Project 3: Use of an IRES-driven N-truncated dystrophin isoform as a clinical therapy for 5 mutations in the dystrophinopathies

项目 3：使用 IRES 驱动的 N 截短肌营养不良蛋白亚型作为肌营养不良蛋白病 5 种突变的临床疗法

• 批准号: 10017028
• 负责人: KEVIN M FLANIGAN
• 金额: $ 29.96万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-14 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017028
• 关键词: Actins; Age; Alleles; Becker Muscular Dystrophy; Binding; Biological Products; CRISPR/Cas technology; Catalogs; Cell Line; Clinical; Clinical Trials; Collaborations; Data; Dependovirus; Duchenne muscular dystrophy; Dystrophin; Exclusion; Exons; Genes; Goals; Guide RNA; Internal Ribosome Entry Site; Link; Mediating; Methods; Modification; Molecular; Mus; Muscle; Muscular Dystrophies; Mutation; Myoblasts; Nonsense Mutation; Open Reading Frames; Outcome; Patients; Phenotype; Principal Investigator; Process; Protein Isoforms; Proteins; RNA Splicing; Reading Frames; Research Personnel; Rod; Severities; Symptoms; Testing; Therapeutic; Translational Research; Translations; Work; adeno-associated viral vector; boys; calponin; clinical development; dystrophinopathy; exon skipping; experience; high throughput screening; improved; mouse model; phosphorodiamidate morpholino oligomer; pre-clinical; programs; small molecule; somatic cell gene editing; therapeutic development; therapy development; vector

Project 3 (Flanigan) Abstract Duchenne muscular dystrophy (DMD) typically results from mutations in the DMD gene that disrupt the open reading frame, resulting in no dystrophin protein, whereas the milder Becker muscular dystrophy (BMD) typically results from mutations that allow expression of a partially functional dystrophin protein. Among the exceptions to this rule are people with mutations in the first few exons of the gene would be predicted to result in DMD but instead are very mild BMD or are even asymptomatic. We have recently shown that this is due to translation of an N- truncated dystrophin which we call ΔCH1, and which results from a cap-independent translational process mediated by a newly described internal ribosome entry site (IRES) within exon 5. We have recently shown that we can activate the IRES by skipping exon 2 using an adeno-associate virus (AAV)-U7snRNA approach, or by antisense oligomer-mediated exon skipping. Our long-term goal is to develop exon 2 skipping as a therapy for patients who carry a duplication of exon 2, which is the most common single exon duplication in DMD patients, as well as for other mutations within the first few exons of the gene. To study this, we have developed a new mouse model of DMD that contains an exon 2 duplication (the Dup2 mouse) and have used it to generate extensive preclinical data showing that AAV-mediated delivery of a modified U7snRNA targeting exon 2 works very well. Our objective in this project is to broaden the applicability of this approach, and to test other ways to stimulated the IRES, as inducing expression would be a meaningful therapeutic approach for up to 5% of dystrophinopathy patients. Under Aim 1, we will assess exon 2 skipping and IRES activation using phosphorodiamidate morpholino oligomers (PMOs) provided by Sarepta Therapeutics. Under Aim 2, we will seek to validate small molecule activators of the dystrophin IRES identified in a high-throughput screen at PTC Therapeutics. Under Aim 3, we will develop a develop a muscle-specific AAV-mediated CRISPR/Cas9 approach to induce somatic IRES activation. As we have already demonstrated the proof-of-concept of IRES activation using an AAV9.U7snRNA approach to exon 2 skipping, the expected outcome will be to demonstrate the broader applicability of our approach to 5' mutations using complementary methods directed at both splicing and translational modification strategies. The immediate impact of our work will be to provide preclinical data that supports rapid clinical development of therapies to provide a clinically meaningful benefit to boys with DMD and BMD, which will be facilitated by our established collaborations with biopharmaceutical companies and by our own extensive experience in investigator-initiated pre-IND interactions

项目 3（弗拉尼根） 抽象的 杜氏肌营养不良症 (DMD) 通常由 DMD 基因突变引起， 破坏开放阅读框，导致没有肌营养不良蛋白，而较温和的贝克尔 肌营养不良症 (BMD) 通常是由突变导致的，这些突变允许部分表达 功能性肌养蛋白突变的人除外。 该基因的前几个外显子预计会导致 DMD，但 BMD 却非常轻微 或者甚至是无症状的，我们最近表明这是由于 N- 的翻译造成的。 截短的肌营养不良蛋白，我们称之为 ΔCH1，它是由帽独立的 由新描述的内部核糖体进入位点（IRES）介导的翻译过程 外显子 5。我们最近表明，我们可以通过使用跳过外显子 2 来激活 IRES 腺相关病毒 (AAV)-U7snRNA 方法，或通过反义寡聚物介导的外显子 我们的长期目标是开发外显子 2 跳跃作为携带 2 号外显子的患者的治疗方法。 外显子 2 重复，这是 DMD 患者中最常见的单外显子重复，如 以及基因前几个外显子内的其他突变为了研究这一点，我们有。 开发了一种新的 DMD 小鼠模型，其中包含外显子 2 重复（Dup2 小鼠） 并用它生成了大量的临床前数据，表明 AAV 介导的递送 靶向外显子 2 的修饰 U7snRNA 效果非常好，我们在此项目中的目标是扩大范围。 该方法的适用性，并测试其他刺激 IRES 的方法，如诱导 表达对于高达 5% 的肌营养不良症来说是一种有意义的治疗方法 在目标 1 下，我们将使用评估外显子 2 跳跃和 IRES 激活。 磷酸二酰胺吗啉寡聚物 (PMO) 由 Sarepta Therapeutics 提供。 目标 2，我们将寻求验证抗肌营养不良蛋白 IRES 的小分子激活剂 PTC Therapeutics 的高通量筛选 在目标 3 下，我们将开发一种 肌肉特异性 AAV 介导的 CRISPR/Cas9 方法诱导体细胞 IRES 激活。 我们已经使用一个方法演示了 IRES 激活的概念验证 AAV9.U7snRNA 外显子 2 跳跃方法，预期结果将证明 我们使用针对 5' 突变的互补方法的方法具有更广泛的适用性 我们工作的直接影响将是剪接和翻译修饰策略。 提供支持疗法快速临床开发的临床前数据 对患有 DMD 和 BMD 的男孩具有临床意义的益处，这将由我们的促进 与生物制药公司建立了合作关系，并通过我们自己的广泛 研究者发起的 IND 前互动经验