GENETICALLY MODIFIED ADENOVIRAL VECTOR CONTAINING A HETEROLOGOUS RGD PEPTIDE IN THE HI LOOP OF THE FIBER KNOB IMPROVES GENE TRANSFER TO VASCULAR ENDOTHELIAL CELLS IN ORGAN CULTURE

Abstract

800 Vascular endothelial cells (VEC) are central to the development of immune and inflammatory processes especially allograft and xenograft rejection. Thus, the genetic manipulation of VEC for transplantation is an attractive option. Adenoviral vectors (Ad) are commonly used for gene therapy, however, very low transfection efficiency has been achieved in intact vasculature. The aim of the present study was to genetically engineer an Ad containing a heterologous RGD peptide epitope in the HI loop of the fiber knob (AdHIRGDLuc) that binds with high affinity to integrin receptors and potentially enhances the capability of gene transfer to VEC in intact blood vessels. Methods: Sections of human and non-human primate blood vessels were obtained during routine organ procurement and preserved in UW solution. Small pieces of aorta, coronary (CA), carotid, pulmonary (PA) and superior mesenteric (SMA) artery, saphena (Saph), vena cava (VC), porta, pulmonary (PV) and superior mesenteric (SMV) vein were placed in individual 48 well plates containing 199 medium supplemented with 10% heat-inactivated fetal calf serum, and cultured at 37°C, in a humidified 5% CO2/air atmosphere. In this organ culture model, described by Fabre et al, the VEC remains intact, and viable as a non-dividing layer for 3-4 days. A non-modified Ad vector (AdCMVLuc), and AdHIRGDLuc encoding a firefly luciferase reported gene, were used. Gene transfer was made with 1 × 109 particle forming units per well in 300 ml of OptiMEM media per well. Complete media was added to the vessels, and incubated 24 hours at 37°C. To assay the gene transfer efficiency, the organ cultures were washed with PBS and 400 mL of Promega 1X cell culture lysis reagent was added. Samples were the homogenized and incubated on ice 1 hour. Luciferase activity was assayed with the Promega kit. The results are expressed as a mean percentage ± SEM. Results:(Figure)FigureConclusions: Significantly higher gene transfer was achieved in intact blood vessels with AdHIRGDLuc compared with the non-modified Ad vector. The technological breakthroughs in generating recombinant Ad vectors with modified fibers containing novel peptides open a broad range of therapeutic opportunities for transplant atherosclerosis and chronic rejection as well as in vascular diseases.