A Cell-based Assay for Botulinum Neurotoxin Detection and Development

描述（由申请人提供）：肉毒杆菌神经毒素（BoNTs）是人类已知的毒性最大的物质，可导致呼吸衰竭。由于其杀伤力和易于生产，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bont具有致命性，但它在化妆品和制药领域仍有应用，目前fda已批准bont用于治疗眉间纹（皱纹）、颈肌张力障碍、慢性偏头痛、眼睑痉挛、颅神经紊乱和鼻窦炎。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的治疗提供了显著和持久的疼痛减轻。BoNTs对神经元的特异性和长时间的作用使其成为治疗神经和肌肉疾病的主要候选药物，而其他治疗方法都失败了。由于缺乏检测BoNT活性的方法，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天后有多少只小鼠死亡。这个过程很慢，需要特殊的设施，而且产量很低；拮抗剂筛选或开发新的bont衍生疗法既昂贵又耗时。BioSentinel最近开发了工程细胞系，稳定表达活细胞中检测BoNT/ a的报告基因。这些细胞系将成为BoCell" A，一种基于细胞的BoNT/A检测方法。该II期提案的总体目标是商业化和验证BoCell”A检测方法，并开发基于细胞的检测方法来检测BoNT/B和e。因为BoNT具有三种活性——细胞受体结合和摄取、囊泡易位和靶标切割，因此需要基于细胞的检测方法来有效测量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BoNT切割SNARE蛋白（BoNT的靶标）来测量BoNT的活性。BioSentinel将验证BoCell“A”测定的生物学相关性和高通量应用，并开始开发第二代BoCell“A”测定，其灵敏度与小鼠生物测定相匹配。此外，bisentinel将在第一阶段的基础上，鉴定出在活细胞中检测BoNT/B的报告基因，并设计出稳定表达报告基因的细胞系。最后，BoCell“A试验将被设计用于BoNT/E的摄取和检测。BioSentinel建议开发、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量化BoNT制剂，执行质量控制措施，筛选新的基于BoNT的治疗方法，以及筛选BoNT拮抗剂。由于对无动物、高通量分析的压力越来越大，研究人员和工业界非常希望基于细胞的BoNT检测分析，并将为开发新的基于BoNT的治疗方法打开大门。

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.