Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides
Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides
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DOI：
10.1093/hmg/7.7.1083
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发表时间：
1998-07-01
影响因子：
3.5
通讯作者：
Dickson, G
中科院分区：
文献类型：
--
作者：
Dunckley, MG;Manoharan, M;Dickson, G
编码细胞骨架蛋白抗肌营养不良蛋白及其亚型的基因的缺失和点突变会导致严重的进行性肌病杜氏肌营养不良症 (DMD) 或较轻的德克肌营养不良症 (BMD)，这很大程度上取决于阅读框是否丢失或保持。移码突变往往会导致肌营养不良蛋白缺失 肌膜、膜不稳定和骨骼肌变性，mdx 小鼠是一种有价值的 DMD 动物模型，因为它在小鼠 DMD 基因的外显子 23 中存在无义点突变，导致肌肉肌膜中肌营养不良蛋白表达缺失和肌营养不良症。 通过用反义RNA转染肌肉细胞来达到转录后水平。本质上，2'-O-甲基寡核糖核苷酸 (2'OMeRNA) 被递送至培养中的原代 mdx 成肌细胞的细胞核。在用与鼠肌营养不良蛋白内含子 22 的 3' 剪接位点互补的寡核苷酸转染后，在转染的 mdx 肌管的肌膜中观察到肌营养不良蛋白表达。对这些细胞的 RT-PCR 产物的直接测序揭示了外显子 22 到外显子 30 的精确剪接，跳过了突变的外显子并创建了新的框内肌营养不良蛋白转录物。由于具有类似框内内部缺失的患者表现出相对轻微的肌病症状，这可能在未来为 DMD 以及其他遗传性疾病提供一种治疗方法。
Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Decker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively, Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle, The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy, This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'-O-methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22, Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.