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: 8299024
• 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/8299024
• 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; Screening procedure; 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; 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（由申请方提供）：肉毒神经毒素（BoNT）是已知对人类毒性最大的物质，可导致呼吸衰竭。由于其致命性和易于生产，国防部（DoD）将BONT指定为“A”类生物恐怖主义制剂。开发有效的暴露后拮抗剂是国防部的首要任务之一。尽管它们具有致命性，但BoNT具有化妆品和药物应用，并且目前FDA批准用于治疗眉间纹（皱纹）、颈部肌张力障碍、慢性偏头痛、眼睑痉挛、颅神经VII障碍和宇宙病。BoNT通过沉默引起肌肉收缩的神经元来缓解肌肉张力。对于许多疾病，基于BoNT的治疗提供了显着和持久的疼痛减轻。BoNTs对神经元的特异性和长时间的作用使其成为治疗其他治疗失败的神经和肌肉疾病的主要候选者。开发新的基于BoNT的疗法受到缺乏测量BoNT活性的测定的阻碍。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天后有多少小鼠死亡。这个过程是缓慢的，需要特殊的设施，是非常低的吞吐量;拮抗剂筛选或开发新的BoNT衍生的治疗是非常昂贵和耗时的。BioSentinel最近开发了稳定表达报告基因的工程细胞系，用于检测活细胞中的BoNT/A。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。本II期提案的总体目标是商业化和验证BoCell”A检测试剂盒，并开发用于检测BoNT/B和E的基于细胞的检测试剂盒。由于BoNT具有三种活性-细胞受体结合和摄取、囊泡易位和靶切割-需要基于细胞的测定来有效地测量BoNT活性。BioSentinel开发了一种基于细胞的检测方法，该方法通过使用荧光发射来监测BoNT对SNARE蛋白（BoNT的靶标）的切割，从而测量BoNT的活性。BioSentinel将验证BoCell”A检测的生物相关性和高通量应用，并开始开发第二代BoCell”A检测，其灵敏度与小鼠生物检测相匹配。此外，BioSentinel将建立在其第一阶段的成就，确定报告，检测BoNT/B在活细胞和工程细胞系，稳定表达的报告。最后，BoCell A测定将被设计用于BoNT/E的摄取和检测。BioSentinel提出开发，验证和商业化检测方法，使研究人员，临床医生和制药公司能够快速定量BoNT制剂，执行质量控制措施，筛选基于BoNT的新疗法，并筛选BoNT拮抗剂。由于无动物、高通量测定的压力增加，研究人员和工业界非常需要基于细胞的BoNT检测测定，这将为开发新的基于BoNT的疗法打开大门。