620. SMC-Specific Transcription Elements for Improved Adenoviral-Mediated Transgene Expression in Saphenous Veins

Abstract

Top of pageAbstract Coronary artery bypass grafting (CABG) using autologous saphenous veins (SV) is an essential procedure to restore blood supply to the heart. However, long-term patency rates remain poor (93%, 74% and 41% at 1, 5 and 10 years post-grafting1) as vascular damage and remodeling often leads to neointima formation and accelerated atherosclerosis. Pharmacological interventions are largely ineffective and since the saphenous vein is accessible for ex vivo modification prior to arterial grafting, this presents an ideal window of opportunity for gene therapy. Several candidate therapeutic genes predicted to counteract vein graft failure have shown success pre-clinically but clinical translation is yet to take place. One of the most important issues is optimisation of transgene expression locally in the vein graft following gene delivery. We therefore assessed the ability of first-generation adenoviral vectors harbouring different promoter and post-transcriptional regulatory elements (Figure 1, ref. 2) for their expression levels in saphenous vein cells in vitro, ex vivo and in vivo. Our results demonstrate that Ad-PREP [in which lacZ expression is driven by the murine CMV promoter (mCMV), the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a fragment of the smooth muscle myosin heavy chain promoter (RE)] produces significantly higher lacZ expression levels in primary saphenous vein smooth muscle cells (20-fold vs. RAd35 at m.o.i. 250) and endothelial cells (8-fold), but not in hepatocytes (1.23-fold), compared to RAd35, a standard Ad5-hCMV-bgal vector. This was particularly evident after short virus:cell incubation times (of 30 minutes), clearly relevant to the clinical setting. High levels of lacZ expression were also achieved with this vector following infection of human saphenous vein segments ex vivo, and 7 days after grafting autologous saphenous veins into pig carotid arteries in vivo. These results demonstrate that high transgene expression levels in saphenous vein can be achieved by the mCMV promoter, in combination with smooth muscle cell-specific and post-transcriptional regulatory elements.