A Cell-based Assay for Botulinum Neurotoxin Detection and Development

肉毒杆菌神经毒素检测和开发的细胞测定法

• 批准号: 8517560
• 负责人: WARD C TUCKER
• 金额: $ 34.56万
• 依托单位: BIOSENTINEL, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517560
• 关键词: Adverse event; Animal Model; Animals; Bacteria; Bacterial Toxins; Biological; Biological Assay; Bioterrorism; Blepharospasm; Bontoxilysin; Botulinum Toxin Type A; Categories; Cell Line; Cell model; Cells; Cervical Dystonia; Cessation of life; Chronic; Clinical; Clostridium; Cosmetics; Department of Defense; Detection; Development; Disease; Drug Industry; Engineering; Ensure; Evaluation; FDA approved; Facial Nerve Diseases; Failure; Fluorescence; Fluorescence Resonance Energy Transfer; Generations; Goals; Government; Half-Life; Human; Industry; Infection; Intoxication; Lead; Licensing; Life; Marketing; Measures; Methods; Migraine; Monitor; Mus; Muscle Contraction; Muscle Tension; Myalgia; Myopathy; Nervous system structure; Neurons; Pain; Performance; Persons; Pharmacologic Substance; Phase; Poison; Preparation; Process; Production; Proteins; Quality Control; Reporter; Research; Research Infrastructure; Research Personnel; Respiratory Failure; Serotyping; Site; Specificity; Stem cells; Therapeutic; Time; Toxin; Vesicle; Work; base; biodefense; botulinum toxin type B; botulinum toxin type E; cellular engineering; conventional therapy; high throughput screening; meetings; nervous system disorder; neurotoxin receptor; neurotransmitter release; novel; overexpression; pressure; receptor binding; scale up; screening; target SNARE proteins; uptake

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans, causing respiratory failure. Because of their lethality and ease of production, the Department of Defense (DoD) designates BoNTs as a category "A" bioterrorism agent. Developing effective, post-exposure antagonists to BoNT is among DoD's top priorities. Despite their lethality, BoNTs have cosmetic and pharmaceutical applications and are currently FDA-approved for treating glabellar lines (wrinkles), cervical dystonia, chronic migraines, blepharospasm, cranial nerve VII disorders, and cosmoses. BoNTs provide relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long lasting pain reduction. BoNTs' exquisite specificity for neurons and long time of action make it a lead candidate for treating neurological and muscle disorders where other treatments have failed. Developing new BoNT-based therapies is hindered by the lack of assays to measure BoNT activity. Currently, the only widely accepted method involves injecting BoNT into mice and counting how many mice die after 1+ days. This process is slow, requires special facilities, and is very low-throughput; antagonist screening or developing new BoNT-derived therapeutics is prohibitively expensive and time-consuming. BioSentinel recently developed engineered cell lines that stably express a reporter for the detection of BoNT/A in living cells. These cell lines will become BoCell" A, a cell-based assay for the detection of BoNT/A. The overall objectives of this Phase II proposal are to commercialize and validate the BoCell" A assay and develop cell-based assays for detecting BoNT/B and E. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-a cell-based assay is required to effectively measure BoNT activity. BioSentinel developed a cell-based assay that measures BoNT activity by using fluorescence emission to monitor BoNT cleavage of SNARE proteins, the target of BoNT. BioSentinel will validate the BoCell" A assay for biological relevance and high-throughput applications, and begin working to develop a second-generation BoCell" A assay with sensitivity that matches that of the mouse bioassay. In addition, BioSentinel will build upon its Phase I accomplishment of identifying reporters that detect BoNT/B in living cells and engineer cell lines that stably express the reporters. Finally, the BoCell" A assay will be engineered for the uptake and detection of BoNT/E. BioSentinel proposes to develop, validate, and commercialize assays that will enable researchers, clinicians, and pharmaceutical companies to rapidly quantify BoNT preparations, perform quality control measures, screen for new BoNT-based therapies, and screen for BoNT antagonists. Because of increased pressure for animal-free, high-throughput assays, a cell-based BoNT detection assay is greatly desired by researchers and industry, and would open a gateway to developing new BoNT-based therapies.

描述（由申请人提供）：肉毒杆菌神经毒素（BoNT）是人类已知的毒性最强的物质，会导致呼吸衰竭。由于 BoNT 的杀伤力和易于生产，美国国防部 (DoD) 将 BoNT 指定为“A”类生物恐怖主义制剂。开发有效的 BoNT 暴露后拮抗剂是国防部的首要任务之一。尽管具有致命性，BoNT 仍具有化妆品和药物应用，目前 FDA 批准用于治疗眉间纹（皱纹）、颈肌张力障碍、慢性偏头痛、眼睑痉挛、第七脑神经疾病和宇宙。 BoNT 通过抑制引起肌肉收缩的神经元来缓解肌肉紧张。对于许多疾病，基于 BoNT 的治疗可显着且持久地减轻疼痛。 BoNT 对神经元的精确特异性和长作用时间使其成为治疗其他治疗方法失败的神经和肌肉疾病的主要候选药物。由于缺乏测量 BoNT 活性的测定方法，开发新的基于 BoNT 的疗法受到阻碍。目前，唯一被广泛接受的方法是将 BoNT 注射到小鼠体内并计算 1 天以上小鼠死亡的数量。这个过程很慢，需要特殊的设施，而且吞吐量非常低；拮抗剂筛选或开发新的 BoNT 衍生疗法非常昂贵且耗时。 BioSentinel 最近开发了工程细胞系，可稳定表达用于检测活细胞中 BoNT/A 的报告基因。这些细胞系将成为BoCell" A，一种用于检测BoNT/A的基于细胞的测定法。该II期提案的总体目标是商业化和验证BoCell" A测定法，并开发用于检测BoNT/B和E的基于细胞的测定法。由于BoNT具有三种活性——细胞受体结合和摄取、囊泡易位和靶标裂解——需要基于细胞的测定法来有效测量BoNT活性。 BioSentinel 开发了一种基于细胞的检测方法，通过使用荧光发射来监测 SNARE 蛋白（BoNT 的靶标）的 BoNT 裂解来测量 BoNT 活性。 BioSentinel 将验证 BoCell" A 测定的生物相关性和高通量应用，并开始致力于开发第二代 BoCell" A 测定，其灵敏度与小鼠生物测定的灵敏度相匹配。此外，BioSentinel 将以其第一阶段的成就为基础，即鉴定可检测活细胞中 BoNT/B 的报告基因，并设计稳定表达报告基因的细胞系。最后，BoCell“ A 检测将被设计用于 BoNT/E 的摄取和检测。BioSentinel 提议开发、验证和商业化检测，使研究人员、临床医生和制药公司能够快速量化 BoNT 制剂、执行质量控制措施、筛选新的基于 BoNT 的疗法以及筛选 BoNT 拮抗剂。由于无动物、高通量检测的压力不断增加，基于细胞的 BoNT 检测方法深受研究人员和业界的欢迎，并将为开发新的基于 BoNT 的疗法打开大门。