Repair of neurological disorders through RNA editing

通过RNA编辑修复神经系统疾病

• 批准号: 10532148
• 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
• 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/10532148
• 关键词: Address; Adenosine; Adolescent; Adult; Amino Acids; Astrocytes; Award; Back; Bacteriophages; Base Pairing; Behavior; Binding; Biological Assay; Brain; Brain region; CRISPR/Cas technology; Catalytic Domain; Cell Line; Cells; Chromatin; Code; Codon Nucleotides; Complementary DNA; Data; Deamination; Disease; Dose; Elements; Enzymes; Exhibits; Female; Funding; Future; Genes; Genetic Enhancer Element; Guanosine; Guide RNA; Hand Strength; Hippocampus; 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; Stereotyping; Symptoms; Technology; Testing; Therapeutic; Time; Transfection; Translations; United States National Institutes of Health; Viral; Virus; Work; base; cellular imaging; chromatin protein; design; disease-causing mutation; dsRNA adenosine deaminase; experimental study; genome editing; human model; improved; in vivo; male; mouse model; mutant; nervous system disorder; neural; next generation; novel; nuclease; posttranscriptional; prevent; promoter; recruit; repaired; tool; transcription factor; transcriptome sequencing; transcriptomics

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.

项目摘要 在这项修订的建议中，我们继续开发一种修复基本突变的方法 RNA，用于减轻人类神经疾病小鼠模型中的症状。实验 是飞行员NIH董事的变革性研究奖的产物，该奖项支持两者 最近发表的和初步的结果证明了这种方法的可行性。我们的工作是 目前专注于Rett综合征，这是由于基因突变引起的毁灭性神经系统疾病 编码转录因子MECP2。我们最初专注于人类患者鸟嘌呤（G） MECP2，MECP2317G> a中的腺苷突变（a）突变，它严重干扰了其与结合的能力 染色质，并导致RETT综合征。我们首次展示了内源性MECP2317G> a RNA可以在从A培养的非分裂神经元中有效地重新编码为野生型氨基酸 MECP2317G>显示出严重RETT样症状的小鼠系。通过站点进行了回收 - 作用于RNA 2（ADAR2）的被劫持的腺苷的催化域的定向死亡 （编辑酶），融合到噬菌体RNA结合肽中，我们针对MECP2突变 通过RNA指南。在此修订的应用程序中，我们提供了新数据，表明还重新编码 在直接海马注射后，在体内发生的3种不同的海马神经元种群 编码混合ADAR2蛋白和MECP2 RNA指南的AAV。此外，重新编码导致 MECP2定位到染色质的量与RNA水平的编辑量一致。我们 已经开发了现在将我们置于解决关键的理想位置的工具和试剂 未解决的问题，用于逆转RETT综合征的神经表型和测试 与站点定向修复有关的假设。在AIM 1中，我们检验了大脑范围维修的假设 MECP2317G>通过站点定向编辑的RNA可以调节小鼠的高效率和特异性，并且可以调整 恢复正确的染色质相互作用。为此，我们执行整个转录组RNA SEQ 外围注射有效的大脑AAV血清型病毒编码后，整个大脑的分析 优化的编辑或控制组件。在AIM 2中，我们测试了新指南的招募能力 内源性ADAR2到体内突变的MECP2 RNA，规避了对该的潜在免疫反应 杂交ADAR2蛋白中的噬菌体部分，并可能最大程度地减少靶向编辑。我们的 初始模型是MECP2311G> A具有适用于这种方法的理想废话密码子。在AIM 3中，我们注入 在外围编码我们当前和优化的编辑组件或控件的病毒，以测试我们的 假设位置定向的RNA编辑可以在MECP2G> A中稳定/反向RETT样症状 鼠标线。除了RETT综合征，我们的方法还可以治愈数千种 其他致病性G> A突变。