Suppression of Dominant-Negative Transcripts Escaping from Nonsense-Mediated mRNA Decay

抑制从无义介导的 mRNA 衰变中逃脱的显性负转录本

• 批准号: 10707154
• 负责人: Tatsuaki Kurosaki
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10707154
• 关键词: 3' Untranslated Regions; APC gene; Affect; Alleles; Aminoglycoside Antibiotics; Cells; Code; Colorectal Cancer; Communicable Diseases; Complex; Data; Deposition; Disease; Dominant-Negative Mutation; Exons; Genes; Genetic Diseases; Goals; Health; Homeostasis; Human; Human Genetics; Inherited; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Methodology; Methods; MicroRNAs; Molecular; Mutate; Nonsense Mutation; Onset of illness; Pharmaceutical Preparations; Phenotype; Physiological; Play; Positioning Attribute; Production; Protein Truncation; Proteins; RNA; RNA Editing; RNA Splicing; Reporter; Repression; Ribosomes; Role; Severities; Severity of illness; Signal Transduction; Somatic Mutation; Specificity; System; Technology; Terminator Codon; Therapeutic; Toxic effect; Transcript; Transfer RNA; Translating; Translations; Tumor Suppressor Proteins; Virus Diseases; Work; beta Globin; beta Thalassemia; cancer cell; cell motility; colon cancer cell line; experience; experimental study; gain of function; human disease; innovation; mRNA Decay; mRNA Precursor; malignant neurologic neoplasms; nervous system disorder; novel; novel therapeutic intervention; novel therapeutics; preference; premature; tumor progression; viral RNA

Abstract Nonsense-mediated mRNA decay (NMD) is a cellular RNA surveillance mechanism that plays a fundamental role in human health and disease. NMD selectively recognizes and degrades aberrant RNAs such as mutated transcripts and many viral RNAs. NMD misregulation is associated with disease onset and severity in various neurological disorders, cancers, and infectious diseases. Therefore, controlling NMD activity is an attractive approach to developing novel therapeutics for many human diseases. Although the suppression of NMD by premature termination codon (PTC) read-through strategies using aminoglycoside antibiotics or suppressor tRNAs has been widely studied, there are significant limitations to their efficacy and specificity. Conceptually, these PTC read-through strategies inhibit NMD and produce limited quantities of functional proteins. In contrast, the concept of NMD induction with the goal of selectively degrading NMD-insensitive targets has barely been studied. The method of NMD induction is critical for a subset of human diseases because about one-fourth of disease-causing PTCs are predicted to be insensitive to NMD. Although most of the NMD-insensitive transcripts are expected to produce truncated proteins and induce a gain-of-function or dominant-negative effect, the underlying molecular mechanisms are largely uncharacterized. Thus, there is no target-specific molecular therapy for NMD-insensitive disorders. Based on over ten years of experience in molecular studies of NMD mechanisms and using cutting-edge CRISPR-Cas13 technology, this proposal aims to establish a novel therapeutic approach, namely, the RNA-Programmed NMD Activation (RP-NMDA) system, to suppress NMD- insensitive dominant-negative transcripts. Aim 1 is a proof-of-concept experiment to develop the RP-NMDA methodology to trigger NMD of dominant-negative transcripts using a well-defined NMD reporter derived from the human beta-globin (HBB) gene. Aim 2 will extend the RP-NMDA approach to human colorectal cancer cell lines to selectively suppress both the expression of APC truncations and cancer progression. This application has high promise to specifically degrade aberrant transcripts derived from a mutated NMD-insensitive allele without any toxic effects on normal transcripts. If successful, my proposed innovative work will not only provide a disease- specific and efficient drug for dominant beta-thalassemia and colorectal cancers but also provide a potential therapeutic strategy for any NMD-insensitive disorders.

抽象的 废话介导的mRNA衰减（NMD）是一种细胞RNA监测机制，它发挥 在人类健康和疾病中的基本作用。 NMD有选择地识别和降解 异常的RNA，例如突变的转录本和许多病毒RNA。 NMD的正直是 与疾病发作和各种神经系统疾病，癌症和严重程度有关 传染病。因此，控制NMD活动是一种有吸引力的开发方法 许多人类疾病的新型治疗学。 虽然过早终止密码子（PTC）抑制NMD读取 使用氨基糖苷抗生素或抑制剂TRNA的策略已广泛研究，那里 是其功效和特异性的重大局限性。从概念上讲，这些PTC读取 策略抑制NMD并产生有限的功能蛋白。相反， NMD诱导的概念的目标是选择性降低NMD不敏感的目标的概念 几乎没有研究。 NMD诱导方法对于人类疾病的一部分至关重要 因为预计大约四分之一的引起疾病的PTC对NMD不敏感。 尽管预计大多数NMD不敏感的转录本都会产生截短的蛋白质和 诱导功能获得或显性阴性效应，潜在的分子机制是 在很大程度上没有特色。因此，没有针对NMD不敏感的靶标特异性分子疗法 疾病。 基于NMD机制分子研究的十多年的经验并使用 尖端的CRISPR-CAS13技术，该建议旨在建立一种新颖的治疗性 即RNA程序编程的NMD激活（RP-NMDA）系统以抑制NMD- 不敏感的显性阴性转录本。 AIM 1是概念验证实验，以开发 使用明确定义的RP-NMDA方法触发主要阴性转录本的NMD NMD报告基因源自人β-珠蛋白（HBB）基因。 AIM 2将扩展RP-NMDA 人类结直肠癌细胞系的方法有选择地抑制APC的表达 截断和癌症进展。该应用程序具有很高的承诺，可以专门降级 源自突变的NMD不敏感等位基因的异常转录本没有对 正常的成绩单。如果成功，我提出的创新工作不仅会提供疾病 - 特定而有效的药物，用于主要的β-甲无血症和结直肠癌，但也提供 任何对NMD不敏感疾病的潜在治疗策略。