High-Throughput NMJ Assay for Botox Potency Screening

项目总结 使用FDA和NCATS的组织芯片计划进行的肉毒杆菌毒素效力分析强调了 能够可靠地评估肉毒杆菌毒素(BOT)效力的新型工程系统。人类诱因 多能干细胞(IPSC)来源的运动神经元和肌肉细胞以前一直维持在共同的 培养并显示形成功能性突触接触，代表体内的神经肌肉连接(NMJ)。 然而，使用符合以下条件体外平台有效地模拟对BOT的NMJ功能反应的能力 高通量筛选尚未实现，因为生成成熟的和功能上的 在文化方面有能力的NMJ。一种可推广NMJ的高通量平台的开发 多重检测的发展将对BOT的生产产生实质性的积极影响，以及 神经元和NMJ的高级治疗发展、药物疗效/毒性筛选和机制研究 神经退行性疾病的病理生理学。根据初步工作和公布的数据，我们假设 集电极刺激神经元放电和磁体测量于一体的培养平台 工程化肌肉收缩将使实时分析NMJ的发展和功能在基线和 作为对机器人暴露的回应。使用IPSC衍生的运动神经元和肌肉细胞，我们将建立有组织的 在与我们公司现有的肌肉收缩试验兼容的培养皿中进行共培养， MantarrayTM.分隔细胞的壁上的微通道将使神经元突起生长到 肌肉隔间，促进突触接触。与BOT参考批次的测试，就其能力而言 来改变我们细胞群体之间的突触通讯，然后将被用来证明 该模型用于体外检测NMJ功能和BOT潜能(阶段1)。一旦确认，我们的NMJ检测将是 进一步评估以确定其在模拟机器人响应时的准确性、精确度、特异性和重复性 在人体组织中(阶段2)。BOT的参考批次将在广泛的供体细胞来源上进行测试 并与一系列对NMJ功能具有已知和可预测影响的参考化合物进行比较。这个 这项工作的中心假设是，工程化骨骼肌和运动之间NMJ功能的差异 用不同剂量的BOT处理的神经元共培养将使表型分层 成功地用于绘制剂量响应曲线，可与小鼠致死率生物测定的输出数据相媲美 目前机器人效力筛选的黄金标准)。这项研究的成功完成将提供一个新的 用于模拟人类周围神经疾病的原型基于人的平台以及一个有价值的临床前 评估新疗法的筛选工具。消耗板将与库里现有的 硬件/软件包等可在验证后快速分发给客户。

PROJECT SUMMARY The Botulinum Toxin Potency Assay using Tissue Chips program from FDA and NCATS highlights the need for novel engineered systems capable of reliably evaluating botulinum toxin (BoT) potency. Human induced pluripotent stem cell (iPSC)-derived motor neurons and muscle cells have been previously maintained in co- culture and shown to form functional synaptic contacts representing in vivo neuromuscular junctions (NMJs). However, the ability to effectively model NMJ functional responses to BoT using in vitro platforms amenable to high-throughput screening has yet to be achieved due to the complexity of generating mature and functionally competent NMJs in culture. The development of a high-throughput platform capable of promoting NMJ development across a multiplexed assay will have a substantial positive impact on BoT production, as well as advanced therapy development, drug efficacy/toxicity screening, and mechanistic studies of neuronal and NMJ pathophysiology in neurodegenerative diseases. Based on preliminary work and published data, we posit that a culture platform integrating electrode-based stimulation of neuronal firing and magnet-based measurement of engineered muscle contraction will enable real-time analysis of NMJ development and function at baseline and in response to BoT exposure. Using iPSC-derived motor neurons and muscle cells, we will establish organized co-cultures within a culture plate that is compatible with our company’s existing muscle contractility assay, MantarrayTM. Microchannels in the walls separating the cells will allow neuronal processes to grow into the muscle compartment, facilitating synaptic contact. Tests with reference batches of BoT, in terms of their ability to alter synaptic communication between our cell populations, will then be used to demonstrate the suitability of this model for assaying NMJ function and BoT potency in vitro (Phase 1). Once validated, our NMJ assay will be further evaluated to determine its accuracy, precision, specificity, and reproducibility in modeling BoT responses in human tissues (Phase 2). Reference batches of BoT will be tested across a wide range of donor cell sources and in comparison to an array of reference compounds with known and predictable effects on NMJ function. The central hypothesis of this work is that differences in NMJ function between engineered skeletal muscle and motor neuron co-cultures treated with different doses of BoT will enable stratification of phenotypes that can be successfully used to plot dose response curves comparable to output data from mouse lethality bioassays (the current gold standard for BoT potency screening). Successful completion of this study will provide a new prototype human-based platform for modeling human peripheral neuropathies as well as a valuable preclinical screening tool for assessing novel therapeutics. The consumable plate will be integrated with Curi’s existing hardware/software packages and so can be quickly disseminated to customers upon validation.