Novel SCID rat models for human cell transplantation studies

用于人类细胞移植研究的新型 SCID 大鼠模型

• 批准号: 7926058
• 负责人: JOSEPH C. RUIZ
• 金额: $ 17.47万
• 依托单位: TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7926058
• 关键词: Animal Model; Animals; B-Lymphocytes; Behavior; Biological Assay; Biological Products; Blood specimen; CD8B1 gene; Cancerous; Cell Count; Cell Transplantation; Cells; Complement; Defect; Detection; Development; Dose; Doxycycline; Drug Industry; Elements; Engraftment; Exhibits; Gene Expression; Genes; Goals; Hepatocyte; Human; Immune; Immune Sera; Immune system; Immunocompromised Host; Indirect Immunofluorescence; Kidney; Knock-out; Knockout Mice; Laboratory Rat; Letters; Liver; Liver Dysfunction; Lung; Lymphocyte; Mediating; Modeling; Monitor; Mus; Mutation; Neural Conduction; Normal tissue morphology; Nude Rats; Operative Surgical Procedures; Patients; Perinatal; Pharmacology; Phase; Phenotype; Physiology; Population; Procedures; Production; Proteins; Public Health; Rat Strains; Rattus; Research; Resources; Reverse Transcriptase Polymerase Chain Reaction; Severe Combined Immunodeficiency; Spleen; System; Testing; Tetanus Helper Peptide; Therapeutic; Therapeutic Agents; Therapeutic Human Experimentation; Thymus Gland; Time; Tissues; Toxic effect; Toxicology; Transgenes; Transgenic Animals; Transgenic Organisms; Transplantation; Xenograft procedure; adenosine deaminase; animal efficacy; base; drinking water; drug candidate; drug discovery; human tissue; innovation; instrumentation; knockout animal; mouse model; mutant; novel; novel therapeutics; peripheral blood; pre-clinical; programs; public health relevance; stem cell population; success

DESCRIPTION (provided by applicant): The laboratory rat is used extensively by the pharmaceutical industry for preclinical animal efficacy and toxicology studies. In addition, the rat has been a valuable animal model for physiology, behavior, and pharmacology research programs, due to ease of experimental manipulation. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, nerve conduction studies, and surgeries. Generating inbred immunodeficient rat strains that can maintain functional human xenografts would provide unique and valuable resources for drug discovery and therapeutic research programs. This project will investigate the use of a novel rat strain carrying a transposon-mediated knockout (KO) insertional mutation within the adenosine deaminase (Ada) locus in the inbred Fischer line. This mutation is an underlying cause of severe combined immunodeficiency (SCID) in humans. In mice, Ada KO mutants have no discernable thymus, two-to-three-fold lower cell numbers in the spleen, and livers that exhibit severe histological defects. This latter defect is the underlying cause of perinatal lethality in Ada KO mice. Each of these defects is also observed in Ada KO rats. Studies in Ada KO mice have shown that low levels of ADA activity can rescue the liver dysfunction, as well as other non-immune defects associated with Ada deficiencies, while maintaining the SCID phenotype. These results indicate that it may be feasible to create a conditional inbred SCID Ada KO rat line suitable for human cell transplantation studies. To achieve this goal, we will assess the feasibility of using doxycycline-inducible systems to conditionally complement the non- immune defects while maintaining the SCID phenotype. In Phase I, we will generate transgenic founders that co-segregate two expression modules: (a) a ubiquitously expressed Tet-repressor; and (b) an Ada mini-gene under the control of a CMV/Tet operator transcriptional element. The Tet-repressor will inhibit the expression of the Ada mini-gene; expression can be rapidly induced by providing doxycycline (dox) in the drinking water of the animals. We will identify founder animals that exhibit dox dose-dependent activation of the Ada mini-gene (referred to as conditional Ada transgenic rats; AdacTG), and determine whether Ada -/- /AdacTG rats are viable and exhibit no overt phenotypes except for immune deficiency. In Phase II, we will transplant primary human cells (e.g., hepatocytes) into the transgenic animals, in proof-of-principal studies to demonstrate the long-term viability and function of human cells in the new SCID rat models. In the long term, we will develop Ada -/- /AdacTG rats with humanized livers for drug discovery and therapeutic applications. This project should provide, for the first time, an inbred rat SCID Ada model for human cell transplantation studies, enabling broad applications for assessing the function of human cells in the context of the rat, which is amenable to a wide variety of experimental manipulations not possible in mice. PUBLIC HEALTH RELEVANCE: The development of a rat strain that can support efficient human cell engraftment would represent a major advance for the production of humanized tissues for drug discovery and therapeutic research applications. In the application "Novel SCID rat models for human cell transplantation studies," studies are aimed at examining the feasibility of using a novel knockout immunodefective (SCID) rat line to generate animals that can serve as hosts to study the function of normal and diseased human tissues. The proposed innovations would further increase the utility of rat models for drug discovery and would aid in the development of pre-clinical animal models that represent more predicative indicators of efficacy and toxicity associated with drug candidates. Being able to generate rats that can permit the long-term propagation of human cells and tissues would provide unique and valuable resources for drug discovery and therapeutic research. In the rat, such transplants would enable a wide variety of experimental manipulations that would not be possible in mice. There is a compelling need for an abundant and accessible supply of animals to screen for new therapeutic agents. Thus, if successful, this project would benefit many goals of public health.

