ADENOVIRUS-MEDIATED GENE DELIVERY TO THE CORNEAL ENDOTHELIUM

Genetic manipulation of donor cornea prior to transplantation has the potential to modulate the allogeneic response, as well as the endothel ial cell function, This study examined the feasibility of gene transfe r to corneal endothelial cells using replication-defective recombinant adenoviral vectors. Adult rabbit corneas were infected with recombina nt adenovirus RAd35, containing the Escherichia coli beta-galactosidas e (lacZ) gene, Localization of gene transfer was assessed by histochem ical staining for beta-galactosidase and recombinant protein productio n was quantified by a soluble assay, In initial experiments, the effic iency of gene transfer and kinetics of expression were studied ex vivo , using organ culture of transfected corneas. Following coculture of w hole corneal fragments with RAd35, high levels of gene expression were evident on days 1-7, diminishing after that time. Gene transfer was f ound to be almost entirely restricted to corneal endothelial cells, wi th scattered expression in epithelial cells. Following these ex vivo s tudies, genetically modified corneas were transplanted as orthotopic a llografts in rabbits, Similar kinetics of gene expression were seen af ter transplantation as in the ex vivo experiment, with maximal levels of gene expression in endothelial cells on days 1-4 after grafting. Co rneal function following transplantation was not affected by the gene transfer, with the corneas attaining clarity within 1 day of grafting, and thereafter showing the expected thinning on ultrasonic pachymetry . in the absence of any immunosuppression, no inflammation was evident in graft recipient eyes, with the exception of allograft rejection in 1 animal 23 days after grafting. In this study we show that gene tran sfer to nonreplicating corneal endothelial cells is feasible using rec ombinant adenovirus vectors, and so may have potential application in the setting of corneal transplantation.