A Tolerance Approach to Xenotransplantation

这项建议代表了一种高度整合的PPG的更新，旨在诱导对猪的耐受性 移植到灵长类动物体内的器官。我们建议将两个独特的耐受性诱导平台结合起来，一个基于 关于混合造血嵌合体和另一种关于带血管胸腺移植。CRISPR/CAS9 利用技术对我们的近交系小型猪进行遗传改良，以增加水平和持续时间 混合嵌合体以及提高器官存活率。在项目1中，“通过以下方式实现异种移植容忍 在《灵长类动物的胸腺编程》一书中，我们将推进带血管胸腺(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，同时也耐受B细胞对近交系SLAhh Galt KO猪肾脏的供体。这 通过将VT移植方法与混合异种嵌合体结合使用，将实现耐受性 最佳转基因猪骨内注射(IBBMTx)猪骨髓。项目2，“实现异种移植” 通过混合嵌合体的宽容“，目标1将建立在我们对长时间混合嵌合体的演示的基础上 在接受hCD47 TG/hCRP TG/GalT KO猪HCS IBBMTx的狒狒中，评估添加人的效用 猪的细胞因子受体基因。目标2将建立在我们的观察基础上，即扩大受体狒狒Tregs可以 延长混合猪的嵌合体和皮肤移植物存活时间。我们将结合目标1中的最优猪和优化后的猪 体外扩增受体Tregs和IBBMTx以实现持久的嵌合体，并随之而来的B细胞和 除T细胞外，还有NK细胞。目标3将测试优化的混合嵌合体方法是否会导致 对异种肾移植的耐受性，包括具有高预先形成的非GalNAB水平的狒狒，将接受延迟的 供体肾移植。项目3，“对适应性和先天人类抗猪免疫反应的耐受性” 在人源化的小鼠中，基于我们对混合异种嵌合体和猪胸腺移植的研究 人类免疫系统(HIS)小鼠。在目标1中，我们试图通过以下方式实现最佳的耐受性和免疫功能 将混合嵌合体与混合胸腺移植或猪+人胸腺移植相结合， 无人胸腺切除猪胸腺移植模型的建立。在目标2中，我们将确定 混合嵌合体和胸腺移植在T、B细胞耐受中的作用机制这两个目标都利用独特的 TCR转基因工具。核心A将提供行政支持，协调调查人员之间的互动 和NIH项目工作人员。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；提供猪和狒狒巨细胞病毒检测； 并为所有项目提供单抗。在核心C中，将使用CRISPR/CAS9来实现高级 转基因猪。生物库和相关的统一数据库将收集、存储和全面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 核心及其相互作用将协同作用，为临床异种移植提供关键进展。

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.