The generation and evaluation of TKO/hCD55/hTM/hEPCR genemodified pigs for clinical organ xenotransplantation

Guoli Huai ¹ Yong Wang ² Jiaxiang Du ² Zhenhui Cheng ² Yuxuan Xie ¹ Jia Zhou ¹ Hongmei Tang ¹ Yanyan Jiang ² Xiangyang Xing ² Shaoping Deng ^1,3 Dengke Pan ^1,2,4*

• ¹ University of Electronic Science and Technology of China, Clinical Immunology Translational Medicine Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan Province, China
• ² Chengdu ClonOrgan Biotechnology, Co., Ltd., Chengdu, China
• ³ Sichuan Taikang Hospital, Chengdu, China
• ⁴ Sichuan ClonOrgan Biotechnology, Co., Ltd., Neijiang, China

Genetically edited pigs modified using CRISPR-Cas9 technology hold promise as potential sources for xenotransplantation. However, determining the optimal or necessary combination of genetic modifications and their expression levels for an initial clinical trial poses a question. We generated the TKO/hCD55/hTM/hEPCR (6GE) pigs from iterative rounds of genome editing and F1 generation's breeding. Comprehensive genotyping, flow cytometry, and immunohistochemical analyses confirmed the complete knockout of GGTA1, CMAH, and B4GALNT2 in these pigs. Additionally, the expression levels of hCD55 and hTM in 6GE pigs were approximately seven and thirteen times higher, respectively, than those in humans, while hEPCR levels were comparable to human counterparts. OurIn vitro experiments revealed that the 6GE aortic endothelial cells (pAECs) had significantly reduced binding of human serum IgM and IgG antibodies compared to wildtype pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a significant reduction in human complement cytotoxicity compared to TKO pAECs (p<0.001), approaching zero. Notably, the co-insertion of hTM and hEPCR in 6GE pigs significantly reduced thrombin-antithrombin (TAT) complex levels following coculture with human whole blood compared to WT (p<0.0001), TKO (p<0.01), and even TKO/hCD55/hTM (p<0.05). Pathophysiological analyses indicated that the kidneys and liver of 6GE pigs demonstrated excellent compatibility with human immune and coagulation systems, fully meeting human physiological requirements. However, the 6GE pigs were found more susceptible to infection compared to other gene-edited pigs, while TKO/hCD55 pigs are considered safe when they were all bred in a general environment. In conclusion, 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.