Experimental Study On Chinese Genetically Engineered Pig-to-Rhesus Monkey Kidney Xenotransplantation

1 Experimental study on different types of Chinese genetically modified pig-to-Rhesus monkey kidney xenotransplantation in vivo【Objective】 1.Different types of genetically engineered pigs independently developed in China were used for the first time in the experimental study of pig to rhesus monkey xenotransplantation to explore whether long-term graft survival can be achieved in pig to non-human primate xenotransplantation under a clinically feasible immunosuppression protocol.2.Based on the above clinically feasible immunosuppressive regimen,anti-CD154 antibody was added to study whether the regimen could further improve the effect of xenotransplantation,which has been reported internationally to significantly prolong the survival time of xenotransplantation.【Method】 The following Chinese gene-edited pigs(Miniature Bama pig)were used as donors:(i)GTKO/h CD55 pigs;(ii)GTKO/h CD55/TM/EPO pigs;(iii)GTKO/4Gal NT2KO/ CMAHKO/h CD55 pigs;(iv)GTKO/4Gal NT2 KO pigs;(v)GTKO/4Gal NT2KO/h CD55 /TM pigs.Rhesus monkeys with low levels of allogeneic natural antibodies screened by donor pig PBMCs were used as receptors,and both kidneys of the recipients were resected.Gene editing pig to rhesus monkey renal transplantation was performed.For this study,immunomodulation therapy with rabbit anti-human thymocyte immunoglobulin(r ATG),anti-CD20 m Ab,and a routine clinical immunosuppression regimen that includes Tac/Cs A,MMF,Pred maintenance therapy were used(Group 1).On the basis of the above regimen,a special immunosuppression regimen with anti-CD154 antibody was used for further anti-rejection therapy(Group 2).The graft survival time of recipient rhesus macaques under different treatment regiments was compared,and the differences in blood biochemistry,blood routine,blood coagulation function,urinary protein and other related physiological indexes during the survival of recipient rhesus macaques were compared between the two groups.The routine pathology of kidney grafts was examined by HE staining.Intragraft CD3,CD20,and CD68 positive cell infiltration were detected by immunohistochemical staining,and Ig G,Ig M,C3 c,C4c,C4 d,C5b-9,and fibrin deposition were determined by immunofluorescence staining.【Results】 Twelve gene-edited pig to rhesus monkey kidney xenotransplantation were successfully performed,of which five were treated with conventional immunosuppressive therapy(Group 1)and seven were treated with anti-CD154 antibody(Group 2).None of the 12 transplanted kidneys had hyperacute rejection.The color,texture,and urinary tract of the transplanted kidneys were normal before abdominal closure,and the renal function of the transplanted kidneys remained normal for at least one week after surgery.The survival time of 12 patients was more than 14 days.Group 1 survived for 18 to 32 days(18,19,20,25,32 days),and Group 2 survived for 14 to 30 days(14,14,16,24,24,27,30 days).There was no significant difference in graft survival time between the two groups(mean: 22.8 ± 5.8 days vs,21.29 ± 6.55 days,P =0.344).Among the 12 recipients,3 showed a significant increase in the level of new xenoantibody after surgery(2 in group 1 and 1 in group 2),and the pathological results of these 3 patients suggested that there was obvious bleeding and congestion in the transplanted kidney and ureter as well as extensive capillary microthrombi.There was obvious deposition of C3 c,C4c,C5B-9,Ig M,and Ig G in immunofluorescence.Immunohistochemistry indicated a small amount of T cell infiltration and extensive infiltration of macrophages,presenting typical acute humoral xenograft rejection.Although the other 9 cases did not produce significant new antibodies in the circulation,their pathological manifestations were similar to those of the 3 cases with significantly increased levels of xenoantibodies,and the degree was relatively mild,presenting as atypical mild acute humoral rejection.There was no significant difference in pathological findings between the two groups.In addition,the levels of serum hydrogen carbonate and sodium were increased and the levels of hemoglobin were decreased in all recipients after the operation,suggesting that there may be some problems in the function of the pig kidney in maintaining the internal environmental balance of rhesus monkeys.【Conclusion】 Hyperacute rejection of pig to rhesus monkey xenotransplantation can be successfully overcome by gene editing strategies with deletion of one or more xenoantigens and transfer of human complement regulatory protein CD55,but long-term graft survival is still difficult to achieve.The addition of anti-CD154 antibodies did not further improve xenograft outcomes.The causes of renal graft failure are mainly related to immune damage mediated by new xenoantibodies and extensive thrombosis caused by coagulation abnormalities.In the future,more in vitro and in vivo experiments are needed to explore new xenoantigens and new transgenic strategies to find more suitable gene-edited pig types and anti-rejection treatment options.2 Both natural and induced anti-Sda antibodies play important roles in GTKO pigto-rhesus monkey xenotransplantation【Objective】 Sda,produced by the B4GALNT2 enzyme,has been recognized as an important xenoantigen for pig-to-nonhuman primate xenotransplantation.However,little is known about Sda expression in pigs and its immunogenicity in xenotransplantation.In view of this,we designed in vitro experiments on the roles of both natural and induced anti-Sda antibodies in GTKO pig-to-rhesus monkey xenotransplantation.【Method】 In this study,peripheral blood mononuclear cells(PBMCs)were isolated from wildtype,GTKO(with high,moderate,and low Sda expression),GTKO/β4Gal NT2 KO,GTKO/CMAHKO,or GTKO/CMAHKO/β4Gal NT2 KO pigs.Anti-pig Ig M/Ig G binding and complement-dependent cytotoxicity(CDC)to pig PBMCs was measured by flow cytometry using pooled rhesus monkey sera(n=20)or human sera(n=20).【Results】 As compared to wild-type pigs(n=12),GTKO pigs(n=17)had a significantly higher mean level of Sda expression on PBMCs and showed a greater individual difference in expression.Both the overall binding of monkey serum Ig M/Ig G antibody to GTKO pig PBMCs and CDC against these PBMCs decreased significantly with a progressive reduction in Sda expression,showing a clear dose-effect relationship.Both the monkey serum antibody binding and CDC decreased significantly after the additional deletion of Sda,whereas the binding of human serum antibody and CDC against the GTKO pig PBMCs were markedly reduced after the deletion of Neu5 Gc in the pigs.In addition,antiSda antibody accounted for > 50% of the induced anti-non-Gal antibody at the time of rejection in two rhesus monkeys that received GTKO/h CD55 pig kidney xenotransplantation,and the anti-Sda antibody showed significant cytotoxic activity against GTKO pig cells.【Conclusion】 We conclude that both natural and induced anti-Sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation,thusproviding further evidence for GTKO/β4Gal NT2 KO pigs as the preferred organ source for rhesus monkeys as a preclinical model of xenotransplantation.