Emerging microRNA Therapeutic Approaches for Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and remains the most common life-shortening diseases affecting the exocrine organs. The absence of this channel results in an imbalance of ion concentrations across the cell membrane and results in more abnormal secretion and mucus plugging in the gastrointestinal tract and in the lungs of CF patients. The direct introduction of fully functional CFTR by gene therapy has long been pursued as a therapeutical option to restore CFTR function independent of the specific CFTR mutation, but the different clinical trials failed to propose persuasive evidence of this strategy. The last ten years has led to the development of new pharmacotherapies which can activate CFTR function in a mutation-specific manner. Although approximately 2,000 different disease-associated mutations have been identified, a single codon deletion, F508del, is by far the most common and is present on at least one allele in approximately 70% of the patients in CF populations. This strategy is limited by chemistry, the knowledge on CFTR and the heterogenicity of the patients. New research efforts in CF aim to develop other therapeutical approaches to combine different strategies. Targeting RNA appears as a new and an important opportunity to modulate dysregulated biological processes. Abnormal miRNA activity has been linked to numerous diseases, and over the last decade, the critical role of miRNA in regulating biological processes has fostered interest in how miRNA binds to and interacts explicitly with the target protein. Herein, this review describes the different strategies to identify dysregulated miRNA opens up a new concept and new opportunities to correct CFTR deficiency. This review describes therapeutic applications of antisense techniques currently under investigation in CF.

Keywords: ANO 1 channel; CFTR (Cystic Fibrosis Transmembrane conductance Regulator); cystic fibrosis; miRNA; oligonucleotides; therapy.

FIGURE 1

Location of miRNAs gene in the genome: (1) miRNAs can be found between two genes (intergenic) or (2) in a gene (intronic). (3) They may be present in a single miRNA gene or (4) in a cluster of miRNA genes. (5) Sometimes intronic miRNAs may exist between two exons (miRtron) or (6) overlapping an exon and an intron of non-coding genes (mixed).

FIGURE 2

Biosynthesis of miRNAs: (1) The biosynthesis begins in the nucleus by transcription of miRNA genes by RNA polymerase II (Pol II). (2) Long transcripts, pri-miRNA, are cleaved by Drosha and DGCR8 protein creating pre-miRNA with hairpin structure. (3) Exportin 5 transfers pre-miRNA into the cytoplasm. (4) Pre-miRNA is cleaved by Dicer into miRNA duplex in mature single-strand miRNA form. This miRNA mature form is incorporated into a miRISC ribonucleoprotein complex. This complex can then act directly on the mRNA in the cell where it is synthesized or out of the cell.

FIGURE 3

Mechanism of action of miRNAs: miRISC complex attenuates mRNA translation and leads to the destabilization of mRNA by deadenylation and/or inhibition of translation.

FIGURE 4

Figure showing the various approaches to inhibit or mimic a miRNA. In physiological condition: the miRNA will bind to the 3′-UTR of its target to inhibit translation resulting to RNA degradation and/or mRNA recycling. To inhibit a miR action, there are three possibilities: transfection of (1) miRNA inhibitor (antagomiR): the miRNA inhibitor binds to the latter by complementarity and prevents its action on its target; (2) TSB: the TSB specifically binds to the 3′-UTR of the target mRNA at the miRNA binding site, the miRNA can then no longer attach to it and exert its action; and (3) mRNA sponge: the miRNA is saturated by the miRNA sponge which contains many miRNA complementary sequences, the miRNA can then no longer attach itself to its target. To increase the expression of a miR, transfection of a mimic of this miR is sufficient to inhibit translation of mRNA.