The objective of this application is to achieve 90-day median survival of life-supporting cardiac xenotransplantation (CXTx) in the pig-to-primate model using genetically-engineered donors, clinical immunosuppression (IS) and modalities to diagnose and treat delayed xenograft rejection (DXR) leading to preclinical studies of CXTx. Prolonged xenograft survival is now limited by non-Gal antibody-mediated DXR (NGDXR). With improved outcomes for orthotopic (OCXTx), it has emerged that early xenograft function is compromised in up to 50% of cases. We have termed this perioperative cardiac xenograft dysfunction (PCXD). For potential future clinical application of CXTx, it is essential to understand the mechanisms and improve the outcomes of both NGDXR and PCXD. All transplants for Specific Aims (SA) 1-3 will use donors transgenic for CD55 with inactivation of the GGTA-1 glycosyltransferase locus (GTKO:CD55+). In SA 1, to better understand PCXD, plasmapheresis will be used to remove preformed non-Gal antibody (PNGA) and the proteasome inhibitor Bortezomib given to delete plasma cells prior to OCXTx. This therapy will also be combined with cariporide cardiac preservation to mitigate preoperative injury. The impact of PNGA depletion and cariporide treatment on PCXD will be assessed by measurement of cardiac function by echocardiography (ECHO), biochemical markers of cardiac injury, recipient antibody and plasma cell levels, and will be correlated with cardiac gene expression in interim biopsies and explanted hearts. In SA 2, to prevent DXR optimal IS based on induction therapy for B- and T-cells, triple drug IS and Bortezomib will be used to control NGA in OCXTx recipients surviving SA 1. We will further study the mechanism(s) of NGDXR through analysis of antibody responses to GTKO PAECs and novel individual non-Gal carbohydrate and protein target antigens we have identified. Changes in cardiac gene expression will be correlated with DXR. In SA 3, we utilize life-supporting intrathoracic heterotopic CXTx, a currently clinically used heart transplant variant which, as well as being a potential technique for initial clinical application of CXTx, will uniquely allow a) observation of recovery of PCXD by ECHO and b) development of methods for the diagnosis and treatment of DXR without loss of the recipient as the native heart supports the circulation during these periods of xenograft dysfunction. In this aim, plasmapheresis and Bortezomib will be used to treat DXR. The objective of this application, to achieve 3-month median survival of circulation-bearing CXTx, if successful, will lay the groundwork for preclinical studies during this grant period to support clinical application of CXTx. RELEVANCE: Successful xenotransplantation offers a potential clinical solution to alleviate the chronic and increasing shortage of donor organs and cells for transplantation. Our previous studies over 12 years have achieved the longest median survival of heterotopic cardiac xenotransplants and the longest survivors of life-supporting orthotopic cardiac xenotransplants. Achievement of the specific aims of this proposal will bring cardiac xenotransplantation to the threshold of clinical application.