Production of four-gene (GTKO/hCD55/hTBM/hCD39)-edited donor pigs and kidney xenotransplantation

Background The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene editing technologies. However, which gene combination is suitable for which organ transplantation remains unclear. Methods In this study, we utilized CRISPR/Cas9 gene editing technology, PiggyBac transposon system and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. Results First, 107 cell colonies were obtained through drug selection, of which 7 were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer, resulting in 7 fetuses, of which 4 were GGTA1 biallelic knockout. Both fetuses had higher expression of hCD55, hTBM and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in a total of 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. Conclusion These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey’s life. ### Competing Interest Statement The authors have declared no competing interest. * ADA : Adenosine deaminase ALB : Albumin ALP : Alkaline phosphate ALT : Alanine aminotransferase (ALT) AST : Aspartate aminotransferase ATG : Antithymocyte globulin cDNA : Complementary DNA COCs : Cumulus-oocyte complexes DAMPs : Damage-associated molecular patterns DBIL : Direct bilirubin ddPCR : Droplet digital PCR GE : Gene-edited GEC : Gene-edited cloned GGTA1 : α1,3-galactosidyl transferase NHPs : Non-human primates NETs : Neutrophil extracellular traps PBMCs : Peripheral blood mononuclear cells PFFs : Pig fetal fibroblasts RT : Room temperature SCNT : Somatic cell nuclear transfer SD : Standard deviation of mean SNPs : Single nucleotide polymorphism TBA : Total bile acid TBIL : Total bilirubin TMB : Thrombomodulin TP : Total protein 4GE : GTKO/hCD55/hTBM/hCD39 gene-edited