540. Genetic Intervention towards Improving the Long Term Outcome of CABG Using a Lipid Peptide DNA Vector System
Authors: Scott A. Irvine, Faiza Afzal, Qing H. Meng, Jimmy Ho, Andrew Wu, Jean R. McEwan, Stephen L. Hart
Journal: Molecular Therapy (2006)

Coronary artery bypass grafting (CABG) involves the engraftment of saphenous vein to re-establish blood flow around atheromatous blockages of the coronary arteries. Although highly effective, long- term failure rates are high due to neointimal hyperplasia (IH) and accelerated atherosclerosis. Anti IH gene therapy strategies mainly focus on preventing smooth muscle cell migration and proliferation and the re-establishment of the endothelium in the vein graft. CABG offers the opportunity for ex vivo transfection with minimal systemic exposure to vector, avoiding inappropriate transfection. However restrictions are imposed by the procedure including a transfection time of 30 mins and exposure to the anti- coagulant, heparin. We are developing novel receptor- targeted, synthetic vectors for the delivery of therapeutic genes to promote vein graft survival. A rabbit vein-graft model involving ex vivo gene transfer to vein tissue prior to engraftment to the carotid artery, to mimic the clinical procedure is used to evaluate in vivo gene transfer and therapeutic efficacy. A series of lipopolyplex-class vector formulations (Lip/Tide) was compared comprising bifunctional peptides with different receptor- targeting and DNA-binding motifs, and a cationic lipid that promotes endosomal release. A specific targeting peptide and lipid combination emerged that displayed greatly enhanced efficiency over first generation integrin-targeted vector complex in transfections of vascular cells and rabbit aortic explants. In cellular transfections, heparin concentrations of 1U/ml completely abrogated the transfection ability of the optimised Lip/ Tide complex. However, at concentrations closer to clinically relevant levels, 0.2 to 0.5 U/ml, transfection efficiency was surprisingly enhanced up to 6-fold. This effect was not observed with lipid alone, Superfect or PEI. Experiments using picogreen fluorescence staining of DNA indicated that the inhibitory effect at higher heparin concentrations involves destabilisation of the Lip/Tide complex. The enhancement effect at lower heparin concentrations may involve interactions with the cell surface or intracellular plasmid release from the Lip/Tide complex. Transfection incubations of rabbit aortic explants were performed from 10 secs up to 30 mins with Lip/Tide complex in isotonic dextrose or saline. The time course study demonstrated that 10 secs incubation was sufficient to produce detectable transfection in lumenal, medial and adventitial cells with more substantial transfection after 10 to 15 mins incubation. GFP reporter gene expression in rabbit aortic explants is first detected 3 days after transfection then peaks at about 7 days, persisting for at least 14 days, matching the kinetics required for the period of therapeutic gene expression following CABG to counter IH. In conclusion, we have optimised a targeted synthetic vector that produces high efficiency gene transfer in primary vascular cells and tissues. The vector is tolerant of heparin up to 0.5 U/ml and peak transfection incubation times (10 to 15 mins) are well within the time window available during the CABG procedure.