<p>Cystic fibrosis (CF) is caused by mutation of CFTR, a cAMP-regulated Cl- channel expressing at the apical PM of epithelia. The most common mutant ΔF508 CFTR induces misfolding, leading to ubiquitin-mediated degradation. The CF drug Trikafta improves the PM expression of ΔF508 CFTR. However, the functional ΔF508 CFTR is still removed from the PM in the presence of Trikafta. Previously, we demonstrated that a ubiquitin ligase RFFL directly interacts with ΔF508 CFTR at the PM and endosomes, thereby facilitating the ubiquitination, endocytosis, and degradation of the CFTR mutant. Notably, RFFL knockdown inhibited the CFTR degradation and enhanced the efficacy of Trikafta in cell lines. </p><p>In this study, we determine whether RFFL KD inhibits the degradation of endogenous ∆F508 CFTR and enhances the efficacy of Trikafta in primary bronchial epithelial cells derived from CF patients (CF-HBE), which is the final evaluation system for CF therapeutics in preclinical stages. To achieve the RFFL KD, antisense oligonucleotides (ASO) containing artificial nucleic acids with high intracellular uptake capacity and excellent nuclease resistance were designed. Our results demonstrated that the introduction of the RFFL-ASO resulted in a 1.4-fold improvement in endogenous ∆F508 CFTR function. In conclusion, our study strongly implies that suppressing RFFL enhances the efficacy of CF therapeutic agents in the CF-HBE, underscoring the genuine potential of RFFL as a viable drug target for CF.</p>