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

摘要 无义介导的mRNA衰变（NMD）是一种细胞RNA监视机制， 在人类健康和疾病中的重要作用。NMD有选择地识别和降解 异常RNA如突变的转录物和许多病毒RNA。国家导弹防御系统的错误管理是 与各种神经系统疾病、癌症和 传染病因此，控制NMD活动是发展 用于许多人类疾病的新疗法。 虽然通过提前终止密码子（PTC）通读抑制NMD， 使用氨基糖苷类抗生素或抑制性tRNA的策略已被广泛研究， 对它们的功效和特异性有很大的限制。从概念上讲，这些PTC通读 策略抑制NMD并产生有限数量的功能蛋白。而反观 以选择性地降低对国家导弹防御系统不敏感的目标为目标的国家导弹防御系统诱导概念， 几乎没有被研究过。NMD的诱导方法对人类疾病的一个子集至关重要 因为据预测，约四分之一的致病PTC对NMD不敏感。 尽管预期大多数NMD不敏感的转录物产生截短的蛋白质， 诱导功能获得性或显性负效应，潜在的分子机制是 基本上没有特征。因此，目前还没有针对NMD不敏感性的靶向特异性分子治疗。 紊乱 基于十多年的NMD分子机制研究经验和应用 尖端的CRISPR-Cas 13技术，该提案旨在建立一种新的治疗方法， 方法，即RNA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。目标1是一个概念验证实验， RP-NMDA方法使用定义明确的 NMD报告基因来源于人β-珠蛋白（HBB）基因。目标2将扩展RP-NMDA 人结肠直肠癌细胞系选择性抑制APC表达的方法 截短和癌症进展。该应用程序具有很高的承诺， 来源于突变的NMD不敏感等位基因的异常转录物，对 正常的成绩单如果成功的话，我所提出的创新工作不仅能提供一种疾病- 用于显性β-地中海贫血和结肠直肠癌特异性和有效的药物， 任何NMD不敏感性疾病的潜在治疗策略。

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.