Developing Splicing-Targeted Therapeutic Strategies for Neurological Diseases

摘要 信使RNA（mRNA）的选择性剪接是控制转录本定位的调节机制， 翻译和稳定性，并且能够从单个基因表达多种蛋白质同种型。在神经元中， 剪接是精细控制的，并且对于调节神经发生、神经元迁移和结构， 突触发生和突触功能。越来越多的证据强调了剪接改变在神经系统疾病中的作用。 疾病，强调需要更好地了解RNA加工的机制，以发展 剪接靶向治疗最近的研究表明，神经元可以控制基因表达在关键 通过将选择性剪接与无义介导的衰变（NMD）偶联，来研究发育阶段。这一机制 依赖于包含盒式外显子，即所谓的毒外显子，来产生框内提前终止密码子 从而触发转录NMD并降低蛋白质表达。重要的是，促进组成型 包含有毒外显子与神经发育有关，最近， 神经退行性疾病然而，该领域缺乏严格的方法来识别和注释毒物外显子 以及影响其剪接的变体。一个公认的例子，其中增加外显子包含可导致 神经系统疾病的一个重要原因是微管相关蛋白tau（MAPT）的异常剪接。致病 促进MAPT外显子10包含的剪接突变导致4 R-tau同种型表达增加， 异常tau蛋白积聚，而外显子10中的错义和缺失功能获得性突变与 突变4 R-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。鉴于疾病中错配的相关性增加，拟议工作的目标 是为了促进对剪接改变在神经系统疾病中的作用的理解， 开发剪接靶向疗法。在目标1中，我们将开发一种转录组学方法来定位毒物 与神经元发育和存活相关的外显子，并通过将鉴定的毒性外显子与 ClinVar致病性变异，我们将对可能影响其剪接并有助于 疾病然后，产生CRISPR/Cas9-工程化的人诱导多能干细胞（iPSC）-衍生的细胞。 神经元模型的选择数量的这些突变将使我们能够评估的影响，异常毒物 神经元表型上的外显子包含。在目标2中，我们将使用一种mRNA靶向策略， 外显子10跳跃作为剪接调节剂化合物的治疗潜力的原理证明， 在额颞叶痴呆（FTD）的患者源性神经元模型中挽救神经元疾病表型。 开发新的mRNA靶向疗法，专门纠正导致 疾病将是一个重大进展，为治疗不可治愈的神经系统疾病所造成的剪接 改变。

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.