Evidence for GTKO/β4GalNT2KO Pigs as the Preferred Organ-source for Old World Nonhuman Primates as a Preclinical Model of Xenotransplantation

Cui, Yehua; Yamamoto, Takayuki; Raza, Syed Sikandar; Morsi, Mahmoud; Nguyen, Huy Quoc; Ayares, David; Cooper, David K. C.; Hara, Hidetaka

doi:10.1097/txd.0000000000001038

Cited by 25 publications

(48 citation statements)

References 17 publications

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“…In our experience, we have rarely identified baboons in which this was the case, although sometimes antibody binding to the two pig cell types is comparable. However, antibody binding to TKO pig cells is associated with a very high degree of cytotoxicity 5,6 . These observations indicate that it will be difficult to identify baboons with lower antibody binding to TKO pig cells.…”

Section: Discussionmentioning

confidence: 99%

“…These observations indicate that it will be difficult to identify baboons with lower antibody binding to TKO pig cells. We have therefore suggested that a different pig (ie, a GTKO/β4GalNT2KO) is required for studies in baboons than those that will be ideal for clinical trials (ie, a TKO) 6 …”

Section: Discussionmentioning

confidence: 99%

“…However, there is a discordance between humans and Old World non‐human primates (NHPs) in their response to TKO pig cells and organs. Baboons and other Old World NHPs have natural IgM antibodies that bind to TKO and GTKO/CMAHKO (double‐knockout, DKO) pig cells, and this binding in vitro may be associated with potent complement‐dependent cytotoxicity 2,4‐6 . These antibodies bind to currently unknown xenoantigen(s) that are believed to be one or more carbohydrate moieties exposed after deletion of expression of N‐glycolylneuraminic acid (Neu5Gc), that is, cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase gene‐knockout (CMAHKO) pigs.…”

Section: Introductionmentioning

confidence: 99%

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Evidence suggesting that deletion of expression of N‐glycolylneuraminic acid (Neu5Gc) in the organ‐source pig is associated with increased antibody‐mediated rejection of kidney transplants in baboons

Iwase

Jagdale

Yamamoto

et al. 2021

Xenotransplantation

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Pigs deficient in three glycosyltransferase enzymes (triple‐knockout [TKO] pigs) and expressing “protective” human transgenes are likely sources of organs for transplantation into human recipients. Testing of human sera against red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs has revealed minimal evidence of natural antibody binding. However, unlike humans, baboons exhibit natural antibody binding to TKO pig cells. The xenoantigen specificities of these natural antibodies are postulated to be one or more carbohydrate moieties exposed when N‐glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival of renal grafts in baboons from pigs that either expressed Neu5Gc (GTKO pigs; Group1, n = 5) or did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group2, n = 5). An anti‐CD40mAb‐based immunosuppressive regimen was administered in both groups. Group1 kidneys functioned for 90‐260 days (median 237, mean 196 days), with histopathological features of antibody‐mediated rejection in two kidneys. Group2 kidneys functioned for 0‐183 days (median 35, mean 57), with all of the grafts exhibiting histologic features of antibody‐mediated rejection. These findings suggest that the absence of expression of Neu5Gc on pig kidneys impacts graft survival in baboon recipients.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evidence suggesting that deletion of expression of N‐glycolylneuraminic acid (Neu5Gc) in the organ‐source pig is associated with increased antibody‐mediated rejection of kidney transplants in baboons

Iwase

Jagdale

Yamamoto

et al. 2021

Xenotransplantation

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“…It has been suggested that TKO pigs could be ideal sources of organs for future clinical xenotransplantation, 10 but they are less suitable for preclinical studies in non‐human primate models 8‐11 . Differences in immune reactivity to CMAHKO pigs (including TKO) between baboons and humans might contribute to variations in the results 9,11 . In baboons, sensitization to TKO pig cells is probably, at least in part, to the 4th xenoantigen (a carbohydrate) exposed after CMAHKO.…”

Section: Discussionmentioning

confidence: 99%

Evidence that sensitization to triple‐knockout pig cells will not be detrimental to subsequent allotransplantation

Hara

Nguyen

Wang

et al. 2021

Xenotransplantation

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The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross‐react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild‐type or α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs. However, it is not known whether recipients of triple‐knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross‐react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti‐TKO pig antibodies could be measured—in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti‐TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

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“…Although A3GALT2 KO pigs rarely had an effect on pig-to-human immune reactivity in our study, it could provide more information on the role of iGb3 on NKT cell activity in preclinical test. Recently, preclinical studies have reported that pigs with a CMAH gene knocked out express new xenoantigens called ''forth xenoantigen'' in pig-to-Old World NHP organ transplantation (Cooper et al 2020;Cui et al 2020;Yamamoto et al 2020). This finding indicates that it is, as yet, almost impossible to confirm the effect of CMAH KO in preclinical tests.…”

Section: Discussionmentioning

confidence: 99%

Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs

Shim

Kim

et al. 2021

Transgenic Res

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In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

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Evidence for GTKO/β4GalNT2KO Pigs as the Preferred Organ-source for Old World Nonhuman Primates as a Preclinical Model of Xenotransplantation

Cited by 25 publications

References 17 publications

Evidence suggesting that deletion of expression of N‐glycolylneuraminic acid (Neu5Gc) in the organ‐source pig is associated with increased antibody‐mediated rejection of kidney transplants in baboons

Evidence suggesting that deletion of expression of N‐glycolylneuraminic acid (Neu5Gc) in the organ‐source pig is associated with increased antibody‐mediated rejection of kidney transplants in baboons

Evidence that sensitization to triple‐knockout pig cells will not be detrimental to subsequent allotransplantation

Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs

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