miR-145 target site blockade is a selective strategy to enhance CFTR restoration and readthrough

项目摘要 囊性纤维化（CF）是一种限制生命的常染色体隐性肺病，影响1/3500 是由囊性纤维化跨膜传导调节因子突变引起的 （CFTR）基因。最近FDA批准的CFTR调节剂部分改善了肺功能， 最普遍的突变（F508 del），但其他常见的基因型，如早熟 终止密码子（PTC）无义突变，缺乏有效干预。即使在F508 del 患者中，25%对最新的三联组合调节剂鸡尾酒低应答。 我们发现一种叫做miR-145的小的非编码microRNA调控CFTR基因， 表达并阻断CFTR调节剂的功效。我们之前的数据显示， miR-145拮抗作用改善F508 del治疗。这个项目将采取更精确的战略 其利用反义寡核苷酸（阿索）仅在空间上阻碍miR-145与 CFTR不中断其他途径。 我们假设ASO指导的miR-145靶位点阻断改善CFTR校正。 我们提出三个目标： 具体目标#1：通过阿索阻断选择性增强F508 del CFTR校正 miR-145的结合位点。 具体目标#2：通过miR-145操作在低水平下支持CFTR校正 响应者。 具体目标#3：采用miRNA抑制来增强PTC中的通读功效 突变。 总之，这些目的研究miR-145抑制作为改善下一代的新策略。 代CFTR校正。目的1：检测阿索靶点阻断的有效性和选择性 增强F508 del CF气道上皮细胞和相关动物中的CFTR调节剂应答 模型目的2使用来自FDA批准的CFTR低应答者的患者源性鼻样本 调节剂来测试miR-145抑制是否增强个体益处。Aim 3研究使用 miR-145抑制增加突变中PTC通读可用的CFTR底物 目前缺乏有效的治疗方法。这些实验将检验一种新颖的、高度选择性的 推进CFTR导向疗法的战略，广泛适用于CFTR干预 和患者基因型。

PROJECT SUMMARY Cystic fibrosis (CF), a life-limiting autosomal recessive lung disease that affects 1 in 3500 children, is caused by mutations in the Cystic Fibrosis Transmembane conductance Regulator (CFTR) gene. Recently FDA-approved CFTR modulators partially improve lung function for the most prevalent mutation (F508del), but other common genotypes, such as premature termination codon (PTC) nonsense mutations, lack an effective intervention. Even in F508del patients, 25% are low responders to the latest triple combination modulator cocktail. We discovered that a small non-coding microRNA called miR-145 regulates CFTR gene expression and blocks efficacy of CFTR modulators. Our previous data show that complete miR-145 antagonism improves F508del therapy. This project will pursue a more precise strategy that utilizes an antisense oligonucleotide (ASO) to sterically impede only miR-145 binding to CFTR without interrupting other pathways. We hypothesize that ASO-directed miR-145 target site blockade improves CFTR correction. We propose 3 Aims: Specific Aim #1: Enhance F508del CFTR correction selectively through ASO blockade of the miR-145 binding site. Specific Aim #2: Bolster CFTR correction through miR-145 manipulation in low responders. Specific Aim #3: Employ miRNA inhibition to augment readthrough efficacy in PTC mutations. Together, these Aims investigate miR-145 inhibition as a novel strategy to improve next- generation CFTR correction. Aim 1 tests the efficacy and selectivity of ASO target site blockade to enhance CFTR modulator response in F508del CF airway epithelial cells and relevant animal models. Aim 2 uses patient-derived nasal samples from low responders to FDA-approved CFTR modulators to test whether miR-145 inhibition boosts individual benefit. Aim 3 investigates using miR-145 inhibition to increase the CFTR substrate available for PTC readthrough in mutations that currently lack an effective therapy. These experiments will examine a novel, highly selective strategy to advance CFTR-directed therapeutics, broadly applicable across CFTR intervention and patient genotype.