A Tolerance Approach to Xenotransplantation

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This proposal represents the renewal of a highly integrated PPG aimed at induction of tolerance to porcine organs transplanted into primates. We propose to combine two unique tolerance induction platforms, one based on mixed hematopoietic chimerism and the other on vascularized thymus transplantation. CRISPR/Cas9 technology is used to modify our inbred miniature swine genetically in order to increase the level and duration of mixed chimerism as well as to enhance organ survival. In Project 1, “Achieving Xenograft Tolerance through Thymic Programming in Primates”, we will advance the vascularized thymus (VT) approach to kidney xenograft tolerance toward a clinical trial (Aim 1). In Aim 2, we will attempt to optimize tolerance by combining deletional and Treg-mediated mechanisms while also tolerizing B cells to the inbred SLAhh GalT KO pig kidney donor. This tolerance will be achieved by combining the VT transplant approach with mixed xenogeneic chimerism using optimal GM pigs with intrabone injection (IBBMTx) of pig bone marrow. In Project 2, “Achieving Xenograft Tolerance through Mixed Chimerism”, Aim 1 will build on our demonstration of greatly prolonged mixed chimerism in baboons receiving IBBMTx of hCD47 Tg/hCRP Tg/GalT KO pig HCs and evaluate the utility of adding human cytokine receptor genes to the pig. Aim 2 will build on our observation that expanded recipient baboon Tregs can prolong mixed porcine chimerism and skin graft survival. We will combine the optimal pig from Aim 1 with optimized ex-vivo expanded recipient Tregs and IBBMTx to achieve durable chimerism and, with it, tolerance of B cells and NK cells in addition to T cells. Aim 3 will test whether or not the optimized mixed chimerism approach leads to tolerance of renal xenografts, including baboons with high pre-formed non-Gal Nab levels, which will receive delayed donor kidney transplantation. Project 3, “Tolerance of Adaptive and Innate Human Anti-Pig Immune Responses in Humanized Mice”, builds on our studies of mixed xenogeneic chimerism and porcine thymic transplantation in human immune system (HIS) mice. In Aim 1, we attempt to achieve optimal tolerance and immune function by combining mixed chimerism with either hybrid thymic transplantation or pig plus human thymic transplantation, modeling pig thymic transplantation without human host thymectomy. In Aim 2, we will determine the mechanisms of T and B cell tolerance via mixed chimerism and thymic transplantation. Both aims utilize unique TCR transgenic tools. Core A will provide administrative support, coordinating interactions among investigators and NIH Program staff. Core B will support large animal needs, supplying baboons; coordinating the production, quality control, phenotyping and genotyping of GM miniature swine; providing swine and baboon CMV assays; and providing mAbs for all Projects. In Core C, CRISPR/Cas9 will be used to enable the construction of advanced GM pigs. A Biobank and associated unified database will collect, store and fully describe cell and tissue samples from all stages of animal production through euthanasia/transplantation. We anticipate that these projects and cores and their interactions will synergize to provide pivotal advances toward clinical xenotransplantation.