Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Airway Epithelial Stem Cells (AESC): an innovative approach to improve Cystic Fibrosis (CF) diagnosis and treatments

Cystic fibrosis (CF) is a multi-system disease caused by pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The corresponding protein is a chloride channel expressed in epithelial cells. The main clinical features are exocrine pancreatic insufficiency, bronchiectasis and lung disease. In CF lungs, defective protein results in a dehydrated surface liquid, compromised mucociliary clearance, thick mucus and consequent chronic infection and inflammation. The damage of the airways eventually leads to respiratory failure. In past years, therapies have been mainly symptomatic, focusing on slowing lung disease progression and compensating for exocrine pancreatic insufficiency. More than 2100 CFTR variants have been described and initially grouped into six functional classes with the following pathogenic consequences: no CFTR protein production (Class I); CFTR protein trafficking defects and premature degradation (Class II); defective regulation of the CFTR channel gating (Class III); decreased conductance of CFTR channel (Class IV); reduced quantity of functional CFTR (Class V); high turnover of CFTR protein at the apical surface (Class VI). However, it has been shown that the majority of CFTR pathogenic variants show multiple biochemical defects and belong to several mutational classes. In addition, most of CFTR variants have a poor functional characterization that prevent a clear classification into mutational classes. These findings have a dramatic translational impact on the so-called precision, mutation-specific, therapies already available to CF patients. For example, drugs targeting class II pathogenic variants (“correctors”) correct protein folding and endoplasmic reticulum export defect, while those targeting class III pathogenic variants (“potentiators”) restore correct gating. Often, the maximal therapeutic effect is reached by combined therapies with a potentiator and different correctors as, for example, in the recent triple therapy (combining one potentiator and two correctors). An emerging class of modulators are the “amplifiers”, defined as compounds that increase the expression of CFTR mRNA and, consequently, the biosynthesis of the CFTR protein. This amplificatory strategy may both enhance the residual functionality of CFTR in mutated genotypes with residual/minimal function and provide a greater amount of mutated CFTR to be corrected or potentiated by other modulators in a synergistic way. However, drugs allowing a full restoration of CFTR function are still needed, and their development and test for all CF patients is challenging. This is especially true for CF patients with rare pathogenic variants, being their low frequency a major obstacle for the accomplishment of valuable clinical trials. Of enormous translational impact is the approach of “theratyping”, which consists in the identification of specific pathogenic variants and genotypes responding to a specific therapy (often combined). This methodology has been approved in USA by the FDA as the unique preliminary step necessary and sufficient for the treatment of patients with the specific responding genotype, by drugs already clinically approved for other pathogenic variants and genotypes. Cell types of various origin have been used as in vitro models of CF, such as immortalized human bronchial epithelial cell lines, which however showed a difficult setup from a large number of patients and the limitations of immortalized cells. In addition, the cellular composition of airways revealed a high complexity, hard to reproduce with a single cellular system. At least 7 different airway epithelial cell types have been demonstrated to contribute to the lung composition: progenitor, secretory club, ciliated, solitary neuroendocrine, goblet and tuft cells, as well as ionocytes. In particular, the ionocyte appears to be a rare cell type characterized by high expression of FOXI1 and CFTR genes and responsible for the greatest part of chloride flux in human airways. The state-of-the-art of precision therapy in CF strongly requires the availability of patient-specific 2D and 3D cellular models to validate personalized therapeutic strategies. Within the most promising patient-specific cellular models that are being evaluated as theratyping tools for CF are the 2D cultures of primary and stem-induced nasal airway epithelial cells, primary intestinal epithelial cells, as well as the corresponding 3D organoids. Patients showing an ex vivo response to theratyping are already eligible to the effective treatments where it is an already approved approach (as in USA) and will become eligible as soon as it will be approved in other Countries (as in Europe). Mutation-specific precision therapies of CF are currently in clinical use for some pathogenic variants. However, CFTR function and relation with clinical status remain poorly understood for a large group of rare ("orphan") variants. This project has the goal to identify, at cellular level, CFTR pathogenic variants which respond to biochemical therapeutic modulators, allowing a quick clinical translation of their use. On the other hand, a more in-depth genetic investigation may be useful to characterize both structurally and functionally rare CFTR variants. Innovative patient-specific cellular models, both differentiated and stem-like, are needed for a meaningful contribution to these aims. Moreover, the possibility to perform theratyping in virtually each CF patient is of great translational impact, with particular relevance for patients with rare CFTR variants. Finally, the Conditionally Reprogrammed Cultures (CRC) technology applied to CF may expand the supply of functional CF respiratory cells for testing personalized therapeutic strategies, including drug testing, cellular and gene therapies.