Suppression of disease causingnonsense mutations by targeted mRNA pseudouridylation

在这次试探性的赠款申请中，我们建议充分开发一种新的方法，即有针对性的 RNA假脱氧核糖核酸，以抑制导致疾病的基因的无义突变(例如， 导致囊性纤维化的CFTR基因无义突变和P53无义突变 导致各种癌症的基因)。这个项目如果成功，将是一个巨大的进步 我们的最终目标是为一些遗传性疾病开发新的治疗方法 以及由无义突变引起的某些类型的癌症。我们建议实施这一项目 在两个具体目标下。 目的1.提高RNA引导的人细胞假性尿嘧啶核糖核酸的效率。我们会改进的 通过确定一种更好的修饰酶来提高靶向信使核糖核酸假脱氢酶的效率 (具体地说，更好的盒H/ACA引导RNA)。我们将重点介绍指南中的三个要素 已经被(我们的实验室和其他实验室)确定为对指导 并构建了完美盒H/ACA引导RNA。我们还将设计指南 通过增加核质定位信号，从而将引导RNA(和RNP)靶向 核质，是合成和成熟信使核糖核酸的地方。最后，我们将增加框H/ACA RNA 表达水平，从而提高其在人类细胞核质中的浓度。为了做到这一点，我们 相信我们将能够创造出一种超级向导RNA，它可以有效地指导特定部位的mRNA 假性尿路。 目的2.在人类疾病细胞系中直接靶向疾病基因中的PTC。使用 设计盒H/ACA RNA(尤其是在AIM 1中鉴定的改良超盒H/ACA RNA )，我们将直接针对提前终止密码子(PTC，由废话引起 突变)在CF细胞系中，在人癌细胞中在P53 mRNA中 排队。引导RNA的转染率及其在转基因细胞中的表达水平将 量过了。还将对mRNAs PTC处的位点特异性假尿酸进行量化。此外， 无意义抑制，包括抑制NMD(无意义介导的mRNA衰变)和 PTC通读，将受到监控。简而言之，这项提议提供了一种潜在的新方法 治疗许多与无义突变相关的遗传病和某些类型的癌症。

In this exploratory grant application, we propose to fully develop a novel approach, namely, targeted RNA pseudouridylation, to suppress nonsense mutations in genes that cause diseases (e.g., nonsense mutations in the CFTR gene that cause Cystic Fibrosis, and nonsense mutations in p53 gene that cause various types of cancer). This project, if successful, will constitute a giant step toward our ultimate goal of developing novel therapeutic treatment for a number of genetic disorders and certain types of cancer caused by nonsense mutations. We propose to carry out this project under two specific aims. Aim 1. To improve the efficiency of RNA-guided pseudouridylation in human cells. We will improve the efficiency of targeted mRNA pseudouridylation by identifying a better modifying enzyme (specifically, a better box H/ACA guide RNA). We will focus on the three elements within the guide RNA that have been identified (by our lab and other labs) to be important for directing pseudouridylation, and construct a perfect box H/ACA guide RNA. We will also engineer the guide RNA by adding a nucleoplasmic localization signal, thus targeting the guide RNA (and RNP) to the nucleoplasm where mRNA is synthesized and matures. Finally, we will increase box H/ACA RNA expression level, thus raising its concentration in the nucleoplasm of human cells. In doing so, we believe we will be able to create a super guide RNA that can efficiently direct site-specific mRNA pseudouridylation. Aim 2. To directly target the PTC within a disease gene in human disease cell lines. Using an designer box H/ACA RNA (especially when the improved super box H/ACA RNA identified in Aim 1 is available), we will directly target the premature termination codon (PTC, resulting from nonsense mutations) within the CFTR mRNA in a CF cell line and within the p53 mRNA in a human cancer cell line. The efficiency of guide RNA transfection and its expression level in transfected cells will be measured. Site-specific pseudouridylation at the PTC of mRNAs will also be quantified. Furthermore, nonsense suppression, including the suppression of NMD (nonsense-mediated mRNA decay) and PTC read-through, will be monitored. In short, this proposal presents a potential novel approach to treat many genetic diseases and certain types of cancer associated with nonsense mutations.