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2.1.1 Gene modificationĬRISPR/Cas9 was used to generate GGTA1, B4GALNT2, and CMAH gene-edited cells. Porcine cells carrying TKO and hTGs were generated and analyzed as reported previously 6 and described in brief below. 2 MATERIALS AND METHODS 2.1 Production of pigs with TKO and multiple human transgenes Further modifications of the porcine genome, refinement of the immunosuppressive protocol, as well as effective infection prophylaxis may improve the consistency of long-term survival.
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In the current study, we transplanted renal xenografts from TKO pigs that expressed multiple human transgenes (hTGs) in cynomolgus monkeys, demonstrating that long-term, rejection-free renal xenograft survival can be achieved with TKO-hTG pigs transplanted in nonhuman primates. Since the binding of human natural antibodies to TKO cells is significantly lower compared to GGTA1 single knock-out (GTKO) or GGTA1/ β4GALNT2 double knock-out (DKO) porcine cells, 4, 5 the TKO pig is expected to be a better donor for human xenotransplantation. 3 With this extremely powerful technology, “triple knock-out” (TKO) pigs devoid of three major carbohydrate xenoantigens (αGal, Neu5GC, and SDa) have recently been developed.
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The recent advent of the CRISPR/Cas9 gene editing technology has made it possible to efficiently inactivate multiple genes that encode enzymes responsible for synthesizing these xenoantigens and to simultaneously address pig-primate coagulation and complement pathway incompatibilities by transgenically expressing various human genes. 2 A major barrier to xenotransplantation exists in the reactivity of human natural antibodies to several carbohydrate xenoantigens expressed on porcine cells. 1 To address this unmet healthcare need, the potential use of porcine organs for human transplantation has been sought. Currently, over 108 000 individuals await organs but fewer than 40 000 transplants are performed yearly in the US. The tremendous success of transplantation as a life-saving therapy for end-stage organ failure has increased organ demand. β1,4-N-acetyl-galactosaminyl transferase 2.α(1,3)galactosyl transferase gene knock-out.glycoprotein α(1,3)galactosyl transferase.cytidine monophospho-N-acetylneuraminic acid hydroxylase.This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs. Prolonged CD4 +T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies.