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 在这个项目中，Electronic BioSciences，Inc. (EBS)将开发和验证其体外，基于芯片， 用于表征肉毒杆菌毒素血清型A（BoNT/A）的完全活性的双层整合测定（BP-IA） 跨膜毒素跨膜毒素是一种分子，其通过介导的免疫反应识别/靶向特定的细胞类型。 受体介导的靶向/内吞作用，在细胞膜上形成孔，并将其自身或另一个 分子进入细胞以破坏细胞功能，例如，肉毒杆菌神经毒素，破伤风，白喉，滋贺，霍乱， 虽然这些毒素天生对人类健康有害，但它们固有的细胞靶向和 酶的能力也可以用于治疗益处。今天，新兴的治疗用途， 毒素包括但不限于治疗肌肉痉挛，皱纹，过度出汗，抑郁， 焦虑、厌食、神经变性疾病（例如，帕金森氏病）和靶向癌症治疗。 然而，目前的毒素活性评估方法的局限性限制了该领域。有必要 对于低成本、易于使用、快速、高灵敏度、高重现性的测定， 量化中毒机制的各个步骤（即，细胞靶向/内吞作用和 细胞内酶活性）以充分理解和利用毒素功能。毒素的现行标准 活性评估是小鼠腹膜内注射测定，其具有许多局限性，包括 价格、可变性、时间、缺乏样本量化以及活动物的利用，此外， 单终点测定排除了毒素机制的评估。未知或较差 已知的含毒素治疗剂的效力（或作用模式）的差异可能混淆临床 剂量发现，导致患者给药过量或剂量不足，并延迟（或禁止）开发和/或可用性 新的/新颖的疗法。EBS的BP-IA技术将能够以前所未有的方式对毒素进行表征， 低成本、易于使用、快速、高灵敏度、高重现性、体外、基于芯片的平台。而且 BP-IA的方法学是可定制的，使得任何跨膜毒素的完整活性都可以被 量化。根据第一阶段计划，BP-IA的开发将通过开发和 构建alpha原型BP-IA设备，并证明BP-IA设备定量 评估商用BoNT/A治疗毒素制剂的效力。BP-IA的开发，a 这项技术目前还没有商业上可获得的同类产品，也是未来毒素的黄金标准， 生物治疗、细胞靶向、摄取/内部活性和因果机制定量，将直接 能够研究和开发基于BoNT/A的治疗剂、新型毒素剂/样品、 抗毒素药物的开发，毒素和抗毒素机制的详细研究， 实验变量对每种特定中毒方式的因果影响，以及毒素的评估 一般的力量。