ENaC (Epithelial Na+ channel) genes and protein have been shown to be involved, although with still undefined mechanisms, in CF and CF-like diseases. The wild-type, but deregulated, ENaC seems a better therapeutic target than the mutated CFTR. Proteolytic activation and epigenetics could play a role both as regulatory mechanism(s) and as therapeutic target(s).
1) To establish whether, in CF or CF-like diseases, mutations or transcriptional alterations in ENaC genes contribute to the pathogenesis and to the phenotype severity.
2) To analyze the mechanism(s) of coordinated transcription of ENaC genes and of ENaC - CFTR interaction, in physiology and pathology.
3) To characterize the functional effect of the ENaC mutations identified.
4) To evaluate therapeutic approaches of ENaC expression downregulation and activity attenuation.
Preliminary results (personal)
Several sequence variations of ENaC genes were already evidenced in CF and CF-like patients. For functional analysis: a) the cloning of tagged sequence of each ENaC gene in mammalian expression vectors was achieved; b) a fluid absorption assay was set up. An inverse correlation between expression and DNA methylation of ENaC genes was found, in particular for the SCNN1A gene. A shRNA approach proved to be useful in order to obtain a decrease of SCNN1A expression and activity.
Project description (experimental plan, methods, timing)
1) Search for mutations and altered expression of ENaC genes in CF and CF-like subjects with at least 1 unidentified CFTR mutation or the same CFTR mutated genotype but different CF severity (0-18th month).
2) Functional characterization of the ENaC mutations found (6th – 24th month).
3) Analysis, in vitro and ex vivo in human cells, of CFTR and ENaC expression, and of ENaC gene DNA methylation (0 – 24th month).
4) Downregulation of ENaC expression and activity by DNA hypermethylation, chromatin condensation, RNA interference and inhibition of proteolytic activation (8th – 24th month).
Relevance and Anticipated output
In CF and CF-like diseases, identification of ENaC mutations and methylation-dependent transcriptional alterations, and clarification of: a) the role of ENaC, b) the ENaC - CFTR relationship, c) the genotype - phenotype relationship. Identification of ENaC as therapeutic target and strategy for CF cure.
This study will add new insight into CF molecular mechanisms offering a new panel of ENaC molecular lesions to be searched. Practical consequences are expected for CF diagnosis, prediction of the clinical course and genetic counseling. ENaC genes may arise as therapeutic targets opening new perspective for CF therapy. We propose the use of small molecules to synergistically influence ENaC at both transcriptional and functional levels. The moving to clinical trials would be greatly facilitated by the fact that some of the proposed molecules are already approved for clinical use in other pathologies.