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Gene Transfer of Adenoviruses Encoding IL-10 and p40 IL-12 to the Corneal Endothelium Prolongs Corneal Graft Survival
1Klebe S., 2Sykes P. J., 1Coster D. J., 2Swinburne S., 1Williams K. A.,
1Department of Ophthalmology, Flinders Medical Centre, Bedford Park (Adelaide)
2Department of Haematology, Flinders Medical Centre, Bedford Park (Adelaide)
Immunological rejection destroys the corneal endothelium and is the main reason for corneal graft. The expression of genes encoding homologous immunosuppressive cytokines by the corneal endothelium might protect the monolayer and prolong graft survival. The aim of this study was to investigate whether expression of ovine IL-10 or p40-IL-12 prolongs orthotopic corneal graft survival in an outbred sheep model. In preliminary experiments, adenovirus-mediated gene transfer was established as the method of choice. Two adenoviral vectors were generated and tested in vitro. The vectors encoded either full-length ovine IL-10 or p40 IL-12 in tandem with green fluorescent protein under the control of two separate CMV promoters. Using RT-PCR, mRNA expression was shown for both cytokines up to 21 days after in-vitro infection of fresh ovine corneas and subsequent culture. Addition of the supernatant of corneas transfected with Adv-IL-10 to MLRs resulted in inhibited stimulation, suggesting suppression by functional IL-10. Similarly, the expression of functional p40 IL-12 was shown by inhibition of IL-12 induced stimulation of monocytes by supernatant of infected corneas. In in-vivo experiments, untreated (n=13) and Mock-virus treated (n=6) grafts rejected at a median of 21 and 20 days, respectively, whereas IL-10 (n=9) and p40 IL-12 (n=9) treated tissue rejected at a median of 55 and 45 and days respectively. The prolongation of corneal graft survival was significant in both cases (p= 0.0011 and p= 0.0032). Immunohistochemistry, histology and iris flat-mounts were used to investigate the mechanism of rejection in these grafts. We conclude that gene transfer of IL-10 and p40 IL-12 to corneal endothelium prolongs corneal graft survival in sheep.