High-Efficiency Gene Transfer to Autologous Rabbit Jugular Vein Grafts Using Adenovirus–Transferrin/Polylysine–DNA Complexes

Abstract

Within the first year, 15–20% of coronary artery saphenous bypass vein grafts (SVGs) occlude because of thrombosis or progressive intimal hyperplasia. One potential new strategy to reduce this complication would be to introduce antithrombotic or antiproliferative genes in vein grafts before implantation. The success of this approach requires an efficient DNA delivery system. In the present study we tested the feasibility of using adenovirus–transferrin/polylysine–DNA complexes (TfAdpl/DNA) to achieve high-efficiency gene transfer into vascular interposition vein grafts. All studies used the Escherichia coli LacZ (β-galactosidase [β-Gal]) reporter gene under the control of the cytomegalovirus (CMV) earlier promoter and enhancer (pCMV/LacZ). Autologous rabbit jugular vein segments were incubated ex vivo for 60 min in a solution of TfAdpl/DNA complexes (1.2 × 10¹⁰ biotinylated adenovirus particles, 2,430 ng of streptavindylated polylysine. 10 μg of plasmid DNA, and 9 μg of transferrin-polylysine per ml), and then reimplanted across the ligated right carotid artery. Control veins were incubated in TfAdpl solution in which DNA was omitted. A total of six grafts were treated with TfAdpl/DNA, and two grafts were treated with TfAdpl. Veins were harvested 3 (n = 3) and 7 (n = 3) days later and β-Gal activity was determined by X-Gal chromogen staining. All six TfAdpl/DNA-treated grafts stained intensely blue, whereas control grafts were negative. Microscopic examination of serial sections revealed intracellular blue granules consistent with β-Gal activity to be present in all of the endothelial cells and in numerous medial and advential cells. β-Gal activity appeared to be stable over the 7 days. From these data, we conclude that ex vivo gene therapy of vein segments prior to reimplantation using TfAdpl/DNA complexes is practical. Further studies will be needed to determine the long-term effects of TfAdpl/DNA exposure on vein grafts and whether graft patency can be improved by the introduction of antithrombotic and/or antiproliferative genes.

Overall Summary

Targeting bypass vein grafts for gene therapy with an antithrombotic gene may help to prolong graft patency. The present study tests the feasibility of using adenovirus–transferrin/polylysine–DNA complexes (TfAdpl/DNA) as the DNA delivery vehicle to achieve high-efficiency gene transfer into vascular interposition vein grafts. Autologous rabbit jugular vein segments were incubated ex vivo for 60 min in a solution of TfAdpl/DNA complexes, where the DNA delivered was the β-galactosidase (β-Gal) reporter gene, and then reimplanted across the ligated right carotid artery. Control veins were incubated in a TfAdpl solution in which DNA was omitted. Veins were harvested 3 (n = 3) and 7 (n = 3) days post-operatively and (β-Gal activity was determined by X-Gal chromogen staining. All six TfAdpl/ DNA-treated grafts stained intensely blue, whereas control grafts were negative. Microscopic examination of serial sections revealed intracellular blue granules consistent with (β-Gal activity to be present in all of the endothelial cells and in numerous medial and advential cells. (β-Gal activity appeared to be stable over the 7 days. No intracellular blue granules were observed in control tissue segments. From these data, we conclude that ex vivo gene therapy of vein segments prior to reimplantation using TfAdpl/DNA complexes is practical. Further studies will be needed to determine the long-term effects of TfAdpl/DNA exposure on vein grafts and whether graft patency can be improved by the introduction of antithrombotic and/or antiproliferative genes.