Browsing by Author "Tector, M."

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    Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes
    (Wiley, 2015-05) Estrada, J.; Martens, G.; Li, P.; Adams, A.B.; Newell, K.A.; Ford, M.L.; Butler, J.R.; Sidner, R.A.; Tector, M.; Tector, A.J.; Department of Surgery, IU School of Medicine
    Background Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes. Methods The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. Results Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Nonhuman primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased nonhuman primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2. Conclusions Inactivation of the β4GalNT2 gene reduces human and nonhuman primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of nonhuman primates towards GGTA1/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the nonhuman primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
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    Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs
    (Wiley Blackwell (Blackwell Publishing), 2014-08) Burlak, C.; Paris, L. L.; Lutz, A. J.; Sidner, R. A.; Estrada, J.; Li, P.; Tector, M.; Tector, A. J.; Department of Surgery, IU School of Medicine
    Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human antibody binding was reduced in preliminary analysis. We analyzed the binding of human IgM and IgG from 121 healthy human serum samples for binding to GGTA1 KO and GGTA1/CMAH KO peripheral blood mononuclear cells (PBMCs). We analyzed a sub population for reactivity toward genetically modified pig PBMCs as compared to chimpanzee and human PBMCs. Deletion of the GGTA1 and CMAH genes in pigs improved the crossmatch results beyond those observed with chimpanzees. Sorting the 121 human samples tested against the GGTA1/CMAH KO pig PBMCs did not reveal a distinguishing feature such as blood group, age or gender. Modification of genes to make pig carbohydrates more similar to humans has improved the crossmatch with human serum significantly.