Generating Exogenic Organs for Transplantation without the Use of Immunosuppression

目前有超过2.5万名患者等待接受肝脏移植。这一数字还在不断增加 随着美国人口老龄化，慢性肝病的发病率不断上升， 酒精性肝病、肝炎、酒精性脂肪肝和非酒精性脂肪肝等疾病。尽管人们努力说服人们 作为器官捐赠者，器官移植越来越供不应求。一种解决方案 这个问题是在动物体内产生人的肝脏用于肝脏和肝细胞移植的能力。 虽然有许多方案来分化人类胚胎干细胞(HESCs)，并且可诱导 多能干细胞(IPSCs)在体外培养成各种类型的细胞，在 翻译到诊所。然而，现在有可能从一个物种再生器官/细胞的复制品 另一个物种体内的动物。这涉及到特定发育基因的敲除(KO) 种二的囊胚；种一至种的多能干细胞的囊胚内注射 产生携带来自该捐赠者的器官/细胞类型的后代。此方法的翻译需要 一种高效的基因编辑技术。事实上，新的TALEN/CRISPR/CAS9技术提供了如此快速、 生产转基因动物的成本效益高的方法。因此，我们建议使用基因编辑 敲除小鼠胚胎中与肝脏发育相关的特定基因的技术。我们假设 通过将大鼠ESCs或PSCs注射到CRISPR中，可以在小鼠体内产生大鼠肝脏--从基因上讲 改造小鼠的囊胚，并将其移植回同基因大鼠体内。这些研究代表着 无免疫抑制的外源器官移植的种间发育。我们有 设计了三个具体目标来检验我们的中心假设。具体地说，我们将描述(1)内部和 HHEX-KO胚胎来源的异种外源性肝脏和内皮；(2)免疫学和功能 来自HHEX KO胚胎的异种外源性肝脏和内皮发育；以及(3)几个 促进种间嵌合体产生的方法，包括形态原配体的人源化- 受体相互作用。由此产生的外源性大鼠肝脏和内皮细胞将被移植回同基因 评估移植物的存活率和功能。主要由大鼠组成的整个肝脏的生成 来自移植的大鼠ESCs或PSCs的肝脏和内皮细胞将代表一种范式转变和 为最终在动物体内创造人类肝脏提供必要的临床前证据。如果成功，则 拟议中的研究将是游戏规则的改变者，可以想象，它可能为替代能源铺平道路 人体肝脏，用于器官和/或肝细胞移植，为特定患者量身定做。此外，这一点 新颖的，尽管有点高风险的方法绕过了与研究 异种移植。对改善美国和世界范围内肝病医疗保健的潜在影响 是伟大的，代表着朝着个体化医疗目标迈出的重要一步。

At present there are more than 25,000 patients waiting to receive liver transplants. The number is increasing due to an aging US population accompanied by an increasing incidence of chronic liver diseases associated with such disorders as alcoholic liver disease, hepatitis, MAFLD and NASH. In spite of efforts to persuade people to serve as organ donors, the demand increasingly outstrips the supply for organ transplantation. One solution to this problem is the ability to generate human livers in animals for liver as well as hepatocyte transplantation. Although there are numerous protocols to differentiate human embryonic stem cells (hESCs), and inducible pluripotent stem cells (iPSCs) ex vivo to a variety of cell types, they have encountered significant challenges in translation to the clinic. However, it is now possible to regenerate the replica of organs/cells from one species of animal within the body of a second species. This involves the knockout (KO) of specific developmental genes in the blastocyst of species two; and the intra-blastocyst injection of pluripotent stem cells from species one to generate offspring that carry organs/cell types derived from that donor. The translation of this approach requires an efficient gene-editing technology. In fact, novel TALEN/CRISPR/Cas9 technologies provide such a rapid, and cost-effective means to generate genetically modified animals. Accordingly, we propose to employ gene-editing technology to knockout specific genes associated with liver development in the mouse embryo. We hypothesize that rat liver can be generated in the mouse by the injection of rat ESCs or PSCs into CRISPR-genetically engineered murine blastocysts and transplanted back into syngeneic rats. The studies represent a first step of interspecies development of exogenic organs for transplantation without immunosuppression. We have designed three Specific Aims to test our central hypothesis. Specifically, we will characterize (1) intra- and interspecies exogenic liver and endothelium derived from HHEX KO embryos; (2) the immunology and function of interspecies exogenic liver and endothelial development derived from HHEX KO embryos; and (3) several approaches to enhance the generation of interspecies chimeras that include humanization of morphogen ligand- receptor interactions. The resulting exogenic rat liver and endothelium will be transplanted back into syngeneic rats to evaluate graft survival and functionality. The generation of whole livers that are comprised primarily of rat hepatic and endothelial cells derived from implanted rat ESCs or PSCs would represent a paradigm shift and provide the necessary preclinical evidence for ultimately creating human livers in animals. If successful, the proposed research would be a game-changer that could conceivably pave the way for an alternate source of human livers for organ and/or hepatocyte transplantation that is tailored to specific patients. In addition, this novel, albeit somewhat high-risk approach circumvents many of the problems associated with research on xenotransplantation. The potential impact on improved health care in the U.S. and worldwide for liver diseases is great and represents a major step towards the goal of individualized medicine.