*Purpose: Genetic engineering (GE) technology has made knockout and multigene expressing animals available. These animals can be used for xenotransplantation (xTX)to treat end stage diseases. We have used GE multigene expressing donor pigs in heterotopic cardiac transplant model and have previously achieved a long term cardiac xenograft survival in non-human primates. In this study we have tested new line of GE donor pigs in combination with or without thrombomodulin (TBM) gene in preclinical (pig to baboon) heterotopic cardiac xTX model.

*Methods: Heterotopic cardiac xTx were performed in specific pathogen-free baboons from GE donor’s pigs which had either five (n=2) or six gene (n=3) manipulation. Five gene GE pigs were triple knockout (GTKO, B4KO.CMAHKO) and over expression of hCD46 and DAF without TBM. Whereas six gene GE pigs were GTKO.CD46.EPCR.HO1 and had low expression of TBM and hCD47 gene. Recipients received Tocilizumab, and Etanercept as an anti-inflammatory drugs along with our established immunosuppression (IS) regime which included (Rituxan, CVF, ATG, anti CD40 antibody, MMF) and tapering doses of steroids. All recipients received continuous intravenous heparin. Xenograft survival was monitored with telemetry, echocardiography (U/S) and manual palpation. CBC, chemistry, troponin, and ACT was performed at 1-2 week intervals.

*Results: Cardiac xTX was performed successfully without any problem. Addition of Tocilizumab, and Etanercept to the IS improved the early recovery of recipient baboon. Cardiac xenograft of triple KO.hCD46.DAF survived only for day 1 and 117. Both the xenograft were rejected and had demonstrated progressive deteriorating function on telemetry and U/S examination. Non-gal IgG antibody was increased and numerous microthrombi were also seen on H& E staining in this group. Whereas, 2 of 3 six gene GE donor pigs in spite of having a low expression of TBM gene, xenograft survived with an excellent function and was electively explanted on day 119 and 121 days respectively. One of three recipient did not receive anti-inflammatory drugs and had to be euthanized due to infection after 42 days.

*Conclusions: Thrombomodulin (TBM) is an anticoagulant which plays an important role in xenotransplantation, Results from this study suggest that hTBM gene expression, even at a lower level, on donor’s hearts is important for the long-term survival of cardiac xenograft.

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