More than 2000 different sequence variations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described. Mutation-specific personalized therapies of cystic fibrosis (CF) is currently in clinical use for the most frequent CFTR pathogenic variant, the F508del, and some other pathogenic variants. However, CFTR function and relation with clinical status remain poorly understood for the large group of remaining and often rare, "orphan" mutations. A new emerging approach for precision medicine in CF is the so-called theratyping. It has the primary goal to identify, at cellular level, which CFTR pathogenic variants respond to certain biochemical therapeutic modulators, allowing a quick clinical translation of their use. On the other hand, the genetic correction of the defect is the final goal. Within the various approaches of gene therapy, the editing of CRTR gene by the Small Fragment Homologous Replacement (SFHR) revealed particularly promising. This proposal has two main objectives related to CF therapy: the theratyping and the gene editing by SFHR approach. From nasal epithelia of CF patients, by the "culture reprogramming condition" (CRC), we setup patient-specific airway epithelial stem cells (CF-CRC-AESC) and corresponding airway organoids. For both objectives, we will use the CF-CRC-AESC and airway organoids with different CFTR mutated genotypes. For theratyping, the pharmacological response will be assessed for: a) therapeutic agents already in clinical use in CF, that will be tested on rare (till now unexplored) CFTR mutated genotypes; b) an 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 studied, on F508del homozygous CF-CRC-AESC, following the manipulation of 4 main cellular pathways: DNA methylation, chromatin remodeling, DNA repair and cell cycle control.
