Impeding transcription of expanded microsatellite repeats using deactivated Cas9

使用失活的 Cas9 阻止扩展微卫星重复序列的转录

基本信息

批准号：
10343730
负责人：
Eric T Wang
金额：
$ 37.78万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-15 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10343730
关键词：
Affect Amyotrophic Lateral Sclerosis Animal Disease Models Animal Model Antisense Oligonucleotides Automobile Driving Bacterial Artificial Chromosomes C9ORF72 CUG repeat Cell Nucleus Cell model Cells Cessation of life Characteristics Complex Corneal Endothelium Corneal dystrophy DNA Dactinomycin Data Defect Dependovirus Disease Dose Elements Enzymes Episome Exhibits Fiber Gait Genetic Genetic Transcription Genome Heart Human Human Characteristics Huntington Disease Induced pluripotent stem cell derived neurons Inherited Length Methods Microsatellite Repeats Modeling Molecular Motor Neurons Muscle Muscle Fibers Myotonia Myotonic Dystrophy Myotonic dystrophy type 1 Neuraxis Nuclear Nuclear Export Nuclear Import Paralysed Pathogenicity Pathologic Pathology Pattern Peptides Phenotype Physiological Play Production Proteins Publishing RNA RNA Polymerase II RNA Processing RNA Splicing RNA-Binding Proteins Repetitive Sequence Symptoms Testing Therapeutic Therapeutic Intervention Tissues Toxic effect Transcript Transgenic Mice Transgenic Model Translations Travel Viral cell type disease phenotype epigenetic silencing experimental study frontotemporal lobar dementia-amyotrophic lateral sclerosis gain of function human disease improved knock-down loss of function motor neuron degeneration mouse model nanomolar neuron loss neurotoxicity premature programs recruit small molecule therapeutically effective tool transcriptome

项目摘要

Transcription of expanded microsatellite repeats is associated with a number of human diseases, including myotonic dystrophy (DM), Fuch's endothelial corneal dystrophy, and C9orf72 ALS/FTD (C9ALS/FTD), among others. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we will test the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellite repeats, in cell and animal models of DM and C9ALS/FTD. We have previously observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat- containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant Muscleblind-dependent splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of C9orf72 ALS/FTD cells was drastically decreased. Pathological CUG-containing RNA foci in DM1 mouse model muscle fibers was reduced by dCas9/gRNA delivered by adeno-associated virus. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention. In this proposal, we will assess the extent to which virally delivered dCas9/gRNA complexes can rescue molecular, cellular, and phenotypic features in to established models of DM1 and C9ALS/FTD. The HSALR model, which exhibits myotonia, centralized nuclei, and altered transcriptomes characteristic of human DM1, will be used to study DM. The C9-500 BAC transgenic model, which exhibits upper and lower motor neuron degeneration, altered gait, paralysis, and premature death, will be used to study C9ALS/FTD. Our proposed experiments will establish whether inhibition of toxic repeat transcription can rescue disease phenotypes, and define a window around which reduction of toxic RNA abundance is therapeutic.

扩展的微卫星重复的转录与许多人类疾病有关，包括 Myotonic营养不良（DM），Fuch的内皮角膜营养不良症和C9orf72 ALS/FTD（C9ALS/FTD）其他的。消除或减少由这些扩展的基因座产生的RNA和蛋白质的产生治疗益处。在这里，我们将检验以下假设：Cas9酶的停用形式阻碍在DM和C9ALS/FTD的细胞和动物模型中，跨扩展的微卫星重复序列的转录。我们以前已经观察到重复长度，pAM和链依赖性的降低。直接将DCAS9靶向重复序列时包含RNA。异常肌肉盲的剪接在DM1细胞中营救了模式，并产生RAN肽的特征C9ORF72 ALS/FTD细胞大幅下降。 DM1小鼠模型肌肉纤维中含有CUG CUG的RNA焦点是由DCAS9/GRNA降低，由腺相关病毒传递。这些观察结果表明转录与其他RNA的转录相比，微卫星重复的RNA对扰动更敏感指示治疗干预的潜在可行策略。在此提案中，我们将评估传递DCAS9/GRNA复合物可以挽救分子，细胞和表型特征已建立的DM1和C9ALS/FTD模型。 HSALR模型，表现出肌瘤，集中核，人DM1的转录组特征的改变将用于研究DM。 C9-500 BAC 转基因模型表现出上部和下运动神经元变性，改变了步态，瘫痪和过早死亡将用于研究C9als/ftd。我们提出的实验将确定是否抑制有毒的重复转录可以挽救疾病表型，并定义一个窗口，该窗口降低了有毒的RNA丰度是治疗性的。