Expression of Naked Plasmid DNA Injected into the Afferent and Efferent Vessels of Rodent and Dog Livers

Abstract

A variety of reporter genes within plasmid constructs were injected into the afferent and efferent vessels of the liver in mice, rats, and dogs. Efficient plasmid expression was obtained following delivery via the portal vein, the hepatic vein, and the bile duct. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels. Combining these surgical approaches with improved plasmid vectors enabled uncommonly high levels of foreign gene expression in which over 15 μg of luciferase protein/liver was produced in mice and over 50 μg in rats. Equally high levels of β-galactosidase (β-Gal) expression were obtained, in that over 5% of the hepatocytes had intense blue staining. Expression of luciferase or β-Gal was evenly distributed in hepatocytes throughout the entire liver when either of the three routes were injected. Peri-acinar hepatocytes were preferentially transfected when the portal vein was injected in rats. These levels of foreign gene expression are among the highest levels obtained with nonviral vectors. Repetitive plasmid administration through the bile duct led to successive events of foreign gene expression. The integration of these Findings into laboratory and clinical protocols is discussed. Previously, we have shown that the intraportal injection of plasmid DNA in hypertonic solution leads to high levels of hepatocyte transfection in mice. This report found efficient expression following the retrograde injection of plasmid DNA into the hepatic vein and bile duct. For mice, the retrograde injection of naked DNA into the hepatic vein with occlusion of the portal vein or retrograde injection into the bile duct exhibits up to 10% of transfected hepatocytes. The same results were obtained in rats and qualitatively similar preliminary results were obtained in dogs. A protocol involving catheterization of the bile duct enabled repeat gene injections without additional surgery. The high efficiency of expression in larger animals and the use of relatively accessible vessels such as the hepatic vein or bile duct demonstrates the potential clinical utility of these gene transfer protocols.