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

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 CR...

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Autores principales: Shim, Joohyun, Ko, Nayoung, Kim, Hyoung-Joo, Lee, Yongjin, Lee, Jeong-Woong, Jin, Dong-Il, Kim, Hyunil, Choi, Kimyung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer International Publishing 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478729/
https://www.ncbi.nlm.nih.gov/pubmed/34232440
http://dx.doi.org/10.1007/s11248-021-00271-w
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author Shim, Joohyun
Ko, Nayoung
Kim, Hyoung-Joo
Lee, Yongjin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
author_facet Shim, Joohyun
Ko, Nayoung
Kim, Hyoung-Joo
Lee, Yongjin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
author_sort Shim, Joohyun
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11248-021-00271-w.
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spelling pubmed-84787292021-10-08 Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs Shim, Joohyun Ko, Nayoung Kim, Hyoung-Joo Lee, Yongjin Lee, Jeong-Woong Jin, Dong-Il Kim, Hyunil Choi, Kimyung Transgenic Res Original Paper 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11248-021-00271-w. Springer International Publishing 2021-07-07 2021 /pmc/articles/PMC8478729/ /pubmed/34232440 http://dx.doi.org/10.1007/s11248-021-00271-w Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Original Paper
Shim, Joohyun
Ko, Nayoung
Kim, Hyoung-Joo
Lee, Yongjin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title_full Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title_fullStr Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title_full_unstemmed Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title_short Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs
title_sort human immune reactivity of ggta1/cmah/a3galt2 triple knockout yucatan miniature pigs
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478729/
https://www.ncbi.nlm.nih.gov/pubmed/34232440
http://dx.doi.org/10.1007/s11248-021-00271-w
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