509. Non-Viral Gene Delivery in the Murine Nasal Epithelium by Hypotonic Shock

Cystic fibrosis is the most frequent genetic disease among Caucasians. It is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulation (CFTR) gene. Several viral and non-viral methods have led to insufficient gene transfers in the nasal epithelium, in animal models and in clinic. Moreover, viral vectors elicit a strong immune response following their first administration in the nose, rending impossible any further delivery. In comparison with an isotonic solution of NaCl, the nasal perfusion of naked DNA in deionized water results in a two-order of magnitude higher luciferase activity (2x105 RLU/mg prot.). Furthermore, the perfusion of deionized water 20 minutes prior the perfusion of naked DNA in deionized water leads to a one-order of magnitude reduction of luciferase activity. We hypothesize that deionized water induces a hypotonic shock of nasal epithelial cells. The first step of hypotonic shock consists of an entry of water triggering an intensive exocytosis, allowing the expansion of plasma membrane. This latter might therefore resist to cell swelling without breaking. The second step is called Regulatory Volume Decrease (RVD) and basically consists in retrieving the excess plasma membrane by intensive endocytosis. It is believed that plasmid DNA, bound on the apical surface of epithelial cells, would be taken up during the RVD. While nasal epithelial cells are still recovering from a first hypotonic shock, a second hypotonic shock would not induce an uptake of plasmid DNA as intensive as in the course of a single hypotonic treatment. Hypotonic shock-mediated local gene delivery might be applicable in tissues other than nasal epithelium. The inhibition of DNases by Aurintricarboxylic acid (20 μg per mouse) led to a 3.4-fold improvement of luciferase activity (6.7x105 RLU/mg prot.), that is the highest achieved with a non-viral gene delivery system in the nasal epithelium, so far.