Full human gene-replacement mouse models of ADRDs

我们的总体目标是开发第一组阿尔茨海默病相关痴呆 (ADRD) 小鼠品系，尽可能模拟 ADRD 的遗传学。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，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。我们使用这项技术生成了 MAPT-GR 小鼠系，其中我们用完整的人类 MAPT 基因组序列（190,081 bp）替换了完整的小鼠 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，并且所有预期的剪接变体都在适当的组织中发现，并且以全功能人 MAPT 基因的预期比例存在。我们该项目 R61 阶段的具体目标是 1) 生成与我们的第一个 wt MAPT-GR 对照品系精确匹配的 5 个小鼠品系，但我们特别引入的致病性额颞叶痴呆帕金森症 17 (FTDP-17) 突变除外； 2) 鉴定致病性 MAPT-GR 变异株系和野生型对照之间显着不同的可量化内表型，无论是否有外部损伤（即头部外伤）； 3）开始产生额外的GR小鼠系，其中涉及ADRD病因学的其他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 R33 阶段的具体目标是： 1）释放匹配的 MAPT-GR 品系集以不受限制地分发； 2) 对已识别的 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表征； 3) 生成与 MAPT-GR 系相似的 ADRD-GR 小鼠系的其他匹配组，即 C9orf72-GR（肌萎缩侧索硬化症-额颞叶痴呆；ALS-FTD）、SNCA-GR（路易体痴呆）、MATR3-GR (ALS-FTD) 和 GRN-GR (FTD)。我们在这里的贡献预计是： a) 一套完整的人类基因替代 ADRD 小鼠模型，这些模型在基因水平上完全定义，具有精确匹配的控制线，并模仿 ADRD 的人类遗传学； b) 鉴定出的、可量化的早期内表型与人类 MAPT 基因的致病性突变密切相关。这些贡献将是意义重大的，因为它们将提供新的工具来探究分子疾病机制、确定治疗靶点并开发有效的疗法。这些精确匹配的动物模型组将使研究界能够在患者身上发生的人类基因组序列背景下评估致病性突变的分子影响，并且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序列到所有RNA转录变体和它们编码的蛋白质产物。

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.