{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T13:03:35Z","timestamp":1791810215168,"version":"4.3.4"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,1,20]],"date-time":"2025-01-20T00:00:00Z","timestamp":1737331200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO\/hCD55\/hTM\/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. <jats:italic>In vitro<\/jats:italic>, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p&amp;lt;0.01, IgM p&amp;lt;0.0001). Similar to TKO\/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p&amp;lt;0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p&amp;lt;0.0001), TKO (p&amp;lt;0.01), and TKO\/hCD55\/hTM pigs (p&amp;lt;0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO\/hCD55 pigs were considered safe when they were all bred in a general environment.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2024.1488552","type":"journal-article","created":{"date-parts":[[2025,1,20]],"date-time":"2025-01-20T07:20:27Z","timestamp":1737357627000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":16,"title":["The generation and evaluation of TKO\/hCD55\/hTM\/hEPCR gene-modified pigs for clinical organ xenotransplantation"],"prefix":"10.3389","volume":"15","author":[{"given":"Guoli","family":"Huai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxiang","family":"Du","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenhui","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuxuan","family":"Xie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongmei","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanyan","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangyang","family":"Xing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaoping","family":"Deng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dengke","family":"Pan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,1,20]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1111\/ajt.16974","article-title":"OPTN\/SRTR 2020 annual data report: introduction","author":"Israni","year":"2022","journal-title":"Am J Transplant"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1093\/cvr\/cvac180","article-title":"Cardiac xenotransplantation: from concept to clinic","volume":"118","author":"Reichart","year":"2023","journal-title":"Cardiovasc Res"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/j.addr.2020.04.001","article-title":"Porcine genome engineering for xenotransplantation","volume":"168","author":"Niu","year":"2021","journal-title":"Adv Drug Delivery Rev"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2004836117","article-title":"Porcine germline genome engineering","volume":"118","author":"Yang","year":"2021","journal-title":"Proc Natl Acad Sci U.S.A"},{"key":"B5","doi-asserted-by":"publisher","first-page":"127055","DOI":"10.1016\/j.ijbiomac.2023.127055","article-title":"Gene-editing technology, from macromolecule therapeutics to organ transplantation: Applications, limitations, and prospective uses","volume":"253","author":"Harisa","year":"2023","journal-title":"Int J Biol Macromol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22052249","article-title":"One-step generation of multiple gene-edited pigs by electroporation of the CRISPR\/cas9 system into zygotes to reduce xenoantigen biosynthesis","volume":"22","author":"Tanihara","year":"2021","journal-title":"Int J Mol Sci"},{"key":"B7","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1111\/xen.2015.22.issue-3","article-title":"Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1\/CMAH\/beta4GalNT2 genes","volume":"22","author":"Estrada","year":"2015","journal-title":"Xenotransplantation"},{"key":"B8","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1016\/j.actbio.2018.03.055","article-title":"Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1\/beta4GalNT2\/CMAH","volume":"72","author":"Zhang","year":"2018","journal-title":"Acta Biomater"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.849711","article-title":"Both natural and induced anti-sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation","volume":"13","author":"Feng","year":"2022","journal-title":"Front Immunol"},{"key":"B10","doi-asserted-by":"publisher","first-page":"9771","DOI":"10.1038\/s41598-020-66311-3","article-title":"Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout)","volume":"10","author":"Yamamoto","year":"2020","journal-title":"Sci Rep"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1586\/eci.09.81","article-title":"Overcoming the barriers to xenotransplantation: prospects for the future","volume":"6","author":"Ekser","year":"2010","journal-title":"Expert Rev Clin Immunol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra1208937","article-title":"Acute liver failure","volume":"369","author":"Bernal","year":"2013","journal-title":"N Engl J Med"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1002\/path.2016.238.issue-2","article-title":"The role of genetically engineered pigs in xenotransplantation research","volume":"238","author":"Cooper","year":"2016","journal-title":"J Pathol"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-6143.2011.03846.x","article-title":"B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months","volume":"12","author":"Mohiuddin","year":"2012","journal-title":"Am J Transplant"},{"key":"B15","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1111\/ajt.16780","article-title":"Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques","volume":"22","author":"Ma","year":"2022","journal-title":"Am J Transplant"},{"key":"B16","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1111\/xen.2013.20.issue-2","article-title":"Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation","volume":"20","author":"Spiezia","year":"2013","journal-title":"Xenotransplantation"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/nri2099","article-title":"Xenotransplantation: current status and a perspective on the future","volume":"7","author":"Yang","year":"2007","journal-title":"Nat Rev