Full human gene-replacement mouse models of ADRDs

ADRD 的完整人类基因替代小鼠模型

• 批准号: 10464809
• 负责人: TIMOTHY J EBNER
• 金额: $ 228.09万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-18 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10464809
• 关键词: Full; human; gene; replacement; mouse; Full; human; gene; replacement; mouse

Our overall objective is to develop the first sets of Alzheimer’s disease related dementia (ADRD) mouse lines that model the genetics of ADRDs as closely as possible. These models will serve as experimental systems for probing the molecular dysfunctions caused by pathogenic ADRD mutations, identifying quantifiable early-stage endophenotypes directly linked to these mutations, and developing and testing therapeutic interventions for correcting these dysfunctions. To make these models, we have developed Gene Replacement (GR) technology that allows us to replace mouse genes with their full human orthologs up to several hundred kb in size. We used this technology to generate a MAPT-GR line of mice in which we replaced the full mouse Mapt genomic coding and regulatory region (156,547 bp) with the full human MAPT genomic sequence (190,081 bp). We have confirmed that mice homozygous for this MAPT-GR allele express human tau at endogenous levels, and that all expected splice variants are found in the appropriate tissues and in ratios expected for the fully functional human MAPT gene. Our specific aims for the R61 phase of this project are to 1) generate five lines of mice that precisely match our first wt MAPT-GR control line except for the pathogenic frontotemporal dementia with parkinsonism-17 (FTDP-17) mutation that we specifically introduce; 2) identify quantifiable endophenotypes that are significantly different between pathogenic MAPT-GR variant lines and the wt control, with and without external insult (i.e., head trauma); and 3) begin to generate additional sets of GR lines of mice in which other genes involved in the etiology of ADRD have been replaced by their human homologs. Once we have achieved these goals, our specific aims for the R33 phase are to: 1) release the matched set of MAPT- GR lines for distribution without restriction; 2) conduct full longitudinal characterization of identified tau- associated endophenotypes, neuropathology and behavior of the MAPT-GR lines; and 3) generate additional matched sets of ADRD-GR mouse lines similar to the MAPT-GR lines, namely C9orf72-GR (amyotrophic lateral sclerosis- frontotemporal dementia; ALS-FTD), SNCA-GR (dementia with Lewy bodies), MATR3-GR (ALS- FTD), and GRN-GR (FTD). Our contributions here are expected to be: a) sets of full human gene-replacement ADRD mouse models that are completely defined at the genetic level, have precisely matched control lines, and mimic the human genetics of ADRD; and b) identified, quantifiable early-stage endophenotypes closely linked to pathogenic mutations in the human MAPT gene. These contributions will be significant because they will provide new tools to interrogate molecular disease mechanisms, identify therapeutic targets, and develop effective therapies. These precisely matched sets of animal models will allow the research community to evaluate the molecular impact of pathogenic mutations within the context of the human genomic sequence in which they occur in patients, and these mouse lines will contain all potential human therapeutic targets ranging from the full genomic DNA sequences to all RNA transcription variants and protein products that they encode.

我们的总体目标是开发第一组老年痴呆症相关的痴呆症（ADRD）小鼠系，将ADRD的遗传学对象尽可能近。这些模型将用作实验系统，用于探测由致病性ADRD突变引起的分子功能障碍，鉴定可定量的与这些突变直接相关的早期阶段的内型型，并开发和测试治疗性干预措施以纠正这些功能障碍。为了制定这些模型，我们开发了基因置换（GR）技术，使我们能够用其全人类直系同源物替换小鼠基因的大小。我们使用这项技术生成了一小鼠的MAPT GR系，其中我们用完整的人类MAPT基因组序列（190,081 bp）代替了完整的小鼠MAPT基因组编码和调节区域（156,547 bp）。我们已经证实，这种MAPT-GR等位基因表达人Tau的小鼠在内源水平上纯合子，并且在适当的组织中发现了所有预期的剪接变体，并且在适当的功能性人类MAPT基因的比率中发现了所有预期的剪接变异。我们对该项目R61阶段的具体目的是1）生成五种小鼠，这些小鼠与我们的第一个WT MAPT-GR控制线完全匹配，除了带有Parkinsonism-17（FTDP-17）突变的致病性额颞痴呆，我们特别引入了； 2）识别可量化的内表型，它们在致病性mapt-gr变异线和WT控制之间有显着差异，有和没有外部侮辱（即头部创伤）； 3）开始生成其他小鼠的GR系，其中其他参与ADRD病因的基因已被其人类同源物取代。一旦我们实现了这些目标，我们对R33阶段的具体目标是：1）释放匹配的MAPT-GR线以进行分发而无需限制； 2）对已鉴定的tau相关的内表型，神经病理学和mapt-gr线的行为进行全面纵向表征； 3）生成类似于MAPT-GR线的ADRD-GR小鼠系的其他匹配集，即C9orf72-gr（肌营养性侧面硬化症 - 额额痴呆; ALS-FTD），SNCA-gr（带有lewy hodies的痴呆症），Matr3-gr（Als-ftd）和Grn-grn-Grn-gr（FDD）。我们在这里的贡献预计将是：a）在遗传水平上完全定义的一组完整的人类基因替代ADRD小鼠模型，具有精确匹配的控制线，并模仿ADRD的人类基因； b）确定的，可量化的早期内表型与人类MAPT基因的致病突变密切相关。这些贡献将是重要的，因为它们将提供新的工具来询问分子疾病机制，确定治疗靶标并开发有效的疗法。这些精确匹配的动物模型集将使研究界能够在患者中发生的人类基因组序列中评估致病突变的分子影响，并且这些小鼠系将包含所有潜在的人类治疗靶标，范围从完整的基因组DNA序列到所有RNA转录变体产物和蛋白质产物，这些靶标具有它们的编码。