A Tolerance Approach to Xenotransplantation

该提案代表了旨在诱导猪对猪流感耐受性的高度整合PPG的更新 移植到灵长类动物体内的器官我们建议联合收割机结合两个独特的公差诱导平台，一个基于 一个是关于混合造血嵌合体，另一个是关于血管化胸腺移植。CRISPR/Cas9 技术被用来修改我们的近交小型猪遗传，以增加水平和持续时间， 混合嵌合体以及增强器官存活。在项目1中，“通过 灵长类动物的胸腺程序化”，我们将推进血管化胸腺（VT）方法用于异种肾移植 对临床试验的耐受性（目标1）。在目标2中，我们将尝试通过结合删除 和Treg介导的机制，同时也使B细胞耐受近交系SLAhh GalT KO猪肾供体。这 通过将VT移植方法与混合异种嵌合体结合， 用猪骨髓的骨内注射（IBBMTx）优化GM猪。在项目2中，“实现异种移植 通过混合嵌合体的耐受性”，目标1将建立在我们的证明，大大延长混合嵌合体 在接受hCD 47 Tg/hCRP Tg/GalT KO猪HC的IBBMTx的狒狒中，并评估加入人 细胞因子受体基因。目标2将建立在我们的观察基础上，即扩大的受体狒狒可以 延长混合猪嵌合体和皮肤移植物存活。我们将联合收割机与目标1中的最佳清管器相结合， 离体扩增的受体T细胞和IBBMTx以实现持久的嵌合状态，并由此实现B细胞的耐受性， NK细胞除了T细胞。目标3将测试优化的混合嵌合体方法是否导致 肾异种移植物的耐受性，包括具有高预形成非Gal Nab水平的狒狒，其将接受延迟的 供肾移植项目3，“适应性和先天性人抗猪免疫应答的耐受性 人源化小鼠”，建立在我们的研究混合异种嵌合体和猪胸腺移植， 人免疫系统（HIS）小鼠。在目标1中，我们试图通过以下方式实现最佳耐受性和免疫功能： 将混合嵌合体与杂交胸腺移植或猪加人胸腺移植相结合， 建立猪胸腺移植模型。在目标2中，我们将确定 通过混合嵌合体和胸腺移植的T和B细胞耐受机制。这两个目标都利用了独特的 TCR转基因工具。核心A将提供行政支持，协调研究者之间的互动 和NIH项目人员。核心B将支持大型动物的需求，供应狒狒;协调生产， 转基因小型猪的质量控制、表型和基因分型;提供猪和狒狒CMV检测; 并为所有项目提供mAb。在核心C中，CRISPR/Cas9将用于构建先进的 转基因猪生物库和相关的统一数据库将收集，存储和完整描述细胞和组织样本 从动物生产的所有阶段通过安乐死/移植。我们预计，这些项目和 核心和它们的相互作用将协同作用，为临床异种移植提供关键的进展。

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.