Immunol"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-6143.2008.02210.x","article-title":"Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI","volume":"8","author":"Roussel","year":"2008","journal-title":"Am J Transplant"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1586\/eci.12.56","article-title":"Immunobiology of liver xenotransplantation","volume":"8","author":"Ekser","year":"2012","journal-title":"Expert Rev Clin Immunol"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0611493104","article-title":"Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells","volume":"104","author":"Bae","year":"2007","journal-title":"Proc Natl Acad Sci U.S.A"},{"key":"B21","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1111\/xen.2014.21.issue-1","article-title":"Regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition","volume":"21","author":"Iwase","year":"2014","journal-title":"Xenotransplantation"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1038\/d41586-024-00853-8","article-title":"First pig liver transplanted into a person lasts for 10 days","volume":"627","author":"Mallapaty","year":"2024","journal-title":"Nature"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1001\/jamasurg.2023.2774","article-title":"Normal graft function after pig-to-human kidney xenotransplant","volume":"158","author":"Locke","year":"2023","journal-title":"JAMA Surg"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2120238","article-title":"Results of two cases of pig-to-human kidney xenotransplantation","volume":"386","author":"Montgomery","year":"2022","journal-title":"N Engl J Med"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1111\/ajt.16930","article-title":"First clinical-grade porcine kidney xenotransplant using a human decedent model","volume":"22","author":"Porrett","year":"2022","journal-title":"Am J Transplant"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1038\/d41586-024-00879-y","article-title":"First pig kidney transplant in a person: what it means for the future","volume":"628","author":"Mallapaty","year":"2024","journal-title":"Nature"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3791\/59673-v","article-title":"Isolation and culture of primary aortic endothelial cells from miniature pigs","volume":"150","author":"Zhao","year":"2019","journal-title":"J Vis Exp"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1111\/xen.12516","article-title":"Justification of specific genetic modifications in pigs for clinical organ xenotransplantation","volume":"26","author":"Cooper","year":"2019","journal-title":"Xenotransplantation"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(23)01349-1","article-title":"Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study","volume":"402","author":"Loupy","year":"2023","journal-title":"Lancet"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-023-02471-9","article-title":"Pig-to-human heart xenotransplantation in two recently deceased human recipients","volume":"29","author":"Moazami","year":"2023","journal-title":"Nat Med"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1038\/s41551-020-00613-9","article-title":"Extensive germline genome engineering in pigs","volume":"5","author":"Yue","year":"2021","journal-title":"Nat BioMed Eng"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1126\/science.aan4187","article-title":"Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9","volume":"357","author":"Niu","year":"2017","journal-title":"Science"},{"key":"B33","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1056\/NEJMoa2201422","article-title":"Genetically modified porcine-to-human cardiac xenotransplantation","volume":"387","author":"Griffith","year":"2022","journal-title":"N Engl J Med"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1097\/TP.0000000000002171","article-title":"Overcoming coagulation dysregulation in pig solid organ transplantation in nonhuman primates: recent progress","volume":"102","author":"Wang","year":"2018","journal-title":"Transplantation"},{"key":"B35","doi-asserted-by":"publisher","first-page":"11138","DOI":"10.1038\/ncomms11138","article-title":"Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft","volume":"7","author":"Mohiuddin","year":"2016","journal-title":"Nat Commun"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1111\/xen.12557","article-title":"Pig endothelial protein C receptor is functionally compatible with the human protein C pathway","volume":"27","author":"Salvaris","year":"2020","journal-title":"Xenotransplantation"},{"key":"B37","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1038\/s41586-023-06594-4","article-title":"Design and testing of a humanized porcine donor for xenotransplantation","volume":"622","author":"Anand","year":"2023","journal-title":"Nature"},{"key":"B38","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1111\/xen.2014.21.issue-1","article-title":"Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model","volume":"21","author":"Mohiuddin","year":"2014","journal-title":"Xenotransplantation"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajt.2022.12.023","article-title":"Milestones on the path to clinical pig organ xenotransplantation","volume":"23","author":"Cooper","year":"2023","journal-title":"Am J Transplant"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1097\/SLA.0000000000004996","article-title":"Anti-C5 antibody tesidolumab reduces early antibody-mediated rejection and prolongs survival in renal xenotransplantation","volume":"274","author":"Adams","year":"2021","journal-title":"Ann Surg"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2024.1488552\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,20]],"date-time":"2025-01-20T07:20:29Z","timestamp":1737357629000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2024.1488552\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,20]]},"references-count":40,"alternative-id":["10.3389\/fimmu.2024.1488552"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2024.1488552","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,20]]},"article-number":"1488552"}}