**6. Conclusion and remarks**

**4.3. Impact of mutations in lncRNA or miRNA genes**

296 Cystic Fibrosis in the Light of New Research

sequences that may affect CF severity and outcome.

**5.1. Could miRNAs help in improving CF treatment?**

**5.2. Assays and molecules**

reconstituted epithelium

**5. Targeting miRNA as new putative therapeutic tool**

To date, no ncRNA mutation has been described in CF. However, alterations in RNA sequence and/or structure can affect the synthesis, maturation and turnover of ncRNAs. Changes in RNA molecules can be introduced in different ways. For instance, SNPs may affect miRNA biogenesis. miRNA tailing can modify pre-miRNAs and mature miRNAs. RNA editing can modify nucleotide sequences of RNA transcripts. NGS technologies, including exome se‐ quencing and complete re-sequencing of the *CFTR* gene, could reveal mutations in lncRNA

A recent work demonstrated that miR-138 mimics might restore CFTR-Phe508del expression and functional chloride transport. However, the authors stressed that miR-138 mimics may also have undesired effects, because miR-138 targets SIN3A, a highly conserved transcriptional repressor that regulates many genes [67]. Another anti-miRNA agent has been exploited as inhibitor of miR-509-3p, which is involved in the regulation of the *CFTR* gene. Recently, we reported that miRNA function can be blocked by targeting the *CFTR* gene with blockers. We designed blockers to prevent the binding of several miRNAs specifically to the 3'UTR-*CFTR* and tested them in well-differentiated primary human nasal epithelial cells from healthy individuals and patients with CF carrying the p.Phe508del *CFTR* mutation. These molecules rescued CFTR chloride channel activity by increasing *CFTR* mRNA and protein levels. This is in agreement with previous studies showing that complementation of just 6–10% of CFTR transcripts leads to the production of enough CFTR to maintain normal chloride transport in epithelia [75]. These data are supported by findings that the presence of a naturally occurring sequence variation in the *CFTR* promoter, in cis of a severe mutation, increases transcription. This allows the production of enough CFTR protein to reach the apical membrane cells and partially restore CFTR channel function, thus leading to a moderate CF phenotype despite the presence of a severe disease-causing mutation [76]. Similarly, stabilization of p.Phe508del CFTR protein has been associated with increased p.Phe508del CFTR channel activity [77].

As depicted in Figure 5Ad, inhibitors or target-site blocker oligonucleotides have been previously used to restore CFTR expression. Tests have been performed by incorporating inhibitors that induce degradation of the targeted endogenous miRNA or with oligonucleoti‐ des that block miRNA binding to the 3'UTR of *CFTR* in cell lines, primary cultures and

The identification of *cis*- and *trans*-regulators and pathways involved in *CFTR* gene expression is essential for developing new CF targeted therapies. Over the past four decades, therapies for CF have focused entirely on symptoms to improve patients' quality of life. The first treatment (VX-770) targeted the basic defect in p.Gly551Asp-CFTR (1.6% of patients with CF worldwide) [78]. The new molecule VX-809 has been evaluated in patients carrying the p.Phe508del CFTR mutation; however, on its own it does not have clear effects [79] and clinical trials testing the combination of different molecules are in progress. The mechanisms respon‐ sible for the phenotype severity are not well understood yet. Mutational heterogeneity and complex alleles influence CF severity. Moreover, the role of few modifier genes has been established [80]. ncRNAs could also contribute to CF progression and severity and their dysregulation in CF opens new perspectives for patient follow-up and treatment.
