Chiara De Santi

Biopolymer-Based Nanoparticles for Cystic Fibrosis Lung Gene Therapy Studies

Lung gene therapy for cystic fibrosis disease has not been successful due to several challenges such as the absence of an appropriate vector. Therefore, optimal delivery of emerging therapeutics to airway epithelial cells demands suitable non-viral systems. In this work, we describe the formulation and the physicochemical investigation of biocompatible and biodegradable polymeric nanoparticles (NPs), including PLGA and chitosan (animal and non-animal), as novel methods for the safe and efficient delivery of CFTR-specific locked nucleic acids (LNAs).

Identification of MiR-21-5p as a functional regulator of mesothelin expression using microRNA capture affinity coupled with next generation sequencing

MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth.

Identification of a novel functional miR-143-5p recognition element in the Cystic Fibrosis Transmembrane Conductance Regulator 3’UTR

MicroRNAs (miRNAs) are small non-coding RNAs involved in regulation of gene expression. They bind in a sequence-specific manner to miRNA recognition elements (MREs) located in the 3′ untranslated region (UTR) of target mRNAs and prevent mRNA translation. MiRNA expression is dysregulated in cystic fibrosis (CF), affecting several biological processes including ion conductance in the epithelial cells of the lung. We previously reported that miR-143 is up-regulated in CF bronchial brushings compared to non-CF. Here we identified two predicted binding sites for miR-143-5p (starting at residues 558 and 644) on the CFTR mRNA, and aimed to assess whether CFTR is a true molecular target of miR-143-5p. Expression of miR-143-5p was found to be up-regulated in a panel of CF vs non-CF cell lines (1.7-fold, P = 0.0165), and its levels were increased in vitro after 20 hours treatment with bronchoalveolar lavage fluid from CF patients compared to vehicle-treated cells (3.3-fold, P = 0.0319). Luciferase assays were performed to elucidate direct miRNA::target interactions and showed that miR-143-5p significantly decreased the reporter activity when carrying the wild-type full length sequence of CFTR 3′UTR (minus 15%, P = 0.005). This repression was rescued by the disruption of the first, but not the second, predicted MRE, suggesting that the residue starting at 558 was the actual active binding site. In conclusion, we here showed that miR-143-5p modestly but significantly inhibits CFTR, improving the knowledge on functional MREs within the CFTR 3′UTR. This could lead to the development of novel therapeutic strategies where miRNA-mediated CFTR repression is blocked thereby possibly increasing the efficacy of the currently available CFTR modulators.

CFTR dysfunction in cystic fibrosis and chronic obstructive pulmonary disease

ABSTRACT Introduction: Obstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are causes of high morbidity and mortality worldwide. CF is a multiorgan genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by progressive chronic obstructive lung disease. Most cases of COPD are a result of noxious particles, mainly cigarette smoke but also other environmental pollutants. Areas covered: Although the pathogenesis and pathophysiology of CF and COPD differ, they do share key phenotypic features and because of these similarities there is great interest in exploring common mechanisms and/or factors affected by CFTR mutations and environmental insults involved in COPD. Various molecular, cellular and clinical studies have confirmed that CFTR protein dysfunction is common in both the CF and COPD airways. This review provides an update of our understanding of the role of dysfunctional CFTR in both respiratory diseases. Expert commentary: Drugs developed for people with CF to improve mutant CFTR function and enhance CFTR ion channel activity might also be beneficial in patients with COPD. A move toward personalized therapy using, for example, microRNA modulators in conjunction with CFTR potentiators or correctors, could enhance treatment of both diseases.

Non-coding RNA in cystic fibrosis

Non-coding RNAs (ncRNAs) are an abundant class of RNAs that include small ncRNAs, long non-coding RNAs (lncRNA) and pseudogenes. The human ncRNA atlas includes thousands of these specialised RNA molecules that are further subcategorised based on their size or function. Two of the more well-known and widely studied ncRNA species are microRNAs (miRNAs) and lncRNAs. These are regulatory RNAs and their altered expression has been implicated in the pathogenesis of a variety of human diseases. Failure to express a functional cystic fibrosis (CF) transmembrane receptor (CFTR) chloride ion channel in epithelial cells underpins CF. Secondary to the CFTR defect, it is known that other pathways can be altered and these may contribute to the pathophysiology of CF lung disease in particular. For example, quantitative alterations in expression of some ncRNAs are associated with CF. In recent years, there has been a series of published studies exploring ncRNA expression and function in CF. The majority have focussed principally on miRNAs, with just a handful of reports to date on lncRNAs. The present study reviews what is currently known about ncRNA expression and function in CF, and discusses the possibility of applying this knowledge to the clinical management of CF in the near future.

