Novel gene based therapy for nemaline myopathy

项目摘要/摘要 线状肌病(NM)是一种儿童起病的骨骼肌疾病，其特征是严重的 残疾，包括(在许多情况下)轮椅和喂养管依赖。超过一年的突变 十几个基因可以引起NM。这些基因中的大多数都编码细丝的成分(细丝的主要部分 肌节)，并且这些基因中相关的突变改变了细丝的结构和/或功能， 导致肌肉收缩受损和全身无力。目前还没有治疗NM的方法。 这项提案的首要目标是开发这种毁灭性疾病的治疗方法。 NEB基因的隐性突变是NM最常见的原因。Neb编码了巨大的蛋白质星云蛋白， 其功能是调节细丝的长度。许多NEB突变导致单个外显子跳过或 单个外显子缺失。这种突变不会改变NEB的RNA阅读框架；然而，尽管 去除一小部分原本巨大的蛋白质，它们出人意料地导致显著减少(或 甚至完全丧失)整个星云蛋白。造成这种情况的原因尚不清楚。我们假设 这一令人惊讶的观察结果的原因是，这些突变移除了重复序列的不完整部分 NEB中的元素，其结果是使星云蛋白失去登记，从而 防止它与细丝结合。我们将在目标1中测试这一假设。 目标1：我们将使用先进的成像和生化技术来研究星云蛋白在一系列 框内星云蛋白突变体。要做到这一点，我们将使用斑马鱼模型系统，这是理想的 这一目的是因为我们可以在活着的有机体中完整的骨骼肌中可视化星云蛋白。 NEB突变患者的星云蛋白水平与疾病严重程度相关(蛋白越少，病情越严重 严重疾病)。这一事实意味着以增加星云蛋白表达为目标的治疗策略 应该是非常有效的。基于上面的假设，我们预测我们可以通过以下方式实现这一点 移除更多的星云RNA以消除完整的重复序列，其结果应该是 使缩短的星云蛋白重新整合到细丝中并得到稳定的维持。我们将测试 在目标2和3中，这一想法，我们称之为“域跳过”。 目的2：使用吗啉介导的多外显子跳过或CRISPR/Cas9基因组缺失，我们将 建立斑马鱼“跳域”的可行性和有效性。斑马鱼使我们能够迅速和 全面研究整个星云蛋白基因的这一策略。 目标3：我们将把我们的发现从斑马鱼转移到小鼠模型和患者细胞上，专注于 特别是两个常见的Neb突变(外显子55缺失和外显子61的无义突变)。对.的使用 小鼠模型将使我们能够在哺乳动物系统中测试疗效，而在人类细胞中的测试将提供 重要的概念证明和临床翻译所需的试剂开发。

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.