Antisense-oligonucleotide-directed inhibition of nonsense-mediated mRNA decay of CFTR gene

项目摘要 囊性纤维化跨膜电导调节基因W1282X无义突变 基因会导致一种严重的囊性纤维化(CF)，这可能导致呼吸衰竭。然而， 对突变引起的CF的治疗不够充分。CFTR-W1282X基因表达水平较低 由于无意义介导的mRNA衰变(NMD)，功能上的、截短的CFTR蛋白处于非常低的水平。在……里面 慢性阻塞性肺疾病的背景下，NMD通过以下方式恶化携带CFTR-W1282X突变的慢性阻塞性肺疾病患者的临床结局 降低部分活性突变蛋白的表达。开发有效的基因特异性治疗方法 Cf，需要对NMD机制有更深入的了解。NMD需要外显子的沉积 剪接mRNA上的连接复合体(EJC)。一个位于提前终止的下游的EJC 密码子(PTC)招募NMD因子，如介导核内裂解的SMG6，这是一个关键 EJC依赖的NMD途径中不可逆转的一步。我们的实验室显示，多个下游的ejc 协同增强NMD；以及合成反义寡核苷酸(ASO)，旨在防止 PTCs下游EjCs的结合可以基因特异性的方式减弱NMD。然而，什么是NMD 介导NMD协同增强的因素尚不清楚。CFTR-W1282X位于三个 推定的EJC站点。这三个ejc对国家导弹防御系统的贡献是否相等尚不得而知。此外，是否 Cftr-W1282X基因的NMD依赖于SMG6的研究尚未见报道。建议的目标是 研究是为了阐明PTC下游的多个JEC在CFTR的NMD中的作用- W1282X基因。开发针对每个下游JEC的ASO将有助于 了解单个EGC增强NMD的机制。首先，我将开发EJC-- 靶向ASO可恢复人呼吸道CFTR-W1282X蛋白的表达和功能 上皮细胞。其次，我将验证EJC靶向ASO减弱NMD的机制。 Cftr-W1282X基因。我将使用RNA免疫沉淀来比较EJC与CFTRmRNA的结合 而且没有接受ASO治疗。第三，虽然SMG6是最有可能的EJC依赖型NMD的介体，但其他 NMD因子可能在CFTR-W1282X基因的NMD中起部分冗余作用。这可能会减少 SMG6在人呼吸道上皮细胞CFTR-W1282X NMD中的作用我将测试SMG6是否播放 使用RNAi敲除在依赖EJC的CFTR-W1282X mRNA的NMD中发挥主要作用。还有，我会的 系统分析每个下游EJC对SMG6介导的内切割的影响，通过 利用基因和反义基因控制下游EjC的数量和位置 方法：研究方法。该项目的结果将提供对NMD机制的见解，并提供支持 将ASO技术确立为通过抑制NMD恢复CFTR功能的策略的证据。

Project abstract The W1282X nonsense mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes a severe form of cystic fibrosis (CF) which can lead to respiratory failure. However, the treatment for CF caused by the mutation is inadequate. The CFTR-W1282X gene expresses poorly functional, truncated CFTR protein at a very low level, due to nonsense-mediated mRNA decay (NMD). In the context of CF, NMD worsens the clinical outcome of CF patients with CFTR-W1282X mutation by reducing the expression of partially active mutant protein. To develop effective gene-specific therapies for CF, a deeper understanding of the NMD mechanism is needed. NMD requires the deposition of the exon junction complex (EJC) on spliced mRNA. An EJC positioned downstream of a premature-termination codon (PTC) recruits NMD factors, such as SMG6 which mediates endonucleolytic cleavage, a key irreversible step in the EJC-dependent NMD pathway. Our lab showed that multiple downstream EJCs cooperatively potentiate NMD; and synthetic antisense oligonucleotides (ASOs) designed to prevent binding of EJCs downstream of PTCs can attenuate NMD in a gene-specific manner. However, what NMD factors mediate cooperative potentiation of NMD is not understood. CFTR-W1282X lies upstream of three presumptive EJC sites. Whether all three EJCs contribute equally to NMD is unknown. Moreover, whether NMD of CFTR-W1282X mRNA depends on SMG6 has not been investigated. The goal of the proposed research is to elucidate the role of multiple EJCs downstream of the PTC contribute to the NMD of CFTR- W1282X mRNA. Developing ASOs that target each of the downstream EJCs will facilitate the understanding of the mechanism by which individual EJCs potentiate NMD. First, I will develop EJC- targeting ASOs that could restore expression and function of CFTR-W1282X protein in human airway epithelial cells. Second, I will validate the mechanism by which the EJC-targeting ASOs attenuate NMD of CFTR-W1282X mRNA. I will use RNA-immunoprecipitation to compare EJC binding to CFTR mRNA with and without ASO treatment. Third, while SMG6 is the most likely mediator of EJC-dependent NMD, other NMD factors may play partially redundant roles in NMD of CFTR-W1282X mRNA. This may diminish the role of SMG6 in the NMD of CFTR-W1282X in human airway epithelial cells. I will test whether SMG6 plays a major role in EJC-dependent NMD of CFTR-W1282X mRNA, using RNAi knockdown. Also, I will systematically analyze the impact of each downstream EJC on SMG6-mediated endocleavage, by controlling the number and location of downstream EJCs, using both genetic and antisense-directed methods. Results from this project will provide insights into the NMD mechanism, and provide supporting evidence to establish ASO technology as a strategy to restore CFTR function by NMD inhibition.