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: 9893130
• 负责人: TIMOTHY J EBNER
• 金额: $ 150.75万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893130
• 关键词: Alleles; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Animal Model; Behavior; Brain imaging; C9ORF72; Catalogs; Code; Communities; Control Animal; Craniocerebral Trauma; DNA Sequence; Dementia; Disease; Etiology; FTD with parkinsonism; Failure; Functional disorder; GRN gene; Generations; Genes; Genetic; Genetic Models; Genetic Transcription; Genomic DNA; Genomics; Goals; Human; Human Genetics; Imaging technology; Lewy Body Dementia; Link; MAPT gene; Modeling; Molecular; Molecular Disease; Molecular Probes; Mus; Mutation; Nucleic Acid Regulatory Sequences; Orthologous Gene; Pathogenicity; Patients; Phase; Proteins; Public Health; RNA; RNA Splicing; Research; Resources; SNCA gene; System; Technology; Tern; Therapeutic Intervention; Time; Tissues; Variant; effective therapy; endophenotype; experimental study; frontotemporal lobar dementia-amyotrophic lateral sclerosis; gene replacement; in vivo; mouse model; neuropathology; novel; tau Proteins; tau dysfunction; therapeutic evaluation; therapeutic target; therapy development; tool

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.

我们的总体目标是发展第一组阿尔茨海默病相关痴呆(ADRD)小鼠系，尽可能接近ADRD的遗传学模型。这些模型将作为实验系统，用于探测致病性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，我们开发了基因替换(GR)技术，允许我们用大小高达数百kb的完整人类同源基因来替换老鼠的基因。我们使用这项技术产生了一个小鼠的MAPT-GR系，其中我们用完整的人MAPT基因组序列(190,081 bp)取代了小鼠MAPT基因组编码和调控区的完整序列(156,547 bp)。我们已经证实，这种MAPT-GR等位基因纯合的小鼠在内源性水平表达人类tau，并且在适当的组织中发现了所有预期的剪接变体，并以完全功能的人类MAPT基因的预期比例进行了研究。我们在这个项目R61阶段的具体目标是：1)产生与我们第一个wt MAPT-GR控制系精确匹配的五个小鼠系，除了我们特别引入的致病性额颞叶痴呆伴帕金森-17(FTDP-17)突变；2)确定在致病MAPT-GR变异系与wt对照之间存在显著差异的可量化的内表型，无论有无外部伤害(如头部创伤)；以及3)开始产生额外的GR系，其中与ADRD的病因有关的其他基因已被它们的人类同源基因取代。一旦我们实现了这些目标，我们在R33阶段的具体目标是：1)释放匹配的MAPT-GR系用于不受限制的分布；2)对已鉴定的tau相关内表型、神经病理学和MAPT-GR系的行为进行全面的纵向鉴定；以及3)产生更多类似于MAPT-GR系的匹配的ADRD-GR小鼠系，即C9orf72-GR(肌萎缩侧索硬化症-额颞部痴呆；ALS-FTD)，SNCA-GR(路易体痴呆)，MATR3-GR(ALS-FTD)和GRN-GR(FTD)。我们在这方面的贡献有望是：a)在基因水平上完全定义的全人类基因替代ADRD小鼠模型，具有精确匹配的控制线，并模拟人类ADRD的遗传学；以及b)与人类MAPT基因的致病突变密切相关的可识别的、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，确定治疗目标，并开发有效的治疗方法。这些精确匹配的动物模型集将使研究界能够在患者出现的人类基因组序列的背景下评估致病突变的分子影响，这些小鼠品系将包含所有潜在的人类治疗靶点，从完整的基因组DNA序列到它们编码的所有RNA转录变体和蛋白质产品。