Gene therapy with p40 IL-12 (but not IL-4) prolongs corneal allograft survival in sheep

Immunological rejection, the major cause of corneal allograftfailure, destroys the corneal endothelium. Aims.We hypothe-sized that transfer of genes encoding immunomodulatorycytokines to corneal endothelium might prolong allograft survival. Our aims were to investigate whether expression ofIL-4 or the p40 subunit of IL-12 by endothelial cells after genetherapy of the donor cornea would prolong sheep corneal graftsurvival. Methods.Adenoviral vectors encoding either full-length ovine IL-4 or p40 IL-12 in tandem with green fluores-cent protein (GFP) under the control of two separate CMVpromoters were generated using the AdEasy system. Ovinecorneas were transfected ex vivo and transgene expressionassessed by RT-PCR and by assays for functional, secretedprotein. Transfected corneas were then transplanted orthotopi-cally to sheep. Results.Using RT-PCR, mRNA expression wasshown for each cytokine for 21 days after in vitroinfection ofovine corneas and subsequent organ-culture. Supernatant fromIL-4-transfected corneas induced proliferation in IL-4-dependent TF1.8 cells. Expression of functional p40 IL-12 wasshown by inhibition of IL-12-induced monocyte stimulation bysupernatant from p40 IL-12 transfected corneas. After trans-plantation of ex vivo-transfected donor corneas, untreated(n=13) and mock-virus treated (n=6) grafts rejected at amedian of 21 and 20 days, respectively, whereas p40 IL-12-treated (n=9) and IL-4-treated (n=6) grafts rejected at a medianof 45 days (p=0.003 compared with controls) and 18.5 days(p>0.05), respectively. GFP was visualized in vivo in survivinggrafts. Immunohistochemistry and histology on corneas andiris flat-mounts demonstrated eosinophils in IL-4–modifiedgrafts. Conclusions.Ex vivo transfer of the gene encoding p40 IL-12 but not IL-4 to donor corneal endothelium prolongscorneal graft survival in sheep.