Correction of Nonsense Mutations in Cystic Fibrosis

Study Overview
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Brief Summary
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The presence of a nonsense mutation leads to the rapid degradation of the carrier mRNA mutation by a mechanism called NMD (nonsense-mediated mRNA decay) [6, 13]. There are currently 3 main strategies at least for correcting nonsense mutations: exon skipping, inhibition of NMD and nonsense mutation readthrough. In the laboratory, we developed a strategy for correcting nonsense mutations combining inhibition of NMD and activation of translecture. For this purpose, we have constructed screening systems to identify NMD-inhibiting and/or readthrough enhancers. The molecules thus identified are then tested on cell lines and in murine models carrying a nonsense mutation. One of our goals is to select a set of molecules that can correct effectively nonsense mutations. For this we have to test these molecules on a great diversity of nonsense mutations. This work will: determine if we can correct all the nonsense mutations tested with at least one of our molecules determine what is common within a group of mutations corrected by a given molecule be able to assign the parameters that make one mutation is corrected by one molecule and not or little by another. This study will therefore improve our theoretical knowledge on the recognition of premature stop codons but also to propose therapeutic approaches for the correction of nonsense mutations of the CFTR gene in cystic fibrosis in a targeted way for a patient.

Official Title
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Optimization of Correcting Molecules of Nonsense Mutations in Epithelial Cells of the Upper Airways of Patients With Cystic Fibrosis With Nonsense Mutations in the CFTR Gene

Conditions
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Cystic Fibrosis

Intervention / Treatment
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* Other: smear of nasal fossae


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male / female adults and minors aged 8 years and over Patients with cystic fibrosis and carry a nonsense mutation on the 2 alleles of the gene coding for the CFTR channel. Patients whose genotype of patients concerning the CFTR gene is known. Patients with social security Major patients who have given their consent Minor patients with parental authorization Exclusion Criteria: Patients who have a mutation other than nonsense in the CFTR gene Patients whose CFTR gene was not sequenced on the 2 alleles Patients not wishing to participate in this study or persons not giving or not able to give consent. Pregnant or lactating women Patients under curatorship or guardianship

Ages Eligible for Study
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Minimum Age: 8 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Intervention/Treatment |
| --- |
|Other: smear of nasal fossae|1 smear of nasal fossae during a usual or scheduled visit|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Transport of iodide ions through the CEVAS membrane | Patient cells with be cultured in BEGM (Lonza) medium and incubated with corrector of nonsense mutations 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. Nitrates bind SPQ without quenching SPQ fluorescence. By placing patient 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 using fluorimeter. 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 corrector of nonsense mutation is able to lead to the synthesis of functional CFTR protein | less than 48hrs after the collect. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Immortalization of patient cells | Immortalization of patient cells will be attempted by transfection of construct expressing the origin-of-replication defective SV40 as described in Gruenert et al.,2004 | an average 12 months | 
| Expression of the CFTR gene at the mRNA and protein level |  | less than 1 week. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Cystic fibrosis, nonsense mutations, CFTR gene, nasal epithelial cells, nonsense mutation readthrough