siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation

作者：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 A K; Garimella P S; Despont A; Rieben R; Niemann H

来源：American Journal of Transplantation, 2015, 15(5): 1407-1414.

DOI：10.1111/ajt.13120

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 94.14.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50ng/mL TNF- was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 +/- 136 vs. WT: 13038 +/- 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 +/- 8.8min, WT: 45.8 +/- 7.7min, 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.

• 出版日期2015-5