Results of the 1-year project FFC#8/2020 (extension of a previous project) consisting in ex vivo expansion, validation and biobanking of 18 long-term cultures of reprogrammed epithelial nasal stem cells (to be added to the 24 banked during the first part of the project). The researchers report that these cells have been obtained from nasal brushings of Cystic Fibrosis (CF) patients with various genotypes (F508del homozygous, compound heterozygous F508del/other, rare genotypes lacking F508del allele), as well as of heterozygotes/carriers and healthy individuals with no CFTR mutation. Based on these stem cells, the research team generated and validated CF models that reproduce the pathologic tissue ex vivo as air liquid interface cultures (ALI-cultures) and nasal organoids. Different assays have been generated and optimized based on these models for the biochemical and functional characterization of CFTR, basal/residual and after pharmacological treatment, to determine the response of different genetic variants (theratyping) to modulators already approved for other variants (Kalydeco, Orkambi, Symkevi, Kaftrio). In particular, a biochemical assay to detect and quantify the corrected CFTR protein in immunoblot, the forskolin-induced swelling of organoids and the fluid re-absorption assay in ALI-cultures to assess and quantify the ability of CFTR modulators to restore CFTR correct protein folding and maturation and function, were applied. These models and assays allowed to demonstrate a general scarce activity of Kalydeco, Orkambi and Symkevi, while a marked Kaftrio response was observed in three heterozygous compound genotypes carrying F508del associated on the second allele to a mutation determining large nucleotide insertions.
Kaftrio response has been evaluated also in rare genetic variants of disease (lacking F508del mutation) with the most promising results for the L1077P CFTR variant.
These cellular models, based on conditionally reprogrammed cells, proved highly precious for theratyping ex vivo and may contribute to therapeutic improvement with clear benefit also for patients with rare variants of CFTR, still lacking approved targeted therapies.