Improved Animal Models for Cell-Specific Regenerative Medicine Paradigms

细胞特异性再生医学范式的改进动物模型

基本信息

批准号：
9206193
负责人：
JEFFREY MUMM
金额：
$ 32.06万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2020-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9206193
关键词：
Ablation Adopted Alpha Cell Animal Model Animals Antibodies Autoimmune Diseases Biological Assay Biological Models Cell Culture Techniques Cell Cycle Kinetics Cell Death Cell model Cells Chemicals Data Degenerative Disorder Development Directed Molecular Evolution Disease Disease model Engineering Enzyme Kinetics Enzymes Escherichia coli Evaluation Exhibits Gene Library Genes Heart Human Imagery Immune response Kinetics Libraries Link Methodology Methods Metronidazole Modeling Molecular Mutagenesis Natural regeneration Nature Nitroreductases Organism Pan Genus Pancreas Performance Pharmaceutical Preparations Positioning Attribute Process Prodrugs Proteins Rana Regenerative Medicine Regimen Reporter Reporter Genes Reporting Research Resources Retina Specificity Stem cells Structure of beta Cell of islet Substrate Specificity System Testing Time Tissues Toxic effect Transgenes Transgenic Animals Transgenic Model Transgenic Organisms Treatment Protocols Variant Whole Organism Zebrafish base cancer cell cell dimension cell type combinatorial comparative curative treatments cytotoxic design dosage expression vector flexibility gene therapy human disease improved in vivo insight novel promoter public health relevance regenerative regenerative therapy selective expression vector

项目摘要

DESCRIPTION (provided by applicant): We propose substantial improvements to a powerful targeted cell ablation system we developed to create novel animal models for regenerative medicine research, and to provide a corresponding set of versatile new toolsets. This system uniquely facilitates: 1) Studies of cell-specific regeneration for nearly any cellular subtype; 2) Inducible degenerative disease modeling; and 3) Whole organism HTS discovery of regenerative therapeutics. Importantly, this approach can be deployed in any transgenic animal model system, and is capable of modeling any disease linked to the loss of specific cell or tissue types. The system employs a prodrug converting nitroreductase (NTR) enzyme, which was first developed to [eradicate human cancer cells via gene therapy.] To create novel cell-specific regeneration paradigms, a NTR enzyme and a fluorescent reporter gene are placed under the control of a cell-type specific promoter in a transgenic organism [(i.e., only expressed in the targeted cells/tissues)]. NTR expression selectively sensitizes those cells to prodrugs that produce cytotoxic derivatives [(e.g., metronidazole), enabling precise] targeted cell ablation, on [demand, without harming surrounding cells]. The reporter allows subsequent regenerative processes to be characterized in detail. This versatile NTR system has been adapted] to multiple species, promoting a wide array of cell-specific regeneration paradigms and degenerative disease models. However, inadequate prodrug activation compromises the full potential of this approach; this limits the types of cells that can be ablated, slows cell loss kinetics, and necessitates semi-toxic prodrug regimens that can confound key data. We propose to develop superior iterations of NTR/prodrug systems that will: 1) Provide additional dimensions of cell targeting; 2) Accelerate cell ablation kinetics, to facilitate better resolutionof regeneration kinetics; and 3) Alleviate systemic semi-toxicity associated with current prodrug treatment regimens. The specific aims of this study are: 1) Identify NTR variants with improved cell-specific ablation efficacy; 2) Define selective NTR prodrug pairs to facilitate independent ablation of multiple cell types; and 3) Develop pan-species transgenic [vectors to create improved] NTR resources. To achieve these aims we have assembled a library of NTR variants from 24 bacterial species, used directed evolution (random mutagenesis at a single-gene level, followed by selection for improved enzyme variants) to tailor desirable NTR activities, identified new prodrug substrates, and developed high-throughput methods to quantify ablation efficacy in comparative assays. We will apply these resources to develop NTR [variants with improved enzymatic activity, and novel NTR/prodrug pairs with enhanced specificity. Our improved NTRs will: 1) Facilitate the creation of improved animal models to yield insights] into cell-specific regeneration; 2) Enable identification of factors that regulate the regenerative potential of discrete stem cell niches; and 3) Aid development of curative therapies for the many degenerative conditions linked to the loss of specific cells.

描述（由申请人提供）：我们建议对我们开发的强大的靶向细胞消融系统进行重大改进，以创建用于再生医学研究的新型动物模型，并提供一套相应的多功能新工具集，该系统独特地促进：1）研究。几乎任何细胞亚型的细胞特异性再生；2）诱导性退行性疾病模型；3）再生疗法的整个有机体 HTS 发现，重要的是，这种方法可以应用于任何转基因动物模型系统。该系统能够模拟与特定细胞或组织类型丧失相关的任何疾病，该系统采用前药转化硝基还原酶（NTR），该酶最初是为了[通过基因疗法根除人类癌细胞]而开发的。在再生范例中，NTR 酶和荧光报告基因被置于转基因生物体中细胞类型特异性启动子的控制下[（即仅在目标细胞/组织中选择性表达）]。使这些细胞对产生细胞毒性衍生物的前药敏感[（例如，甲硝唑），从而能够根据需要进行精确的]靶向细胞消融，而不伤害周围细胞。报告者可以详细描述随后的再生过程。已适应多种物种，促进了广泛的细胞特异性再生范例和退行性疾病模型。然而，前药激活不足会损害该方法的全部潜力，这限制了其类型。我们建议开发 NTR/前药系统的高级迭代，这将： 1) 提供额外的细胞靶向维度； 2) 加速细胞靶向。细胞消融动力学，以促进更好地解析再生动力学；以及 3) 减轻与当前前药治疗方案相关的全身半毒性。是：1）识别具有改进的细胞特异性消融功效的NTR；2）定义选择性NTR前药对以促进多种细胞类型的独立消融；以及3）开发泛物种转基因[载体以创建改进的]NTR资源。组装了来自 24 个细菌物种的 NTR 变体文库，使用定向进化（在单基因水平上随机诱变，然后选择改进的酶变体）来定制所需的 NTR 活性，确定了我们将利用这些资源来开发 NTR [具有改进的酶活性的变体，以及具有增强特异性的新型 NTR/前药对]。促进创建改进的动物模型，以深入了解细胞特异性再生；2) 识别调节离散干细胞生态位再生潜力的因素；3) 帮助开发针对与特定细胞损失有关的许多退行性疾病的治疗方法。