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 低得令人不安。造成这一不幸局面的潜在因素之一是， 体外分析的过于简单的条件不能反映大脑的精致复杂性。例如，大脑不断地 从我们周围的世界接收信号，并在处理这些信号后控制许多器官的功能。 然而，典型的体外测定通常不并入在药物治疗期间动态激活神经元的任何外部信号。 筛选，而不是新的候选药物的神经系统疾病正在定期评估其影响 自发神经元活动或终点试验。 先进的体外试验已开始纳入细胞刺激能力，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，可能会影响药物筛选 结果是不可预测的例如，光遗传学需要通过表达外源基因来遗传改变神经元。 光敏感离子通道，使神经元适合被光学刺激。除了时间和费用之外， 纯粹为了细胞刺激能力而进行的消耗性遗传修饰在hIPSC中是特别不希望的。 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，不需要基因修饰， 神经元，因此，当基于hIPSC的疾病模型用于药物筛选测定时，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的光刺激（Gramos）技术， 石墨烯材料独特的光电性能的优势，并提供了细胞的非侵入性调制 通过石墨烯-神经元生物界面附近的外部光控制电场来激活。 在这里，我们建议将我们的GramOS技术整合到神经科学药物的药物筛选试验中。 的发现我们将使用2-D和3-D hiPSC为基础的模型，这些模型是从携带突变的患者中产生的，已知这些突变会改变hiPSC的功能。 神经元活动，如缺乏MECP 2基因。对于经典的2-D模型，我们将制作石墨烯基衬底， 以通过专门的细胞培养板的底部实现神经元的光学激活。我们将继续进行 全光学分析的发展，该分析结合了联合收割机的GramOS-使能光学刺激与神经元活动的记录 使用荧光指示剂。为了评估神经元网络的突触连接性，我们将开发图案化的石墨烯 在一些实施方案中，G-底物能够选择性地刺激仅与G-底物介导的神经元的子集。在3D模型中，例如 神经元球体和大脑皮质类器官，我们将评估功能性神经元活动，无论是使用全光学 GramOS使能测定或将GramOS与基于MEA的细胞外电活动记录相结合。格拉莫斯- 将使用影响突触传递和神经元兴奋性的基准化合物来验证启用的测定。 提出的非遗传光学刺激技术有望大大提高转化神经科学 研究和支持发现具有新作用机制的神经药物。

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.