An AQP4-focused, HTS-compatible, BBB-on-a-chip model

专注于 AQP4、HTS 兼容、BBB 片上模型

• 批准号: 2711834
• 金额: --
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2711834
• 关键词: AQP4; focused; HTS; compatible; BBB

"Background: Aquaporin-4 (AQP4) is the main water channel protein in the brain, where it is expressed in astrocytes and enriched at the blood-brain barrier, and allows water to move both between the blood and the brain tissue and through perivascular spaces as part of the recently-described glymphatic system. Following stroke or head injury, water homeostasis is disrupted, which can lead to influx of water into the brain. This excess water causes the brain to swell, increasing intracranial pressure, which can be fatal or lead to long-term disability. AQP4 is therefore established as a drug target for cerebral oedema following stroke and head injury. Despite this, little progress has been made on development of direct AQP4 inhibitors. A recent discovery by my supervisor, Philip Kitchen, has shown that AQP4 can rapidly relocalise from intracellular vesicles to the plasma membrane, and that targeting this relocalisation is a viable therapeutic strategy to prevent or minimise brain and spinal cord oedema in rodent models of CNS injury (Kitchen et al., 2020). However, the drugs used in this study have clear potential for side-effects if used in patients. Further exploitation of this discovery for patient benefit will therefore require a screening campaign to discover novel AQP4 trafficking inhibitors.2-dimensional (2D) astrocyte monocultures have been used to make some progress in understanding the molecular biology of AQP4. However, a key weakness is that monocultured astrocytes are not polarised in the same way as astrocytes in vivo, do not develop endfeet, and AQP4 is not localised to specific vasculature-facing membrane sub-domains. Therefore, an intermediate between simple 2D astrocyte monocultures and physiologically relevant in vivo experiments, using human cells in a high-throughput screening (HTS)-compatible system, would be an ideal platform for AQP4 drug discovery. Such an in vitro system does not currently exist for the study of AQP4. The goal of this project is to develop this system. Research Plan/Methods: We will develop a BBB model using the HTS- compatible microfluidic organ-on-a-chip platform developed by Mimetas BV (Wevers et al, 2018), who are collaborators on my BBSRC Discovery fellowship. Unlike most organ-on-a-chip platforms, the Mimetas system is compatible with existing high-throughput imaging, microplate and robotics technologies, making it ideal for screening projects. In addition, it incorporates a simple, gravity-driven perfusion system, which is crucial for development of full endothelial barrier function. The Mimetas system's "phaseguide" technology means support membranes are not required and that adjacent microfluidic channels allow direct cell-cell contact (here between astrocytes, endothelial cells and pericytes).Cell seeding densities, extracellular matrix composition, and media compositions will be optimised for formation of astrocyte endfeet and endfoot localisation of AQP4. This will be measured in chemically fixed co-cultures using primary astrocytes by immunofluorescence, or by live-cell imaging using iPSC-derived astrocytes which have been edited using CRISPR/Cas9 to have a 3' (C-terminal) eGFP tag on one copy of the AQP4 gene. These cells have already been produced and partially characterised with support from the Joint Research Group Fund. Full characterisation will form part of this project.Expected outcomes: Several publications describing our AQP4-eGFP iPSC astrocytes, and our optimised BBB-on-a-chip model. More importantly, we will have a model to use for screening for novel inhibitors of AQP4 trafficking. My supervisor is a founding shareholder of Estuar Pharmaceuticals which has received significant VC investment to fund several screening projects. There is therefore a clear path to exploitation for any discoveries made during this studentship.ReferencesWevers et al., Fluids Barriers CNS. 2018; 15: 23.Kitchen et al., Cell. 2020; 181(4): 784-799.e19."

“背景：Aquaporin-4 (AQP4) 是大脑中主要的水通道蛋白，在星形胶质细胞中表达并在血脑屏障处富集，并允许水在血液和脑组织之间以及通过血管周围移动作为最近描述的类淋巴系统一部分的空间，在中风或头部受伤后，水的稳态会被破坏，这可能会导致水流入大脑，从而导致大脑肿胀，从而增加大脑的压力。因此，AQP4 可能致命或导致长期残疾，因此被确定为治疗中风和头部损伤后脑水肿的药物靶点，但最近发现直接 AQP4 抑制剂的开发进展甚微。我的导师 Philip Kitchen 的研究表明，AQP4 可以快速从细胞内囊泡重新定位到质膜，并且针对这种重新定位是预防或最小化大脑和脊髓损伤的可行治疗策略中枢神经系统损伤啮齿动物模型中的水肿（Kitchen et al., 2020）。然而，本研究中使用的药物如果用于患者，显然有可能产生副作用。因此，为了患者利益而进一步利用这一发现将需要开展筛选活动，以发现新型 AQP4 运输抑制剂。二维 (2D) 星形胶质细胞单一培养物已被用于在了解 AQP4 分子生物学方面取得一些进展。然而，一个关键的弱点是单一培养的星形胶质细胞的极化方式与体内星形胶质细胞不同，不发育终足，并且 AQP4 不定位于特定的面向血管的膜子域。因此，介于简单 2D 星形胶质细胞单一培养和生理相关体内实验之间的中间体，在高通量筛选 (HTS) 兼容系统中使用人体细胞，将是 AQP4 药物发现的理想平台。目前尚不存在用于 AQP4 研究的这样的体外系统。该项目的目标是开发该系统。研究计划/方法：我们将使用 Mimetas BV（Wevers 等人，2018）开发的 HTS 兼容微流控片上器官平台开发 BBB 模型，他们是我的 BBSRC 发现奖学金的合作者。与大多数器官芯片平台不同，Mimetas 系统与现有的高通量成像、微孔板和机器人技术兼容，使其成为筛选项目的理想选择。此外，它还包含一个简单的重力驱动灌注系统，这对于完整内皮屏障功能的发展至关重要。 Mimetas 系统的“相导”技术意味着不需要支撑膜，并且相邻的微流体通道允许直接细胞与细胞接触（此处为星形胶质细胞、内皮细胞和周细胞之间）。细胞接种密度、细胞外基质成分和培养基成分将针对星形胶质细胞终足的形成和 AQP4 的终足定位。这将在使用原代星形胶质细胞的化学固定共培养物中通过免疫荧光进行测量，或通过使用 iPSC 衍生的星形胶质细胞进行活细胞成像来测量，这些星形胶质细胞已使用 CRISPR/Cas9 进行编辑，在一个副本上具有 3'（C 端）eGFP 标签AQP4 基因。在联合研究小组基金的支持下，这些细胞已经生产出来并进行了部分表征。完整的表征将构成该项目的一部分。预期结果：多篇出版物描述了我们的 AQP4-eGFP iPSC 星形胶质细胞以及我们优化的 BBB 芯片模型。更重要的是，我们将有一个模型用于筛选 AQP4 运输的新型抑制剂。我的主管是 Estuar Pharmaceuticals 的创始股东，该公司已获得大量风险投资来资助多个筛选项目。因此，在学生期间取得的任何发现都有一条明确的利用途径。参考文献Wevers 等人，流体屏障 CNS。 2018； 15: 23.Kitchen 等人，Cell。 2020； 181(4)：784-799.e19。”