Antisense oligonucleotide suppression of non-deletion DMD causing mutations

描述(由申请人提供)： Duchenne肌营养不良症(DMD)是一种致命的X连锁肌肉萎缩疾病，由dystrophin基因的蛋白质截断突变引起。反义寡聚体导致携带无义突变的外显子的移除，或最常见的DMD突变类型-移帧缺失的外显子的移除，已被证明产生框架内信息和内部删除的但有功能的蛋白质。Becker肌营养不良症(BMD)是一种等位基因疾病，通常由一个或多个外显子的框内缺失引起，最常见的是基因的前三分之二。BMD的严重程度从交界性DMD到无症状的DMD不等，轻度BMD患者的dystrophin基因提供了功能外显子组合的迹象。至少三分之一的DMD病例是由重复、微插入/缺失和单碱基改变引起的，这些改变改变了剪接位点的识别或导致翻译的提前终止。本项目将针对由非缺失突变引起的DMD病例，设计和应用用于诱导外显子跳跃的反义寡聚体。将测试化合物导入患者细胞系，并评估外显子跳跃。外显子跳过策略将被修改，以最大限度地提高诱导的抗肌营养不良蛋白的质量和数量，如每个特定的抗肌营养不良蛋白基因损伤的背景所允许的。具体目标是：7优化反义低聚物以去除携带序列变异的外显子(致病或中性多态)，否则会影响外显子跳过。7开发适用于假外显子整合或一个或多个外显子重复所致的DMD病例的外显子跳过策略。7建立瞬时动物模型，根据外显子边界确定有重要功能的抗肌营养不良蛋白结构域，以便于设计最优的外显子跳过策略。 公共卫生相关性： Duchenne肌营养不良症是一种持续发展的致命疾病，目前还没有有效的治疗方法。去除特定的外显子有可能极大地减轻DMD的严重程度，DMD患者Dstrophin表达的恢复，甚至部分功能的恢复，有望导致类似BMD的表型，减少发病率和延长寿命。这项申请寻求为三分之一具有非缺失突变的DMD患者开发个性化的外显子跳过疗法。外显子跳过应该提供给所有可能受益的患者，而不仅仅是那些具有更常见的外显子缺失突变的患者。

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by protein truncating mutations in the dystrophin gene. Antisense oligomer induced removal of an exon carrying a nonsense mutation, or exons flanking frame-shifting deletions, the most common type of DMD mutation, has been shown to generate an in-frame message and an internally deleted, but functional protein. Becker muscular dystrophy (BMD) is an allelic disorder typically caused by in-frame deletions of one or more exons, most commonly in the first two thirds of the gene. The severity of BMD varies from borderline DMD to asymptomatic, and the dystrophin genes in mildly affected BMD patients provide an indication of functional exon combinations. At least one third of DMD cases result from duplications, micro-insertions/deletions and single base changes that alter splice site recognition or cause premature termination of translation. This project will address the design and application of antisense oligomers for induced exon skipping, for those DMD cases caused by non-deletion mutations. Patient cell lines will be transfected with test compounds and exon skipping assessed. Exon skipping strategies will be modified to maximize induced dystrophin quality and quantity, as permitted by the context of each particular dystrophin gene lesion. The specific aims are to: 7 Optimise antisense oligomers to remove exons carrying sequence variations (disease-causing or neutral polymorphims) that would otherwise compromise exon skipping. 7 Develop exon skipping strategies appropriate to DMD cases caused by pseudo-exon incorporation or duplications of one or more exons. 7 Develop transient animal models to identify functionally significant dystrophin domains, according to exon boundaries, to facilitate design of optimal exon skipping strategies. PUBLIC HEALTH RELEVANCE: Duchenne muscular dystrophy is a relentlessly progressive, fatal disease for which there is no effective treatment. Specific exon removal has the potential to greatly reduce the severity of DMD, and restoration of dystrophin expression, even of partial function in a DMD patient is expected to result in a BMD-like phenotype, and reduce morbidity and extend life expectancy. This application seeks to develop personalised exon skipping therapies for the one third of DMD patients who have non-deletion mutations. Exon skipping should be made available to all patients who could benefit, not only those with the more common exon deletion mutations.