426 TRANSGENIC PIGS EXPRESSING HIGH LEVELS OF HUMAN THROMBOMODULIN ON VASCULAR ENDOTHELIUM

Abstract

The development of a1,3-galactosyltransferase deficient pigs along with overexpression of complement regulatory proteins was an important step to overcome hyperacute rejection of pig organs transplanted into primates. However, the lack of galactose-α1,3-galactose (αGal) epitopes does not protect against subsequent rejection mechanisms, such as acute vascular rejection, which plays a crucial role to achieve prolonged graft survival in pig-to-human xenotransplantation. Acute vascular rejection involves incompatibilities between human blood and porcine endothelial cells, leading to coagulation disorders. Indeed, thrombotic microangiopathy is one of the key characteristics of rejected pig-to-primate grafts from aGal-deficient donor animals. One of the strategies to overcome this coagulation problem by genetic modification of donor pigs is expression of human thrombomodulin (TM) on the porcine endothelium. Although porcine TM is able to bind circulating human thrombin, the resulting heterodimer is unable to cleave human protein C, which promotes the coagulation cascade. We addressed this problem by the development of a vector placing the single-exon open reading frame of human TM under the control of a 6-kb promoter fragment obtained from the porcine TM gene. In addition, we replaced the 3′-untranslated region (UTR) by a polyadenylation box from the bovine growth hormone gene to avoid interferon-y-dependent destabilization of thrombomodulin transcripts. This vector was linked to a floxed neomycin cassette and transfected into porcine fetal fibroblasts. Positive cell clones were selected with G418, mixed, and then used for nuclear transfer. Donor fibroblasts were electrofused with enucleated oocytes resulting in 358 reconstructed embryos (91.1% fusion rate). One or two days later, 294 embryos were transferred to 3 synchronized gilts. In total, 8 cloned transgenic piglets with 1 or 2 independent integration sites of the transgene were born. Heart, kidney, lung, and liver of 5 founder animals were analyzed by immunohistochemistry They were found to express thrombomodulin strongly and strictly limited to the vascular endothelium of at least heart and kidney. The staining intensity was estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Cells from founder pigs expressing TM strongly have been used for recloning. The effect of human TM to normalize blood coagulation will be tested in experiments involving ex vivo perfusion of transgenic hearts and kidneys with human blood. Supported by the Deutsche Forschungsgemeinschaft (FOR 535).