Introduction: The contradiction between global blood supply and demand is growing, and the shortage of clinical blood has become a key public health challenge. Previous research indicates that the use of TKO-pRBC can mitigate immune rejection in pig-to-monkey blood transfusions study, but with limited effect. In this study, we aimed to test the effect of combined approach by over-expressing human-derived complement inhibitor (CD55 transgene) on TKO-pRBC in preventing human natural antibody mediated hemolysis.
Methods: The knockout of xeno-antigens and expression of transgene were designed using CRISPR-Cas9 system, the gRNA targeting GGTA1, B4GALNT2, CMAH, Rosa26 locus, Cas9 protein and the expression construct vector was transferred to pig fibroblasts by electroporation. Positive clones were obtained through PCR identification, and then used for obtaining cloned piglets. The red blood cells of cloned pigs were isolated and expression was identified by flow cytometry. In vitro experiments, blood agglutination reaction and IgM/IgG antibody binding level were detected after incubation with human serum. The complement dependent cytotoxicity assay was used to measure the level of hemolysis.
Results: The TKO/hCD55 porcine red blood cells demonstrated successful knockout of α-Gal Neu5Gc, Sda, and exhibited about 10 times higher expressions of hCD55 than human red blood cells. In vitro studies demonstrated that IgM and IgG binding to TKO pRBCs was significantly lower than WT and GTKO pRBCs (p < 0.001) and the binding of TKO/CD55 pRBCs was only slightly higher than human ABO-C RBCs (p<0.05). There was no difference in complement dependent cytotoxicity between TKO/hCD55 and human ABO-C group, demonstrates that after knockout of three major antigens, there are still existing other xeno-antigens which can induce IgG/IgM antibody binding, but the over-expression of human CD55 can effectively reduce human antibody mediated hemolytic reactions in vitro.
Conclusion: Over-expression of human CD55 on the basis of TKO porcine red blood cells can further reduce the antibody-mediated hemolysis reaction with human serum. Future in vivo study, will be done to demonstrate the potential of such combined strategy to further improve the viability of porcine red blood cells in human blood transfusion, which could relieve the shortage of human blood supply.