Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

中文翻译：

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外显子45-55使用DMD基因中反义Morpholinos突变修饰的鸡尾酒跳过。

肌营养不良蛋白（DMD）基因的突变和肌营养不良蛋白的丧失导致了杜氏肌营养不良症（DMD）。DMD的一种有前途的治疗方法是使用反义二氨基磷酸二酰胺吗啉代寡聚物（PMO）进行单外显子跳跃，目前面临的主要问题是，一种反义药物会诱导功能不确定的肌营养不良蛋白的产生，并且在具有不同突变的患者中可能无效。因此，这种方法的适用性仅限于帧外突变。在这里，我们使用外显子跳跃效率预测工具，为外显子45-55设计了三种不同的PMO鸡尾酒组合，目的是产生具有保留功能的肌营养不良蛋白变体，如在框架内外显子45-55缺失的轻度或无症状个体中所见。在他们当中 最有效的一组由选择的PMO组成，每个PMO在基于细胞的筛选中均有效跳过指定的外显子。这些适合于永生化DMD患者肌肉细胞不同缺失的组合PMO显着诱导外显子45-55跳跃，同时去除3、8或10个外显子和肌营养不良蛋白恢复，如Western blotting所示。在人源化的小鼠中证实了体内最大11个人类DMD外显子的跳过。该发现表明，我们的PMO套装可用于创建针对45-55外显子跳跃的突变量身定制的鸡尾酒，并治疗超过65％的进行框外或框内缺失的DMD患者。或10个外显子和肌营养不良蛋白恢复，如Western blotting所示。在人源化的小鼠中证实了体内最大11个人类DMD外显子的跳过。该发现表明，我们的PMO套装可用于创建针对45-55外显子跳跃的突变量身定制的鸡尾酒，并治疗超过65％的进行框外或框内缺失的DMD患者。或10个外显子和肌营养不良蛋白恢复，如Western blotting所示。在人源化的小鼠中证实了体内最大11个人类DMD外显子的跳过。该发现表明，我们的PMO套装可用于创建针对45-55外显子跳跃的突变量身定制的鸡尾酒，并治疗超过65％的进行框外或框内缺失的DMD患者。