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

项目摘要 囊性纤维化跨膜传导调节因子（CFTR）中的W1282 X无义突变 这种基因会导致严重的囊性纤维化（CF），从而导致呼吸衰竭。但 对突变引起的CF的治疗是不充分的。CFTR-W1282 X基因表达差 由于无义介导的mRNA衰变（NMD），功能性的、截短的CFTR蛋白在非常低的水平。在 CF的背景下，NMD通过以下方式评估了CFTR-W1282 X突变的CF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疗法， CF，需要对NMD机制有更深入的了解。NMD需要外显子的沉积 连接复合物（EJC）。位于提前终止下游的EJC 密码子（PTC）招募NMD因子，如SMG 6，其介导核酸内切裂解，这是一个关键的 EJC依赖性NMD途径中的不可逆步骤。我们的实验表明，多个下游EJC 协同增强NMD;和合成反义寡核苷酸（ASO），旨在防止 PTC下游的EJC结合可以以基因特异性方式减弱NMD。然而，NMD 影响NMD协同增效的因素尚不清楚。CFTR-W1282 X位于三个 推定的EJC地点。这三个EJC对NMD的贡献是否相等尚不得而知。此外，无论 CFTR-W1282 X mRNA依赖SMG 6的NMD尚未研究。建议的目标 研究旨在阐明PTC下游的多个EJC对CFTR NMD的作用， W1282X mRNA。开发针对每个下游EJC的ASO将促进 了解个体EJC增强NMD的机制。首先，我将开发EJC- 靶向恢复人气道CFTR-W1282 X蛋白表达和功能的ASO 上皮细胞其次，我将验证EJC目标ASO削弱NMD的机制， CFTR-W1282X mRNA。我将使用RNA免疫沉淀比较EJC与CFTR mRNA的结合， 没有阿索治疗。第三，虽然SMG 6是最有可能的中介EJC依赖NMD，其他 NMD因子可能在CFTR-W1282 X mRNA的NMD中起部分冗余作用。这可能会减少 SMG 6在人气道上皮细胞中CFTR-W1282 X的NMD中的作用。我将测试SMG 6是否播放 使用RNAi敲低，在CFTR-W1282 X mRNA的EJC依赖性NMD中起主要作用。我必 系统地分析每个下游EJC对SMG 6介导的内切割的影响， 控制下游EJC的数量和位置，使用遗传和反义定向 方法.这一项目的成果将为深入了解NMD机制提供依据， 有证据表明，阿索技术可作为通过NMD抑制恢复CFTR功能的策略。

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.