473. Magnetofection To Enhance Airway Gene Transfer

Abstract

Gene delivery to the airway epithelium (AE) is very inefficient due to many extracellular barriers such as mucus, cilia, glycocalyx, and in the case of Cystic Fibrosis (CF) a thick layer of sputum. In an attempt to overcome these we assessed “magnetofection”, a method shown to increase transfection efficiency in vitro and in gut epithelium in vivo. This requires the formation of magnetic gene transfer complexes by coupling superparamagnetic iron oxide nanoparticles to vectors, which are then attracted to strong magnets. Magnetofection is thought to increase the contact time and the local concentration of the vectors on the cell surface, thus increasing gene transfer efficiency. A variety of magnetic particles have been described. Here we used TransMAGPEI (Chemicell, Germany), a polyethylenimine (PEI) – coated iron oxide particle, complexed to Genzyme Lipid 67 (GL67), which has already been used in several clinical trials. GL67 was mixed with luciferase plasmid DNA (pLux) at a previously optimised molar ratio of 1:4 (GL67:pLux). This complex was then coupled to TransMAGPEI at 1:1 up to 4:1 w/w ratios (transMAGPEI:pLux) in the presence of 0.9% NaCl. The complexes were tested in C127 cells (mouse mammary epithelial cells) in vitro (0.2 μg pDNA/well in 96-well plate). After a 15 min exposure to the magnet, lux expression was increased up to 3-fold (magnet: 45,199 ± 11,063 RLU/mg protein, no magnet: 14,102 ± 3,278 RLU/mg protein; p < 0.05; n = 6). These complexes were also tested in mouse nasal epithelium in vivo. We complexed our standard vector dose (80 μg/100 μl of GL67/pLux) to TransMAGPEI at a 4:1 w/w ratio (TransMAGPEI:pLux). The complexes were slowly perfused (6.7 μl/min) into the nasal cavity via a thin catheter placed 2.5 mm into the nostrils of anaesthetised mice. However, at this concentration the formulation precipitated resulting in a ~50-fold decrease in gene expression, when compared to the standard GL67 formulation without addition of magnetic particles (TransMAGPEI: 0.9 ± 0.7 RLU/mg protein, no TransMAGPEI: 47.7 ± 4.1; p < 0.01; n = 8). Exposure of the nostril to the magnet (NeoDelta circular disc attached to an AlNiCo rod magnet, IBS Magnet, Germany) for 15 min during perfusion did not increase gene expression. We attempted to overcome the precipitation problem by changing the ratio of TransMAGPEI to pDNA, the total volume and the mixing order. The optimised formulation (TransMAGPEI-GL67/pLux 80 μg/500 μl; 1:1 w/w TransMAGPEI:pDNA) did not precipitate and gave a 6-fold increase in gene expression in vitro in the presence of the magnet. However, Lux expression in the nasal tissue after in vivo transfection was still reduced compared to GL67/pDNA alone (TransMAGPEI: 1.3 ± 0.8 RLU/mg protein, GL67/pDNA: 26.3 ± 6.6 RLU/mg protein; p < 0.01; n = 8). Again, exposure to the magnetic field during perfusion did not make a difference. Although TransMAGPEI in combination with magnetic forces increased gene transfer in vitro, it did not improve transfection efficiency of GL67/pDNA in airway epithelial cells in vivo.