Background: Gene therapy, delivered directly to the vascular wall, could potentially protect against atherosclerotic lesion growth or regress existing lesions. Previously, we demonstrated that infusion of HDAdApoAI [a helper-dependent adenoviral vector (HDAd) expressing apolipoprotein (apo) A-I] in carotid arteries of fat-fed rabbits reduced early carotid atherosclerosis development (4 wk after vector infusion). Here we tested whether the same HDAd could protect long-term against more severe atherosclerosis. Methods: 25 fat-fed rabbits underwent bilateral carotid gene transfer [HDAdApoAI on one side; control vector (HDAdNull) on the other, with sides randomized]. Postoperatively, diets were adjusted to maintain plasma cholesterols of 200-800mg/dl. This protocol yields large lipid- and macrophage-rich lesions. 6 mo later, carotids were harvested for analyses of DNA, RNA, protein, cholesterol, and histology (H&E, oil red O), or immunostaining for macrophages, muscle actin, T-cells, ICAM-1 and VCAM-1. Results: Vector genomes persisted equally in HDAdApoAI and HDAdNull-infused arteries; however, apoA-I mRNA was detected only in HDAdApoAI-infused arteries. HDAdApoAI-infused arteries had ~60% less median intimal lesion volume than HDAdNull-infused arteries, with concomitant reductions (40-75%) in intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1. We used within-rabbit paired analyses to control for high correlations of lesion size and composition between left and right carotids of the same rabbit. 19 of 25 rabbits had smaller lesions in HDAdApoAI-infused vs HDAdNull-infused arteries; median decreased intimal area between paired carotids in the same rabbit was 30% ( P <0.03). Within individual rabbits, intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1 were all less in HDAdApoAI arteries (median 23-36% less; P <0.05 for all except ICAM-1). Conclusions: Vascular gene therapy with HDAdApoAI reduced lesion development and decreased intimal lipid, macrophage, and adhesion molecule expression 6 mo after treatment. Vector genomes and apo A-I mRNA remained high at 6 mo. A single application of vascular gene therapy durably retards development of atherosclerosis, even in a setting of severe hyperlipidemia.