High-Throughput NMJ Assay for Botox Potency Screening

用于 Botox 效力筛选的高通量 NMJ 检测

• 批准号: 10745380
• 负责人: Nicholas Andrew Geisse
• 金额: $ 27.58万
• 依托单位: CURI BIO INC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10745380
• 关键词: Acetylcholine; Adoption; Animals; Area; Assessment tool; Axon; Biological Assay; Biomedical Engineering; Body Regions; Botox; Botulinum Toxins; Cell Culture Techniques; Cells; Coculture Techniques; Communication; Computer software; Cosmetics; Data; Development; Devices; Disease model; Dose; Drug Screening; Electrodes; Engineering; Ethics; Exposure to; Frequencies; Functional disorder; Human; In Vitro; Induced pluripotent stem cell derived neurons; Interview; Marketing; Measurement; Measures; Medical; Methods; Modeling; Modernization; Motor Neurons; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscle Fibers; Muscle function; National Center for Advancing Translational Sciences; Nerve; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Neurotoxins; Output; Patients; Peripheral Nervous System Diseases; Pharmaceutical Preparations; Phase; Phenotype; Physiology; Population; Process; Production; Protocols documentation; Publishing; Reproducibility; Safety; Sales; Screening procedure; Skeletal Muscle; Source; Specificity; Stratification; Synapses; Synaptic Cleft; System; Technology; Testing; Time; Tissue Microarray; Toxic effect; Validation; Voice; Work; Writing; animal facility; cell type; commercialization; culture plates; design; driving force; drug development; drug efficacy; high throughput screening; human model; human tissue; in vitro Model; in vivo; induced pluripotent stem cell; instrumentation; manufacture; multiplex assay; muscle engineering; nerve supply; neuromuscular; neuronal cell body; next generation; novel; novel therapeutics; organ on a chip; pre-clinical; preclinical study; predictive modeling; prevent; programs; prototype; response; screening; skills; stem cell model; therapy development

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.

项目摘要 使用FDA和NCAT的组织芯片程序的肉毒杆菌毒素效力测定强调了需要 能够可靠评估肉毒杆菌毒素（BOT）效力的新型工程系统。人类诱导 多能干细胞（IPSC）衍生的运动神经元和肌肉细胞先前一直保持在共同体中 培养并证明形成代表体内神经肌肉连接（NMJS）的功能突触接触。 但是，能够使用可使用的体外平台有效地对机器人建模功能响应的能力 由于产生成熟和功能的复杂性，尚未实现高通量筛选 在文化中有能力的NMJ。能够促进NMJ的高通量平台的开发 多路复用测定的开发将对机器人生产产生重大积极影响，以及 晚期治疗开发，药物效率/毒性筛查以及神经元和NMJ的机理研究 神经退行性疾病中的病理生理。基于初步工作和发布的数据，我们海报 培养平台集成基于电极的神经元放电和基于磁铁的测量的模拟 工程肌肉收缩将实时对基线和基线的NMJ开发和功能进行实时分析 响应机器人暴露。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 与我们公司现有的肌肉收缩力测定法兼容的文化板中的共同培养 Mantarraytm。分隔细胞的墙壁中的微通道将使神经元过程生长到 肌肉室，支持突触接触。用引用批次进行机器人的测试，就其能力而言 为了改变我们的细胞群体之间的突触通信，然后将用于证明 该模型用于在体外测定NMJ功能和机器人效力（第1阶段）。一旦验证，我们的NMJ分析将是 进一步评估以确定其在建模机器人响应时的准确性，精度，特异性和可重复性 在人体组织中（第2阶段）。机器人的参考批次将在各种供体细胞源中进行测试 与具有已知且可预测的NMJ功能影响的参考化合物相比。这 这项工作的中心假设是，工程骨骼肌和运动之间的NMJ功能差异 用不同剂量的机器人治疗的神经元共培养将使可以分层的表型分层 成功用于绘制剂量响应曲线与小鼠致死性生物测定的输出数据相当的剂量响应曲线（ 机器人效力筛选的当前金标准）。这项研究的成功完成将提供新的 基于人类的原型平台，用于建模人类外周神经病以及有价值的临床前 评估新疗法的筛查工具。可消耗板将与Curi的现有 硬件/软件包，因此可以在验证后快速将其传播给客户。