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 FDA和NCATS的使用组织芯片的肉毒杆菌毒素效价测定项目强调了以下需求： 能够可靠地评估肉毒杆菌毒素（BoT）效力的新型工程化系统。人诱导 多能干细胞（iPSC）衍生的运动神经元和肌肉细胞先前已经维持在共- 培养并显示形成代表体内神经肌肉接头（NMJ）的功能性突触接触。 然而，使用体外平台有效地模拟NMJ对BoT的功能性应答的能力是可行的， 由于产生成熟的和功能性的 有能力的NMJ。能够促进NMJ的高通量平台的开发 跨多重测定的开发将对BoT生产产生实质性的积极影响， 先进的治疗开发，药物疗效/毒性筛选，以及神经元和NMJ的机制研究 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，我们认为， 培养平台，其整合了神经元放电的基于电极的刺激和神经元放电的基于磁体的测量， 工程化肌肉收缩将能够实时分析基线时的NMJ发育和功能， 以应对BoT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 在与我们公司现有的肌肉收缩性测定相容的培养板内共培养， MantarrayTM.细胞壁中的微通道将允许神经元突起生长到细胞中。 肌肉区室，促进突触接触。BoT参比批次的检测，就其能力而言 来改变我们细胞群之间的突触通讯，然后将被用来证明 该模型用于体外测定NMJ功能和BoT效力（阶段1）。一旦验证，我们的NMJ检测将是 进一步评价，以确定其在模拟BoT反应中的准确性、精密度、特异性和再现性 人体组织（第2阶段）。BoT的参考批次将在广泛的供体细胞来源中进行检测 并与一系列对NMJ功能具有已知和可预测作用的参比化合物进行比较。的 这项工作的中心假设是，工程化骨骼肌和运动肌之间NMJ功能的差异， 用不同剂量的BoT处理的神经元共培养物将使得能够对表型进行分层， 成功地用于绘制与小鼠致死率生物测定的输出数据相当的剂量反应曲线（ 目前BoT效价筛选的金标准）。这项研究的成功完成将提供一个新的 原型基于人类的平台，用于模拟人类周围神经病变，以及有价值的临床前 用于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，因此可以在验证时快速地分发给客户。