Porcine iGb3s gene silencing provides minimal benefit for clinical xenotransplantation

The Galα(1,3)Gal epitope (-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyper-acute rejection (HAR) in pig-to-primate and pig-tohuman xenotransplantation. In response GGTA1 gene deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1-/pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an -GAL terminal disaccharide (iGb3) creating the possibility that iGb3s may be a source of -GAL epitopes in GGTA1-/animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross reactivity by creating and comparing GGTA1-/pigs to GGTA1-/and A3GalT2-/double knockout pigs. We used the CRISPR/Cas system to target the GGTA1, and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single and double-gene knockout piglets in a single pregnancy. Once grown to maturity the glycoshingolipid profile (including iGb3) was assayed in renal tissue by normal phase liquid chromatography. In addition Peripheral Blood Mononuclear Cells (PBMC) were subjected to 1) comparative cross match cytotoxicity analysis against human and baboon serum and 2) IB4 staining for -GAL/iGB3. Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and -GAL/iGb3 staining was unchanged by iGb3s silencing. Our data suggests that iGb3s is not a significant contributor to HAR in pig-to-primate or pig-to-human xenotransplantation. Introduction Galα1,3Gal (αGal) is a disaccharide synthesized in pigs but not in humans and old world monkeys. (1)(Figure 1a) Primarily, α-GAL is formed by the catalytic activity of the enzyme α-galactotransferase (GGTA1) by which galactose is added to Gal1–4GlcNAc structures present on glycoproteins or glycosphingolipids. (2) (Figure 1c) In humans the GGTA1 gene is inactive and humans develop antibodies when exposed to -GAL epitopes. (3) Following xenotransplantation, α-GAL antibodies promote hyperacute rejection (HAR). (4) To address the issue of -Gal epitopes, GGTA1 null animals have been generated in order to eliminate the α-GAL epitope and eliminate HAR. (5-8) However, a residual amount of α-GAL epitope reactivity has been recognized in biallelic GGTA1 knockout pig cells and implicated as a possible contributor to chronic rejection of GGTA1-/organs seen in nonprimate models of xenotransplantation. (9-11) One reason suggested for this is that the αGAL epitope can be biosynthesized by other members of the galactotransferase enzyme family. (Figure 1c) In particular the enzyme isoglobotrihexosylceramide synthase (iGb3S) is known to generate the galα1,3gal disaccharides epitopes on glycosphingolipids by the addition of galactose to lactosyl ceramide. (Figure 1b) In a similar manner to GGTA1, the human genome includes iGb3s (NM001080438) and the gene is thought to be silent due to several mutations that prevent the formation of iGb3. (12). Studies in other animals demonstrate that iGb3 levels are species specific. In rat thymus iGb3 levels are moderate whereas in mouse and human thymus iGb3 is undetectable. (13) Moreover, iGb3 is tissue specific. iGb3 is not detectable in brain, liver, kidney, spleen, thymus, testis, lung, stomach, intestine, eye, spinal cord, and plasma of mice but is detected in murine dorsal root ganglia (DRG). (13) Further studies, demonstrate that in mice iGb3 is an immune modulatory glycosphingolipid, whereby iGb3 binds to CD1d and is presented to the T-cell receptor (TCR) of invariant natural killer T (iNKT) cells. (14, 15) In addition, iGb3-primed bone marrow-derived dendritic cells exert a significant iNKT cell-mediated anti-tumor activity. (16) Consequently, both tissueand species-specific differences in iGb3 levels make information gained from rodent models difficult to translate into the porcine-human or porcine-primate xenotransplant models. (13) This translation is made more difficult because iGb3s gene expression does not match iGb3 epitope levels. In mice, iGb3s gene expression is ubiquitous within mouse tissues but iGb3 is detected only in the DRG. This disparity is potentially due to translational regulation or because of continuous flux in glycosphingolipid pathways. In particular iGb3 is a substrate for iGb4 synthase which converts iGB3 to iGb4. iGb4 does not contain the terminal Galα(1,3)gal epitope. Consequently, there is a conflict regarding the importance of iGb3/iGb3s in the literature. (12, 17) Within the context of pig-to-primate xenotransplantation, some argue for the importance of iGb3 while others have failed to detect iGb3 in porcine tissues. (18, 19) To address this we have generated iGb3s gene (A3GalT2) knockout pigs in a GGTA1/background and compared α-GAL epitope expression, antibody binding profiles, and antibody-mediated complement depended cytotoxicity between GGTA1-/and GGTA1-//A3GalT2-/double knockout pigs. Materials and Methods