Developing Choroid Plexus-Based Tools and Drug Screens

开发基于脉络丛的工具和药物筛选

基本信息

批准号：
9304373
负责人：
Michael Jackson
金额：
$ 24.19万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9304373
关键词：
Alpha Cell Alzheimer&apos s Disease Animals Attention Biological Assay Biology Blood Circulation Brain Cell Line Cell physiology Cell secretion Cells Cellular biology Central Nervous System Diseases Cerebrospinal Fluid Clinical Cognitive Defect Development Disease Drug Delivery Systems Drug Targeting Enzyme-Linked Immunosorbent Assay Epithelial Cells Fenestrated Capillary Future Gap Junctions Generations Goals Growth Factor Hormones Human Hydrocephalus Intercellular Fluid Legal patent Libraries Link Literature Methods Molecular Target Mus Nerve Degeneration Neurodegenerative Disorders Pathogenesis Patients Peripheral Pharmacology Physiology Prealbumin Preclinical Drug Evaluation Proteins Protocols documentation Proxy Publishing Quality Control Readiness Reagent Reproducibility Schizophrenia Scientist Source Specificity Spinal Cord Stroke Structure of choroid plexus Technology Testing Therapeutic Intervention Time Tissues Toxin Validation Waste Products Work analog assay development base blood cerebrospinal fluid barrier cell type cytokine effective therapy high throughput screening human disease human embryonic stem cell in vitro Assay neglect scale up screening stem tool

项目摘要

Although famous for secreting the cerebrospinal fluid (CSF), the choroid plexus has drawn relatively little attention from basic and clinical scientists. Every day, human choroid plexus epithelial cells (CPECs) secrete about two cups of protein-rich CSF, which clears out waste products from and delivers beneficial molecules to every cell in the brain and spinal cord. CPEC defects have been implicated in a large number of major CNS disorders, including hydrocephalus, psychiatric conditions, and neurodegenerative diseases. However, almost all of the literature related to CPEC biology and disease is descriptive due to a lack of CPEC-targeted tools. Importantly, if such tools existed, delivery of compounds to CPECs - in either experimental animals or patients - is straightforward, because CPECs exchange freely with the peripheral circulation. This greatly simplifies drug delivery and strengthens the appeal of CPECs as a new target for CNS studies and disease therapies. Despite this appeal, CPEC-targeted drug screens have not been possible due to difficulties in propagating and deriving them in culture. However, we recently developed a method for generating derived CPECs (dCPECs) from mouse and human embryonic stem cells. This method provides, for the first time, a scalable means to produce dCPECs in the large numbers required for high-throughput screening (HTS). In this R21 proposal, we combine the dCPEC technology inventor with experts in assay development and drug screening to develop a high-throughput dCPEC screening platform. In preliminary studies, we describe dCPEC generation, candidate cell lines to be screened for HTS compatibility, and a sensitive 96-well ELISA for secreted human TTR, an ideal proxy for CPEC secretion and an important molecular target in its own right. The TTR ELISA will be converted into an HTS-compatible AlphaLISA, which will be used for a pilot screen to determine assay readiness. Hits and analogs from the pilot screen will then be screened and validated via "testing funnels" that include new and already-established assays of CPEC function, mechanism, and specificity. This validated high-throughput dCPEC platform will set the stage for full-scale screening proposals to generate first-in-class tool and therapy leads for a brand-new CPEC-based biomedicine.

尽管脉络丛以分泌脑脊液（CSF）而闻名，但它的作用相对较少受到基础科学家和临床科学家的关注。每天，人类脉络丛上皮细胞（CPEC）都会分泌大约两杯富含蛋白质的脑脊液，可清除体内废物并提供有益分子大脑和脊髓中的每个细胞。 CPEC缺陷与大量主要中枢神经系统有关疾病，包括脑积水、精神疾病和神经退行性疾病。然而，几乎由于缺乏针对 CPEC 的工具，所有与 CPEC 生物学和疾病相关的文献都是描述性的。重要的是，如果存在这样的工具，可以将化合物递送至实验动物或患者的 CPEC - 很简单，因为 CPEC 与外围循环自由交换。这大大简化了药物交付并增强了 CPEC 作为中枢神经系统研究和疾病治疗新目标的吸引力。尽管在此呼吁中，由于传播和衍生困难，针对 CPEC 的药物筛选尚未成为可能他们在文化中。然而，我们最近开发了一种从生成 CPEC（dCPEC）的方法小鼠和人类胚胎干细胞。该方法首次提供了一种可扩展的方法来生产高通量筛选 (HTS) 所需的大量 dCPEC。在此 R21 提案中，我们将 dCPEC 技术发明者与检测开发专家结合起来和药物筛选，开发高通量 dCPEC 筛选平台。在初步研究中，我们描述 dCPEC 生成、要筛选 HTS 兼容性的候选细胞系以及敏感的 96 孔用于分泌型人 TTR 的 ELISA，它是 CPEC 分泌的理想代表，也是其重要的分子靶点自己的权利。 TTR ELISA 将转换为兼容 HTS 的 AlphaLISA，用于试点筛选以确定测定准备情况。然后将筛选来自试播屏幕的点击和类似内容通过“测试漏斗”进行验证，其中包括新的和已经建立的 CPEC 功能、机制、和特异性。这个经过验证的高通量 dCPEC 平台将为全面筛选奠定基础为基于 CPEC 的全新生物医学开发一流工具和治疗线索的建议。