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Improved precision of nucleic acid based therapy of cystic fibrosis

Final Report Summary - IMPROVED PRECISION (Improved precision of nucleic acid based therapy of cystic fibrosis)

This project represents a combined effort to develop a novel therapeutic approach for the treatment of cystic fibrosis lung disease. Cystic fibrosis is an autosomal-recessive inherited disease, with an incidence of 1:3 000 newborns in Central Europe. It is one of the most common monogenic diseases with lethal outcome in our population. Mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene, localised on the long arm of chromosome 7, lead to a lack of or dysfunctional CFTR protein on the apical membrane of ciliated epithelial cells and serous cells of the submucosal glands in the airways. The water- and salt-content of the liquid protecting the airways (Airway surface liquid, ASL) is regulated by CFTR. CFTR is a chloride channel exchanging chloride ions between the cytoplasm and the airway lumen, but the crucial effect for water- and salt-content of airway secretion is based on an inhibition of the Epithelial sodium channel ENaC. The missing function of CFTR causes an increased ENaC-mediated sodium uptake from the luminal secretions of the airways, depleting the airway surface liquid and leading to a defective mucociliary clearance. This benefits bacterial infections, resulting in a slow progressive destruction of the airway tissue. Approximately 90 % of cystic fibrosis patients die from lung complications. The anticipated average lifespan of cystic fibrosis patients in Central Europe is 32 years.

Delivery of the wild type CFTR gene to the lung epithelium appears to be the most straightforward approach towards gene therapy of cystic fibrosis airway disease. So far, clinical studies stressing this approach have failed due to very limited transfection and transduction efficiency in vivo. A different and novel approach to modulate key factors in cystic fibrosis lung disease would be the delivery of inhibitory RNA (RNAi) to the airways. In this proposal, the aim was to develop a novel therapeutic approach for the treatment of cystic fibrosis lung disease by targeting the ENaC of the respiratory epithelium by RNA interference. The epithelial sodium channel is assumed to play a major role in the pathogenesis of chronic lung disease in cystic fibrosis patients. Its natural regulation by the cystic fibrosis transmembrane conductance regulator appears to be compromised by the impaired function of CFTR. The missing down-regulation of the channel results in increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and decreased mucociliary clearance.

Several observations suggested that down-regulation of ENaC restores the perciliary liquid layer, thereby rehydrating the mucus and improving ciliary clearance in the lung. Therefore, it was proposed to specifically down-regulate ENaC expression by RNA interference. For this purpose, new means of nucleic acid precision targeting either at molecular and at macroscopic level were developed. The first level of precision was introduced by the use of ENaC-specific siRNA, a technology known for its high down-regulating specificity. The second level of precision was brought about by loco-regional precision contributed by the administration of such constructs via the airways upon aerosolisation. Yet another and novel level of precision and targeting was introduced by the association of constructs for siRNA delivery and / or expression with magnetic nanoparticles, such that lung-specific accumulation and retention could be mediated by external magnetic fields. For this purpose, novel magnetic vector formulations, viral as well as non-viral, and magnetic field generating equipment were developed. These novel constructs and technologies had been evaluated in foetal and postnatal animal models in order to demonstrate their efficacy.

Work performed in the first funding period was focused on the synthesis and selection of sequences for siRNA to down-regulate ENaC expression in different cell lines. The most successful sequences resulted in a down-regulation of mRNA ENaC subunits to 90 % of the normal value, and functional analysis several days after transfection with siRNA sequences showed a significant inhibition of ENaC activity in cell culture. Efficient carrier systems for synthetic siRNA were developed and tested. Integrating lentiviral vectors for siRNA expression and ENaC down-regulation displaying high transduction efficiency in respiratory epithelial cells had been constructed. A major focus was the development of magnetic nanoparticle formulations for DNA and siRNA delivery. Efficient high-gradient magnetic devices for the delivery of aerosolised magnetic fine particles to the lungs had been developed. In utero vector delivery to the airways of foetal mice had been optimised using first generation and HD-adenovirus vectors systems and tests on VSVG-lentiviral vectors suggested a need for alternative pseudotypes.

Work performed in the second funding period was focused on the application of selected specific sequences which had been developed in the first funding period. Newly developed synthetic carriers had been applied in vitro and in vivo. Efficient down-regulation of ENaC mRNA was achieved in murine and human cell lines. Functional analysis after transfection with siRNA sequences showed a significant inhibition of ENaC activity in cell culture models. A major focus also in the second funding period was the development of magnetic nanoparticle formulations and chitosan-nanoparticles for DNA and siRNA delivery. Novel high-gradient magnetic devices for the delivery of aerosolised magnetic particles to the lungs and to the nose of mice have been developed. In utero vector delivery to the airways of foetal mice has been optimised using VSVG-lentiviral vectors.

During the last reporting period, the consortium made substantial progress to confirm the hypothesis that RNA interference could be of clinical relevance in the treatment of cystic fibrosis lung disease. In an approach to use synthetic siRNA sequences, the consortium had analysed the possibility to down-regulate ENaC function on bronchial epithelia and examined the resulting effects on fluid transport. siRNA sequences complementary to any of the three ENaC subunits had been used to establish whether single subunit down-regulation might be enough to reduce Na+ absorption. Transfection was performed by exposure to siRNA for 24 h at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells it could be demonstrated that:
i) siRNA sequences complementary to any of the ENaC subunit are able to reduce ENaC transcripts and Na+ channel activity by 50 - 70 % that transepithelial fluid absorption decreases that these functional effects last at least eight days. A decrease in ENaC mRNA resulted in a significant reduction of ENaC protein function and of fluid absorption through the bronchial epithelium, indicating that a siRNA approach may improve the airway hydration status in cystic fibrosis patients.

In conclusion, the three and a half year study demonstrates that siRNA sequences complementary to any of the ENaC subunits cause a long-term reduction of ENaC activity in human bronchial epithelia for at least eight days after treatment. The long duration of this effect might depend on the fact that cells forming part of a differentiated epithelium are not dividing; therefore, there is no dilution effect. The reduction of ENaC activity does have functional consequences on the airways, as demonstrated by the fluid measurements. These findings suggest that the periciliary fluid volume would increase after treatment with siRNA for ENaC and, as a consequence, there might be an improvement in mucociliary clearance on the airways of cystic fibrosis patients. In addition, in vivo studies showed that administration of siRNA sequences complexed with cationic liposomes resulted in a reduction of the amiloride-sensitive epithelial Na+ channel activity. However, before applying our results to patients, delivery of the interfering nucleic acid requires further optimisation.