Novel gene based therapy for nemaline myopathy

线状肌病的新型基因疗法

• 批准号: 10216977
• 负责人: JAMES J DOWLING
• 金额: $ 33.68万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-18 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216977
• 关键词: Actins; Back; Behavior; Biochemical; Biological Models; Biology; CRISPR/Cas technology; Cell Line; Cell model; Cells; Childhood; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Contracture; Dependence; Development; Disease; Elements; Ensure; Enteral Feeding; Event; Excision; Exons; Generations; Genes; Genomics; Germ Lines; Goals; Human; Image; Impairment; Infusion procedures; Interruption; Intramuscular Injections; Knowledge; Length; Mediating; Modeling; Molecular; Mus; Muscle; Muscle Contraction; Muscle Fibers; Mutant Strains Mice; Mutation; Myoblasts; Myopathy; Myosin ATPase; Nemaline Myopathies; Neonatal; Nonsense Mutation; Organism; Patients; Phenotype; Post-Translational Protein Processing; Preclinical Drug Development; Preclinical Testing; Proteins; RNA; RNA Splicing; Reading Frames; Reagent; Sarcomeres; Series; Severities; Severity of illness; Site; Skeletal Muscle; Specificity; Splice-Site Mutation; Structure; Swimming; System; Technology; Testing; Therapeutic; Thin Filament; Transcript; Translating; Translations; Wheelchairs; Work; Zebrafish; base; clinical translation; disability; efficacy testing; exon skipping; experimental study; gene therapy; imaging capabilities; improved; in vivo; mouse model; mutant; nebulin; novel; novel therapeutic intervention; photo switch; physically handicapped; prevent; protein expression; success; therapy development; treatment strategy

PROJECT SUMMARY/ABSTRACT Nemaline myopathy (NM) is a childhood-onset skeletal muscle disease that is characterized by severe disabilities, including (in many cases) wheelchair and feeding tube dependence. Mutations in more than a dozen genes can cause NM. Most of these genes encode components of the thin filament (a principle part of the sarcomere), and mutations associated in these genes alter the structure and/or function of the thin filament, resulting in impaired muscle contraction and generalized weakness. There are currently no treatments for NM. The overarching goal of this proposal is to develop therapies for this devastating disease. Recessive mutations in NEB are the most common cause of NM. NEB encodes the giant protein Nebulin, which functions to regulate the length of the thin filament. Many NEB mutations cause single exon skipping or single exon deletion. Such mutations do not alter the RNA reading frame of NEB; however, despite only removing a very small part of an otherwise giant protein, they unexpectedly result in significant reduction (or even complete loss) of the whole Nebulin protein. The reason for this is not known. We hypothesize that the reason for this surprising observation is that these mutations remove an incomplete portion of repeat elements within NEB, the consequence of which is to make the Nebulin protein out of register, thereby preventing it from incorporating into the thin filament. We will test this hypothesis in Aim 1. Aim 1: we will use cutting edge imaging and biochemical strategies to study the Nebulin protein in a series of in-frame nebulin mutants. To accomplish this, we will employ the zebrafish model system, which is ideal for this purpose because we can visualize the Nebulin protein in intact skeletal muscle in a living organism. Nebulin protein levels in patients with NEB mutations are correlated with disease severity (less protein = more severe disease). This fact means that a therapeutic strategy targeted at increasing Nebulin protein expression should be very effective. Based on our hypothesis above, we predict that we can accomplish this by removing more of the Nebulin RNA to take out complete repeats, the result of which should be to enable a shortened Nebulin to re-integrate into the thin filament and be stably maintained. We will test this idea, which we call “domain skipping”, in Aims 2 and 3. Aim 2: Using either morpholino mediated multi exon skipping or CRISPR/Cas9 genomic deletion, we will establish the feasibility and efficacy of “domain skipping” in zebrafish. Zebrafish allow us to rapidly and comprehensively examine this strategy across the entire nebulin gene. Aim 3: We will translate our findings from zebrafish to the mouse model and to patient cells, focusing specifically on two common Neb mutations (exon 55 deletion and a nonsense mutation in exon 61). The use of the mouse model will enable us to test efficacy in a mammalian system, and testing in human cells will provide vital proof of concept and reagent development necessary for clinical translation.

项目摘要/摘要 Nemaline肌病（NM）是一种儿童期骨骼肌疾病，其特征是严重 残疾，包括（在许多情况下）轮椅和喂养管依赖性。突变不仅仅是 十几个基因可能导致NM。这些基因中的大多数编码细丝的成分（原理部分 这些基因中的肌膜）和突变改变了细丝的结构和/或功能， 导致肌肉收缩和普遍的无力。目前没有针对NM的治疗方法。 该提案的总体目标是开发这种毁灭性疾病的疗法。 NEB中的隐性突变是NM的最常见原因。 NEB编码巨型蛋白质烟丝， 哪个功能可以调节细丝的长度。许多NEB突变会导致单个外显子跳过或 单个外显子删除。这种突变不会改变NEB的RNA阅读框。但是，只有dospite 去除原本巨型蛋白的一小部分 甚至完全损失）整个云蛋白蛋白。原因尚不清楚。我们假设 令人惊讶的观察的原因是，这些突变消除了重复的不完整部分 NEB内的元素，其结果是将云蛋白蛋白从寄存器中脱离出来，从而使 防止将其掺入细丝中。我们将在AIM 1中检验这一假设。 目的1：我们将使用前沿成像和生化策略来研究一系列中的云蛋白蛋白 框内雾蛋白突变体。为此，我们将采用斑马鱼模型系统，这是理想的选择 这个目的是因为我们可以在活生物体中可视化完整骨骼肌肉中的树星蛋白。 NEB突变患者中的云蛋白蛋白水平与疾病的严重程度相关（蛋白质较少=更多 严重疾病）。这一事实意味着一种针对增加雾蛋白蛋白表达的治疗策略 应该非常有效。基于上面的假设，我们预测我们可以通过 去除更多的nebulin RNA以完全重复，其结果应为 使缩短的树云蛋白可以重新整合到细丝中并保持稳定。我们将测试 在目标2和3中，我们称之为“域跳过”的想法。 AIM 2：使用Metpholino介导的多外显子跳过或CRISPR/CAS9基因组删除，我们将 在斑马鱼中建立“跳过域”的可行性和易用性。斑马鱼允许我们迅速和 全面检查整个云蛋白基因的策略。 AIM 3：我们将我们的发现从斑马鱼转化为鼠标模型和患者细胞，聚焦 特别是在两个常见的NEB突变（外显子缺失和外显子61中的废话突变）上。使用 小鼠模型将使我们能够在哺乳动物系统中测试效率，在人类细胞中进行测试将提供 临床翻译所必需的概念和试剂开发的重要证明。