Production and functional verification of 8-gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-edited donor pigs for xenotransplantation

Gene-edited pig-to-human xenotransplantation continues to make breakthroughs and is expected to enter clinic to solve the global shortage of donor organs. However, which gene combination is suitable for which organ transplantation remains unclear. In this study, we utilized CRISPR/Cas9 gene editing technology, PiggyBac transposon system and somatic cell cloning to construct GTKO/CMAHKO/{beta}4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs, and performed pig to non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The multiple vectors were co-transfected into fetal fibroblasts of Diannan miniature pig with O blood type, and 25 colonies were screened out, and one of them carried GGTA1, CMAH and {beta}4GalNT2 biallelic knockout and integration of hCD46, hCD55, hCD59, hTBM and hCD39 genes, which was used as a donor cell for cloning, and a 33-day-old viable fetus was obtained. The fetus was identified and confirmed for normal karyotype and the absence of three xenogeneic antigens -Gal, Neu5Gc and Sda, and expression of hCD46, hCD55, hCD59, hTBM and hCD39 genes, then the recloning was carried out and 28 cloned piglets were obtained by natural delivery. Molecular identification at DNA, mRNA and protein levels showed that 8 gene editing (GE) was successful in these GEC piglets. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8GE effectively reduced the immune incompatibility and kidney xenograft survived up to 15 and 17 days into two NHPs, respectively. During this period, the recipient serum antibodies IgA and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. The 24-hour urine output and serum creatinine remained normal at a period of post-transplantation. These results indicated that the 8GEC pigs effectively alleviated immune rejection and exerted life-supporting kidney function in the recipient.