Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse
Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse
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DOI：
10.1073/pnas.011408598
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发表时间：
2001-01-02
影响因子：
11.1
通讯作者：
Wilton, SD
中科院分区：
文献类型：
--
作者：
Mann, CJ;Honeyman, K;Wilton, SD
杜氏肌营养不良症（DMD）是一种严重的肌肉萎缩性疾病，由肌营养不良蛋白基因缺陷引起，通常是无义突变或移码突变，妨碍了功能蛋白的合成。一种较温和的等位基因形式的疾病，贝克肌营养不良症，通常是由框内缺失引起的，框内缺失允许合成较短但仍具有半功能的蛋白质。迄今为止，通过细胞或基因替代将功能性肌营养不良蛋白引入营养不良组织的疗法尚未成功。我们报告了一种替代方法，其中2 '-O-甲基反义寡核苷酸已被用于修改DMD的mdx小鼠模型中的肌营养不良蛋白前mRNA的加工。通过靶向2 '-O-甲基反义寡核苷酸阻断参与正常肌营养不良蛋白前mRNA剪接的基序，我们诱导了外显子23的切除和mdx无义突变，而不破坏阅读框架。首先在转染的H-2K（B）-tsA 58 mdx成肌细胞中体外优化外显子23跳跃，然后在体内诱导。免疫组织化学染色证明肌内递送反义寡核糖核苷酸：脂质体复合物后mdx小鼠中肌营养不良蛋白和γ-肌聚糖的合成和正确的肌膜下定位。这种方法应该通过允许营养不良基因转录物被修饰来降低DMD的严重程度，使得它可以被翻译成贝克尔-肌营养不良蛋白样蛋白。
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma -sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.