Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD
Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD
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DOI：
10.1038/sj.gt.3302800
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发表时间：
2006-10-01
期刊：
影响因子：
5.1
通讯作者：
Wilton, S. D.
中科院分区：
文献类型：
--
作者：
McClorey, G.;Moulton, H. M.;Wilton, S. D.
通过反义寡核苷酸（AO）操纵前mRNA剪接为许多遗传疾病提供了相当大的潜力。其中之一是杜氏肌营养不良症（DMD），其中肌营养不良蛋白基因中的突变通常导致翻译的过早终止，从而导致功能蛋白的丧失。AO可以诱导外显子跳跃，从而绕过突变并恢复阅读框架，产生类似于在轻度贝克尔肌营养不良症中发现的内部缺失蛋白质。迄今为止，这种方法已被应用于mdx小鼠模型在体外和体内，并在人成肌细胞培养。在这里，我们报告的应用AO指导的外显子跳跃诱导dystrophin在DMD，金毛猎犬肌营养不良症（GRMD）犬模型体外表达。评估了2'-O-甲基硫代磷酸酯（2OMe）、磷酰二胺吗啉代寡聚物（PMO）和肽连接的PMO（PMO-Pep）诱导肌营养不良蛋白表达的功效。2OMe化学仅对短期诱导校正的转录物有效，并且不能诱导可检测的肌营养不良蛋白。PMO化学通常仅在高浓度下诱导有限的外显子跳跃;然而，在处理的细胞中产生低水平的肌营养不良蛋白。在此首次应用于DMD模型的PMO-Pep的使用能够诱导高水平和持续水平的外显子跳跃，并诱导最高水平的肌营养不良蛋白表达，对细胞没有明显的不利影响。在GRMD模型中肌营养不良蛋白的诱导为在更大的DMD动物模型中进一步测试AO递送方案提供了可能性，为在人类临床试验中的应用做准备。
Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2'-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.