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Generating Exogenic Organs for Transplantation without the Use of Immunosuppression

不使用免疫抑制生成用于移植的外源器官

基本信息

批准号：
10708928
负责人：
WALTER C LOW
金额：
$ 75.8万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-21 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708928
关键词：
Ablation Aging Alcoholic Liver Diseases Animal Organ Animals Back Biological Biological Markers CRISPR/Cas technology Cell Differentiation process Cells Chimera organism Clinic Clustered Regularly Interspaced Short Palindromic Repeats Complement Development Developmental Gene Disease Down-Regulation Education Embryo Endoderm Endothelial Cells Endothelium Exhibits Family suidae Fetal Development Fetus Flow Cytometry Future Gene Expression Generations Genes Genetic Engineering Genetically Modified Animals Genomics Goals Graft Survival Healthcare Hematopoiesis Hematopoietic Hepatic Hepatitis Hepatocyte Hepatocyte transplantation Homeobox Homeobox Genes Homologous Transplantation Human Immune Immune response Immune system Immunohistochemistry Immunologics Immunology Immunosuppression Implant Incidence Incubators Individual Injections Knock-out Ligands Liver Liver diseases Methods Mus Natural regeneration Organ Organ Donor Organ Transplantation Pancreas Patients Persons Persuasive Communication Pilot Projects Pluripotent Stem Cells Population Process Production Property Protocols documentation Rattus Receptor Cell Regulation Reporter Reproducibility Research Resistance Sheep Source Systems Development Technology Testing Thymus Gland Time Transcript Translations Transplantation Visual Xenograft procedure blastocyst cell type chronic liver disease cost effective design donor stem cell graft vs host disease high risk human embryonic stem cell human stem cells improved individualized medicine induced pluripotent stem cell innovation knockout gene liver development liver transplantation metabolic-associated fatty liver disease morphogens mouse model nonalcoholic steatohepatitis novel offspring organ growth permissiveness pre-clinical preimplantation receptor single-cell RNA sequencing stem cells success transcription activator-like effector nucleases transcriptomics translational approach

项目摘要

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.

目前还有超过25000名患者等待接受肝移植。由于数量正在增加随着美国人口老龄化，慢性肝病发病率不断增加酒精性肝病、肝炎、MAFLD 和 NASH 等疾病。尽管努力说服人们作为器官捐献者，器官移植的需求日益超过供应。一种解决方案这个问题是如何在动物体内生成人类肝脏用于肝脏以及肝细胞移植。尽管有许多方案可以分化人类胚胎干细胞 (hESC)，并且可诱导多能干细胞 (iPSC) 离体形成多种细胞类型，但它们在以下方面遇到了重大挑战：翻译到诊所。然而，现在可以从一种物种中再生器官/细胞的复制品。第二个物种体内的动物。这涉及到特定发育基因的敲除（KO）物种二的囊胚；以及囊胚内注射从物种一到物种一的多能干细胞产生携带来自该捐赠者的器官/细胞类型的后代。这种方法的翻译需要高效的基因编辑技术。事实上，新颖的 TALEN/CRISPR/Cas9 技术提供了如此快速、且以具有成本效益的方式生产转基因动物。因此，我们建议采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 ESC 或 PSC 注射到 CRISPR 基因中，可以在小鼠体内产生大鼠肝脏改造小鼠囊胚并移植回同基因大鼠体内。这些研究代表了第一步在没有免疫抑制的情况下进行移植的外源器官的种间发育。我们有设计了三个具体目标来检验我们的中心假设。具体来说，我们将描述（1）内部和来自 HHEX KO 胚胎的种间外源肝脏和内皮； (2)免疫学与功能来自 HHEX KO 胚胎的种间外源肝脏和内皮发育；和（3）几个增强种间嵌合体生成的方法，包括形态发生素配体的人源化受体相互作用。由此产生的外源大鼠肝脏和内皮将被移植回同基因大鼠评估移植物的存活和功能。主要由大鼠组成的完整肝脏的产生来自植入的大鼠 ESC 或 PSC 的肝细胞和内皮细胞将代表一种范式转变，为最终在动物身上创造人类肝脏提供必要的临床前证据。如果成功的话，拟议的研究将改变游戏规则，可以想象为替代来源铺平道路用于针对特定患者的器官和/或肝细胞移植的人类肝脏。此外，这新颖但风险较高的方法规避了与研究相关的许多问题异种移植。对改善美国和全球肝病医疗保健的潜在影响这是伟大的，代表着朝着个体化医疗目标迈出的重要一步。