Targeted exon skipping to correct exon duplications in the dystrophin gene.
Targeted exon skipping to correct exon duplications in the dystrophin gene.
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DOI：
10.1038/mtna.2014.8
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发表时间：
2014-03-18
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--
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杜氏肌营养不良症是一种严重的肌肉消耗性疾病，由肌营养不良蛋白基因突变引起，消除功能性蛋白质表达。虽然外显子缺失是最常见的杜氏肌营养不良症病变，重复占10-15%的报告致病突变，外显子2是最常见的重复外显子。在这里，我们描述了在体外评估的磷酰二胺吗啉寡聚体耦合到一个细胞穿透肽和2′-O-甲基硫代磷酸寡核苷酸，使用三种不同的策略，在患者细胞携带外显子2复制重构肌营养不良蛋白转录。在相同序列的寡聚体类似物之间观察到体外外显子跳跃效率的差异，其中与细胞穿透肽偶联的磷酰二胺吗啉代寡聚体证明是最有效的。正常和外显子2复制细胞之间外显子2切除效率的差异是明显的，表明外显子背景影响寡聚体诱导的剪接转换。在携带外显子2复制的细胞中诱导外显子2的单个拷贝的跳过，这是恢复阅读框并产生正常肌营养不良蛋白转录物的最简单策略。相比之下，外显子2-7的多外显子跳跃以产生贝克尔肌营养不良样肌营养不良蛋白转录物更具挑战性，并且只能用磷酸二酰胺吗啉代寡聚物化学有效诱导。
Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10–15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2′-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2–7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry.
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