Alpha-1 antitrypsin augmentation therapy decreases MIR-199a-5p, MIR-598 and MIR-320a expression in monocytes via inhibition of NFκB

Alpha-1 antitrypsin (AAT) augmentation therapy involves infusion of plasma-purified AAT to AAT deficient individuals. Whether treatment affects microRNA expression has not been investigated. This study’s objectives were to evaluate the effect of AAT augmentation therapy on altered miRNA expression in monocytes and investigate the mechanism. Monocytes were isolated from non-AAT deficient (MM) and AAT deficient (ZZ) individuals, and ZZs receiving AAT. mRNA (qRT-PCR, microarray), miRNA (miRNA profiling, qRT-PCR), and protein (western blotting) analyses were performed. Twenty one miRNAs were differentially expressed 3-fold between ZZs and MMs. miRNA validation studies demonstrated that in ZZ monocytes receiving AAT levels of miR-199a-5p, miR-598 and miR-320a, which are predicted to be regulated by NFκB, were restored to levels similar to MMs. Validated targets co-regulated by these miRNAs were reciprocally increased in ZZs receiving AAT in vivo and in vitro. Expression of these miRNAs could be increased in ZZ monocytes treated ex vivo with an NFκB agonist and decreased by NFκB inhibition. p50 and p65 mRNA and protein were significantly lower in ZZs receiving AAT than untreated ZZs. AAT augmentation therapy inhibits NFκB and decreases miR-199a-5p, miR-598 and miR-320a in ZZ monocytes. These NFκB-inhibitory properties may contribute to the anti-inflammatory effects of AAT augmentation therapy.

Identification of miR-21 as a regulator of mesothelin expression using microRNA capture affinity coupled with next generation sequencing

Mesothelin (MSLN) protein levels are increased in malignant pleural mesothelioma (MPM). Whether this is due to alterations in microRNAs that regulate MSLN mRNA has not been studied. Here, the miR-CATCH technique (Hassan T, et al. Nucleic Acids Res. 2013; 41:e71) was applied to the MSLN gene and coupled with next generation sequencing (NGS) to identify miRNAs that regulate MSLN mRNA, and that may be responsible for its increased protein levels in MPM. Biotinylated antisense- MSLN mRNA oligonucleotides were employed to isolate miRNA:: MSLN mRNA complexes from a normal mesothelial cell line, Met5A. Library construction was performed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1) and sequencing was performed on an Illumina MiSeq Platform using the Miseq Reagent Kit v2. Thirteen miRNAs were captured in the MSLN- vs scrambled-captured samples. miR-21 and miR-100 were selected for further validation in the Mero-14 mesothelioma cell line. A miR-21, but not miR-100, mimic inhibited MSLN protein expression in these cells (P MSLN mRNA (starting at residues 1237, 1302, 1512 and 1507). Thus miR-21 is a novel regulator of MSLN expression and may have a role in MSLN expression in MPM.

The Biology of MicroRNA

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in mRNA regulation at a post-transcriptional level. The first miRNA was discovered in 1993, and since then many branches of research have been explored to fully understand the miRNA world. Studies regarding the biogenesis process have highlighted different pathways according to miRNA gene localization, although the biochemical mechanism is not completely clear yet. In animals, miRNAs act mainly as negative regulators through translation inhibition, but recent evidence has shown their ability to stimulate mRNA degradation through recruiting decapping enzymes and nucleases. The “canonical” binding site of miRNAs is located within the 3′UTR of the mRNA target, but the coding sequence and the 5′UTR can also be bound by miRNAs. Although they mainly play a negative role at a post-transcriptional level, a few miRNAs have been reported to actually enhance mRNA expression. Altered patterns of miRNAs, due to genetic alterations, defects in the biogenesis process, epigenetic modification or aberrant expression of miRNA genes, are associated with many pathological contexts, including cancer and inflammatory diseases. Although the wider world of small RNAs has to be further explored, these regulators have already been shown to play a crucial role in all biological processes.