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
试验速览
- 阶段
- 不适用
- 状态
- 招募中
- 发起方
- 入组人数
- 85
- 试验地点
- 8
- 主要终点
- Transport of iodide ions through the CEVAS membrane
研究概览
简要总结
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.
研究设计
- 研究类型
- Observational
- 观察模型
- Case Control
- 时间视角
- Prospective
入排标准
- 年龄范围
- 8 Years 至 —（Child, Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •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
排除标准
- •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
结局指标
主要结局
Transport of iodide ions through the CEVAS membrane
时间窗: less than 48hrs after the collect.
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
次要结局
- Immortalization of patient cells(an average 12 months)
- Expression of the CFTR gene at the mRNA and protein level(less than 1 week.)