Therapeutic target discovery in Drosophila models of Nemaline Myopathy

描述(申请人提供)：我们的长期目标是找到治疗线状肌病(NM)的有效疗法。肌萎缩侧索硬化症是最常见的非营养不良先天性肌病，在某些人群中的发病率为1/20000。它是一种慢性或非进行性疾病，其特征是在受影响的肌肉组织中出现线状小体，肌肉无力，肌肉缺乏再生。NM是一组不同严重程度的肌病：不同类型的NM可从新生儿死亡到轻度成人发病。到目前为止，还没有有效的治疗这种疾病的方法。九个不同基因的突变与NM有关，其中六个基因编码骨骼肌肌节细丝相关蛋白，包括Cofilin 2(CFL2)。基因突变与疾病严重程度或其他表型之间没有关系。一组不同的NM患者的基因表达谱揭示了NM的一致特征；这些基因表达的变化如何有助于疾病的发病和/或严重程度尚不清楚。我们利用果蝇CFL2直系同源基因Twinstar(TSR)的突变，开发了一种新的果蝇NM模型。这个模型显示了NM的特征：线状身体，肌肉无力，在严重情况下，过早死亡。在我们不太严重的情况下，肌肉功能下降，肌节没有明显变化。我们将使用我们的新模型来解决这种疾病的现有治疗方法以及我们对NM特征基因对疾病发展和严重程度的贡献的理解方面的关键差距。利用表型的快速生成时间、重复性和渗透性、可用的遗传工具以及进行成本效益高、相对高通量筛选的能力，我们提出了两个目标：第一，我们将筛选FDA批准的药物文库，以识别延缓或消除NM症状的药物。这些研究依赖于我们稳健的模型，我们最近开发的药物应用方法，以及我们为如此高通量的筛查而简化的分析。我们希望找到FDA批准的影响NM形成或严重程度的药物。在第二部分中，我们将确定NM特征基因如何影响NM疾病的严重程度。我们将首先评估在轻度和重度近视条件下的基因表达谱。验证后，我们将把这组基因与我们的合作者艾伦·贝格斯博士产生的患者NM数据进行比较。然后，在我们的苍蝇模型中，将分析受类似调控的保守基因对疾病的贡献。这些数据将加深我们对NM异质性的了解，为药物有效性提供洞察，并为未来的药物开发提供靶点。结合本提案中概述的工作和NM的新亮点：我们将确定可以通过我们的 与贝格斯博士合作，首先是在脊椎动物和哺乳动物的疾病模型上，最终是在患者身上。

DESCRIPTION (provided by applicant): Our long-term goal is to find effective therapeutics for treatment of Nemaline myopathy (NM). NM is the most common form of non-dystrophic congenital myopathy, having an incidence of 1/20,000 in some populations. It is a slow or non-progressive disease, characterized by the presence of Nemaline bodies in the affected muscle tissue, by muscle weakness and by the absence of muscle regeneration. NM is a heterogeneous group of myopathies of variable severity: different types of NM can range from neonatal lethal to mild adult onset. To date, there is no effective treatment for this disease. Mutations in nine different genes have been linked to NM, with six of these encoding skeletal muscle sarcomere thin filament-associated proteins, including Cofilin 2 (CFL2). No relationships exist between gene mutations and disease severity or other phenotypes. Gene expression profiling of a heterogeneous group of NM patients uncovered a consistent signature for NM; how these changes in gene expression contribute to disease onset and/or severity is not clear. We have developed a novel Drosophila model of NM, using mutations in the Drosophila CFL2 ortholog, twinstar (tsr). This model displays the hallmarks of NM: Nemaline bodies, muscle weakness and, in the severe condition, premature death. In our less severe condition, muscle function is reduced without gross alterations to the sarcomere. We will use our novel model to address the critical gaps in both available treatments for this disease and in our understanding of the contributions of NM signature genes to disease development and severity. Taking advantage of the fast generation time, reproducibility and penetrance of the phenotype, the genetic tools available, and the ability to perform cost effective, relatively high throughput screens, we propose two aims: In the first, we will screen an FDA approved drug library to identify drugs that delay or eliminate NM symptoms. These studies depend on our robust model, our recently developed methods for drug application, and our assays streamlined for such a high throughput screen. We expect to find FDA approved drugs that impact the formation or severity of NM. In the second, we will determine how NM signature genes contribute to NM disease severity. We will first assess gene expression profiles in our mild and strong NM conditions. After validation, we will compare this set of genes with patient NM data generated by our collaborator, Dr. Alan Beggs. Conserved genes that are similarly regulated will then be analyzed for their contribution to disease in our fly model. These data will enhance our understanding of NM heterogeneity, provide insights to drug effectiveness and supply targets for future drug development. Together the work outlined in this proposal with shed new light on NM: we will identify a short list of drugs and clinically relevant genes that can be validated, via our collaboration with Dr. Beggs, first in vertebrate and mammalian disease models and, ultimately, in patients.