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.

PROJECT NARRATIVE In cystic fibrosis (CF), a life-limiting lung disease caused by mutations in the CFTR gene, recently FDA-approved therapies still only partially restore the basic defect for some mutations, while other genotypes have no available intervention. We have discovered that small non- coding nucleotide sequences called microRNA are targetable inhibitors of CFTR restoration, applicable across mutation and therapeutic approach. In the proposed experiments, we will test whether antisense oligonucleotide (ASO) steric blockade of miR-145 binding to CFTR selectively improves the effectiveness of current FDA-approved and future investigational CFTR-directed therapies to improve the pulmonary health and quality of life for all patients with the disease.