Improved Precision of thystic fibrosys therapy .::. Oral Presentations

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Joseph Rosenecker, RNA Interference Therapy by Targeting Cystic Fibrosis, European Cystic Fibrosis Conference - New Frontiers in Basic Science of Cystic Fibrosis, Èvora/ Portugal, 14 - 17 April 2005

The epithelial sodium channel (ENaC) 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 (CFTR) appears to be compromised based on the impaired function of CFTR. The missing downregulation of the channel results in increased absorbtion of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the depression of mucus clearance.

Several observations suggest that a downregulation of ENaC restores the perciliary liquid layer, thereby rehydrating the mucus and improving ciliary clearance in the lung. Therefore, specifically downregulating ENaC expression by RNA interference is a promising approach.

In this talk new means of nucleic acid precision targeting both on the molecular and macroscopic level will be presented tp target ENaC-Expression in the airways.

Christa Schmidt and Joseph Rosenecker, Nucleic acid based therapy of cystic fibrosis, Vaincre La Mucoviscidose/Mukoviszidose ev, Young Researcher Meeting 2006, Paris, 7th April 2006

The amiloride-sensitive epithelial sodium channel (ENaC) 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 (CFTR) appears to be compromised, based on the impaired function of CFTR. The missing inhibition of the channel results in increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the airway surface liquid (ASL) layer and to the depression of mucociliar clearance.

Several observations suggest that a downregulation of ENaC restores the periciliar liquid layer, thereby rehydrating the mucus and improving ciliary clearance in the lung. Therefore, we attempt to specifically downregulate ENaC expression by RNA interference.

ENaC is a tetrameric ion channel localized in the apical membrane of epithelia. It is comprised of three subunits alpha, beta and gamma. To study the downregulation of the subunits by RNAi we designed ENaC-specific synthetic and plasmid based siRNA sequences. These sequences are transfected in ENaC expressing epithelial cells; the knockdown is analyzed on mRNA and protein level via quantitative real-time PCR and Western blotting. To test the downregulation of sodium transport we established a fluorescence assay capable of measuring changes in the cells membrane potential. We identified a first siRNA sequence against the alpha ENaC subunit that results in an mRNA knockdown of down to 30%. This sequence has been adapted to the murine ENaC mRNA from Li and Folkesson who tested it in the rat model. Collaborating partners will perform electrophysiological measurements using Ussing chambers and patch clamp technology to demonstrate the functional activity.

Other groups integrated in the project develop new means of nucleic acid delivery and targeting to the lung. Constructs will be administered to the airways via aerosolization and/or nebulization. To enhance and precise the administration, constructs will be associated with magnetic nanoparticles. For this purpose novel magnetic vector formulations and magnetic field generating equipment are developed. These novel constructs will be evaluated in fetal and postnatal mouse models in order to demonstrate their efficacy.