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

项目3（Flanigan） 摘要 杜氏肌营养不良症（DMD）通常由DMD基因突变引起， 破坏开放阅读框架，导致没有肌营养不良蛋白，而温和的贝克尔 肌营养不良症（BMD）通常是由允许部分 功能性肌营养不良蛋白这一规则的例外是在基因突变的人。 该基因的前几个外显子将被预测为导致DMD，但相反是非常轻微的BMD 或者甚至没有症状。我们最近表明，这是由于翻译的N- 截短的肌营养不良蛋白，我们称之为Δ CH 1，它是由一个不依赖于帽的 一个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。我们最近已经证明，我们可以通过使用一种新的方法跳过外显子2来激活IRES。 腺相关病毒（AAV）-U 7snRNA方法，或通过反义寡聚体介导的外显子 跳跃。我们的长期目标是开发外显子2跳读作为一种治疗携带 外显子2的重复，这是DMD患者中最常见的单外显子重复， 以及基因前几个外显子中的其他突变。为了研究这个问题， 开发了一种新的DMD小鼠模型，其中包含外显子2重复（Dup 2小鼠） 并且已经使用它产生了大量的临床前数据，表明AAV介导的递送 靶向外显子2的修饰的U 7snRNA非常有效。我们在这个项目中的目标是扩大 这种方法的适用性，并测试其他方式来刺激IRES，作为诱导 表达将是一种有意义的治疗方法，用于高达5%的肌营养不良蛋白病 患者在目标1下，我们将使用以下方法评估外显子2跳跃和IRES激活： 由萨雷普塔Therapeutics提供的磷酰二胺吗啉代寡聚物（PMO）。下 目的2，我们将寻求验证在一个研究中鉴定的抗肌萎缩蛋白IRES的小分子激活剂。 PTC Therapeutics高通量筛选。根据目标3，我们将开发一个 肌肉特异性AAV介导的CRISPR/Cas9方法诱导体细胞IRES活化。作为 我们已经证明了IRES激活的概念验证， AAV9.U7snRNA方法进行外显子2跳读，预期结果将是证明 更广泛的适用性，我们的方法5'突变使用互补的方法，针对 剪接和翻译修饰策略。我们工作的直接影响将是 提供临床前数据，支持快速临床开发治疗方法， 对DMD和BMD男孩有临床意义的益处，这将通过我们的 与生物制药公司建立了合作关系，并通过我们自己的广泛 药物启动的IND前相互作用的经验

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