Biocompatibility of genetically-engineered pig cornea as corneal xenograft for the use of human clinical trial

초록

Purpose : To evaluate functional compatibility of endothelial cells in genetically-engineered (GE) pig corneas to suppress immune reaction as corneal xenograft for the use of human clinical trial. Methods : α1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β1,4-N-acetylgalactosaminyltransferase (β4GalNT2) were inactivated to block Galα(1,3)Gal, N-glycolylneuraminic acid (Neu5Gc), and β4GalNT2 antigens and human CD55 (complement modulatory protein) and CD39 were introduced (hCD55+hCD39 double knock-in (DKI)) into those triple knock-out (TKO) pigs. Quadruple KO (QKO) pigs included inactivated GGTA1, B4galNT2, CMAH and isoglobotrihexosylceramide synthase (iGB3s) genes. Seven pairs of eyeballs obtained from three TKO/DKI and QKO pigs were used to evaluate the physical and functional properties as a suitable donor cornea. Corneal endothelial cell density (ECD) was serially measured with confocal microscope until 7 days of preservation in the storage solution. Doubling time for corneal endothelial cell proliferation and immunofluorescence staining was evaluated for corneal endothelial cell pump function or tight barrier function. Results : Mean age of TKO/DKI and QKO pigs was 355.7 and 636.8 days, respectively. After 7 days of preservation, mean ECD was 4065/mm2 and 2554/mm2 of TKO/DKI and QKO pigs, respectively. Mean ECD loss/week was 36.2% for GE pigs younger than 6 months while, it was 8.0% for GE pigs older than 6 months during 1 week of preservation. Mean doubling time was 81.9 hours for TKO/DKI pigs and 71.6 hours for QKO pigs. ZO-1, N-cadherin, ATPase, SLC4A1, and aquaporin 1 were well expressed on most of the endothelial cells in both GE pigs. Conclusions : It suggests that these TKO/DKI and QKO pig corneas show acceptable endothelial cell functional capacities in older than 6 month-aged pigs as a suitable donor for corneal xenotransplantation.