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

• 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
• 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

 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实质性改进，以创建用于再生医学研究的新动物模型，并提供一套相应的通用新工具集。该系统独特地促进了：1)几乎任何细胞亚型的细胞特异性再生的研究；2)诱导性退行性疾病建模；以及3)整个生物体HTS再生疗法的发现。重要的是，这种方法可以部署在任何转基因动物模型系统中，并能够模拟与特定细胞或组织类型丧失有关的任何疾病。该系统使用了一种前药物转化硝基还原酶(NTR)酶，这种酶最初是为了[通过基因疗法根除人类癌细胞]而开发的。为了创造新的细胞特异性再生范例，在转基因生物中，NTR酶和荧光报告基因被置于细胞类型特异性启动子的控制下[即，仅在目标细胞/组织中表达]。NTR的表达选择性地使这些细胞对产生细胞毒性衍生物[(如甲硝唑)]的前体药物敏感，从而能够根据[需要]进行精确的靶向细胞消融，而不会损害周围细胞。该报告允许详细描述后续的再生过程。这一多功能的NTR系统已被适应于多种物种，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，不充分的前药物激活会损害这一方法的全部潜力；这限制了可被消融的细胞类型，减缓了细胞丢失动力学，并需要可能混淆关键数据的半毒性前药方案。我们建议开发更好的NTR/前药系统迭代，它将：1)提供更多的细胞靶向；2)加速细胞消融动力学，以促进更好地分辨再生动力学；以及3)减轻与当前前药治疗方案相关的全身半毒性。这项研究的具体目的是：1)确定具有改进的细胞特异性消融效率的NTR变体；2)定义选择性的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。为了实现这些目标，我们从24个细菌物种中组装了一个NTR变异体文库，使用定向进化(在单基因水平上进行随机突变，然后选择改进的酶变体)来定制所需的NTR活性，确定新的前药物底物，并开发高通量方法来在比较分析中量化消融效果。我们将应用这些资源来开发酶活性提高的NTR[变体]，以及具有增强特异性的新型NTR/前药对。我们改进的NTR将：1)促进创建改进的动物模型，以深入了解细胞特异性再生；2)能够识别调节离散干细胞壁龛再生潜力的因素；以及3)帮助开发与特定细胞丧失相关的许多退行性疾病的根治疗法。