872. Highly Efficient Virus-Mediated Gene Transfer to Human Vein Grafts Via Intraluminal Pressure Delivery

Abstract

Introduction Long term patency rates for coronary artery bypass grafting (CABG) using autologous saphenous vein are poor, showing 10 years post-CABG patency rates of 41%. To date, effective and proven pharmacologic interventions to prolong vein graft patency are lacking. Gene therapy seems particularly well suited for the prevention or postponement of vein graft failure since (1) the stimulation of smooth muscle cell proliferation appears to largely be an early and transient process, and (2) the target tissue is easily accessible ex vivo during the surgical procedure. However, a major obstacle in efficient gene transfer to human vein grafts is the heterogenity of vessel quality due to age, preexisting veinous disease, and surgical trauma. In this study we developed a clinically relevant viral delivery technique to achieve a reproducible and efficient gene transfer in human vein grafts. Methods 46 intact human saphenous veins (age 68+/−7) from patients undergoing coronary bypass surgery were cut into rings 15 mm in length. A small piece of each vein was tested for endothelial viability with trypan blue. Individual vein rings were endoluminally incubated with adenoviral vectors expressing two reportergenes (Ad.CMV.lacZ/GFP, 1x10E11 pfu/mL) with different intraluminal pressures (50, 100, 150mmHg) for 30 or 60 minutes. Veinsegments were cultured for 6days after transfection and harvested for cryopreservation. Transfection efficiency was evaluated by X-gal staining and GFP- Westernblot; endothelial integrity and neointima-hyperplasia were evaluated by CD31-staining, Elastica Van Gieson-staining and H-E-staining. Results Veins which underwent endoluminal gene transfer without pressure showed only poor transfection efficiency (12%+/−7). In contrast, veins wich were pressure-transfected with either 100 or 150 mmHg showed high transfection efficiency throughout the vessel wall (75%−+/−13 and 91+/−7% respectively, p<0.02). Veins transfected for 60 minutes showed no significant difference in transfection efficiency versus 30 minute incubation. However, CD31 staining revealed a significant loss of endothelium in the high pressure group (150 mmHg), whereas the 100 mmHg pressure group showed only minor endothelial damage compared to control veins, irrespective of incubation time. Discussion This study presents an optimized transfection protocol for gene transfer in human vein grafts. We here show for the first time that viral transfection using carefully regulated supraphysiological endoluminal pressure greatly enhances transfection efficiency in human veins. Using continuous intraluminal pressure-monitoring and careful evaluation of endothelial integrity, we found optimal conditions for highly efficient and reproducible transfection using 100 mmHg intraluminal pressure for 30 minutes. Establishing a safe and efficient delivery method in a clinically relevant model for human cardiovascular gene therapy is an important aspect of translation from pre-clinical to clinical gene therapy.