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

摘要 无意义介导的信使核糖核酸衰变(NMD)是一种细胞RNA监测机制，它发挥着 在人类健康和疾病中起着基础性作用。NMD选择性地识别和降级 变异的RNA，如突变的转录本和许多病毒RNA。NMD监管不善是 与各种神经疾病、癌症和疾病的发病和严重程度有关 传染病。因此，控制NMD活动是一种有吸引力的发展途径 治疗许多人类疾病的新疗法。 尽管提前终止密码子(PTC)通读抑制NMD 使用氨基糖苷类抗生素或抑制tRNAs的策略已经被广泛研究， 对它们的疗效和特异性有很大的限制。从概念上讲，这些PTC通读 抑制NMD的策略和产生有限数量的功能蛋白。相比之下， 以选择性降解NMD不敏感靶为目标的NMD诱导概念已经 几乎没有人研究过。诱导NMD的方法对于人类疾病的一个子集是至关重要的 因为据预测，大约四分之一的致病PTC对NMD不敏感。 尽管大多数NMD不敏感的转录本预计会产生截短的蛋白质和 诱导一种功能增益或显性负效应，潜在的分子机制是 在很大程度上没有特征。因此，目前还没有针对NMD不敏感的靶向特异性分子疗法。 精神错乱。 基于十多年来对NMD机制和用途的分子研究经验 尖端CRISPR-Cas13技术，这项提议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- 不敏感的显性负向转录本。目标1是一个概念验证实验，旨在开发 使用明确定义的转录本触发显性阴性转录本的NMD的RP-NMDA方法 NMD报告来源于人类β-珠蛋白(HBB)基因。AIM 2将扩展RP-NMDA 选择性抑制人结直肠癌细胞株APC表达的方法 截断和癌症进展。这个应用程序有很高的前景，可以专门降级 来自突变的NMD不敏感等位基因的异常转录本对 正常的成绩单。如果成功，我提议的创新工作不仅将提供一种疾病- 治疗主要的β-地中海贫血和结直肠癌的特效药物，但也提供了一种 任何NMD不敏感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.