Chimeric RNA/ethylene-bridged nucleic acids promote dystrophin expression in myocytes of Duchenne muscular dystrophy by inducing skipping of the nonsense mutation-encoding exon
Chimeric RNA/ethylene-bridged nucleic acids promote dystrophin expression in myocytes of Duchenne muscular dystrophy by inducing skipping of the nonsense mutation-encoding exon
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DOI：
10.1089/1043034041648444
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发表时间：
2004-08-01
影响因子：
4.2
通讯作者：
Matsuo, M
中科院分区：
文献类型：
--
作者：
Surono, A;Van Khanh, T;Matsuo, M
已经提出使用反义寡核苷酸通过诱导外显子跳跃来编辑肌营养不良蛋白mRNA，作为在杜氏肌营养不良症（DMD）患者中产生肌营养不良蛋白表达的一种方式。在此，测试由RNA和新修饰的核酸组成的反义嵌合寡核苷酸诱导含有无义突变的外显子跳读的活性。在一例日本DMD病例中，鉴定出了由于肌营养不良蛋白基因（C5899 T）外显子41中的单核苷酸变化导致的无义突变（R1967 X）。设计了由2 '-O-甲基RNA和一种新的2'-O，4 '-C-乙烯桥核酸（ENA）组成的寡核苷酸，以结合外显子41的突变位点，并评估它们诱导dystrophin mRNA中外显子41跳跃的能力。最后，在测试的特定寡核苷酸中，发现18聚体RNA/ENA嵌合体具有最强的活性，可诱导近90%的肌营养不良蛋白mRNA的41号外显子跳跃。因此，近90%的培养的肌细胞被证明是肌营养不良蛋白阳性的免疫组织化学分析。Western印迹分析揭示了转染后1周内存在几乎正常大小的抗肌萎缩蛋白。我们的研究结果表明，RNA/ENA嵌合体可用于表达肌营养不良蛋白的DMD。
Editing of dystrophin mRNA by induction of exon skipping, using antisense oligonucleotides, has been proposed as one way to generate dystrophin expression in Duchenne muscular dystrophy (DMD) patients. Here, antisense chimeric oligonucleotides consisting of RNA and a new modified nucleic acid are tested for activity to induce skipping of an exon containing a nonsense mutation. In a Japanese DMD case, a nonsense mutation (R1967X) due to a single nucleotide change in exon 41 of the dystrophin gene (C5899T) was identified. Oligonucleotides consisting of 2'-O-methyl RNA and a new 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA) were designed to bind the mutation site of exon 41, and their ability to induce exon 41 skipping in dystrophin mRNA was evaluated. Finally, among the specific oligonucleotides tested, an 18-mer RNA/ENA chimera was found to have the strongest activity, inducing exon 41 skipping in nearly 90% of dystrophin mRNA. Accordingly, nearly 90% of cultured myocytes were shown to be dystrophin positive by immunohistochemical analysis. Western blot analysis disclosed the presence of nearly normal-sized dystrophin up to 1 week after the transfection. Our results suggest that an RNA/ENA chimera can be used to express dystrophin in DMD.