144. Duration of Reporter Gene Expression from Naled pDNA in the Mouse Lung Following Direct Electroporation and Development or Wire Electrodes for Sheep Lung Electroporation Studies

Abstract

Current non-viral vectors for lung gene transfer are limited by inefficient uptake, and low levels of expression in cells throughout the lung. The use of electroporation to enhance reporter gene expression from naked plasmid DNA (pDNA) in the mouse lung has been assessed. A direct electroporation model has been established, in which following delivery of pDNA and electroporation of the left lung, luciferase (Lux) reporter gene expression has been enhanced by up to 500 fold compared to the non-electroporated right lung depending on the combination of plasmid, field strength (V/cm) and pulse length employed. Furthermore, following delivery of a GFP expressing plasmid, up to 5% of the left lung cells were positive for GFP after electroporation. Now, the duration of Lux activity following electroporation of the mouse lung has been assessed. Female BALB/c mice were anaesthetised with isoflurane and received 100μg pUbLux (human ubiquitin C promoter, Lux transgene) in 150μl water. The left lungs of mice were exposed by performing a lateral thoracotomy. Electroporation of the left lung (8 pulses of 800V/cm, 2ms with a 1sec interval) was carried out using the BTX ECM®830 Generator with 2-Needle electrodes with 5mm gap with the right lungs remaining non-electroporated. Lux activity was measured at 2, 14 and 28 days post dosing. At day 2 post dosing, mean Lux activity in the left lungs of mice was 40 fold higher than the right lung (Mann-Whitney U: P = 0.016). The Lux activity in the left lungs at day 28 post dosing was no different to the level at day 2 (P=0.512) and still significantly higher than the activity observed in the right lung (P = 0.050). Therefore this study has established that direct electroporation of the mouse lung can result in high levels of Lux activity from naked pDNA and that this level persists for at least 28 days post dosing. In similar studies with the plasmid pCIKLux (CMV promoter, Lux transgene), electroporation resulted in far greater levels of expression at day 2 post dosing, but by day 14, the Lux activity had fallen to background. The safety and efficacy of lung electroporation has therefore been well established in the mouse lung. Further development of lung electroporation is now planned in the sheep lung, as a larger animal model will be more relevant for clinical development of this approach. A series of wire electrodes have been constructed from Teflon insulated stainless steel up to 80cm in length with a diameter of just 0.3mm. In an ex vivo mouse lung model, electroporation with these wires following pCIKLux intranasal delivery (100μg pDNA/150μl) resulted in equivalent 200 fold increases in Lux activity to commercially available needle electrodes. These wire electrodes will be placed within the sheep airways with a bronchoscope allowing for a range of electroporation studies to be carried out. These studies will determine if the electric field can be efficiently and safely applied over a large area to allow electroporation to be used for improving gene transfer throughout the whole lung.