Nonsense mutations are found in about 10% of cystic fibrosis patients. The consequence of the presence of a nonsense mutation in an mRNA is its rapid degradation by a mechanism called nonsense-mediated mRNA decay (NMD). To correct nonsense mutations, we developed a combined strategy involving NMD inhibition and nonsense mutation readthrough which corresponds to the incorporation of an amino-acid at the nonsense mutation position. For that, we screened molecule libraries in order to identify compounds capable of not only NMD inhibition but also readthrough activation. Until now, we selected 10 molecules with this double feature. We now would like to determine which molecules are the most efficient in correcting nonsense mutation in CFTR gene among the 10 molecules that we identified by our screening process. In order to determine this ranking list, we plan to measure the efficiency of nonsense mutation correction of these molecules in epithelial upper respiratory tract (EURT) cells collected from cystic fibrosis patients harboring a nonsense mutation in CFTR gene. In France, 85 patients harbor a nonsense mutation on both CFTR alleles and represent 34 different mutations. For experimental convenience, we will focus on patients with nonsense mutation on both CFTR alleles in order to be able to measure the NMD inhibition without interference by the expression of a CFTR allele harboring another type of mutation that would be strongly expressed.
We will first culture these EURT cells and then incubate them with our molecules for 20 hours and with a fluorescent molecule called SPQ (for 6-methoxy-N-3’-sulfopropylquinolinium). Iodine can bind SPQ and will quench the SPQ fluorescence. By placing EURT cells first into an iodine-rich medium to quench the SPQ fluorescence and second into a nitrate-rich medium, we will be able to measure the level of functional CFTR protein present in these cells by measuring the re-apparition of fluorescence. Indeed, nitrate will be able to replace iodine on SPQ without quenching SPQ fluorescence only if iodine exits cells through CFTR channels. This assay allows determining whether a molecule is able to lead to the synthesis of functional CFTR protein. With this quantifiable SPQ assay, we will be able to give a score to all our molecules according to their capacity at correcting one or all tested CFTR nonsense mutations.
In a second time, we will purify RNA and protein from molecules –incubated cells in order to measure the level of mRNA or protein CFTR and determine the NMD inhibition level and the readthrough activation level. These results will validate the SPQ assay results by giving a molecular demonstration of the capacity of the molecule.
Overall, our project will provide a list of ranked molecules with the capacity of rescuing the functional CFTR expression from nonsense mutation containing gene. These molecules will be studied in vivo in parallel of this project in order to complete their characterization and prepare them to enter in clinical phase trials. This project represents a case of a targeted medicine development based on an extensive test on 85 different cell lines harboring a nonsense mutation in CFTR gene. Additionally, the experimental procedure used in this project might be useful to develop a pre-treatment test in the future in order to determine the most appropriated molecules from EURT patient cells.