Novel SCID rat models for human cell transplantation studies

描述(由申请人提供)：实验室大鼠被制药行业广泛用于临床前动物疗效和毒理学研究。此外，由于实验操作简单，大鼠一直是生理学、行为学和药理学研究项目的有价值的动物模型。它们的较大尺寸有利于在小鼠身上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。培育出能够维持人类异种移植物功能的近交系免疫缺陷大鼠品系，将为药物发现和治疗研究项目提供独特而有价值的资源。本项目将研究一种新型大鼠品系的使用，该品系在近交系Fischer系中携带转座子介导的基因敲除(KO)插入突变的腺苷脱氨酶(Ada)基因。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突变体没有可识别的胸腺，脾中的细胞数量减少了两到三倍，肝脏表现出严重的组织学缺陷。后一种缺陷是导致Ada KO小鼠围产期死亡的根本原因。这些缺陷中的每一个也在Ada KO大鼠中观察到。对Ada KO小鼠的研究表明，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，建立一个适合于人类细胞移植研究的条件近交系SCID Ada KO大鼠系是可行的。为了实现这一目标，我们将评估使用多西环素诱导系统在维持SCID表型的同时有条件地补充非免疫缺陷的可行性。在第一阶段，我们将产生共分离两个表达模块的转基因创建者：(A)普遍表达的Tet抑制因子；(B)CMV/Tet操纵子转录元件控制下的Ada微型基因。Tet抑制因子会抑制Ada-mini基因的表达；在动物的饮用水中提供强力霉素(Dox)可以快速诱导其表达。我们将确定表现出Dox剂量依赖的Ada微型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大鼠是否存活并且除了免疫缺陷外没有明显的表型。在第二阶段，我们将把原代人类细胞(如肝细胞)移植到转基因动物中，进行原则验证研究，以证明人类细胞在新的SCID大鼠模型中的长期活性和功能。从长远来看，我们将培育Ada-/-/AdacTG大鼠的人源化肝脏，用于药物开发和治疗应用。该项目将首次为人类细胞移植研究提供近亲交配的大鼠SCID Ada模型，使其能够广泛应用于在大鼠环境中评估人类细胞的功能，这是小鼠不可能进行的各种实验操作的结果。 公共卫生相关性：能够支持高效人类细胞植入的大鼠品系的开发将代表着为药物发现和治疗研究应用生产人性化组织的重大进展。在《用于人类细胞移植研究的新型SCID大鼠模型》的应用中，研究旨在检验使用新型免疫缺陷(SCID)基因敲除大鼠系产生动物的可行性，这些动物可以作为宿主来研究正常和疾病的人体组织的功能。拟议的创新将进一步增加大鼠模型用于药物发现的实用性，并将有助于开发临床前动物模型，这些模型代表与候选药物相关的疗效和毒性的更具预测性的指标。能够产生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有价值的资源。在大鼠身上，这样的移植将使各种各样的实验操作成为可能，而这在小鼠身上是不可能的。迫切需要有充足和可获得的动物供应来筛选新的治疗剂。因此，如果该项目成功，将使公共卫生的许多目标受益。

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.