A Cell-based Assay for Botulinum Neurotoxin Detection and Development

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)是人类已知的最有毒的物质，会导致呼吸衰竭。由于它们的杀伤力和生产简便性，国防部(DoD)将BoNTs指定为“A”类生物恐怖主义毒剂。开发有效的、暴露后的BONT拮抗剂是国防部的首要任务之一。尽管BoNTs具有致命性，但它具有美容和制药用途，目前已获FDA批准用于治疗眉间皱纹(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。BoNTs通过沉默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缓解。BoNTs对神经元具有精致的特异性和较长的作用时间，使其成为治疗其他治疗方法失败的神经和肌肉疾病的首选方案。由于缺乏衡量BONT活性的分析方法，基于BONT的新疗法的开发受到阻碍。目前，唯一被广泛接受的方法是向小鼠体内注射BONT，并计算1+天后有多少小鼠死亡。这个过程很慢，需要特殊的设备，而且产量很低；拮抗剂筛选或开发新的BONT衍生疗法昂贵得令人望而却步，而且耗时。BioSentinel最近开发了稳定表达报告基因的工程化细胞系，用于在活细胞中检测BONT/A。这些细胞系将成为BoCell“A，这是一种基于细胞的检测BONT/A的方法。这个第二阶段提案的总体目标是将BoCell”A方法商业化并验证其有效性，并开发基于细胞的检测BONT/B和E的方法。由于BONT有三种活动--细胞受体结合和摄取、囊泡转位和靶向切割--因此需要一种基于细胞的分析来有效地测量BONT的活性。BioSentinel开发了一种基于细胞的分析方法，通过使用荧光发射来监测BONT靶标SNARE蛋白的BONT切割来测量BONT的活性。BioSentinel将验证BoCell“一种生物相关性和高通量应用的检测方法，并开始开发第二代BoCell”一种灵敏度与小鼠生物检测相匹配的检测方法。此外，BioSentinel将在其第一阶段成果的基础上，识别在活细胞中检测到BONT/B的记者，并设计稳定表达这些记者的细胞系。最后，BoCell“A分析将被设计用于吸收和检测BONT/E。BioSentinel建议开发、验证和商业化分析，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、筛选基于BONT的新疗法和筛选BONT拮抗剂。由于无动物、高通量分析的压力增加，基于细胞的BONT检测分析受到研究人员和产业界的极大渴望，这将为开发基于BONT的新疗法打开大门。 公共卫生相关性：肉毒杆菌神经毒素是一种极具致命性的细菌毒素，可攻击神经系统，被认为是重大的生物恐怖主义威胁。肉毒杆菌神经毒素还被广泛用于化妆品和制药，缓解肌肉收缩和疼痛。BioSentinel建议开发一种用于测量活细胞中肉毒杆菌神经毒素活性的高通量测试方法，为筛选BONT的拮抗剂和开发基于BONT的新疗法提供一个亟需的平台。

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. PUBLIC HEALTH RELEVANCE: Botulinum neurotoxins are extremely lethal bacterial toxins that attack the nervous system and are considered a significant bioterrorism threat. Botulinum neurotoxins are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. BioSentinel proposes to develop a high- throughput assay for measuring botulinum neurotoxin activity in living cells, providing a much needed platform for screening antagonists to BoNT and developing new BoNT-based therapies.