Peptide Nucleic Acids as miRNA Target Protectors for the Treatment of Cystic Fibrosis

Abstract

Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3’UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3’UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.

Peptide Nucleic Acids as a Tool for Site-Specific Gene Editing

“…These favorable biological properties have spurred many therapeutic applications for PNAs, such as inhibiting transcription, translation, or the activity of microRNAs, that have been previously reviewed [ 6 ]. PNAs have also recently been described as microRNA target protectors [ 7 ]. Further, the ability of PNAs to form triplexes with genomic DNA (gDNA) in a site-directed manner has also been harnessed as a strategy to achieve targeted gene editing at endogenous loci.…”

Protecting Pax6 3′ UTR from MicroRNA-7 Partially Restores PAX6 in Islets from an Aniridia Mouse Model

“…Further optimization of TP length, including evaluating the use of peptide nucleic acids, could be used to further enhance specificity. 47 , 48 , 49 However, our results show that derepression of the WT Pax6 allele by a TP targeting the miR-7 site increased PAX6 expression without resulting in overexpression.…”

“…In principle, this approach could be applied to other haploinsufficiency disorders, including monogenetic diabetes (MODY) and neurofibromatosis type 1, or to upregulate homozygous mutants where the mutant protein retains some function, as was recently shown for cystic fibrosis transmembrane conductance regulator (CFTR). 47 …”

Non-Coding RNAs in Pediatric Airway Diseases

“…Another interesting strategy was reported recently by Zarilli et al [ 75 ] who used peptide nucleic acids (PNAs) to prevent miRNA binding to the 3’UTR sequences of CFTR mRNA. The authors synthesized 7- and 13-base-long PNAs with the tetrapeptide Gly-SerP-SerP-Gly at their C-end that was complementary to the sequence recognized by miR-509-3p and blocked its binding to CFTR , thus enabling its expression.…”