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

• 批准号: 10576631
• 负责人: WALTER C LOW
• 金额: $ 75.2万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 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/10576631
• 关键词: Ablation; Aging; Alcoholic Liver Diseases; Animal Organ; Animals; Back; Biological; Biological Markers; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; 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; Homologous Transplantation; Human; Immune; Immune response; Immune system; Immunohistochemistry; Immunologics; Immunology; Immunosuppression; Implant; Incidence; Incubators; Individual; Injections; Knock-out; Lead; Ligands; Liver; Liver diseases; Methods; Mus; Natural regeneration; Organ; Organ Donor; Organ Transplantation; Pancreas; Patients; Persons; 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 function; graft vs host disease; high risk; human embryonic stem cell; 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; pre-clinical; preimplantation; receptor; single-cell RNA sequencing; stem; stem cells; success; transcription activator-like effector nucleases; transcriptomics

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.

目前有超过25，000名患者等待接受肝脏移植。由于数量不断增加， 美国人口老龄化伴随着慢性肝病的发病率增加， 例如酒精性肝病、肝炎、MAFLD和NASH。尽管努力说服人们 器官移植的需求日益超过供应。的一个解决方案 这个问题是在动物中产生人肝用于肝以及肝细胞移植的能力。 虽然有许多方案来分化人胚胎干细胞（hESC），并且诱导性分化是不可能的。 尽管多能干细胞（iPSC）离体转化为多种细胞类型，但它们在以下方面遇到了重大挑战： 翻译到诊所然而，现在有可能从一个物种再生器官/细胞的复制品。 第二个物种体内的动物。这涉及敲除（KO）特定发育基因， 种2的胚泡;以及种1至种2的多能干细胞的胚泡内注射。 产生携带来自该供体的器官/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，新的TALEN/CRISPR/Cas9技术提供了这种快速， 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 技术敲除小鼠胚胎中与肝脏发育相关的特定基因。我们假设 大鼠肝脏可以通过将大鼠ESC或PSC注射到CRISPR基因中在小鼠中产生， 改造小鼠胚泡并移植回同基因大鼠。这些研究代表了 种间发育的外源器官移植无免疫抑制。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 HHEX KO胚胎的异种外源性肝和内皮细胞;（2）免疫学和功能 来自HHEX KO胚胎的种间外源性肝脏和内皮发育;和（3）几种 增强种间嵌合体产生的方法，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性大鼠肝脏和内皮细胞将被移植回同基因的 大鼠以评估移植物存活和功能。主要由大鼠组成的整个肝脏的产生 来自植入的大鼠ESC或PSC的肝和内皮细胞将代表一种范式转变， 为最终在动物体内制造人类肝脏提供了必要的临床前证据。如果成功， 拟议中的研究将是一个游戏规则改变者，可以想象为替代来源铺平道路。 用于器官和/或肝细胞移植的人类肝脏，其为特定患者定制。另外这款 一种新颖的，尽管有点高风险的方法规避了许多与研究相关的问题， 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，代表着向个体化医疗目标迈出的重要一步。