描述（由申请方提供）：制药行业广泛使用实验室大鼠进行临床前动物有效性和毒理学研究。此外，由于易于实验操作，大鼠一直是生理学、行为学和药理学研究计划的有价值的动物模型。它们较大的尺寸有助于在小鼠中进行其他困难的程序，包括使用仪器的研究，血液采样，神经传导研究和手术。产生可以维持功能性人类异种移植物的近交系免疫缺陷大鼠品系将为药物发现和治疗研究计划提供独特且有价值的资源。本项目将研究使用一种新的大鼠品系携带转座子介导的敲除（KO）插入突变内的腺苷脱氨酶（Ada）基因座在近交系费舍尔。这种突变是人类严重联合免疫缺陷（SCID）的潜在原因。在小鼠中，Ada KO突变体没有可辨别的胸腺，脾脏中的细胞数量减少2 - 3倍，肝脏表现出严重的组织学缺陷。后一种缺陷是Ada KO小鼠围产期死亡的根本原因。在Ada KO大鼠中也观察到这些缺陷中的每一种。在Ada KO小鼠中的研究表明，低水平的ADA活性可以挽救肝功能障碍，以及与Ada缺陷相关的其他非免疫缺陷，同时保持SCID表型。这些结果表明，它可能是可行的，以建立一个条件近交SCID Ada KO大鼠系适合人类细胞移植研究。为了实现这一目标，我们将评估使用强力霉素诱导系统来条件性地补充非免疫缺陷同时维持SCID表型的可行性。在I期，我们将产生共分离两个表达模块的转基因创始人：（a）普遍表达的Tet-repressor;和（B）在CMV/泰特操纵转录元件控制下的Ada微型基因。Tet-repressor将抑制Ada mini-gene的表达;通过在动物的饮用水中提供强力霉素（dox）可以快速诱导表达。我们将鉴定表现出dox剂量依赖性Ada迷你基因激活的创始动物（称为条件性Ada转基因大鼠; AdacTG），并确定Ada -/- /AdacTG大鼠是否存活，以及是否表现出除免疫缺陷外的明显表型。在第二阶段，我们将移植原代人类细胞（例如，肝细胞）注入到转基因动物中，以证明人类细胞在新的SCID大鼠模型中的长期存活力和功能。从长远来看，我们将开发具有人源化肝脏的Ada -/- /AdacTG大鼠，用于药物发现和治疗应用。该项目将首次提供用于人类细胞移植研究的近交系大鼠SCID Ada模型，使其能够广泛应用于评估大鼠背景下人类细胞的功能，这适用于小鼠中不可能进行的各种实验操作。 公共卫生关系：能够支持有效的人类细胞移植的大鼠品系的开发将代表用于药物发现和治疗研究应用的人源化组织的生产的重大进展。在“用于人类细胞移植研究的新型SCID大鼠模型”申请中，研究的目的是检查使用新型敲除免疫缺陷（SCID）大鼠系来产生可用作研究正常和患病人体组织功能的宿主的动物的可行性。拟议的创新将进一步提高大鼠模型在药物发现中的实用性，并将有助于开发临床前动物模型，这些模型代表了与候选药物相关的疗效和毒性的更具预测性的指标。能够培育出能够长期繁殖人类细胞和组织的大鼠，将为药物发现和治疗研究提供独特而宝贵的资源。在大鼠中，这种移植将使各种各样的实验操作成为可能，而这些操作在小鼠中是不可能的。迫切需要大量和可获得的动物供应来筛选新的治疗剂。因此，如果成功，该项目将有利于公共卫生的许多目标。