B28-09 Phenotype Evaluation and Messenger RNA Therapeutic Exploration of CFTR P.G970D Mutation

Abstract Background Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by variants in the CFTR gene. While common variants have been extensively studied, rare variants like CFTR: c.2909GA; p.G970D(G970D) — the most prevalent CFTR variant in Chinese CF patients — remain poorly understood due to insufficient functional validation. This knowledge gap limits both clinical application of CFTR modulators and development of targeted therapies to rare variants. To bridge the gap, we generated cell lines expressing G970D-CFTR and a novel mouse model to assess the phenotypic impact of this variant and evaluated its response to CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) in primary airway epithelial cells in vitro. Furthermore, we developed a mutation-agnostic mRNA therapy that may offer a new treatment avenue for patients carrying this and other similar variants. Methods The pathogenicity of the G970D variant was investigated using Western blot and patch-clamp assays in HEK293 cells transiently transfected with WT or mutant CFTR expression plasmids. A Cftrp.G965D/p.G965D (G965D) mouse model (corresponding to human G970D) was generated via CRISPR/Cas9 for phenotyping. Human nasal and bronchial epithelial cells (hN/BECs) from homozygous and heterozygous G970D patients were collected and subjected to primary cell culture and air-liquid interface differentiation. Following complete cellular differentiation, Ussing chamber measurements were performed to evaluate CFTR-mediated ion transport and assess the functional response of this variant to ELX/TEZ/IVA and our novel mRNA therapy, which delivers full-length CFTR mRNA to cells via a polymeric complex delivery system. Results Western blot and patch-clamp analysis revealed that the G970D variant exhibited normal protein expression and maturation but moderately reduced channel activity compared to WT-CFTR. G965D mice showed growth impairment, reduced survival, and intestinal abnormalities. Ussing chamber assays demonstrated that both homozygous and heterozygous G970D hN/BECs retained only 25-50% of healthy CFTR function. ELX/TEZ/IVA treatment effectively restored CFTR function in these cells to 100-140% of healthy control levels. Our novel mRNA therapy achieved comparable efficacy to ELX/TEZ/IVA. Conclusion G970D is a pathogenic CFTR variant, it does not impair protein expression but causes a gating defect. Differentiated hN/BECs carrying this variant retain partial CFTR function, measuring 25-50% of that in healthy controls, suggesting its classification as a Class III mutation. Therapeutically, the triple-combination modulator therapy ELX/TEZ/IVA was effective in treating this defect, mutation-agnostic mRNA-based therapy demonstrated comparable efficacy to ELX/TEZ/IVA, showing promise as a optional treatment for this and other similar variants. This abstract is funded by: National Natural Science Foundation of China