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

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and 3) begin to generate additional sets of GR lines of mice in which other genes involved in the etiology of ADRD have been replaced by their human homologs.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阿尔茨海默病相关痴呆（ADRD）小鼠系，尽可能接近ADRD的遗传模型。这些模型将作为实验系统，用于探测由致病性ADRD突变引起的分子功能障碍，识别与这些突变直接相关的可量化的早期内表型，并开发和测试纠正这些功能障碍的治疗干预措施。为了制作这些模型，我们开发了基因替代（GR）技术，使我们能够用它们的完整的人类同源物替换小鼠基因，其大小可达几百kb。我们使用该技术生成了一个Mapt - gr小鼠系，我们用完整的人类Mapt基因组序列（190,081 bp）替换了小鼠完整的Mapt基因组编码和调控区（156,547 bp）。我们已经证实，这种MAPT- gr等位基因的纯合小鼠在内源性水平上表达人类tau，并且所有预期的剪接变体都在适当的组织中被发现，其比例与完全功能的人类MAPT基因预期的相同。我们在这个项目的R61阶段的具体目标是：1)产生5种与我们的第一个wt MAPT-GR控制系精确匹配的小鼠，除了我们特别引入的致病性额颞叶痴呆伴帕金森病-17 （FTDP-17）突变；2)确定致病MAPT-GR变异系与wt对照之间存在显著差异的可量化的内部表型，无论是否有外部损伤（即头部创伤）；3)开始产生额外的小鼠GR系，其中涉及ADRD病因学的其他基因已被其人类同源基因所取代。一旦我们实现了这些目标，我们在R33阶段的具体目标是：1)不受限制地发布匹配的MAPT- GR系列；2)对已鉴定的MAPT-GR系的tau相关内表型、神经病理学和行为进行全面的纵向表征；3)生成与MAPT-GR相似的额外匹配组ADRD-GR小鼠系，即C9orf72-GR（肌萎缩性侧索硬化症-额颞叶痴呆；ALS-FTD）、SNCA-GR（伴Lewy体痴呆）、mat3 - gr （ALS- FTD）和GRN-GR （FTD）。我们在这里的贡献预计是：a)在遗传水平上完全定义的完整的人类基因替代ADRD小鼠模型，具有精确匹配的控制系，并模拟ADRD的人类遗传学；b)与人类MAPT基因致病性突变密切相关的已确定的、可量化的早期内表型。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，确定治疗靶点，并开发有效的治疗方法。这些精确匹配的动物模型集将允许研究界在人类基因组序列的背景下评估致病性突变在患者身上发生的分子影响，这些小鼠系将包含所有潜在的人类治疗靶点，从完整的基因组DNA序列到所有RNA转录变体和它们编码的蛋白质产物。