610 Transfer of PAI 1 Gene Prevents Abdominal Aortic Aneurysm Development in Mice Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ����������� �!����� ����"� �������� S237 ���[.]
CARDIOVASCULAR all of the animals examined (gene transfer and controls) EGFP expression was clearly evident by direct whole mount fluorescent microscopy in the lung lobes of all gene transferred animals, demonstrating transgene expression throughout the postadministration gestational period (2-3 mos post-gene transfer) These findings are significant because they indicate that (1) direct administration of a lentiviral vector to the developing lung lobes during early gestation and (2) subsequent transgene expression postadministration did not impair lung growth or development in this nonhuman primate model Further studies with the rhesus monkey model will be crucial for assessing the safety and efficiency of gene transfer for the treatment of human congenital lung disease 608 Localized Gene Delivery from Polyurethane Heart Valve Cusps and Vascular Implants Using Antibody Tethered Adenovirus Stanley J Stachelek,1 Cunxian Song,1 Ivan Alferiev,1 Suzanne DeFelice,1 Xiumin Cui,1 Jeanne M Connolly,1 Richard W Bianco,2 Robert J Levy.1 Cardiology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Experimental Surgery, University of Minnesota, Minneapolis, MN Favorable biocompatibility and durability make polyurethanes an attractive material for fashioning prosthetic heart valves and vascular implants We recently reported that bromoalkylated polyurethane films, modified with covalently attached bisphosphonate, function as a calcification resistant composite material for replacement heart valve leaflets We test here the hypothesis that gene vectors can also be appended to polyurethane using a variant of our process chemistry, and thereby the prosthetic leaflet could serve as a gene delivery platform Pendant bromoalkyl groups on either Carbothane or Tecothane were activated with a thiol-to-thiol crosslinker (1,4-di-[3’-(2’-pyridyldithio)propionamido]butane), and an adenovirus specific antibody (antiknob) was covalently linked either directly to the polyurethane film (Tecothane), or to a collagen buffer layer covalently bound to the polyurethane (Carbothane) Replication incompetent adenoviral vectors (type V) containing the gene encoding green fluorescent protein (AdGFP) or a beta galactosidase reporter molecule (AdLacZ) were then tethered to the modified polyurethane surface In vitro analysis of both polyurethane configurations showed that rat arterial smooth muscle cells (A10), grown on polyurethane with either tethered AdLac Z or AdGFP, was successfully transduced with the virus and expressed the appropriate marker gene within 24 hours In vivo analysis using implantable vascular “buttons” composed of Delrin, coated on the blood exposed surface with AdGFP appended to the collagen coated polyurethane matrix, showed GFP gene expression in 14.3% ± 2.5% of attached cells after days Similarly, single pulmonary valve leaflet replacement in juvenile sheep showed that leaflets composed of polyurethane with antibody complexed AdGFP demonstrated GFP expression in 17.8% ± 1.8% of cells in the adjacent myocardium, and in 25.1% ± 5.7% of adherent cells In both in vivo models, FITC autofluorescence was not noted in control animals, and PCR analyses for the GFP gene noted an absence of the GFP in the bloodstream and in distal tissues of both experimental and control animals Cells interacting with the polyurethane surfaces were immunochemically identified as largely leukocyte common antigen positive These data represent a marked improvement in localized transduction efficiency over other vascular gene therapies We conclude that chemically modified polyurethane vascular implants are suitable gene delivery devices 609 Delivery of p27 to Rat Carotid Arteries Using Electroporation Protects from Intimal Hyperplasia Following Balloon Injury Jennifer L Young,1 David A Dean.1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, United States 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 and 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 including larger arteries Using a square wave electroporator (200V/cm, pulses of 10msec duration), we delivered a CMV driven luciferase plasmid to the common carotid arteries of rats In animals receiving DNA plus electroporation, we obtained an average of 56.38 pg/mg total protein with values as high as 180 pg/mg protein at 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 rat model for restenosis Other labs have demonstrated that early expression of the small cell cycle inhibitor p27 can inhibit intimal hyperplasia following vascular injury Therefore we hypothesized that delivering a p27 expressing plasmid to the common carotid arteries of rats by electroporation immediately following balloon injury would decrease the degree of intimal thickening after injury, providing protection from intimal hyperplasia and vessel occlusion Indeed, we found that delivering as little as 50 μg of plasmid expressing p27 from the CMV immediate early promoter was sufficient to decrease the intimal to medial area ratio four-fold compared to untreated carotid arteries, (balloon injury only) at 14 days post-injury Further, injured arteries treated with the p27 plasmid without electroporation had the same degree of intimal hyperplasia as control balloon-injured arteries Taken together, these results demonstrate that electroporation is an efficient method for gene transfer and expression in rat carotid arteries and can be effectively used to deliver therapeutic genes for the treatment of smooth muscle proliferative disease in vivo 610 Transfer of PAI-1 Gene Prevents Abdominal Aortic Aneurysm Development in Mice Hu Sheng Qian,1 Ana Freay,2 Perry Liu,1 Katalin Kauser,2 Linda Cashion,3 Jon Morser,2 Ron Vergona,1 William P Dole,2 Mark E Sullivan,1 Gary G Deng.2 Department of Pharmacology, Berlex Biosciences, Inc, Richmond, CA, United States; 2Department of Cardiovascular Research, Berlex Biosciences, Inc, Richmond, CA, United States; Department of Gene Therapy, Berlex Biosciences, Inc, Richomnd, CA, United States Vessel wall matrix destruction is a key event in abdominal aortic aneurysm (AAA) formation and rupture We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy S237 CARDIOVASCULAR AAA and is critical for its development In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), through gene delivery on the development of AAA Methods: Angiotensin II (Ang II) (1.44 mg/kg/day) was administered to 6-month old male mice deficient in ApoE (apoE-/-) Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad-PAI-1) or the luciferase gene (Ad-Luc) by localized delivery through intra-advential injection or systemic delivery through tail vein injection In vivo gene expression and biodistribution of luciferase was monitored by a cooled charge coupled device (CCCD) camera imaging system in living mice The expression of human PAI-1 was determined by both ELISA and immunohistology Abdominal aortas were examined after 30 days and the tissues were harvested for histology and protein or enzyme activity measurement Results: After 30 days of Ang II treatment, there were no significant differences in general appearance and cholesterol levels between the Ad-PAI-1 treated and the AdLuc treated groups through localized or systemic gene delivery Systemic delivery of the PAI-1 gene did not affect AAA incidence (78% vs 90% in Ad-Luc controls, p=0.125) In contrast, AAA development was completely prevented in the localized delivery group (0% in Ad.PAI-1 vs 55.6% in Ad-Luc controls, p