Development of marmoset models of neurodegenerative disease using embryonic stem cell-based gene-editing approaches

项目摘要 该项目的长期目标是通过基因- 胚胎干细胞（ESCs）鼠标系统是医学研究的强大工具， 操纵小鼠基因组的能力。然而，相当多的解剖，生理，认知， 小鼠和人类之间的行为差异限制了小鼠模型的洞察力 人类疾病的光。这反映在大量有效药物的临床试验失败上 治疗人类疾病的小鼠模型。有几条证据表明绒猴代表了 一种改进的动物系统，用于研究一系列人类疾病，包括中风和与年龄相关的疾病。 神经退行性疾病，如阿尔茨海默病（AD）。绒猴是最短命的 灵长类动物（平均寿命为5-7年，而恒河猴为25年）， 表现出与人类相似的年龄相关的变化，包括β-淀粉样蛋白沉积， 大脑皮层，胆碱能神经支配的丧失和神经发生减少，如在AD中观察到的。此外，本发明还提供了一种方法， 绒猴具有高度的社会性和交际性，并表现出学习复杂知识的能力。 认知行为因此，绒猴代表了一个理想的遗传平台，用于产生模型， 神经退行性疾病，更准确地反映了人类的状况，并使测试 潜在的自体（同种）干细胞为基础的再生疗法。初期工作将侧重于 产生AD的绒猴模型。最近出现的基因编辑和干细胞技术， 灵长类动物为产生绒猴疾病模型铺平了道路，但这两个领域的改进都是 使这种方法可行是必要的。在此，常规同源重组和 CRISPR/Cas9基因组编辑技术将用于修饰绒猴ESC。作为遗传证据 表明淀粉样前体蛋白（APP）基因突变导致β-淀粉样蛋白增加， 生产，斑块的形成，认知障碍，绒猴APP将被编辑携带 人类点突变用于研究认知神经元细胞类型特异性回路的遗传工具 还将通过将Cre重组酶盒插入AD中3 ′端， 小清蛋白和胆碱乙酰转移酶基因的末端非翻译区。这些Cre驱动程序线将 使这些细胞类型的可视化和功能操作成为可能。圆满完成拟议的 目的将产生一个大大改善的AD动物模型，使测试基于干细胞的再生 治疗AD的方法，并为应用这些遗传工具分析神经元回路铺平道路 健康的大脑。在绒猴胚胎干细胞中建立基因编辑也将使其他基因的开发成为可能。 人类疾病的灵长类动物模型，提供关键的实验资源， 多个NIH研究所（例如，NINDS、NIA、NIMH、NEI、NIAAA、NIDA、NICHG、NIGMS）。

PROJECT SUMMARY The long-term goal of this project is to model human neurodegenerative diseases in marmosets via gene- editing in embryonic stem cells (ESCs). The mouse system is a powerful tool for medical research due to the ability to manipulate the mouse genome. However, considerable anatomical, physiological, cognitive, and behavioral differences between mice and humans limit the degree to which insights from mouse models shed light on human diseases. This is reflected in the high number of failed clinical trails for drugs that were effective in treating mouse models of human disease. Several lines of evidence suggest that the marmoset represents an improved animal system for studying a range of human diseases, including stroke and age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Marmosets are the shortest-lived of the anthropoid primates (average lifespan of 5–7 years compared with 25 years for the rhesus macaque) and exhibit age-related changes that are similar to those seen in humans, including β-amyloid deposition in the cerebral cortex, loss of cholinergic innervation, and reduced neurogenesis, as observed in AD. In addition, marmosets are highly social and communicative and have demonstrated the capacity to learn sophisticated cognitive behaviors. Therefore, marmosets represent an ideal genetic platform for generating models of neurodegenerative diseases that more accurately reflect the human condition and enable the testing of potential autologous (the-same-species) stem cell-based regenerative therapies. Initial efforts will focus on generating a marmoset model of AD. The recent emergence of gene-editing and stem-cell technologies in primates pave the way toward generating marmoset disease models, but improvements in both areas are necessary to make this approach viable. Here, both conventional homologous recombination and CRISPR/Cas9 genome-editing technologies will be employed to modify marmoset ESCs. As genetic evidence demonstrates that mutations in the amyloid precursor protein (APP) gene result in increased β-amyloid production, the formation of plaques, and cognitive impairment, the marmoset APP will be edited to carry human point mutations. Genetic tools for studying neuronal cell type-specific circuits underlying cognitive impairment and neuropathology in AD will also be generated by inserting a Cre recombinase cassette into 3' end non-translated regions of the parvalbumin and choline acetyltransferase genes. These Cre driver lines will enable the visualization and functional manipulation of these cell types. Successful completion of the proposed Aims will generate a greatly improved animal model of AD, enable testing of stem cell-based regenerative methods for treating AD, and pave the way toward applying these genetic tools for analyzing neuronal circuitry of healthy brains. Establishing gene-editing in marmoset ESCs will also enable the development of additional primate models of human diseases, providing critical experimental resources for research supported by multiple NIH Institutes (e.g., NINDS, NIA, NIMH, NEI, NIAAA, NIDA, NICHG, NIGMS).