Novel gene based therapy for nemaline myopathy

线状肌病的新型基因疗法

• 批准号: 10665673
• 负责人: JAMES J DOWLING
• 金额: $ 34.72万
• 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/10665673
• 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; Visualization; Wheelchairs; Work; Zebrafish; clinical translation; disability; efficacy evaluation; efficacy testing; exon skipping; experimental study; gene therapy; imaging capabilities; improved; in vivo; mortality; mouse model; mutant; nebulin; novel; novel therapeutic intervention; 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.

项目总结/摘要 线状体肌病（NM）是一种儿童期发作的骨骼肌疾病，其特征是严重的 残疾，包括（在许多情况下）依赖轮椅和饲管。基因突变超过 十几个基因可以导致NM。这些基因中的大多数编码细丝的组分（细丝的主要部分）。 肌节），并且这些基因中相关的突变改变细丝的结构和/或功能， 导致肌肉收缩受损和全身无力。目前还没有针对NM的治疗方法。 该提案的总体目标是为这种毁灭性疾病开发治疗方法。 NEB中的隐性突变是NM的最常见原因。NEB编码巨大的蛋白质Nebulin， 其作用是调节细丝的长度。许多NEB突变导致单外显子跳跃或 单外显子缺失。这种突变不会改变NEB的RNA阅读框架;然而，尽管只有 除去一小部分巨大的蛋白质，它们出乎意料地导致显著减少（或 甚至完全丧失）整个Nebulin蛋白。原因不明。我们假设 这一令人惊讶的观察结果的原因是这些突变去除了重复序列的不完整部分， NEB中的元素，其结果是使Nebulin蛋白质失去注册，从而 从而防止其结合到细丝中。我们将在目标1中检验这一假设。 目标1：我们将使用尖端成像和生化策略来研究一系列Nebulin蛋白 框内星云蛋白突变体为了实现这一点，我们将采用斑马鱼模型系统，这是理想的， 这是因为我们可以在生物体的完整骨骼肌中观察到Nebulin蛋白。 NEB突变患者的Nebulin蛋白水平与疾病严重程度相关（蛋白质较少=较多 严重疾病）。这一事实意味着以增加Nebulin蛋白表达为目标的治疗策略 应该非常有效。基于我们上面的假设，我们预测我们可以通过以下方式实现这一目标： 移除更多的星云蛋白RNA以取出完整的重复序列，其结果应该是 使缩短的Nebulin重新整合到细丝中并保持稳定。我们将测试 这个想法，我们称之为“域跳跃”，在目标2和3。 目的2：使用吗啉代介导的多外显子跳跃或CRISPR/Cas9基因组缺失，我们将 在斑马鱼中建立“结构域跳跃”的可行性和有效性。斑马鱼让我们能够迅速 全面检查整个星云蛋白基因的这种策略。 目标3：我们将把我们的发现从斑马鱼转化到小鼠模型和患者细胞， 特别是两种常见的Neb突变（外显子55缺失和外显子61中的无义突变）。使用 小鼠模型将使我们能够在哺乳动物系统中测试功效，而在人类细胞中的测试将提供 临床翻译所需的重要概念验证和试剂开发。