Full human gene-replacement mouse models of ADRDs

我们的总体目标是开发第一批阿尔茨海默病相关痴呆（ADRD）小鼠品系，尽可能接近ADRD的遗传学模型。这些模型将作为实验系统，用于探测由致病性ADRD突变引起的分子功能障碍，鉴定与这些突变直接相关的可量化的早期内在表型，以及开发和测试用于纠正这些功能障碍的治疗干预措施。为了制造这些模型，我们开发了基因替换（GR）技术，使我们能够用大小高达数百kb的完整人类直系同源物替换小鼠基因。我们使用该技术来产生MAPT-GR小鼠系，其中我们用完整的人MAPT基因组序列（190，081 bp）替换完整的小鼠Mapt基因组编码和调控区（156，547 bp）。我们已经证实，该MAPT-GR等位基因纯合的小鼠在内源水平表达人tau蛋白，并且所有预期的剪接变体都存在于适当的组织中，并且以全功能人MAPT基因预期的比例存在。本项目R61阶段的具体目标是：1）产生五个品系的小鼠，除了我们特别引入的致病性额颞叶痴呆伴帕金森综合征-17（FTDP-17）突变外，这些品系的小鼠与我们的第一个wt MAPT-GR对照品系精确匹配; 2）鉴定在致病性MAPT-GR变体系和wt对照之间显著不同的可定量的内表型，有和没有外部损伤（即，头部创伤）;和3）开始产生另外的小鼠GR系组，其中参与ADRD病因学的其它基因已被其人类同源物取代。一旦我们实现了这些目标，我们对于R33阶段的具体目标是：1）释放匹配的MAPT-GR系组用于不受限制地分布; 2）对所鉴定的tau相关内在表型、神经病理学和MAPT-GR系的行为进行完全纵向表征;和3）产生与MAPT-GR系相似的ADRD-GR小鼠系的额外匹配组，即C9 orf 72-GR（肌萎缩侧索硬化-额颞痴呆; ALS-FTD）、SNCA-GR（路易体痴呆）、MATR 3-GR（ALS-FTD）和GRN-GR（FTD）。我们在此的贡献预计是：a）在遗传水平上完全定义的完整人类基因置换ADRD小鼠模型集，具有精确匹配的对照系，并模拟ADRD的人类遗传学;和B）与人类MAPT基因中的致病性突变密切相关的鉴定的、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组将允许研究界评估致病性突变在患者中发生的人类基因组序列背景下的分子影响，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物。

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.