5.2. Gene transfer by closed-circuit perfusion ex vivo

To increase the time of contact between the adenovirus vector and the target organ cells, we developed a closed-circuit perfusion method. Perfusion of two explanted kidneys with the adenoviral vector ex vivo at room temperature for up to 17 hours did not result in β -galactosidase-positive kidney cells. Therefore, the ex vivo perfusion was carried out at body temperature, 37° C, for 12 hours in four separate experiments, to allow viral entry into the target cells and to create optimal conditions for gene expression and protein synthesis. For this reason, MEM amino acids were also added to the ex vivo perfusate to provide enough material for protein synthesis. After 12 hours of ex vivo closed-circuit perfusion, effective and strong expression of the lacZ gene could be observed in glomerular cells. In a histological analysis, lacZ gene expression was observed in approximately 80% of the glomeruli (Fig. 2), and most mesangial and endothelial cells as well as epithelial podocytes appeared to be positive in several glomeruli. Only scant staining was seen in endothelial cells elsewhere in the blood vessels, and the epithelial cells of the tubuli did not exhibit any staining.

The pilot experiment by ex vivo lung perfusion for ten hours at 37°C resulted in transgene expression in capillary endothelial cells and alveolar epithelial cells (data not shown).