Targeting tissue factor and GGTA1 expression in pigs for improved xenograft survival

Pig-to-human xenotransplantation could contribute to alleviate the growing shortage of human donor organs. Genetically modified pigs are crucial for overcoming the severe immune rejections resulting from the phylogenetic distance between pigs and humans. The hyperacute rejection can be prevented by knocking out the α1,3-galactosyltransferase gene (GGTA-1) and/or transgenic expression of human complement regulators in the porcine genome. Anti-apoptotic, anti-inflammatory, and/or anticoagulant strategies are considered necessary to overcome the acute vascular rejection with associated endothelial cell activation and microvascular thrombosis. Tissue factor (TF) is upregulated by activated endothelium and initiates the extrinsic coagulation cascade. Therefore, it constitutes a promising target for genetic modification. We have recently produced pigs with decreased TF expression by siRNA mediated silencing to ameliorate the dysregulated coagulation during AVR (1). Here, we describe the generation of pigs with a TF knockdown (TFkd) in combination with a knockout of the GGTA1 gene. TFkd pigs were generated from porcine wild-type fibroblasts transfected with a plasmid expressing an siRNA targeting the TF gene. Seven surviving piglets were born after somatic cell nuclear transfer and embryo transfer. All piglets expressed the siRNA. TF mRNA levels were decreased by 94.1 ± 4.7 % and TF protein levels were decreased and only slightly up-regulated upon TNF-α stimulus compared to a significant up-regulation in wild-type (WT) controls (mean fluorescence intensity TFkd: 6408 ± 35.4 vs. WT: 8362 ± 495.0 unstimulated; TFkd: 7136 ± 136 vs. WT: 13038 ± 1672 with 50ng/ml TNF-α). TF down-regulation significantly increased clotting time in a microcarrier bead clotting assay (TFkd 73.3 ± 8.8 minutes vs. WT 45.8 ± 7.7 minutes, p < 0.0001) and significantly decreased thrombus formation in a flow chamber assay (thrombus coverage per viewing field TFkd: 3.18 ± 3.75 % vs. WT: 23.5 ± 13.01 %, p < 0.0001). One TFkd boar was mated to two heterozygous GGTA1-KO (GGTA1+/-) sows produced earlier (2) and a wild-type pig. Seventeen out of 35 piglets born carried the TFkd plasmid as detected by PCR. Surveyor mutation detection analysis revealed that six of the seventeen TFkd pigs had a heterozygous GGTA1-KO. The TFkd/GGTA1+/- pigs will soon be bred to produce TFkd/GGTA1-KO-/- pigs. Expression of the siRNA could be proven in all chosen founder animals. Further expression analysis of these pigs is ongoing. We anticipate that this approach will generate pigs with genetic properties that prolong xenograft survival in pig-to-primate xenotransplantation. This project was funded by DFG TR-CRC 127 and ‘REBIRTH’ Cluster of Excellence..