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

使用基于胚胎干细胞的基因编辑方法开发狨猴神经退行性疾病模型

基本信息

批准号：
9209904
负责人：
KUO-FEN LEE
金额：
$ 74.21万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2021-01-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9209904
关键词：
Abeta synthesis Age Alleles Alzheimer&apos s Disease Alzheimer&apos s disease model Amyloid beta-Protein Amyloid beta-Protein Precursor Anatomy Animal Model Animals Area Autistic Disorder Autologous Behavior Behavioral Brain CRISPR/Cas technology Callithrix Cell Therapy Cerebral cortex Choline O-Acetyltransferase Chronic Kidney Failure Clinical Clinical Trials Cognitive Degenerative Disorder Dementia Development Diabetes Mellitus Disease Disease model Embryo Enterobacteria phage P1 Cre recombinase Ethics Exhibits Functional disorder Gene Proteins Gene Targeting Genes Genetic Genomics Goals Haplorhini Hereditary Disease Human Human Amyloid Precursor Protein Imagery Impaired cognition Institutes Internal Ribosome Entry Site Interneurons Knock-in Lead Learning Light Longevity Macaca mulatta Maintenance Malignant Neoplasms Mediating Medical Research Methodology Methods Modeling Modification Morphology Mosaicism Mus Mutation National Institute of Drug Abuse National Institute of General Medical Sciences National Institute of Mental Health National Institute of Neurological Disorders and Stroke National Institute on Alcohol Abuse and Alcoholism Nerve Degeneration Neurobiology Neurodegenerative Disorders Neurologic Neurons Parkinson Disease Parvalbumins Pharmaceutical Preparations Physiological Point Mutation Prevalence Primates Production Protein Overexpression Research Research Personnel Research Support Resources Schizophrenia Stem cells Stroke Symptoms System Technology Testing Thinness United States National Institutes of Health abeta deposition age related basal forebrain base blastocyst cartilaginous cell type cholinergic cholinergic neuron efficacy testing embryonic stem cell genetic manipulation genome editing hearing impairment homologous recombination human disease improved insight mouse genome mouse model muscle form nerve supply neural circuit neurogenesis neuromechanism neuronal circuitry neuropathology nonhuman primate novel therapeutic intervention pluripotency promoter protein expression regenerative regenerative therapy screening social stem cell technology tool

项目摘要

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).

项目概要该项目的长期目标是通过基因模型在狨猴中模拟人类神经退行性疾病小鼠胚胎干细胞 (ESC) 编辑系统是医学研究的强大工具。然而，操纵小鼠基因组的能力还需要相当大的解剖学、生理学、认知和能力。小鼠和人类之间的行为差异限制了小鼠模型的洞察力的程度这反映在大量有效药物的临床试验失败上。多项证据表明狨猴代表了治疗人类疾病的小鼠模型。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，如阿尔茨海默氏病（AD）是狨猴中寿命最短的。类人灵长类动物（平均寿命为 5-7 年，而恒河猴的平均寿命为 25 年）和表现出与人类相似的与年龄相关的变化，包括β-淀粉样蛋白沉积大脑皮层、胆碱能神经支配的丧失以及神经发生的减少，如 AD 中所观察到的。狨猴具有高度的社交性和沟通性，并表现出学习复杂知识的能力因此，狨猴代表了生成认知行为模型的理想遗传平台。神经退行性疾病能够更准确地反映人类状况并能够进行测试潜在的基于自体（同种）干细胞的再生疗法将集中于。生成 AD 狨猴模型最近出现的基因编辑和干细胞技术。灵长类动物为狨猴疾病模型的产生铺平了道路，但这两个领域的改进都在为了使这种方法可行，传统的同源重组和 CRISPR/Cas9 基因组编辑技术将用于修改狨猴 ESC 作为遗传证据。表明淀粉样蛋白前体蛋白 (APP) 基因的突变导致 β-淀粉样蛋白增加生产、斑块的形成、认知障碍，狨猴APP将被编辑携带用于研究认知神经细胞类型特异性回路的人类点突变。 AD 中的损伤和神经病理学也可以通过将 Cre 重组酶盒插入 3' 来产生这些 Cre 驱动系将终止小清蛋白和胆碱乙酰转移酶基因的非翻译区域。使这些细胞类型的可视化和功能操作能够成功完成。目标将产生一个大大改进的 AD 动物模型，从而能够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回路铺平道路在狨猴胚胎干细胞中建立基因编辑也将有助于开发更多的功能。人类疾病的灵长类动物模型，为以下支持的研究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）。