Adenovirus mediated gene transfer into rat lung grafts at the time of harvest

Objective: New methods to introduce genetic material into cells in vivo may revolutionize current treatment modalities. Expression of functional genes in lung allografts could be used as a prophylactic strategy for reperfusion injury and rejection. We studied the feasibility of ex vivo adenovirus mediated transfection of rat lung allografts. Methods: In group I (n = 3) donor rat lungs (Fisher) were flushed with Low Potassium Dextran Glucose (LPDG) solution (20 ml, 20°C). 4 x 10¹¹ viral particles of adenovirus 5 containing the E. coli lacZ reporter gene coding for β- galactosidase (AdCMV-β-Gal) were added to the last milliliter of the flash solution. Lung grafts were stored for 3.5 h at room temperature followed by syngeneic orthotopic transplantation (Fisher to Fisher) using a microsurgical cuff technique. On postoperative day 5 the heart lung block was extracted and flushed with x-Gal (β-Gal substrate) and kept in x-Gal for 3 h at 37°C. Color development was observed macroscopically and plastic embedded sections were used for histologic examination. Group II grafts (n = 3) served as controls and were flushed without adenovirus. Results: X-Gal stained the transfected lung grafts blue, indicating high reporter gene expression. Control lungs did not stain with x-Gal. In group I histological examination demonstrated transfection predominantly in type II pneumocytes. Surprisingly endothelial cells showed no β-Gal activity. Conclusion: This study demonstrates that ex vivo transfection of lung grafts at the time of harvest is a feasible method of gene transfer and results in gene expression after transplantation.