The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation

作者：Guoli Huai, Yong Wang, Jiaxiang Du, Zhenhui Cheng, Yuxuan Xie, Jia Zhou, Hongmei Tang, Yanyan Jiang, Xiangyang Xing, Shaoping Deng, Dengke Pan · 发表于：Frontiers in Immunology · 年份：2025 · DOI：10.3389/fimmu.2024.1488552 · 被引用次数：16 · 研究领域：Xenotransplantation and immune response、Animal Genetics and Reproduction、Pluripotent Stem Cells Research

Introduction: 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. Methods: 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. Results: , 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<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<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excell...