203. Electroporation-Mediated Gene Delivery to Mouse Femoral Arteries in a Model of Vascular Injury

Abstract

Vascular diseases such as atherosclerosis, restenosis and vein graft stenosis affect millions of Americans each year. The pathophysiologies of these diseases all consist of unregulated smooth muscle cell proliferation that results in intimal hyperplasia and ultimately vascular occlusion. Gene therapy is a promising treatment option for smooth muscle proliferative diseases with several genes identified as potential therapies. However, the efficient delivery of these genes to the target cells remains a major limiting factor. Our lab recently developed a non-viral delivery method for gene transfer to the vasculature using electroporation. We have demonstrated that plasmid delivery by electroporation results in high level gene expression in rat mesenteric arteries with expression peaking between 1 and 3 days post transfer. Based on our results in the mesenteric arteries, we reasoned that electroporation could be used to deliver genes to other vascular beds. Using a square wave electroporator (200 V/cm, 8 pulses of 10msec duration) we delivered GFP and luciferase reporter plasmids to femoral arteries of mice. Delivery of a GFP-expressing plasmid results in expression not unlike that we have previously observed in the mesenteric arteries, and histological analysis was used to determine the localization of gene expression. To quantify the levels of gene expression, we delivered a CMV driven luciferase-expressing plasmid. In animals receiving DNA plus electroporation, we obtained an average of 200 pg/mouse with up to nanogram levels at 2 days post-transfer. In contrast, luciferase expression was undetected in arteries receiving DNA but no electroporation. These levels are similar to those achieved in the mesenteric vessels, thus providing evidence that our previously described gene delivery method for the vasculature can be successfully applied to multiple vascular beds. To extend these findings to a vascular disease model, we have focused on a mouse model of restenosis where a polyethylene cuff placed around the femoral artery results in significant intimal hyperplasia at 21 days post-injury. Based on results from other labs demonstrating that early expression of the cell cycle inhibitor p27 can inhibit intimal hyperplasia following vascular injury, we hypothesized that delivering a p27 expressing plasmid to the mouse femoral artery by electroporation immediately before cuff placement would decrease the degree of intimal thickening after injury, providing protection from intimal hyperplasia and vessel occlusion. To test this hypothesis we delivered a CMV driven p27 expressing plasmid by electroporation, placed the cuff, and assayed the degree of intimal hyperplasia by morphometric analysis on hematoxylin and eosin stained thin sections at 21 days post-injury. As expected, injury was less severe in the treated animals. Taken together with our previous results using a rat carotid angioplasty model, these results suggest that electroporation-mediated gene transfer of cytostatic genes can protect from restenosis.