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：
7849556
负责人：
JOEL R CHAMBERLAIN
金额：
$ 7.72万
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/7849556
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（由应用提供）：肌发育症是成年人最常见的肌肉营养不良的形式，其特征是多种临床特征，包括肌瘤（肌肉过敏性），进行性肌病，心脏传导缺陷，高胰岛胰岛素血症和神经精神疾病。 DM1的多系统表现归因于从MRNA的3'未翻译区域（3'-utr）的一部分中，由肌发育症蛋白激酶基因（DMPK）表达的CTG重复膨胀的主要负面影响。提出的实验计划的目的是进一步开发RNA干扰（RNAI）技术，以减少含有重复膨胀的DM1 RNA。最初的努力是为在记者小鼠（Rosa26小鼠）中全身传递短发的RNA（shRNA）向肌肉的肌肉的最初努力，导致表达水平，有效地敲低心脏和骨骼肌中的靶向 - 半乳糖苷酶的表达。然而，由于shRNA的过表达，在某些细胞类型中遇到了毒性，这使细胞活力所需的天然microRNA途径饱和。我们当前针对DM1的序列目标的新版本将结合基于microRNA的设计策略，以产生从前体完全处理的RNAi序列，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。将基于microRNA的矢量与肌肉特异性启动子表达的shRNA的水平低于先前使用强RNA聚合酶III启动子的较低水平。预计DM的HSALR小鼠模型中这些RNAi表达盒的测试有望建立适合通过AAV6进行全身传递的条件，并确定热RNAi对DM的可行性。重要的是，使用具有不同表达敲低水平的目标序列调节突变RNA表达，应揭示消除肌肉病理所需的还原水平。这些方法可能会导致HSALR小鼠中身体营养不良病理的全身减轻，并为这些方法适应人DM1基因DMPK提供了线索。这种方法的成功可能导致人类对DM1的治疗，并且无论RNA还是蛋白质是致病性的，都将提供一种可行的方法来治疗其他主要遗传性疾病。我们建议开发一种方法，该方法将靶向并使导致肌动症障碍（DM）的细胞因子失活。 DM是一种主要的肌肉疾病，会导致不受控制的僵硬。我们的治疗剂将被设计为靶向肌肉，以消除患有疾病的小鼠的疾病影响，以建立治疗人类疾病的疾病。