Inhibiting miRNAs as a new therapeutic strategy for cystic fibrosis

Selective inhibition of miRNAs: An effective therapeutic strategy for improving CFTR dysfunction

Cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.

The CFTR gene encodes a channel that facilitates chloride and anion transport across the membrane of epithelial cells. In order to develop CF, individuals must carry two mutant CFTR alleles, mutations which can result in altered protein synthesis, folding, trafficking or function.

While CF is a multi-organ disorder, the vast majority of morbidity and mortality experienced by patients with this illness relates to chronic lung disease. Absent or poorly functioning CFTR results in impaired mucociliary clearance, chronic infection and inflammation.

CFTR is post-transcriptionally regulated by microRNAs (miRNA), with previous research indicating that inhibitors of certain miRNAs can enhance CFTR expression or function in CF bronchial epithelial cells (BECs). Target site blockers (TSBs) specifically compete with miRNAs for binding to individual miRNA elements (MREs) and hence prevent them from gaining access to those sites.

In the study available here, Professor Catherine Greene and her team at RCSI University of Medicine and Health Sciences demonstrate the effects of CFTR-specific TSBs and how they can block miRNA mediated inhibition of CFTR, This research was conducted alongside colleagues from University College Dublin, University Hospital Limerick, University of Pittsburgh, Boston University and Aerogen Limited.

The RCSI team began by measuring endogenous levels of five lead miRNAs that were previously shown to be direct regulators of CFTR, preforming this step in a range of in-vitro, ex-vivo and clinical samples. They demonstrated that levels of several of the miRNAs were increased in the different models studied.

Nine TSBs were designed to target MREs for the five lead miRNAs. The efficacy of these TSBs was judged based on increasing CFTR 3’UTR luciferase expression. Luciferase was found to increase after transfection with TSB4, TSB6, TSB7 and TSB8.

Cell lines were then transfected with each of these four TSBs and levels of endogenous CFTR protein were measured. TSB4 targeting a specific miR-223–3p MRE at position 166–173 of the CFTR 3’UTR and TSB6 targeting the miR-145–5p MRE at position 298–305 significantly increased CFTR protein levels and hence subsequent studies focused on these TSBs.

Using a fluorescence-based test and a CFTR functional assay, the authors demonstrated that inhibition of CFTR-specific miRNAs could improve CFTR anion channel activity. The team went on to illustrate how TSB4 and TSB6 enhanced the CFTR modulator effects in BECs.

In the final component of the study, Professor Greene’s team finished by encapsulating TSB4 and TSB6 in poly-lactic-co-glycolic acid (PLGA) nanoparticles, proving how these encapsulated TSBs could be successfully aerosolised and increase CFTR expression in primary CF BECs.

This last step was crucial given the importance of aerosolised treatments as a mechanism of rapid drug delivery in patients with CF.

In this study, the research team at RCSI provide the first evidence of a synergistic effect of specific miRNA binding site inhibition combined with CFTR correctors to enhance anion channel activity. The TSBs can enhance up to three-fold the corrector effects of medications such as Ivacaftor or Tezacaftor on the CFTR channel.

This means that there may be therapeutic value in selectively inhibiting the activity of specific miRNAs in CF to potentiate the effectiveness of CFTR modulators. The most effective approach to CF treatment will likely require combinations of CFTR correctors and adjunct therapies.

The authors illustrate how CFTR-specific TSBs can modulate CFTR in CF BECs to enhance its expression and anion permeability. Furthermore, aerosolised PLGA nanoparticles containing the TSBs retained desirable aerodynamic and biophysical characteristics indicating their potential use as an inhalation therapy.

In summary, the study suggests that aerosolised TSBs encapsulated in PLGA nanoparticles used alone or in combination with systemic CFTR modulators might represent a promising therapeutic strategy for the treatment of CFTR dysfunction in CF bronchial epithelium, specifically in patients carrying the p.Phe508del mutation.

This research discovery enhances the field of cystic fibrosis research, bringing us closer to providing effective therapeutic treatment and care to patients.

Journal Article Information:
Precise targeting of miRNA sites restores CFTR activity in CF bronchial epithelial cells.
Molecular Therapy 2020, 28 (4)
https://doi.org/10.1016/j.ymthe.2020.02.001