**7. Conclusion**

be expected that new biomarkers of the course of the CF lung disease will be identified in the

It can also be foreseen that single nucleotide polymorphisms (SNPs) in the 3'UTR of miRNAtargeted genes, in particular CFTR, may explain phenotype variability. As an illustration, an SNP (c.\*1043A>C) was identified in the 3'UTR of CFTR in a patient with a CFTR-related disease [38]. CFTR-related diseases are clinical entities associated with CFTR dysfunction but that do not fulfil diagnostic criteria for classical CF (e.g., Congenital Bilateral Absence of Vas Deferens (CBAVD), chronic pancreatitis and disseminated bronchiectasis). This SNP was located within the binding site of two miRNAs including miR-509-3p (shown otherwise to directly target CFTR mRNA), and experimental data suggested that it might impair the regulation of gene expression. That could explain the mild phenotype, as this SNP would act as a mild mutation. Consequently, polymorphisms in the CFTR 3'UTR may play a role in the observed heteroge‐ neous phenotype. Molecular analysis of the 3'UTR of CFTR could therefore be performed as a differential diagnostic tool in patients presenting with suggestive clinical symptoms of CF

Modulating miRNA expression in vivo looks very appealing for developing new CF therapies [38]. Indeed miRNAs are short and need to be delivered only to the cytoplasm, as opposed to nucleus delivery required for DNA-based constructs. Moreover, miRNA-based therapies have several advantages over gene therapy strategies aiming at restoring CFTR expression. First, miRNA modulators are likely to target multiple genes in the context of a deregulated network. However, this might also be a major drawback as potential off-target effects may cause adverse phenotypes. Second, as in all cases of gene therapies, tissue-specific delivery remains a major issue in miRNA-based therapies. An interesting approach has been used by McKiernan et al. to successfully deliver miRNA replacement therapy in CFBE41o- cells [39]. They demonstrated that polyethyleneimine nanoparticles complexed with pre-miR-126 resulted in significant knock-down of TOM1, previously described as a direct target of miR-126 [8]. This result shows that polymeric nanoparticles may be used to effectively deliver miRNA replacement therapy with no adverse effects and may present a strong advantage in comparison to virus-based delivery strategies. As for any other disease for which miRNA-based therapy is considered as an attractive new option, factors controlling the stability of the miRNAs, the delivery systems and the off-target effects of miRNA-based therapies represent strong challenges for the future

**6. Limitations in translating miRNAs from deeper knowledge of their role**

Altogether, it is crucial to highlight important experimental considerations regarding miRNA investigations in CF. Expression profiles of miRNAs in human CF bronchial tissue often differ from study to study. The differences can be explained in part by the selection of tissue material. Indeed, the presence of inflammatory cytokines or non-resident, migratory cells (neutrophils, macrophages, etc.) or both infiltrating airway epithelium generate a non-negligible degree of heterogeneity in biopsy samples. Similar issues can be faced when analyzing miRNA expres‐

**in pathogenesis to development of new therapies**

coming years.

242 Cystic Fibrosis in the Light of New Research

but no mutation in the *CFTR* gene per se.

of development of such drugs.

The study of miRNA in CF is still at an embryonic stage. To date, several studies have determined profiles of miRNAs in samples from target tissues such as lung and nasal biopsies, primary bronchial epithelial cell cultures or epithelial tissues from relevant mouse models. They have generated some indirect data suggesting a significant role of miRNA in controlling lung development, CFTR expression, as well as inflammation signaling pathways in CF.

The preliminary findings reviewed here form a solid basis for growing interest in miRNAs in CF. The possibility that they may act as phenotype modifiers and that they could be used as diagnostic and prognostic biomarkers looks very attractive. With no doubt, further research on miRNA biogenesis and function in CF will expand widely in the coming years, shedding new light on disease variability and paving the way to innovative therapies.
