siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Ahrens, H E; Petersen, B; Herrmann, D; Lucas-Hahn, A; Hassel, P; Ziegler, M; Kues, W A; Baulain, U; Baars, W; Schwinzer, R; Denner, J; Rataj, D; Werwitzke, S; Tiede, A; Bongoni, Anjan; Garimella, Sai Pavan; Despont, Alain; Rieben, Robert; Niemann, H (2015). siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation. American journal of transplantation, 15(5), pp. 1407-1414. Wiley-Blackwell 10.1111/ajt.13120

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Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

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