相同eteplirsen靶向区域的较短的25-mer PMO，并比较了它们在mdx体内和体外的功效52鼠模型。我们的研究结果表明，特替普生的30-mer PMO序列与较短的PMO之一之间的肌营养不良蛋白的诱导作用相当，而其他25-mer PMO的外显子跳过效率较低。较短的PMO将使高剂量在经济上可行，并且高剂量将导致更好的药物吸收到肌肉中，在DMD肌肉中诱导更高水平的肌营养不良蛋白恢复，并最终提高临床疗效。