Abstract
Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts. To overcome biological incompatibility between pigs and humans, one strategy is to remove the major antigens [Gal, Neu5Gc, and Sd(a)] present on pig cells and tissues. Triple gene (GGTA1, CMAH, and β 4GalNT2) knockout (TKO) pigs were produced in our laboratory by CRISPR-Cas9 targeting. To investigate the antigenicity reduction in the TKO pigs, the expression levels of these three xenoantigens in the cornea, heart, liver, spleen, lung, kidney, and pancreas tissues were examined. The level of human IgG/IgM binding to those tissues was also investigated, with wildtype pig tissues as control. The results showed that αGal, Neu5Gc, and Sd(a) were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs. Compared to wildtype pigs, the liver, spleen, and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding, whereas corneas, heart, lung, and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding. These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.
Highlights
Clinical transplantation has been improved enormously in recent decades; there is a major disparity between the number of patients awaiting transplantations and the available donor organs and tissues such as the hearts[1], livers[2– 3], kidneys[4–6], lungs[7–8], islets[9–10], and corneas[11–12]
The overall staining of αGal epitopes was low in the cornea with weak signals distributed in several keratocytes in the anterior-most part of the corneal stroma of WT pigs, whereas GTKO and triple gene knockout (TKO) porcine keratocytes did not show any expression of the αGal epitopes (Fig. 1B)
Due to its similarity with humans, the pig has been studied as a donor for xenotransplantation
Summary
Clinical transplantation has been improved enormously in recent decades; there is a major disparity between the number of patients awaiting transplantations and the available donor organs and tissues such as the hearts[1], livers[2– 3], kidneys[4–6], lungs[7–8], islets[9–10], and corneas[11–12]. Galactose-α1,3-galactose (αGal), the most abundant immunogenic glycan in pigs to which the human immune system is highly responsive, has long been known as the causative xenoantigen associated with hyperacute rejection of a xenograft. Antibody-mediated rejection is not eliminated even in GGTA1-deficient porcine tissues harboring complement inhibitory receptor transgenes, revealing the significance of non-Gal antigens expressed on pig tissues[19–21]. Continued pursuit of xenoantigens in pigs has led to the identification of other glycans associated with xenograft injury induced by highly specific circulating human antibodies, including N-glycolylneuraminic acid (Neu5Gc) encoded by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene and DBAreactive glycans ( named Sd(a) antigen) produced by β-1,4-N-acetylgalactosaminyl transferase 2 (β4GalNT2)[22– 23]. Modified pigs lacking αGal and Neu5Gc carbohydrate modifications have subsequently been produced, including GTKO/CMAH knockout (KO) pigs[24–25], GTKO/CD46/CMAH KO pigs[24], and GGTA1/CMAH/ASGR1 KO pigs[26], in which human antibody binding is dramatically reduced
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