Xu, K.; Zhao, H.; Jia, B.; Wang, J.; Siddig, N. A. M. A.; Jamal, M. A.; Mao, A.; Liu, K.; Cheng, W.; Yang, C.; Wei, T.; Zhu, F.; Huo, X.; Jiao, D.; Guo, J.; Zhao, H.; Cheng, W.; Zhang, Y.; Zhang, X.; Jiang, L.; Zhang, Z.; Zhang, W.; Liang, T.; Zhao, H.-Y.; Sun, B.-C.; Wei, H.-J.

Xenotransplantation has entered the clinical phase to fulfill the global organ shortage. However, recent clinical studies revealed that the xenograft from current gene-edited (GE) pigs still poses the risk of immune rejection, and biosafety concerns. In this study, we successfully constructed a large batch of 10-(GTKO/CMAHKO/ {beta}4GalNT2KO/hCD46/hCD55/hCD59/hTBM/hEPCR/hCD39/hCD47) GE cloned (GEC) donor pigs by utilizing gene editing and somatic cell cloning technology, and successfully obtained F1 generation. Phenotypic characterization of 10-GEC pigs showed the deletion of three xenoantigens along with expression of seven human transgenes in various tissues. Digital droplet polymerase chain reaction, and whole genome sequencing indicated 2 copies of hCD46/hCD55/hCD59/hTBM/hCD39 and 1 copy of hEPCR/hCD47 in pig genome without significant off-target and damage to the porcine own functional genes. The validation results showed that 10-GEC pigs effectively inhibited the attacks of human antibodies, complement, and macrophages on porcine endothelial cells and alleviated the coagulation abnormalities between pigs and humans. 10-GEC pigs were negative for all zoonotic pathogens (48) including cytomegalovirus, except streptococcal infections. Kidney, heart, and liver xenografts from these 10-GE pigs were transplanted to non-human primates (NHP), which started working normally without hyperacute rejection. Among them, the heart and liver transplant recipient died without resuscitation due to unexpected interruption of oxygen supply, while the 2 kidney transplant recipients survived for 23 and 16 days, respectively. Pathological analysis showed that 10-GE pig kidney xenografts showed mesenchymal congestion, and fibrosis, cellular hyperplasia, with minor antibody and complement deposition, and significantly reduced the infiltration of CD68+ macrophage. In summary, we successfully produced a group of specific pathogen free GEC donor pigs that effectively mitigated immune rejection upon multi-organ transplantation to NHP.