Generation of Transgenic Pigs Co-Expressing Human Thrombomodulin and CD39

Background: Intravascular thrombosis remains a problem in pig-to-primate xenotransplantation, even with donors that are α1,3-galactosyltransferase knockout (GTKO) and express human complement regulatory proteins (hCRPs). Thrombomodulin (TBM) is a key endothelial anticoagulant but pig TBM interacts poorly with human thrombin; CD39 has potent antithrombotic activity but is lost from the endothelium under inflammatory conditions. We propose that co-expression of human TBM and CD39 on the GTKO/hCRP platform will inhibit the development of thrombosis in solid organ xenografts. In this study, we investigated the feasibility of generating viable transgenic pigs expressing both of these antithrombotic genes. Aim: To generate and characterise GTKO/hCD55-hCD59 pigs coexpressing hTBM and hCD39. Methods: Early passage fibroblasts were transfected with a 2A-linked HygroR-hTBM-hCD39 construct driven by the mouse H-2Kb promoter. Stable transfectants expressing hTBM were sorted by flow cytometry and used for somatic cell nuclear transfer. The resulting piglets were screened by PCR, immunohistochemistry and Western blot. Results: HygroR fibroblasts sorted on the basis of hTBM expression also strongly expressed hCD39. Transfers performed at 2 different sites (Adelaide University and NSRRC) produced a total of 10 litters. Although there was a high early mortality rate in the offspring, this was related to the cloning process rather than to bleeding problems. Pigs surviving beyond the first month appeared to be healthy and normal. Immunohistochemical analysis revealed expression of both transgenes in various tissues including heart, kidney, lung and liver, with hCD39 expressed more strongly than hTBM. Expression was most pronounced on lymphoid cells. Western blot analysis of fibroblast lysates showed bands for hTBM and hCD39 at the expected molecular weights, confirming efficient 2A-mediated processing of the 3 genes in the construct. Conclusion: We have successfully generated viable GTKO/hCD55-hCD59/hTBM-hCD39 pigs. These pigs are healthy and do not show any overt bleeding phenotype. They will be tested in pig-to-baboon renal and cardiac preclinical models to determine the impact of antithrombotic transgene expression on the development of intravascular thrombosis.