*Equal first authorship

In experimental models of corneal transplantation, gene therapy of the donor cornea has modulated rejection and prolonged graft survival in a proportion of cases. Our objective was to construct multiple vectors for use in a cocktail to prolong corneal allograft survival in all animals treated. We constructed recombinant adenoviral or lentiviral vectors encoding therapeutic proteins that target the host inflammatory response, neovascularisation, or apoptosis. Interleukin 10, an immunomodulatory cytokine that significantly prolongs sheep corneal allograft survival, was encoded in an adenoviral vector (AdV-IL10). Lentiviral vectors encoding soluble vascular endothelial growth factor receptor-1  and endostatin-kringle 5, both of which are anti-angiogenic, were constructed (LV-sFlt, LV-EK5), together with lentivectors encoding indoleamine 2,3-dioxygenase, an inhibitor of T-cell proliferation (LV-IDO), and B-cell lymphoma-extra large protein, which is anti-apoptotic (LV-BclxL). To test the biological function of transgenic proteins expressed by the vectors, human umbilical vein endothelial cells (HUVEC) were treated with conditioned medium from LV-sFlt or LV-EK5-treated cells. A significant reduction in HUVEC proliferation was observed compared with controls. The function of IDO was assessed by detection of L-kynurenine, a stable down-stream product of tryptophan degradation catalysed by IDO. Over-expression of kynurenine in the supernatant of LV-IDO treated cells was detected, compared with controls. A LV-BclxL-treated cell line exhibited significantly fewer cells in early apoptosis compared with controls, when assessed by flow cytometry. Transduction of sheep corneas with each lentivector in vitro showed expression of each transgene compared with controls at the protein or mRNA level, as detected by ELISA or RT-PCR, respectively. Co-transduction with AdV-IL10 and LV-EK5 resulted in expression of both IL10 and EK5, and levels were not affected by the combined treatment. We conclude that each of our constructs produces functional transgenic protein, and a cocktail will be suitable for testing in our sheep model of corneal transplantation.