Antisense Mediated Suppression of Dystrophin Mutations

描述（由申请人提供）： X连锁肌营养不良蛋白基因的突变导致杜氏肌营养不良症（DMD），或较温和的贝克尔肌营养不良症（BMD）。自从对导致这种毁灭性儿童疾病的遗传缺陷进行初步表征以来，一些潜在的治疗方法受到了极大的关注。一些实例包括递送抗肌萎缩蛋白的野生型或微基因V形式的基因疗法、引入抗肌萎缩蛋白产生细胞的成肌细胞或干细胞疗法、校正突变的himeric寡核苷酸和设计用于在剪接期间修饰mRNA的反义寡核苷酸。在该提议中提出的另一种不同的方法是针对改变突变基因的解码（翻译），使得活性全长蛋白质的表达得以恢复。由于翻译是由核糖体的分子机制介导的，因此恢复性解码疗法将针对在一定水平上改变核糖体功能。本项目的总体目标是（1）确定使用反义技术和靶向核糖体的小分子药物以位点特异性方式破坏翻译装置的保真度的方法，以及（2）探索这种方法用于抑制抗肌萎缩蛋白基因中的终止密码子和移码突变的实用性。由于目前的恢复性解码方法利用引起广泛翻译错误的化合物，因此通过将阅读框恢复或终止密码子通读引导至引起疾病的突变位点的能力将实现该领域的明显进步。最后，正如本研究所提出的，证明从肌营养不良蛋白基因中携带移码和终止密码子突变的mdx小鼠中分离的原代细胞中有效和位点特异性恢复性解码，将为这些动物和肌营养不良症的人类细胞培养模型的未来临床前研究奠定基础。

DESCRIPTION (provided by applicant): Mutations in the X-linked dystrophin gene result in Duchenne Muscular Dystrophy (DMD), or the milder Becker Muscular Dystrophy (BMD). Since the initial characterization of the genetic defect responsible for this devastating childhood disease, a number of potential therapeutic approaches have received significant attention. Some examples include, gene therapy to deliver a wild type or mini-gene v version of dystrophin, myoblast or stem-cell therapy to introduce dystrophin producing cells, himeric oligonucleotides to correct the mutation, and antisense oligonucleotides designed to modify the mRNA during splicing. Another distinct approach, addressed in this proposal, is directed toward altering decoding (translation) of the mutant gene, such that expression of active full length protein is restored. As translation is mediated by the molecular machinery of the ribosome, restorative decoding therapies will be directed toward altering ribosomal function at some level. The overall goals of this project are to (1) identify ways to subvert the fidelity of the translational apparatus in a site-specific manner using antisense technology and small molecule drugs which target the ribosome, and (2) explore the utility of this approach for suppressing stop codon and frameshift mutations in the dystrophin gene. As current approaches to restorative decoding utilize compounds which cause wide-spread translational errors, a clear advancement in the field would be achieved by the ability to direct either reading frame restoration or stop codon readthrough to the site of a disease causing mutation. Finally, as proposed in this grant, demonstrating efficient and site-specific restorative decoding in primary cells isolated from mdx mice carrying frameshift and stop codon mutations in the dystrophin gene, will establish the rationale for future preclinical studies in these animals and in human cell culture models of muscular dystrophy.