**6. Limitations in translating miRNAs from deeper knowledge of their role in pathogenesis to development of new therapies**

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‐ sion in human primary HBEs cultures. miRNA expression is strongly dependent on cell origin and differentiation state and on culture conditions. Accordingly, the air-liquid interface condition, which favors differentiation towards the epithelial phenotype, might influence miRNA expression. Moreover, although cultured, undifferentiated HBEs are quite homoge‐ nous, when switching to air-liquid interface condition, not only ciliated cells but also basal and goblet cells are found. In summary, any heterogeneity in cell population is likely to bring bias in miRNA expression profiles.

The large discrepancy among miRNA studies on CF disease may also indicate tissue- or organspecific expression patterns of miRNAs [40]. Consequently, miRNA gene targeting may be variable in different tissues and organs as well. This point is particularly relevant in CF, a multisystemic disease. Similarly, studies aimed at profiling miRNAs in established CF mouse models may show differences that could be species specific. Substantial differences in technical approaches and statistical analyses are also important factors of variability and lack of reproducibility among studies.

Moving towards translation from bench to bedside, practical limitations including degradation and inactivation by nucleases, efficacy of intracellular delivery, short plasma elimination rates, renal and dose-limiting hemodynamic toxicities may hamper development of miRNA-based therapies. As miRNAs have pleiotropic intracellular effects on multiple signal transduction pathways, either single or combined therapies targeting CFTR expression, modulation of inflammation and cell differentiation might eventually be considered. This development could prove lengthy and full of traps and pitfalls before reaching a stage of translation to the clinical setting.
