This proposal is the continuation and development of researches of the previous year. The cystic fibrosis transmembrane conductance regulator (CFTR) gene has more than 2000 different sequence variations. Precision (mutation-specific) therapy of cystic fibrosis (CF) is currently in clinical use for the most frequent CFTR pathogenic variant, the F508del, and some others. However, CFTR function and exact pathogenic effect remain poorly understood for a large group of rare "orphan" mutations. Theratyping is an approach of precision medicine aimed to identify, at cellular level, which CFTR pathogenic genotypes respond to specific biochemical therapeutic modulators, allowing a quick clinical translation "from bench-to-bedside". On the other hand, also the editing of CFTR gene by the Small Fragment Homologous Replacement (SFHR) is particularly promising. This proposal extends the two main objectives of the previous year related to CF precision therapy: the theratyping and the gene editing by SFHR approach. For both objectives, we will use the CF patient-specific airway epithelial stem cells (CF-CRC-AESC) and corresponding airway organoids, with different CFTR mutated genotypes, obtained by the "culture reprogramming condition" (CRC) from nasal epithelium. This cellular and organoid systems were completely setup and characterized during the previous years. For theratyping, the case series of rare CFTR mutated genotypes will be expanded and the pharmacological response will be assessed for: a) therapeutics already in clinical use in CF, to be tested on rare (till now unexplored) genotypes; b) an epigenetic experimental drug (not in clinical use and until know unexplored in CF). The molecular mechanism and the efficacy of the CFTR gene editing by SFHR approach will be further studied, on F508del homozygous CF-CRC-AESC, pointing to the manipulation of 4 main cellular pathways: DNA methylation, chromatin remodeling, DNA repair and cell cycle control.
