Novel gene based therapy for nemaline myopathy

项目概要/摘要 线状肌病 (NM) 是一种儿童期发病的骨骼肌疾病，其特征是严重的 残疾，包括（在许多情况下）轮椅和饲管依赖。突变超过 有十几个基因可以导致 NM。这些基因中的大多数编码细丝的成分（细丝的主要部分） 肌节），这些基因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。目前没有针对 NM 的治疗方法。 该提案的总体目标是开发针对这种毁灭性疾病的疗法。 NEB 隐性突变是 NM 的最常见原因。 NEB 编码巨型蛋白 Nebulin， 它的作用是调节细丝的长度。许多 NEB 突变会导致单外显子跳跃或 单外显子缺失。此类突变不会改变 NEB 的 RNA 阅读框；然而，尽管只有 去除原本巨大的蛋白质的一小部分，它们会意外地导致显着减少（或 甚至完全丧失）整个 Nebulin 蛋白。其原因尚不清楚。我们假设 这一令人惊讶的观察结果的原因是这些突变删除了重复序列的不完整部分 NEB 中的元素，其结果是使 Nebulin 蛋白失控，从而 防止其融入细丝中。我们将在目标 1 中检验这个假设。 目标 1：我们将使用尖端成像和生化策略来研究 Nebulin 蛋白的一系列 框内星云蛋白突变体。为了实现这一目标，我们将采用斑马鱼模型系统，该系统非常适合 这个目的是因为我们可以看到活体完整骨骼肌中的星云蛋白。 NEB 突变患者的星云蛋白水平与疾病严重程度相关（较少的蛋白质 = 更多的蛋白质） 严重疾病）。这一事实意味着针对增加 Nebulin 蛋白表达的治疗策略 应该是非常有效的。根据上述假设，我们预测可以通过以下方式实现这一目标 去除更多的 Nebulin RNA 以去除完整的重复序列，其结果应该是 使缩短的星云蛋白重新整合到细丝中并保持稳定。我们将测试 这个想法，我们在目标 2 和目标 3 中称之为“域跳跃”。 目标 2：使用吗啉代介导的多外显子跳跃或 CRISPR/Cas9 基因组删除，我们将 建立斑马鱼“域跳跃”的可行性和有效性。斑马鱼使我们能够快速、 全面检查整个 nebulin 基因的这一策略。 目标 3：我们将把斑马鱼的发现转化为小鼠模型和患者细胞，重点关注 特别是两个常见的 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测试哺乳动物系统中的功效，而人体细胞中的测试将提供 临床转化所需的重要概念证明和试剂开发。

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.