Developing Choroid Plexus-Based Tools and Drug Screens

虽然以分泌脑脊髓液而闻名，但脉络丛相对较少 基础和临床科学家的关注。每天，人类脉络丛上皮细胞（CPECs）分泌 大约两杯富含蛋白质的CSF，它可以清除废物，并提供有益的分子， 大脑和脊髓的每一个细胞CPEC缺陷与大量主要CNS疾病有关。 疾病，包括脑积水、精神疾病和神经退行性疾病。但几乎 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，将化合物递送到CPEC-在实验动物或患者中 - 是直接的，因为CPEC与外周循环自由交换。这大大简化了药物 这将有助于提高药物递送的效率，并增强CPEC作为CNS研究和疾病治疗新靶点的吸引力。尽管 这种呼吁，CPEC靶向药物筛选由于传播和衍生困难而不可能 他们在文化。然而，我们最近开发了一种方法，用于从 小鼠和人类胚胎干细胞。这种方法首次提供了一种可扩展的生产方法， 高通量筛选（HTS）所需的大量dCPEC。 在该R21提案中，我们将dCPEC技术发明者与检测开发专家联合收割机 和药物筛选，建立高通量dCPEC筛选平台。在初步研究中，我们 描述了dCPEC的产生、待筛选HTS相容性的候选细胞系以及敏感的96孔板。 分泌型人TTR的ELISA，CPEC分泌的理想替代物和其治疗中的重要分子靶标 自己的权利。TTR ELISA将转换为与HTS兼容的AlphaLISA，用于试点 屏幕，以确定检测试剂盒准备就绪。然后将筛选来自试点屏幕的命中和类似物， 通过“测试漏斗”进行验证，包括新的和已经建立的CPEC功能，机制， 和特异性。这种经过验证的高通量dCPEC平台将为全面筛选奠定基础 为全新的基于CPEC的生物医学产生一流的工具和治疗线索的建议。

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.