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

项目总结 囊性纤维化是一种限制生命的常染色体隐性遗传性肺部疾病，每3500人中就有一人患病 儿童，是由囊性纤维化传导性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 最常见的突变(F508del)，但其他常见的基因型，如早产儿 终止密码子(PTC)无义突变，缺乏有效干预。即使在F508del中也是如此 患者中，25%对最新的三重组合调节剂鸡尾酒反应较低。 我们发现，一种名为miR-145的非编码小RNA调控CFTR基因 Cftr调节子的表达和阻断效应。我们之前的数据显示， MIR-145拮抗剂改善了F508del的治疗效果。这个项目将采取更精确的战略。 它利用反义寡核苷酸(ASO)仅立体地阻止miR-145与 在不中断其他路径的情况下进行CFTR。 我们假设ASO指导的miR-145靶点阻断可以改善CFTR矫正。 我们提出三个目标： 具体目标#1：通过ASO封锁有选择地增强F508del CFTR校正 MiR-145结合位点的位置。 具体目标#2：在低位通过miR-145操作支持CFTR校正 响应者。 具体目标#3：使用miRNA抑制来增强PTC的通读效率 突变。 总之，这些目标研究了miR-145抑制作为一种新的策略来改善下一步- 生成CFTR校正。目的1测试ASO靶点封锁的有效性和选择性 增强F508del CF呼吸道上皮细胞及相关动物对CFTR调节剂的反应 模特们。AIM 2使用来自低应答者的患者鼻样进行FDA批准的CFTR 测试miR-145抑制是否提高个体利益的调节器。AIM 3调查使用 在突变中抑制MIR-145增加可用于PTC通读的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指导治疗的战略，广泛适用于CFTR干预 和病人的基因型别。

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.