Elucidating the protective effects of the KL-VS variant using isogenic hiPSCs

使用同基因 hiPSC 阐明 KL-VS 变体的保护作用

• 批准号: 10354135
• 负责人: DAVID A BRAFMAN
• 金额: $ 43.18万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10354135
• 关键词: 3-Dimensional; AD transgenic mice; Address; Aging; Alzheimer disease prevention; Alzheimer' s Disease; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid; Amyloid beta-Protein; Animal Model; Attenuated; Biochemical; Biomedical Engineering; Calcium; Cell Line; Cells; Coculture Techniques; Cognitive; Data; Development; Disease; Disease model; Ensure; Environment; Exhibits; Future; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Gold; Haplotypes; Heterozygote; Human; Human Engineering; Impaired cognition; Individual; Inflammatory; Inflammatory Response; Investigation; Karyotype; Knock-out; Learning; Link; Mediating; Methods; Modeling; Molecular; Mus; Mutation; Nature; Neuraxis; Neurodegenerative Disorders; Nitric Oxide; Onset of illness; Oxidative Stress; Pathologic; Pathology; Phenotype; Probability; Process; Production; Regulation; Reporter; Reporting; Research; Risk; Risk Factors; Series; Signal Pathway; Signaling Protein; Single Nucleotide Polymorphism; Stimulus; Synapses; System; Testing; Therapeutic Intervention; Variant; Veins; Work; age related; attenuation; base; base editing; biological adaptation to stress; design; gene regulatory network; genetic variant; genome editing; genome wide association study; in vivo; induced pluripotent stem cell; loss of function; molecular targeted therapies; mouse model; neuroprotection; neurotoxic; non-demented; overexpression; pluripotency; protective effect; relating to nervous system; stem cell model; stem cells; tau Proteins; tau phosphorylation; therapeutic target; transcriptomics; uptake; β-amyloid burden

PROJECT SUMMARY ABSTRACT Genome-wide association studies (GWAS) studies have identified several risk factors associated with altered probability of sporadic AD (SAD) onset. However, the majority of these studies have identified genetic variants that increase risk of AD and, consequently, downstream therapeutic interventions have focused on mitigating these effects. On the other hand, the study of variants that decrease risk of AD might provide an alternative to identifying promising therapeutic target for AD prevention. In this regard, polymorphisms in the Klotho (KL) gene have been associated with a reduced risk of AD onset and age-related progression. KL was first identified as suppressing numerous age-related processes as KL deficient mice exhibited accelerated aging. With regards to AD, KL overexpression in AD transgenic mouse models significantly ameliorates AD-related pathological and cognitive phenotypes. Interestingly, it has been shown that two single nucleotide polymorphisms in the KL gene, rs9536314 (F352V) and rs9527025 (C370S) segregate together to form a haplotype KL-VS. Specifically, KL-VS heterozygosity has not only been associated with greater cortical volumes and elevated synaptic connectivity but also attenuation of AD-related phenotypes. As such, understanding the mechanism by which the KL-VS variant mitigates AD onset and age-related progression will have a significant impact on therapeutic interventions. In this proposal, we will use our collective expertise in stem cell bioengineering, neurodegenerative disease modeling, and highly efficient genome editing to elucidate the specific mechanisms by which KL-VS heterozygosity mitigates AD risk. In the first aim, we will use our recently developed gene editing methods to introduce the KL-VS variant into isogenic hiPSCs from healthy non-demented control (NDC) and AD patients. In the second aim, we will employ these isogenic hiPSC lines in a 3-D co-culture model to (i) determine if KL-VS heterozygosity mitigates the presence of AD-related phenotypes and (ii) exerts global transcriptional changes in signaling pathways and gene regulatory networks that would enhance neuroprotection against AD. In addition, through the use of KL knockout hiPSCs we will be able to determine if these KL-VS heterozygosity induces its effects occur through gain- or loss-of-function mechanisms. Overall, a better understanding of potential genetic factors, such as KL-VS heterozygosity, that enhance neuroprotection against AD will provide new avenues for therapeutic interventions.

项目摘要 全基因组关联研究（GWAS）研究已经确定了与改变的基因型相关的几个风险因素。 散发性AD（SAD）发作的可能性。然而，这些研究中的大多数已经确定了遗传变异， AD风险增加，因此，下游治疗干预的重点是减轻 这些影响。另一方面，对降低AD风险的变异的研究可能提供一种替代方法， 为AD预防确定有希望的治疗靶点。在这方面，Klotho（KL）基因的多态性 与AD发作和年龄相关进展的风险降低相关。KL最初被确定为 抑制许多与年龄相关的过程，因为KL缺陷小鼠表现出加速老化。关于 在AD转基因小鼠模型中，AD、KL过表达显著改善AD相关的病理学和免疫学改变。 认知表型有趣的是，已经表明KL基因中的两个单核苷酸多态性， rs 9536314（F352 V）和rs 9527025（C370 S）分离在一起以形成单倍型KL-VS。 杂合性不仅与更大的皮质体积和更高的突触连接有关， 而且还可以减轻AD相关表型。因此，了解KL-VS的机制 变异缓解AD发作和年龄相关进展将对治疗产生显著影响 干预措施。在这项提案中，我们将利用我们在干细胞生物工程、神经退行性疾病 疾病建模和高效的基因组编辑，以阐明KL-VS的具体机制， 杂合性降低AD风险。在第一个目标中，我们将使用我们最近开发的基因编辑方法， 将KL-VS变体引入来自健康非痴呆对照（NDC）和AD患者的同基因hiPSC中。在 第二个目的，我们将在3-D共培养模型中使用这些等基因hiPSC系，以（i）确定KL-VS是否 杂合性减轻了AD相关表型的存在，并且（ii）在AD相关表型中产生全局转录变化。 信号通路和基因调控网络，将增强对AD的神经保护。此外，本发明还提供了一种方法， 通过使用KL敲除的hiPSC，我们将能够确定这些KL-VS杂合性是否诱导其表达。 作用通过功能获得或丧失机制发生。总的来说，更好地了解潜在的遗传 增强对AD的神经保护作用的因素，如KL-VS杂合性，将为AD的治疗提供新的途径。 治疗干预。