Transcription therapeutics for myotonic dystrophy type 1

1 型强直性肌营养不良的转录疗法

基本信息

批准号：
8822685
负责人：
Peter B Dervan
金额：
$ 48.1万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8822685
关键词：
3&apos Untranslated Regions Actins Action Potentials Affect Age of Onset Alleles Animals Binding Biochemical Markers Biological Availability CAG repeat Cardiac Myocytes Cell Culture Techniques Cell Nucleus Cells Characteristics Chemicals DNA Binding DNA Sequence Defect Disease Dose Down-Regulation Drug Kinetics Drug or chemical Tissue Distribution Fibroblasts Gene Expression Gene Expression Profile Genes Genetic Genetic Transcription Health Human Imidazole Inherited Laboratories Ligand Binding Maximum Tolerated Dose Messenger RNA Methods Minor Groove Modification Molecular Monitor Mus Muscle Muscle Cells Muscular Dystrophies Myotonia Myotonic Dystrophy Neuromuscular Diseases Neurons Northern Blotting Nuclear Nuclear RNA Nylons Pathogenesis Patients Pattern Phenotype Preparation Proteins Pyrroles RNA RNA Splicing Series Severity of illness Specificity Spices Symptoms Synthesis Chemistry Therapeutic Toxic effect Transcript Translating Trinucleotide Repeats Universities Untranslated Regions Vertebral column base cell type design efficacy testing gain of function human disease improved induced pluripotent stem cell mouse model prevent programs research study restoration safety study skeletal small molecule transcription factor uptake

项目摘要

DESCRIPTION (provided by applicant): We seek to develop DNA-binding pyrrole-imidazole (Py-Im) polyamides as therapeutics for the neuromuscular disorder myotonic dystrophy type 1 (DM1). Py-Im polyamides are a class of synthetic minor groove-binding ligands that can be programmed to bind predetermined DNA sequences, such as the CTG/CAG triplet repeats that are causative of DM1. Long repeats in the DMPK gene are transcribed into a toxic gain-of-function poly(CUG) RNA that sequesters essential mRNA splicing factors, and may also be translated into toxic protein species. We have generated human induced pluripotent stem cell (hiPSC) clones of identical genetic background but different CTG/CAG triplet-repeat size from DM1 patient fibroblasts by transcription factor reprogramming, and these cells have been differentiated into the relevant cell types in the human disease DM1, such as neurons, cardiomyocytes and myocytes. We have made the important observation that a specific Py-Im polyamide targeting the CTG/CAG triplet-repeats is able to virtually abolish nuclear foci in patient cells. This molecule is without effect on genes that harbor short, non-pathogenic CTG/CAG repeats but shows a decrease in DMPK mRNA in DM1 cells, consistent with down regulation of the pathogenic allele. We will establish the therapeutic potential of the polyamides with the following specific aims: (1) We will fully characterize DM1 and control hiPSC-derived neurons, cardiomyocytes and myocytes, for studies of polyamide efficacy. DM1 signatures will be investigated, including CUG nuclear RNA foci formation and defects in RNA spicing. The efficacy of the Py-Im polyamide in these differentiated cells will be assessed by monitoring CUG RNA foci, transcription of the pathogenic DMPK allele and restoration of normal splicing patterns of genes that are affected in DM1 cells. Improvements in cellular uptake and compound stability will be achieved by synthetic chemistry efforts. (2) Synthetic methods will be optimized for preparation of sufficient quantities of the active compound for animal studies. The pharmacokinetics, maximum tolerated dose and tissue distribution of the most efficacious molecules will first be determined in normal mice. (3) Efficacy in a mouse model that expresses a long CUG RNA will be explored with Dr. Charles Thornton of the University of Rochester. Manifestations of the disease, such as myotonia, as well as biochemical markers (splicing defects and nuclear RNA foci) in affected muscle will be monitored.

描述（由申请人提供）：我们寻求开发 DNA 结合吡咯咪唑 (Py-Im) 聚酰胺作为神经肌肉疾病强直性肌营养不良 1 型 (DM1) 的治疗药物 Py-Im 聚酰胺是一类合成的小沟结合。可以编程结合预定 DNA 序列的配体，例如导致 DM1 的 CTG/CAG 三联体重复序列。 DMPK 基因中的长重复序列被转录成有毒的功能获得性聚 (CUG) RNA，隔离必需的 mRNA 剪接因子，也可能被翻译成有毒的蛋白质种类。我们已经生成了人类诱导多能干细胞 (hiPSC) 克隆。通过转录因子重编程从DM1患者成纤维细胞中获得相同遗传背景但不同CTG/CAG三联体重复大小的细胞，并且这些细胞已分化为人类疾病中的相关细胞类型DM1，例如神经元、心肌细胞和肌细胞，我们发现，针对 CTG/CAG 三联体重复序列的特定 Py-Im 聚酰胺能够几乎消除患者细胞中的核灶。具有短的非致病性 CTG/CAG 重复序列，但显示 DM1 细胞中 DMPK mRNA 减少，与致病等位基因的下调一致，我们将确定聚酰胺的治疗潜力。以下具体目标：（1）我们将全面表征DM1并控制hiPSC衍生的神经元、心肌细胞和肌细胞，以研究DM1特征，包括CUG核RNA焦点形成和RNA拼接缺陷的功效。这些分化细胞中的 Py-Im 聚酰胺将通过监测 CUG RNA 焦点、致病性 DMPK 等位基因的转录以及 DM1 细胞中受影响的基因正常剪接模式的恢复来评估。通过合成化学努力，将实现细胞摄取和化合物稳定性的改善（2）将优化合成方法，以制备足够数量的活性化合物，用于动物研究的药代动力学、最大耐受剂量和最有效分子的组织分布。 (3) 将与罗切斯特大学的 Charles Thornton 博士一起探讨表达长 CUG RNA 的小鼠模型的功效，例如肌强直。以及受影响肌肉中的生化标记（剪接缺陷和核 RNA 焦点）将受到监测。