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）： 肌营养不良症是肌肉营养不良的第二大最常见原因，也是成人发作类型的最常见原因。主要形式DM 1型（DM1）是由DMPK基因的3'未翻译区（UTR）的CTG扩展引起的，该基因被转录并形成核RNA焦点。小鼠和细胞培养中膨胀的RNA（DMPK-CUG）的表达再现了DM1的细胞和分子方面。突变体RNA逆转了受发展调控的替代剪接过渡，直接引起患者经历的肌瘤和胰岛素抵抗。但是，肌肉浪费是造成DM1死亡率和发病率最常见原因的原因，尚未解释。来自患者的培养的肌源性卫星细胞表现出明显的分化缺陷，可能与该症状直接相关。该项目将：（i）建立可诱导的小鼠C2C12成肌细胞系，表达DMPKCUG RNA，以研究突变RNA抑制分化的机制，并且（ii）使用反义寡核苷酸（ASOS）和内源性RNase RNase RNase H酶H活性开发了一种新的方法来逆转这些效果。在特定的目标1中，将使用现有的TetracyCline反式反激活线和TET-RESPONS构建体生成C2C12的C2C12细胞系在DMPK 3'UTR（DMPK-CUG960）的背景下诱导的RNA。将确定具有低基础表达，高诱导和鲁棒分化的克隆线； DMPK-CUG960 RNA抑制分化的能力将得到证实，并将分析培养物的DM1细胞和分子特征。在特定的目标2中，这些线将用于研究DMPK-CUG960抑制骨骼肌分化的机制。 DMPK-CUG960 RNA将在分化之前或之后诱导，以确定对成肌细胞和分化后肌管的影响；对肌原性和细胞周期因子的分析将用于确定肌细胞分化被阻断的点。在特定的目标3中，嵌合锁定的核酸ASO和无处不在的RNase H活性将用于降解这些线中的DMPK-CUG960 RNA，以恢复肌肉分化。优化的ASO将在DM1细胞培养物和表达在赞助商实验室中开发的DMPK-CUG960 RNA的小鼠模型中测定，以逆转惊人的肌肉浪费。该项目的目的是阐明肌营养不良症中肌肉浪费的机制，这是成人发作肌肉营养不良的最常见原因，并在细胞培养和小鼠模型中扭转了这种症状。这些研究还可能有助于理解其他疾病，例如脆弱的X综合征和相关的FXTAS综合征，亨廷顿的舞蹈舞，弗里德里希共济失调和脊柱小脑共济失调。