Extended Survival of 9-and 10-Gene-edited Pig Heart Xenografts with Ischemia Minimization and CD154 Costimulation Blockade-Based Immunosuppression

Background Xenotransplantation has made significant advances recently using pigs genetically engineered to remove carbohydrate antigens, either alone or with addition of various human complement, coagulation, and anti-inflammatory ‘transgenes’. Here we evaluated results associated with gene-edited (GE) pig hearts transplanted in baboons using an established costimulation-based immunosuppressive regimen and a cold-perfused graft preservation technique. Methods Eight baboons received heterotopic abdominal heart transplants from 3-GE (GalKO.β4GalNT2KO.hCD55, n=3), 9-GE (GalKO.β4GalNT2KO.GHRKO.hCD46.hCD55. TBM.EPCR.hCD47.HO-1, n=3) or 10-G (9-GE+CMAHKO, n=2) pigs using Steen’s cold continuous perfusion for ischemia minimization. Immunosuppression (IS) included induction with anti-thymocyte globulin and αCD20, ongoing αCD154, MMF, and tapered corticosteroid. Results All three 3-GE grafts functioned well initially, but failed within 5 days. One 9-GE graft was lost intraoperatively due to a technical issue and another was lost at POD 13 due to antibody mediated rejection (AMR) in a baboon with a strongly positive pre-operative cross-match. One 10-GE heart failed at POD113 with combined cellular and antibody mediated rejection. One 9-GE and one 10-GE hearts had preserved graft function with normal myocardium on protocol biopsies, but exhibited slowly progressive graft hypertrophy until elective necropsy at POD393 and 243 respectively. Elevated levels of IL-6, MCP-1, C-reactive protein, and human thrombomodulin were variably associated with conditioning, the transplant procedure, and clinically significant postoperative events. Conclusion Relative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, 9- or 10-GE pig hearts exhibit promising performance in the context of a clinically applicable regimen including ischemia minimization and αCD154-based IS, justifying further evaluation in an orthotopic model.