Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy
Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy
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DOI：
10.1002/jgm.295
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发表时间：
2002-11-01
影响因子：
3.5
通讯作者：
Wilton, SD
中科院分区：
文献类型：
--
作者：
Mann, CJ;Honeyman, K;Wilton, SD
研究背景杜氏肌营养不良症（DMD）是一种致死性遗传病。由肌营养不良蛋白基因突变引起的妨碍功能蛋白合成的疾病。该疾病的一种潜在治疗方法是利用反义寡核苷酸（AO）在前mRNA加工过程中诱导去除疾病相关外显子。诱导的框内mRNA转录物编码较短但功能性的肌营养不良蛋白。我们已经研究和改进了AOs的设计，能够删除外显子23，从而致病的无义突变，从mRNA中的mdx小鼠模型DMD.Methods H-2K（B）-tsA 58 mdx培养物转染脂质体和AOs的复合物。RT-PCR检测外显子跳跃。并通过Western印迹证明随后的蛋白质产生。AOs被交付在一系列的剂量，以比较相对efficiency.Results我们描述了有效的和可重复的外显子23跳跃与几个AOs，包括一个小到17个核苷酸。此外，敏感的外显子23靶位点的位置已被细化，而最小有效剂量已在体外估计。这些剂量显着低于以前报道的，并与合成的dystrophin蛋白在vitro.Conclusions这些结果表明，越来越多的可行性的AO为基础的治疗DMD的治疗。通过改进AO设计，我们已经能够减小AO的尺寸和有效剂量，并显着提高了该技术的效率。版权所有（C）2002约翰威利父子有限公司
Background Duchenne muscular dystrophy (DMD) is a fatal genetic. disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx Mouse model of DMD.Methods H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR. and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies.Results We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro.Conclusions These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique. Copyright (C) 2002 John Wiley Sons, Ltd.