Cargando…

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Abl...

Descripción completa

Detalles Bibliográficos
Autores principales: Ko, Nayoung, Shim, Joohyun, Kim, Hyoung-Joo, Lee, Yongjin, Park, Jae-Kyung, Kwak, Kyungmin, Lee, Jeong-Woong, Jin, Dong-Il, Kim, Hyunil, Choi, Kimyung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9187654/
https://www.ncbi.nlm.nih.gov/pubmed/35688851
http://dx.doi.org/10.1038/s41598-022-13536-z
_version_ 1784725211783888896
author Ko, Nayoung
Shim, Joohyun
Kim, Hyoung-Joo
Lee, Yongjin
Park, Jae-Kyung
Kwak, Kyungmin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
author_facet Ko, Nayoung
Shim, Joohyun
Kim, Hyoung-Joo
Lee, Yongjin
Park, Jae-Kyung
Kwak, Kyungmin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
author_sort Ko, Nayoung
collection PubMed
description Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.
format Online
Article
Text
id pubmed-9187654
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-91876542022-06-12 A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model Ko, Nayoung Shim, Joohyun Kim, Hyoung-Joo Lee, Yongjin Park, Jae-Kyung Kwak, Kyungmin Lee, Jeong-Woong Jin, Dong-Il Kim, Hyunil Choi, Kimyung Sci Rep Article Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients. Nature Publishing Group UK 2022-06-10 /pmc/articles/PMC9187654/ /pubmed/35688851 http://dx.doi.org/10.1038/s41598-022-13536-z Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This 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 Article
Ko, Nayoung
Shim, Joohyun
Kim, Hyoung-Joo
Lee, Yongjin
Park, Jae-Kyung
Kwak, Kyungmin
Lee, Jeong-Woong
Jin, Dong-Il
Kim, Hyunil
Choi, Kimyung
A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title_full A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title_fullStr A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title_full_unstemmed A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title_short A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
title_sort desirable transgenic strategy using ggta1 endogenous promoter-mediated knock-in for xenotransplantation model
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9187654/
https://www.ncbi.nlm.nih.gov/pubmed/35688851
http://dx.doi.org/10.1038/s41598-022-13536-z
work_keys_str_mv AT konayoung adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT shimjoohyun adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kimhyoungjoo adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT leeyongjin adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT parkjaekyung adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kwakkyungmin adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT leejeongwoong adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT jindongil adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kimhyunil adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT choikimyung adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT konayoung desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT shimjoohyun desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kimhyoungjoo desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT leeyongjin desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT parkjaekyung desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kwakkyungmin desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT leejeongwoong desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT jindongil desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT kimhyunil desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel
AT choikimyung desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel