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功效和毒理学研究。此外，由于易于实验操作，大鼠已成为生理学，行为学和药理学研究项目的有价值的动物模型。它们更大的尺寸有助于在小鼠身上进行其他困难的研究，包括使用仪器的研究、血液取样、神经传导研究和手术。产生能够维持人类异种移植物功能的免疫缺陷大鼠系将为药物发现和治疗研究计划提供独特而宝贵的资源。本项目将研究在近交系Fischer中携带转座子介导的敲除（KO）插入突变的一种新型大鼠菌株。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，脾脏细胞数量减少2 - 3倍，肝脏表现出严重的组织学缺陷。后一种缺陷是Ada KO小鼠围产期死亡的潜在原因。在Ada KO大鼠中也观察到这些缺陷。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缺乏相关的其他非免疫缺陷，同时保持SCID表型。这些结果表明，建立适合人类细胞移植研究的条件近交SCID Ada - KO大鼠系是可行的。为了实现这一目标，我们将评估使用强力霉素诱导系统在维持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，我们将产生转基因创始人，共同分离两个表达模块：(a)一个无处不在表达的tet抑制因子；(b)一个由CMV/Tet操作子转录元件控制的Ada迷你基因。tet抑制因子会抑制Ada mini基因的表达；在动物的饮用水中添加强力霉素（dox）可快速诱导表达。我们将鉴定Ada迷你基因表现出dox剂量依赖性激活的创始动物（称为条件Ada转基因大鼠；AdacTG），并确定Ada -/- /AdacTG大鼠是否可以存活，并且除了免疫缺陷外没有表现出明显的表型。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，在新的SCID大鼠模型中进行初步研究，以证明人类细胞的长期生存能力和功能。从长远来看，我们将开发具有人源肝脏的Ada -/- /AdacTG大鼠，用于药物发现和治疗应用。该项目将首次为人类细胞移植研究提供一个近交系大鼠SCID Ada模型，为评估人类细胞在大鼠环境下的功能提供广泛的应用，这可以适应小鼠无法进行的各种实验操作。

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.