Developing Splicing-Targeted Therapeutic Strategies for Neurological Diseases

开发针对神经系统疾病的剪接靶向治疗策略

基本信息

批准号：
10669256
负责人：
Elisabetta Morini
金额：
$ 42万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10669256
关键词：
Address Affect Alternative Splicing Atlases Biological Biological Assay Brain CRISPR/Cas technology Catalogs Cell model ClinVar Clustered Regularly Interspaced Short Palindromic Repeats Coupling Data Data Set Defect Development Disease Equipment and supply inventories Event Excision Exclusion Exons Familial Dysautonomia Frontotemporal Dementia Gene Expression Gene Expression Regulation Generations Genes Genetic Diseases Genotype Goals Human Human Engineering In Vitro Induced pluripotent stem cell derived neurons Knowledge Lead MAPT gene Maps Measures Mediating Messenger RNA Methods Modeling Molecular Mutation Nerve Degeneration Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Nonsense-Mediated Decay Pathogenesis Pathogenicity Pathology Patients Phenotype Play Poison Protein Isoforms Proteins Proteome RNA Processing RNA Splicing RNA, Messenger, Splicing RNA-targeting therapy Regulation Research Role Structure Tauopathies Terminator Codon Testing Therapeutic Therapeutic Intervention Tissues Transcript Translating Translations Untranslated RNA Variant Work disease phenotype exon skipping gain of function gain of function mutation genetic variant genomic data in silico migration mutant nervous system disorder neurogenesis neuron development neuronal survival novel novel therapeutic intervention premature protein expression synaptic function synaptogenesis targeted treatment tau Proteins tau aggregation therapeutic development therapeutic evaluation transcriptome transcriptome sequencing transcriptomics

项目摘要

Abstract Alternative splicing of messenger RNA (mRNA) is a regulatory mechanism that controls transcript localization, translation, and stability, and enables the expression of multiple protein isoforms from a single gene. In neurons, splicing is finely controlled and is critical for regulation of neurogenesis, neuronal migration and structure, synaptogenesis, and synaptic function. Growing evidence highlights a role for splicing alterations in neurological diseases, emphasizing the need to better understand the mechanisms of RNA processing in order to develop splicing-targeted therapies. Recent studies have shown that neurons can control gene expression during critical developmental stages by coupling alternative splicing with nonsense mediated decay (NMD). This mechanism relies on inclusion of cassette exons, known as poison exons, to create in-frame premature termination codons that trigger transcript NMD and reduce protein expression. Importantly, genetic variants promoting constitutive inclusion of poison exons have been associated with neurodevelopmental and, more recently, neurodegenerative diseases. However, the field lacks rigorous methods to identify and annotate poison exons and variants affecting their splicing. A well-established example in which increased exon inclusion can lead to neurological diseases is the aberrant splicing of the microtubule associated protein tau (MAPT). Pathogenic splicing mutations that promote MAPT exon 10 inclusion lead to increased 4R-tau isoform expression and aberrant tau accumulation, whereas missense and deletion gain-of-function mutations in exon 10 are associated with mutant 4R-tau pathology. In both contexts, an approach that promotes MAPT exon 10 exclusion would be therapeutically beneficial. Given the increase relevance of misplicing in disease, the goal of the proposed work is to advance the understanding of the role of splicing alterations in neurological diseases with the objective of developing splicing-targeted therapeutics. In Aim 1, we will develop a transcriptomic approach to map poison exons relevant for neuronal development and survival, and by intersecting the identified poison exons with ClinVar pathogenic variants, we will catalog mutations that are likely to affect their splicing and contribute to disease. Then, generation of CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC)-derived neuronal models for a selective number of these mutations will allow us to evaluate the impact of aberrant poison exon inclusion on neuronal phenotypes. In Aim 2, we will use a mRNA-targeted strategy that promotes MAPT exon 10 skipping as proof-of-principle for the therapeutic potential of splicing modulator compounds, by showing rescue of neuronal disease phenotypes in patient-derived neuronal models of frontotemporal dementia (FTD). The development of novel mRNA-targeted therapies that specifically correct the molecular defects leading to disease will be a major advance for the treatment of incurable neurological diseases caused by splicing alterations.

抽象的 Messenger RNA的替代剪接（mRNA）是控制转录本定位的调节机制，翻译和稳定性，并能够从单个基因中表达多种蛋白质同工型。在神经元中，剪接受到细微的控制，对于调节神经发生，神经元迁移和结构，至关重要，突触发生和突触功能。越来越多的证据突出了神经系统剪接改变的作用疾病，强调需要更好地了解RNA处理的机制以发展针对性的疗法。最近的研究表明，神经元可以控制临界过程中的基因表达发育阶段通过将替代剪接与胡说八道介导的衰减（NMD）耦合。这种机制依靠包含盒式外显子（称为毒药外显子）来创建框架内终止密码子这会触发转录本NMD并降低蛋白质表达。重要的是，促进本构的遗传变异包括毒药外显子与神经发育有关，最近，神经退行性疾病。但是，该领域缺乏识别和注释毒药外显子的严格方法以及影响其剪接的变体。一个公认的例子，其中增加的外显子纳入可以导致神经疾病是微管相关蛋白TAU（MAPT）的异常剪接。致病性促进MAPT外显子10包含的剪接突变导致4R-TAU同工型表达和异常的tau积累，而外显子10中的错义和删除功能突变是相关的与突变4R-TAU病理学。在这两种情况下，促进MAPT外显子10排除的方法将是治疗上有益。鉴于疾病中错误解释的相关性增加，拟议工作的目标是为了促进对神经疾病中剪接改变的作用的理解，目的开发针对剪接的治疗剂。在AIM 1中，我们将开发一种转录组方法来绘制毒药与神经元发育和生存相关的外显子，并通过与确定的毒药外显子与 Clinvar致病性变体，我们将分类可能影响其剪接的突变，并有助于疾病。然后，生成CRISPR/CAS9工程的人类诱导多能干细胞（IPSC）衍生选择性数量的这些突变的神经元模型将使我们能够评估异常毒物的影响外显子纳入神经元表型。在AIM 2中，我们将使用以mRNA为目标的策略来促进MAPT 外显子10跳过作为剪接调制器化合物的治疗潜力的原则证明，通过显示在额颞痴呆（FTD）的患者衍生神经元模型中营救神经元疾病表型。新型mRNA靶向疗法的发展，该疗法专门纠正了导致的分子缺陷疾病将是治疗因剪接引起的无法治愈的神经系统疾病的重大进步改变。