Modifying the Glycome in Pigs for Xenotransplantation.

I of glycosyltransferase genes during human evolution is a fundamental cause of differential glycosylation in humans and pigs, resulting in the presence of carbohydrate structures on pig cells that are recognized by natural (preexisting) human antibodies. αGal, the first of these epitopes to be identified, is synthesized by α1,3galactosyltransferase, encoded by theGGTA1 gene. The crucial importance of αGal in solid organ xenotransplantation was demonstrated by the finding that knockout of GGTA1 prevented hyperacute rejection in the pig-to-nonhuman primate (NHP) preclinical model. However, there is evidence to suggest that knockout of additional glycosyltransferase genes will further improve the “compatibility” of porcine xenografts. One such gene is CMAH, which is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc). The development of efficient genome editing techniques (ZFNs, TALENs, and CRISPR/Cas9) has revolutionized the genetic engineering of donor pigs, allowing precise and rapid targeted mutagenesis in cultured primary cells, which can then be cloned by somatic cell nuclear transfer. The Tector group was the first to adopt this technology to knock out multiple glycosyltransferase genes, namely, GGTA1 and CMAH. When tested against 121 human serum samples, peripheral blood mononuclear cells from double-knockout (DKO) pigs generally bound less antibody than GGTA1 single-KO cells. In this issue of Transplantation, the group presents preliminary evidence that the elimination of Neu5Gc may have benefits in pig liver xenotransplantation beyond reducing the binding of natural antibodies. In vivo and ex vivo models have shown that pig livers rapidly sequester human and NHP platelets, resulting in the development of lifethreatening thrombocytopenia in the pig-to-baboon model (reviewed by Ekser et al). Inhibition and knockout studies indicate that the asialoglycoprotein receptor-1 (ASGR-1) plays an important role in the binding of human and NHP platelets to porcine endothelium, whereas deletion of αGal and/or expression of human CD46 have no effect. In the