Novel Combined approaches to PTC mutation correction in CFTR

Tese de mestrado, Biologia Molecular e Genética, 2024, Universidade de Lisboa, Faculdade de CiênciasCystic Fibrosis (CF) is a life-threatening genetic disease caused by deleterious mutations in the CFTR gene. Of all variants, 8,4% are nonsense mutations (class I) which are associated with premature termination codons (PTCs), which produce severe phenotypes of CF. In this work, a HEK cell line expressing a G542X CFTR minigene (HEK-GX) was used to test compounds for their PTC readthrough and NMD inhibitory activities. Preliminary screening demonstrated that G418 and SMG1i were the most useful control compounds at concentrations 500uM and 1uM, respectively. The library of novel compounds’ screen identified 7 compounds with Z-score > 1, with each having a p-value < 0,01 (**). Compound 17 showed predominantly mCherry fluorescence, while compounds 7, 10, 13 and 15 showed GFP fluorescence. Finally, compounds 11 and 40 had significant fluorescence at both wavelengths. An autofluorescence screen conducted by comparing fluorescence intensity in HEK-GX cells and HEKWild Type (WT CFTR) cell lines, following identical treatments, showed mostly constant autofluorescence for all compounds, although exceptionally high for compound 17, being absent for compounds 40 and 46. qPCR analysis was performed in 16-HBE cell lines expressing gene edited CFTR with PTC variant (Y122X, G542X, or W1282X legacy names) using wild-type line as control. Compounds 11 and 17 were tested at concentrations of 2 and 10uM. Compound 17 failed to significantly raise the abundance of CFTR mRNA in all three variants, ruling it as a false positive. Compound 11 was the most promising of the compound library so far (Fold change mean 2,822 ± 0,010 at the concentration of 2uM). In summary, Compound 11 was the most promising out of the tested compounds, but a wider range of concentrations should be tested, and protein analysis by Western Blotting should be used to assess PTC readthrough potential. Additionally, compounds’ 40 and 47 tests should be repeated