Repair of neurological disorders through RNA editing

通过RNA编辑修复神经系统疾病

基本信息

批准号：
10055971
负责人：
Gail Mandel
金额：
$ 33.69万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
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：
2019-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10055971
关键词：
Address Adenosine Adolescent Adult Amino Acids Astrocytes Attenuated Award Back Bacteriophages Base Pairing Bees Behavior Binding Biological Assay Brain Brain region CRISPR/Cas technology Catalytic Domain Cell Line Cells Chromatin Codon Nucleotides Complementary DNA Data Deamination Disease Dose Elements Enzymes Exhibits Female Funding Future Genes Genetic Enhancer Element Genetic Transcription Guanosine Guide RNA Hand Strength Hippocampus (Brain)Human Hybrids Immune response In Vitro Injections Inosine Length Measures Methods Methyl-CpG-Binding Protein 2 Mismatch Repair Modeling Molecular Morphology Mus Mutation Neonatal Neurologic Neurons Nonsense Codon Nonsense Mutation Pathogenicity Patients Peptides Peripheral Phenotype Population Positioning Attribute Process Proteins Publishing RNA RNA Binding RNA Editing RNA Phages Reagent Research Respiration Rett Syndrome Serotyping Site Specificity Structure Symptoms Technology Testing Therapeutic Time Translations United States National Institutes of Health Viral Virus Work adenosine deaminase base cellular imaging chromatin protein design disease-causing mutation experimental study genome editing human model improved in vivo in vivo evaluation male mouse model mutant nervous system disorder next generation novel nuclease prevent programs promoter recruit relating to nervous system repaired tool transcription factor transcriptome sequencing transcriptomics

项目摘要

Project Summary In this revised proposal, we continue to develop an approach to repair base mutations at the level of RNA, for attenuating symptoms in mouse models of human neurological disease. The experiments are an outgrowth of a pilot NIH Director’s Transformative Research Award that supported both recently published and preliminary results attesting to feasibility of this approach. Our work is currently focused on Rett syndrome, a devastating neurological disease due to mutations in the gene encoding the transcription factor, MECP2. We focused initially on a human patient guanosine (G) to adenosine (A) mutation in MECP2, MECP2317G>A, which interferes severely with its ability to bind to chromatin and results in Rett syndrome. We showed, for the first time, that endogenous Mecp2317G>A RNA can be recoded to the wild type amino acid efficiently in non-dividing neurons cultured from a Mecp2317G>A mouse line that exhibits severe Rett-like symptoms. The recoding occurred through site- directed deamination by a hijacked catalytic domain of Adenosine Acting on RNA 2 (ADAR2) (Editase), fused to a bacteriophage RNA binding peptide, which we targeted to the Mecp2 mutation by an RNA guide. In this revised application, we present new data indicating that recoding also occurs in vivo, in 3 different hippocampal neuronal populations, after direct hippocampal injection of AAV encoding the hybrid ADAR2 protein and Mecp2 RNA guides. Moreover, recoding resulted in amount of MeCP2 localization to chromatin consistent with amount of editing at the RNA level. We have developed the tools and reagents that now place us in an ideal position to address critical unanswered questions for reversing neurological phenotypes of Rett syndrome, and for testing hypotheses related to site-directed repair. In Aim 1, we test the hypotheses that brain-wide repair of Mecp2317G>A RNA, by site-directed editing, can be tuned to high efficiency and specificity in mice, and restores proper chromatin interaction. For this purpose, we perform whole transcriptomic RNA seq analysis across the brain after peripheral injections of an efficient brain AAV serotype virus encoding optimized editing or control components. In Aim 2, we test new guides for the ability to recruit endogenous ADAR2 to mutant Mecp2 RNA in vivo, circumventing potential immune responses to the bacteriophage moiety in the hybrid ADAR2 protein and potentially minimizing off-target editing. Our initial model is Mecp2311G>A that has the ideal nonsense codon for this approach. In Aim 3, we inject peripherally the virus encoding our current and optimized editing components, or controls, to test our hypothesis that site-directed RNA editing can stabilize/reverse Rett-like symptoms in both Mecp2G>A mouse lines. In addition to Rett syndrome, our approach has the potential to cure thousands of additional pathogenic G>A mutations.

项目概要在这个修订后的提案中，我们继续开发一种修复碱基突变的方法 RNA，用于减轻人类神经疾病小鼠模型的症状。是 NIH 主任变革性研究奖试点项目的产物，该奖支持最近发表的初步结果证明了这种方法的可行性。目前专注于雷特综合征，这是一种由于基因突变而导致的毁灭性神经系统疾病我们最初关注的是人类患者的鸟苷 (G)。 MECP2 中的腺苷 (A) 突变，MECP2317G>A，严重干扰其结合能力染色质和 Rett 综合征的结果我们首次证明内源性 Mecp2317G>A。在从非分裂神经元培养的非分裂神经元中，RNA 可以有效地重新编码为野生型氨基酸。 Mecp2317G>表现出严重雷特样症状的小鼠品系通过 site- 进行记录。通过劫持的腺苷催化结构域作用于 RNA 2 (ADAR2) 进行定向脱氨 (Editase)，与噬菌体 RNA 结合肽融合，我们将其靶向 Mecp2 突变在这个修订后的应用程序中，我们提供了新的数据，表明记录也可以。直接海马注射后，发生在体内 3 个不同的海马神经元群中此外，记录结果还包括编码混合 ADAR2 蛋白和 Mecp2 RNA 向导的 AAV。 MeCP2 定位到染色质的量与 RNA 水平的编辑量一致。开发了工具和试剂，现在使我们处于解决关键问题的理想位置逆转雷特综合征神经表型和测试的未解答问题与定点修复相关的假设在目标 1 中，我们测试了全脑修复的假设。 Mecp2317G>A RNA，通过定点编辑，可以在小鼠中调整为高效率和特异性，并且恢复正确的染色质相互作用为此，我们进行了全转录组 RNA seq。外周注射高效脑 AAV 血清型病毒编码后的大脑分析在目标 2 中，我们测试了新的招募能力指南。内源性 ADAR2 体内突变 Mecp2 RNA，规避潜在的免疫反应混合 ADAR2 蛋白中的噬菌体部分，并有可能最大限度地减少脱靶编辑。初始模型是 Mecp2311G>A，它具有适合此方法的理想无义密码子。在目标 3 中，我们注入。外围病毒编码我们当前和优化的编辑组件或控件，以测试我们的假设定点 RNA 编辑可以稳定/逆转 Mecp2G>A 中的 Rett 样症状除了雷特综合征之外，我们的方法还有可能治愈数千种小鼠系。其他致病性 G>A 突变。