Non-genetic optical stimulation platform for the next-generation neuroscience drug discovery assays

摘要 尽管基础神经科学研究取得了巨大进展，但神经科学药物发现的成功率 一直低得令人失望。造成这一不幸局面的潜在因素之一是，公平地 体外分析的简单化条件并不能反映大脑的精致复杂性。例如，大脑不断地 接收来自我们周围世界的信号，并在处理这些信号后控制许多器官的功能。 然而，典型的体外试验通常不包含任何在药物过程中动态激活神经元的外部信号。 筛选，取而代之的是神经疾病的新药候选药物，正在根据它们的效果进行常规评估 无论是自发的神经元活动还是终点分析。 先进的体外分析已经开始包含细胞刺激能力，以解决这一关键缺陷。 然而，现有的细胞刺激技术存在一些固有的缺陷，可能会影响药物的筛选。 以一种不可预测的方式产生结果。例如，光遗传学要求通过表达外源基因来改变神经元。 光敏离子通道，使神经元适合光刺激。除了时间和费用外， 在HiPSC中，纯粹为了细胞刺激能力而进行消耗、基因修改尤其不可取。 基于神经疾病模型，因为它们可能会改变模型本身。 我们最近开发了一种开创性的光学刺激平台，它不需要对 因此，当基于HiPSC的疾病模型用于药物筛选分析时，可以提供巨大的优势。 我们的平台基于我们突破性的石墨烯介导光刺激(Gramos)技术，该技术采用 石墨烯材料独特的光电性能优势，并提供对电池的非侵入性调制 通过石墨烯-神经元生物界面附近的外部光控电场进行活动。 在这里，我们建议将我们的Gramos技术集成到神经科学药物的药物筛选分析中 发现号。我们将使用基于2-D和3-D HiPSC的模型，这些模型来自携带突变的患者，已知的突变会改变 神经元活性如MECP2基因缺失等。对于经典的2-D模型，我们将制造基于石墨烯的衬底 通过专门的细胞培养板底部实现神经元的光学激活。我们将继续进行 将Gramos使能光刺激与神经元活动记录相结合的全光学分析的发展 使用荧光指示器。为了评估神经元网络的突触连接，我们将开发图案化的石墨烯 底物只能选择性地刺激与G-底物连接的神经元的子集。在3D模型中，例如 神经元球体和大脑皮质器体，我们将使用全光技术来评估功能神经元的活动 启用Gramos的分析或将Gramos与基于MEA的细胞外电活动记录相结合。格拉莫斯- 启用的分析将使用影响突触传递和神经元兴奋性的基准化合物进行验证。 提出的非遗传光学刺激技术有望极大地加强翻译神经科学 研究和支持具有新作用机制的神经药物的发现。

ABSTRACT Despite tremendous advances in fundamental neuroscience research, the success rate of neuroscience drug discovery has been disappointingly low. Among potential factors contributing to this unfortunate situation is the reality that fairly simplistic conditions of in vitro assays do not reflect the exquisite complexity of the brain. For example, the brain constantly receives signals from the world surrounding us, and controls the functions of many organs after processing of these signals. Yet, typical in vitro assays often do not incorporate any external signals dynamically activating neurons during drug screening, and instead novel drug candidates for neurological disorders are being routinely evaluated based on their effects on either spontaneous neuronal activity or in end-point assays. Advanced in vitro assays have started incorporating cell stimulation capabilities to address this critical shortcoming. However, existing cell stimulation technologies have some inherent shortcomings that might affect the drug screening results in an unpredictable way. For example, optogenetics requires to genetically change neurons by expressing exogenous light-sensitive ion channels to make neurons fit to be optically stimulated. In addition to being time- and expense- consuming, genetic modifications purely for the sake of the cell stimulation capability are especially not desirable in hIPSC- based neurological disease models, because they might change the model itself. We have recently developed a pioneering optical stimulation platform that do not require genetic modifications of neurons, and, thus, can provide enormous advantages when hIPSC-based disease models are used in drug screening assays. Our platform is based on our breakthrough graphene-mediated optical stimulation (GraMOS) technology that takes advantage of unique optoelectronic properties of graphene materials and provides non-invasive modulation of the cell activity via an external light-controlled electrical field near the graphene-neuron biointerface. Here we are proposing to integrate our GraMOS technology into drug screening assays for neuroscience drug discovery. We will use 2-D and 3-D hiPSC-based models generated from patients carrying mutations, known to alter the neuronal activity such as the lack of MECP2 gene. For classical 2-D models, we will fabricate graphene-based substrates to enable optical activation of neurons via the bottoms of specialized cell culture plates. We will proceed with the development of all-optical assays that combine GraMOS-enable optical stimulation with recordings of the neuronal activity using fluorescent indicators. To evaluate the synaptic connectivity of neuronal networks, we will develop patterned graphene substrates to be able to selective stimulate only a subset of neurons interfacing with G-substrates. In 3-D models, such as neuronal spheroids and brain cortical organoids, we will evaluate the functional neuronal activity by either using all-optical GraMOS-enabled assays or combining GraMOS with MEA-based recording of extracellular electric activity. GraMOS- enabled assays will be validated using benchmark compounds affecting synaptic transmission and neuronal excitability. The proposed non-genetic optical stimulation technology is expected to dramatically enhance translational neuroscience studies and support the discovery of neurological drugs with novel mechanism of actions.