Comprehensive Botulinum Characterization via the Bilayer Nanowell Integrated Assay

通过双层纳米井综合测定进行全面的肉毒杆菌表征

• 批准号: 10480376
• 负责人: Eric Ervin
• 金额: $ 40万
• 依托单位: ELECTRONIC BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480376
• 关键词: Acetylcholine; Animals; Anorexia; Anxiety; Area; Binding; Biological Assay; Biological Response Modifier Therapy; Biological Sciences; Bontoxilysin; Botox; Botulinum Toxins; Breathing; Cell Wall; Cell membrane; Cell physiology; Cells; Cessation of life; Cholera; Clinical; Complex; Cosmetics; Cytosol; Development; Devices; Diphtheria; Disease; Dose; Electrodes; Endocytosis; Endpoint Determination; Ensure; Enzymes; Etiology; Evaluation; Face; Fatigue; Fluorescence; Focal Dystonias; Formulation; Future; Gold; Growth; Guidelines; Health; Healthcare; Hour; Human; In Vitro; Individual; Integral Membrane Protein; Intoxication; Intraperitoneal Injections; Ions; Lead; Lethal Dose 50; Light; Lipid Bilayers; Manufacturer Name; Measurement; Measures; Mediating; Membrane; Mental Depression; Methodology; Methods; Modality; Movement Disorders; Mus; Muscle; Muscle Contraction; Muscle Weakness; Neurodegenerative Disorders; Neurologic; Neurons; Noise; Paralysed; Parkinson Disease; Patient Care; Patients; Peptides; Permeability; Pertussis; Phase; Pore Proteins; Presynaptic Terminals; Price; Process; Production; Proteins; Proteolysis; Reproducibility; SNAP receptor; Sampling; Serotyping; Signal Transduction; Silicon Dioxide; Spasm; Standardization; Surface; Sweating; System; Technology; Testing; Tetanus; Therapeutic; Therapeutic Uses; Time; Toxin; Transmembrane Transport; United States National Institutes of Health; Validation; Variant; Vertebrates; Work; antitoxin; aqueous; base; botulinum; cell type; cost; improved; in vivo; ineffective therapies; neurotransmission; novel; novel therapeutics; polypeptide; potency testing; prevent; programs; prototype; receptor; research and development; screening; stability testing; targeted cancer therapy; uptake

Project Summary During this program, Electronic BioSciences, Inc. (EBS) will develop and validate its in-vitro, chip-based, picowell bilayer integrated assay (BP-IA) for characterizing the complete activity of botulinum toxin serotype A (BoNT/A) transmembrane toxin. A transmembrane toxin is a molecule that recognizes/targets a specific cell type via receptor-mediated targeting/endocytosis, forms a pore in the cell membrane, and transports itself or another molecule into the cell to disrupt cellular function, e.g., botulinum neurotoxin, tetanus, diphtheria, shiga, cholera, pertussis, etc. While these toxins are innately hazardous to human health, their inherent cell targeting and enzymatic capabilities can also be harnessed for therapeutic benefit. Today, the emerging therapeutic uses of toxins include but are not limited to the treatment of muscle spasms, wrinkles, excessive sweating, depression, anxiety, anorexia, neurodegenerative disorders (e.g., Parkinson’s disease), and targeted cancer therapy. However, the limitations of current toxin activity assessment methods have constrained the field. There is a need for a low cost, easy-to-use, rapid, highly sensitive, highly reproducible assay that is capable of individually quantifying the separate steps of the intoxication mechanism (i.e., the cell targeting/endocytosis and the intracellular enzymatic activity) to fully understand and utilize toxin functionality. The present standard for toxin activity assessments is the mouse intraperitoneal injection assay, which has numerous limitations, including price, variability, time, lack of sample quantification, and the utilization of live animals, in addition relying on a single endpoint determination that precludes assessment of the toxin’s mechanism. Unknown or poorly understood differences in the potency (or mode of action) of toxin-containing therapeutics can confound clinical dose findings, result in over or under dosing patients, and delay (or prohibit) the development and/or availability of new/novel therapeutics. EBS’ BP-IA technology will be capable of unprecedented toxin characterization in a low cost, easy-to-use, rapid, highly sensitive, highly reproducible, in vitro, chip-based platform. Furthermore, the methodology of the BP-IA is customizable such that the complete activity of any transmembrane toxin could be quantified. The development of the BP-IA under this Phase I program will be accomplished by developing and building an alpha prototype BP-IA device, and demonstrating the capability of the BP-IA device to quantitatively assess the potency of commercial BoNT/A therapeutic toxin formulations. Development of the BP-IA, a technology for which there is no equivalent commercially available and the future gold standard in toxin, biotherapeutics, cell-targeting, uptake/internal activity, and causation mechanism quantification, will directly enable the research and development of BoNT/A-based therapeutics, novel toxin agents/samples, the development of antitoxin agents, the detailed study of toxin and antitoxin mechanisms, the evaluation of the causative effects of experimental variables on each specific intoxication modality, and the assessment of toxin potency in general.

项目摘要 在这项计划中，电子生物科学公司(EBS)将开发和验证其体外、基于芯片的微微管 A型肉毒毒素(BONT/A)完全活性的双分子层结合试验(BP-IA) 跨膜毒素。跨膜毒素是一种识别/靶向特定细胞类型的分子，通过 受体介导的靶向/内吞作用，在细胞膜上形成一个孔，并自行或另一种转运 进入细胞内扰乱细胞功能的分子，例如肉毒杆菌神经毒素、破伤风、白喉、志贺氏菌、霍乱、 百日咳等。虽然这些毒素天生危害人类健康，但它们固有的细胞靶向和 酶的能力也可以被利用来获得治疗效益。今天，新出现的治疗用途 毒素包括但不限于治疗肌肉痉挛、皱纹、多汗、抑郁、 焦虑症、厌食症、神经退行性疾病(如帕金森氏病)和靶向癌症治疗。 然而，目前毒素活性评估方法的局限性制约了该领域的研究。有必要 用于低成本、易于使用、快速、高灵敏度、高重复性的能够单独 量化中毒机制的不同步骤(即细胞靶向/内吞作用和 细胞内酶活性)，以充分了解和利用毒素的功能。现行的毒素标准 活动性评估是小鼠腹膜内注射试验，它有许多局限性，包括 价格、可变性、时间、缺乏样本量化以及活体动物的利用，此外还依赖于 单一终点测定排除了对毒素机制的评估。未知或很差 了解含毒素疗法的效力(或作用模式)的差异可能会令临床感到困惑 剂量发现，导致患者服药过量或不足，并延迟(或禁止)药物的开发和/或供应 新的/新的疗法。EBS的BP-IA技术将能够在 低成本、易用、快速、高灵敏度、高重复性、体外芯片平台。此外， BP-IA的方法学是可定制的，因此任何跨膜毒素的完整活性都可以是 量化的。在此第一阶段计划下，BP-IA的开发将通过开发和 建立了BP-IA装置的阿尔法原型，并展示了BP-IA装置的定量能力 评估商业BONT/A治疗毒素制剂的效力。BP-IA的开发，A 没有同等商业价值的技术和未来毒素的黄金标准， 生物疗法、细胞靶向、摄取/内部活性和致病机制量化，将直接 支持研究和开发基于BONT/A的疗法、新的毒素制剂/样本、 抗毒素药物的发展，对毒素和抗毒素机制的详细研究，对 实验变量对每种特定中毒方式的致病作用，以及毒素的评估 一般说来就是效力。