Potential deleterious role of anti‐Neu5Gc antibodies in xenotransplantation

Human beings do not synthesize the glycolyl form of the sialic acid (Neu5Gc) and only express the acetylated form of the sugar, whereas a diet‐based intake of Neu5Gc provokes a natural immunization and production of anti‐Neu5Gc antibodies in human serum. However, Neu5Gc is expressed on mammal glycoproteins and glycolipids in most organs and cells. We review here the relevance of Neu5Gc and anti‐Neu5Gc antibodies in the context of xenotransplantation and the use of animal‐derived molecules and products, as well as the possible consequences of a long‐term exposure to anti‐Neu5Gc antibodies in recipients of xenografts. In addition, the importance of an accurate estimation of the anti‐Neu5Gc response following xenotransplantation and the future contribution of knockout animals mimicking the human situation are also assessed.     

Pig kidney graft survival in a baboon for 136 days: longest life‐supporting organ graft survival to date

The longest survival of a non‐human primate with a life‐supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α‐1,3‐galactosyltransferase gene‐knockout pig transgenic for two human complement‐regulatory proteins and three human coagulation‐regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti‐CD20mAb (induction) and anti‐CD40mAb+rapamycin+corticosteroids (maintenance). Anti‐TNF‐α and anti‐IL‐6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein‐losing nephropathy were observed. There was no evidence of an elicited anti‐pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti‐inflammatory agents prevented immune injury and a protein‐losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.     

siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation

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.