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，有25,000多名患者正在等待接受肝移植。该数字越来越多与美国老年人口相关的慢性肝病的发生率越来越多酒精性肝病，肝炎，黑手党和纳什等疾病。尽管努力说服人们作为器官捐献者，需求越来越多地超过了器官移植的供应。一种解决方案这个问题是能够在动物中产生人类肝脏以及肝细胞移植的能力。尽管有许多分化人类干细胞（HESC）和可诱导的方案多能干细胞（IPSC）在各种细胞类型中，它们在翻译到诊所。但是，现在可以从一种物种中再生器官/细胞的复制品动物在第二种物种的体内。这涉及特定发育基因的敲除（KO）物种二的胚泡；以及从物种到一个物种到多能干细胞的爆破内注射生成携带从该供体衍生的器官/细胞类型的后代。这种方法的翻译需要有效的基因编辑技术。实际上，新颖的Talen/CRISPR/CAS9技术提供了如此迅速的具有成本效益的方法来产生转基因动物。因此，我们建议采用基因编辑与小鼠胚胎中肝发育相关的敲除特定基因的技术。我们假设可以通过将RAT ESC或PSC注入CRISPR-GENETENE-GENETING中的老鼠中产生大鼠肝脏设计的鼠胚泡，并将其移植回合元大鼠。研究是第一步外部器官的种间发展，用于移植而无需免疫抑制。我们有设计了三个特定的目的，以检验我们的中心假设。具体而言，我们将表征（1）内部和源自HHEX KO胚胎的种间外生肝和内皮；（2）免疫学和功能源自HHEX KO胚胎的种间外源肝脏和内皮发育；（3）几个增强种间嵌合体的产生的方法，包括人性化配体的人性化受体相互作用。由此产生的外源性大鼠肝脏和内皮将被移植到同步性中大鼠评估移植物的生存和功能。主要由大鼠组成的整个肝脏的产生源自植入的大鼠ESC或PSC的肝和内皮细胞将代表范式转移，并且提供必要的临床前证据，以最终在动物中形成人类肝脏。如果成功，拟议的研究将是一种改变游戏规则的人，可以想象可以为替代来源铺平道路针对特定患者量身定制的器官和/或肝细胞移植的人肝。此外，这新颖的，尽管有些高风险的方法阐明了与研究有关的许多问题异种移植。对美国和全球肝病的改善医疗保健的潜在影响很棒，是朝着个性化医学目标迈出的重要一步。