A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model
Abstract 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 devel...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2022-06-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-13536-z |
| _version_ | 1811251486128078848 |
|---|---|
| author | Nayoung Ko Joohyun Shim Hyoung-Joo Kim Yongjin Lee Jae-Kyung Park Kyungmin Kwak Jeong-Woong Lee Dong-Il Jin Hyunil Kim Kimyung Choi |
| author_facet | Nayoung Ko Joohyun Shim Hyoung-Joo Kim Yongjin Lee Jae-Kyung Park Kyungmin Kwak Jeong-Woong Lee Dong-Il Jin Hyunil Kim Kimyung Choi |
| author_sort | Nayoung Ko |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-12T16:21:10Z |
| format | Article |
| id | doaj.art-9af5897a9cd04f4cb6fd32739454dc65 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T16:21:10Z |
| publishDate | 2022-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-9af5897a9cd04f4cb6fd32739454dc652022-12-22T03:25:34ZengNature PortfolioScientific Reports2045-23222022-06-0112111110.1038/s41598-022-13536-zA desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation modelNayoung Ko0Joohyun Shim1Hyoung-Joo Kim2Yongjin Lee3Jae-Kyung Park4Kyungmin Kwak5Jeong-Woong Lee6Dong-Il Jin7Hyunil Kim8Kimyung Choi9Department of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and BiotechnologyDepartment of Animal Science and Biotechnology, Chungnam National UniversityDepartment of Transgenic Animal Research, Optipharm, Inc.Department of Transgenic Animal Research, Optipharm, Inc.Abstract 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.https://doi.org/10.1038/s41598-022-13536-z |
| spellingShingle | Nayoung Ko Joohyun Shim Hyoung-Joo Kim Yongjin Lee Jae-Kyung Park Kyungmin Kwak Jeong-Woong Lee Dong-Il Jin Hyunil Kim Kimyung Choi A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model Scientific Reports |
| 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 |
| url | https://doi.org/10.1038/s41598-022-13536-z |
| work_keys_str_mv | AT nayoungko adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT joohyunshim adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT hyoungjookim adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT yongjinlee adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT jaekyungpark adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT kyungminkwak adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT jeongwoonglee adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT dongiljin adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT hyunilkim adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT kimyungchoi adesirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT nayoungko desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT joohyunshim desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT hyoungjookim desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT yongjinlee desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT jaekyungpark desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT kyungminkwak desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT jeongwoonglee desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT dongiljin desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT hyunilkim desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel AT kimyungchoi desirabletransgenicstrategyusingggta1endogenouspromotermediatedknockinforxenotransplantationmodel |