Development of RNA interference for treatment of myotonic dystrophy in the HSALR

用于治疗 HSALR 强直性肌营养不良的 RNA 干扰的开发

基本信息

批准号：
7620909
负责人：
JOEL R CHAMBERLAIN
金额：
$ 7.8万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-10 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7620909
关键词：
3&apos Untranslated Regions ACTA1 gene Adult Bacteria Cardiac Cell Culture Techniques Cell Line Cell Survival Cell physiology Cells Clinical Cloning Complementary DNA DNA Polymerase II DNA Polymerase III DNA Sequence Defect Dependovirus Development Disease Dominant-Negative Mutation Dose Double-Stranded RNA Engineering Galactosidase Gene Targeting Genes Genetic Transcription Goals Hereditary Disease Human Hyperinsulinism Impairment Inborn Genetic Diseases Injection of therapeutic agent Intramuscular Injections Lead Liver Messenger RNA Methods MicroRNAs Modeling Mus Muscle Muscular Dystrophies Mutate Myopathy Myotonia Myotonic Dystrophy Obstruction Oligonucleotides Pathology Pathway interactions Phenotype Plasmid Cloning Vector Plasmids Preparation Process Production Proteins RNA RNA Interference RNA Polymerase II RNA Polymerase III RNA Splicing Recombinants Relative (related person)Reporter Reporter Genes Reporting Safety Skeletal Muscle Structure System Technology Testing Therapeutic Therapeutic Agents Tissues Toxic effect Toxicity Tests Transfection Tropism Virus base cell type design disease phenotype improved in vivo intravenous injection knock-down mouse model mutant myotonic dystrophy protein kinase neuropsychiatry overexpression promoter response small hairpin RNA success tissue/cell culture vector

项目摘要

DESCRIPTION (provided by applicant): Myotonic dystrophy is the most common form of muscular dystrophy in adults, and is characterized by a wide range of clinical features including myotonia (muscle hyperexcitability), progressive myopathy, cardiac conduction defects, hyperinsulinemia, and neuropsychiatric impairment. The multi-systemic manifestations of DM1 are attributable to dominant negative effects of a CTG repeat expansion expressed from the myotonic dystrophy protein kinase gene (DMPK) as part of the 3' untranslated region (3'-UTR) of the mRNA. The goal of the proposed experimental plan is to further develop RNA interference (RNAi) technologies to reduce the DM1 RNA containing the repeat expansion. Initial efforts to establish conditions for systemic delivery of short-hairpin RNAs (shRNAs) to muscles bodywide in a reporter mouse, the ROSA26 mouse, resulted in a level of expression that was effective in knocking down targeted ¿-galactosidase expression in cardiac and skeletal muscle. However, toxicity was encountered in some cell types due to overexpression of the shRNAs which saturated the natural microRNA pathways necessary for cell viability. New versions of our current sequence targets for DM1 will incorporate microRNA-based design strategies to produce RNAi sequences that are fully processed from precursors and expressed at a level that is both efficacious and non-toxic, and can be expressed in a tissue-specific manner. The microRNA-based vectors will be compared to shRNAs that are expressed from muscle-specific promoters at a lower level than previously seen with strong RNA polymerase III promoters. Tests of these RNAi expression cassettes in the HSALR mouse model of DM are expected to establish conditions appropriate for systemic delivery by AAV6 and to establish the feasibility of therapeutic RNAi for DM. Importantly, modulating mutant RNA expression by using target sequences with different levels of expression knockdown should reveal the level of reduction necessary to eliminate muscle pathology. These approaches could result in a body-wide alleviation of dystrophic pathology in the HSALR mouse and provide clues for the adaptation of these methods to target the human DM1 gene DMPK. The success of this approach may lead to a treatment in humans for DM1 and would offer a viable approach for treating other dominantly inherited disorders, regardless of whether RNA or protein is pathogenic. We propose to develop a method that will target and inactivate the cell factor that causes myotonic dystrophy (DM). DM is a disease mainly of muscle that causes uncontrolled stiffening. Our therapeutic agent will be engineered to target muscle to eliminate the disease effects in mice with the disease to establish conditions to treat the disease in humans.

描述（由申请人提供）：强直性肌营养不良是成人肌营养不良最常见的形式，具有多种临床特征，包括肌强直（肌肉过度兴奋）、进行性肌病、心脏传导缺陷、高胰岛素血症和神经精神障碍。 DM1 的多系统表现可归因于强直性肌营养不良蛋白激酶基因 (DMPK) 表达的 CTG 重复扩增的显着负面影响所提出的实验计划的目标是进一步开发RNA干扰(RNAi)技术以减少含有DM1 RNA的重复扩增的条件。用于将短发夹 RNA (shRNA) 系统性递送至报告小鼠 ROSA26 小鼠的全身肌肉，产生的表达水平可有效击倒目标 ¿ - 心肌和骨骼肌中的半乳糖苷酶表达然而，由于 shRNA 的过度表达，导致细胞活力所需的天然 microRNA 途径饱和，因此，我们当前的 DM1 序列目标的新版本将纳入基于 microRNA 的设计。产生从前体完全加工并以有效且无毒的水平表达并且可以以组织特异性方式表达的RNAi序列的策略将与基于microRNA的载体进行比较。肌肉特异性启动子表达的 shRNA 水平低于之前使用强 RNA 聚合酶 III 启动子所见的水平。在 DM 的 HSALR 小鼠模型中对这些 RNAi 表达盒进行测试，有望建立适合 AAV6 全身递送的条件。重要的是，通过使用具有不同表达敲低水平的靶序列来调节突变体 RNA 表达应该揭示消除肌肉病理学所需的减少水平。减轻 HSALR 小鼠的营养不良病理，并为这些方法适应人类 DM1 基因 DMPK 提供线索。这种方法的成功可能会导致人类治疗 DM1，并为治疗其他显性遗传提供可行的方法。无论 RNA 还是蛋白质是否致病，我们都建议开发一种方法来靶向并灭活导致强直性肌营养不良 (DM) 的细胞因子，DM 是一种主要导致失控的肌肉疾病。我们的治疗剂将被设计为针对肌肉，以消除患有该疾病的小鼠的疾病影响，从而为治疗人类的疾病创造条件。