Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole-exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells, and it restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping, and it is potentially applicable to 10.1% of patients with DMD.

杜兴氏肌营养不良症（DMD）是最常见的致命性遗传性儿童期疾病，是由DMD基因突变导致的，该突变导致缺乏功能性肌营养不良蛋白。最近，反义寡核苷酸介导的外显子跳跃作为恢复肌营养不良蛋白的有效方法已经出现，DMD外显子51的跳跃获得了加速批准。识别可提供最高可能跳过效率的反义序列对于未来的临床应用至关重要。在本文中，我们系统地测试了人类DMD上的两步反义寡核苷酸序列为了确定反义寡核苷酸结合位点在人横纹肌肉瘤细胞系中的序列依赖性效应，使用外显子53。第一个粗略的全外显子53步移能够识别目标区域，该区域的第二个步移用于确定外显子53跳跃的最佳反义寡核苷酸序列（NS-065 / NCNP-01）。该寡核苷酸在横纹肌肉瘤和DMD患者来源的细胞中，在预mRNA剪接过程中以剂量依赖性方式强烈促进外显子53跳跃，并恢复了患者来源的细胞中的肌营养不良蛋白水平。NS-065 / NCNP-01，一种二氨基磷酸吗啉代低聚物，似乎是治疗外显子53跳跃的有前途的候选药物，它可能适用于10.1％的DMD患者。