Therapeutic application for a cell culture model of myotonic dystrophy

强直性肌营养不良细胞培养模型的治疗应用

• 批准号: 7405771
• 负责人: Misha Vladislavovich Koshelev
• 金额: $ 3.28万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405771
• 关键词: 3' Untranslated Regions; Actins; Adult; Affect; Alleles; Alternative Splicing; Antisense Oligonucleotides; Arrhythmia; Biological Assay; Cardiomyopathies; Cell Cycle; Cell Fraction; Cell Line; Cells; Cerebellar Ataxia; Characteristics; Clinical Trials; Cultured Cells; Cytomegalovirus Retinitis; Defect; Disease; Disruption; Elements; Employee Strikes; Event; Exhibits; FXTAS; Fragile X Syndrome; Friedreich Ataxia; Gel; Genes; Genetic Transcription; Goals; Green Fluorescent Proteins; Heart; Human; Huntington Disease; Hybrids; In Situ Hybridization; In Vitro; Insulin Resistance; Introns; Investigation; Laboratories; Length; Manuscripts; Maps; Mediating; Modeling; Modification; Molecular; Morbidity - disease rate; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscular Dystrophies; Myoblasts; Myogenic Regulatory Factors; Myotonia; Myotonic Dystrophy; Natural regeneration; Normal Range; Northern Blotting; Nuclear; Nuclear RNA; Oligonucleotides; Pathway interactions; Patients; Persons; Phenotype; Preparation; RNA; RNA Splicing; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease H; Skeletal Muscle; Skeletal Muscle Satellite Cells; Skeletal system; Spinal; Symptoms; Syndrome; System; Tamoxifen; Testing; Tetanus Helper Peptide; Tetracycline; Tetracyclines; Therapeutic; Time; Tissues; Trans-Activators; Transcript; Transgenes; Undifferentiated; United States Food and Drug Administration; Untranslated Regions; Viral; Western Blotting; body system; design; established cell line; experience; gain of function; in vivo; locked nucleic acid; mortality; mouse model; mutant; novel strategies; phosphorothioate; satellite cell; stable cell line; wasting

DESCRIPTION (provided by applicant): Myotonic dystrophy is the second most common cause of muscular dystrophy and the most common cause of the adult-onset type. The major form, DM type 1 (DM1), is caused by a CTG expansion in the 3' untranslated region (UTR) of the DMPK gene, which is transcribed and forms nuclear RNA foci. Expression of expanded RNA (DMPK-CUG) in mice and in cell culture reproduces cellular and molecular aspects of DM1. The mutant RNA reverses a developmentally regulated alternative splicing transition, directly causing the myotonia and insulin resistance that patients experience. However, the muscle wasting, which is responsible for the most common cause of DM1 mortality and morbidity, has yet to be explained. Cultured myogenic satellite cells from patients exhibit a significant differentiation defect likely to be directly relevant to this symptom. This project will: (i) establish inducible mouse C2C12 myoblast cell lines that express DMPKCUG RNA to investigate the mechanism by which mutant RNA inhibits differentiation, and (ii) develop a novel approach to reverse these effects using antisense oligonucleotides (ASOs) and endogenous RNase H activity. In Specific Aim 1, C2C12 cell lines inducibly expressing RNA containing 960 CUG repeats in the context of the DMPK 3' UTR (DMPK-CUG960) will be generated using existing tetracycline transactivator lines and tet-responsive construct. Clonal lines with low basal expression, high induction, and robust differentiation will be identified; the ability of DMPK-CUG960 RNA to inhibit differentiation will be confirmed and the cultures will be analyzed for cellular and molecular features of DM1. In Specific Aim 2, these lines will be used to investigate the mechanism by which DMPK-CUG960 inhibits skeletal muscle differentiation. DMPK-CUG960 RNA will be induced before or after differentiation to determine the effects on myoblasts and post-differentiation myotubes; analysis of myogenic and cell cycle factors will be used to determine the point at which myoblast differentiation is blocked. In Specific Aim 3, chimeric locked nucleic acid ASOs and ubiquitous RNase H activity will be used to degrade DMPK-CUG960 RNA in these lines to restore muscle differentiation. Optimized ASOs will be assayed in DM1 cell cultures and in a mouse model expressing DMPK-CUG960 RNA developed in the sponsor's lab for their ability to reverse the striking muscle wasting. The goal of this project is to elucidate the mechanism of muscle wasting in Myotonic Dystrophy, the most common cause of adult-onset muscular dystrophy, and to reverse this symptom in cell culture and a mouse model. These investigations may also help understand other disorders such as Fragile X Syndrome and the related FXTAS Syndrome, Huntington's chorea, Friedreich's Ataxia, and Spinal Cerebellar Ataxia.

描述（由申请人提供）： 强直性肌营养不良是肌营养不良的第二常见原因，也是成人发病型最常见的原因。主要形式 1 型 DM (DM1) 是由 DMPK 基因 3' 非翻译区 (UTR) 的 CTG 扩增引起的，该基因被转录并形成核 RNA 灶。扩展 RNA (DMPK-CUG) 在小鼠和细胞培养物中的表达再现了 DM1 的细胞和分子方面。突变的RNA逆转了发育调节的选择性剪接转变，直接导致患者出现肌强直和胰岛素抵抗。然而，肌肉萎缩是导致 DM1 死亡和发病的最常见原因，目前尚未得到解释。从患者体内培养的肌源性卫星细胞表现出明显的分化缺陷，可能与这种症状直接相关。该项目将：(i) 建立表达 DMPKCUG RNA 的诱导型小鼠 C2C12 成肌细胞系，以研究突变 RNA 抑制分化的机制，以及 (ii) 开发一种使用反义寡核苷酸 (ASO) 和内源性 RNase 逆转这些影响的新方法H 活动。在具体目标 1 中，将使用现有的四环素反式激活剂系和 tet 响应构建体生成在 DMPK 3' UTR (DMPK-CUG960) 背景下诱导表达包含 960 个 CUG 重复的 RNA 的 C2C12 细胞系。将鉴定具有低基础表达、高诱导和强分化的克隆系；将确认 DMPK-CUG960 RNA 抑制分化的能力，并分析培养物的 DM1 细胞和分子特征。在具体目标 2 中，这些细胞系将用于研究 DMPK-CUG960 抑制骨骼肌分化的机制。分化前或分化后诱导DMPK-CUG960 RNA，以确定对成肌细胞和分化后肌管的影响；肌源性和细胞周期因子的分析将用于确定成肌细胞分化被阻断的点。在具体目标 3 中，嵌合锁核酸 ASO 和普遍存在的 RNase H 活性将用于降解这些细胞系中的 DMPK-CUG960 RNA，以恢复肌肉分化。优化的 ASO 将在 DM1 细胞培养物和表达 DMPK-CUG960 RNA 的小鼠模型中进行分析，DMPK-CUG960 RNA 是由申办者实验室开发的，因为它们具有逆转显着肌肉萎缩的能力。该项目的目标是阐明强直性肌营养不良症（成人发病性肌营养不良症最常见的原因）中肌肉萎缩的机制，并在细胞培养和小鼠模型中逆转这种症状。这些研究还可能有助于了解其他疾病，例如脆性 X 综合征和相关的 FXTAS 综合征、亨廷顿舞蹈病、弗里德赖希共济失调和脊髓小脑性共济失调。