Engineered cell-based assays for detection of SNAP25-cleaving botulinum neurotoxins

用于检测 SNAP25 裂解肉毒杆菌神经毒素的工程化细胞分析

• 批准号: BB/W017210/1
• 负责人: Bazbek Davletov
• 金额: $ 32.14万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW017210%2F1
• 关键词: Engineered; cell; based; assays; detection

This project proposes to establish quantitative animal-free assays for detection of Botulinum Neurotoxins (BoNTs) type A and C. Botulism is a severe paralytic disease affecting both humans and animals. The paralytic botulinum neurotoxins types A-G are produced by anaerobic bacteria which are ever-present in the soil, and also in decaying matter and poorly prepared food. Unlike other botulinum serotypes, type A and C BoNTs target specifically intraneuronal protein SNAP25 and cause the longest muscle paralysis lasting up to 6 months. Botulism type A was originally identified in humans but also afflicts animals, whereas type C botulism is an animal disease. There is a long-standing need for sensitive detection of toxic material in afflicted animals and environmental samples. To prevent botulism outbreaks managed animals receive botulism vaccines that incorporate inactivated BoNTs. These are being tested for the lack of active toxic material in live mouse toxicity assays. In addition, anti-toxins used in human and veterinary medicine are also tested for their potency in live mouse toxicity assays. Finally, due to the longest muscle paralysis type A BoNT is widely used in medicine for local muscle treatments and as an aesthetic, while BoNT/C is being investigated as an additional medical option in the cases where repeated BoNT/A use leads to immunological resistance. The manufacturing of pharmaceutical BoNT preparations unfortunately also relies on live mouse toxicity assays. The currently used live mouse toxicity assays are unethical due to the slow mode of animal death by asphyxiation.There is a long-standing need, and recently governmental pressure, to replace live mouse toxicity assays with animal-free detection of botulinum activities. This project aims to develop cell-based assays which would accurately measure the activities of BoNT type A and C. This opportunity arose from our recently acquired detailed knowledge of molecular mechanisms of BoNT actions and our identification of a new continuous cell line that exhibits a robust binding of BoNTs. Using synthetic biology approaches we will engineer this cell line to report the activity of BoNT type A and C in a simple multiwell light-emitting assay. Successful implementation of this project will contribute to better animal health through easier production of veterinary and human medicines, will save hundreds of thousands of mice from unethical testing, and also provide a long-sought-after test for BoNT presence during botulism outbreaks both in farmed animals and in wild environments.

本项目拟建立无动物源的定量分析方法，用于检测A型和C型肉毒神经毒素（BoNT）。肉毒杆菌中毒是一种严重的麻痹性疾病，影响人类和动物。麻痹性肉毒杆菌神经毒素A-G型是由厌氧细菌产生的，这些细菌一直存在于土壤中，也存在于腐烂的物质和准备不好的食物中。与其他肉毒杆菌血清型不同，A型和C型BoNT特异性靶向神经元内蛋白SNAP 25，并导致持续时间最长达6个月的肌肉麻痹。A型肉毒杆菌中毒最初在人类中发现，但也折磨动物，而C型肉毒杆菌中毒是一种动物疾病。长期以来，人们一直需要对患病动物和环境样品中的有毒物质进行灵敏检测。为了防止肉毒杆菌中毒爆发，管理的动物接受包含灭活BoNT的肉毒杆菌中毒疫苗。目前正在对这些产品进行活体小鼠毒性试验，以确定其是否不含活性有毒物质。此外，人类和兽医学中使用的抗毒素也在活小鼠毒性试验中测试其效力。最后，由于最长的肌肉麻痹A型BoNT被广泛用于局部肌肉治疗和美容医学，而BoNT/C正在研究作为一个额外的医疗选择的情况下，重复BoNT/A使用导致免疫抗性。不幸的是，药物BoNT制剂的制造也依赖于活小鼠毒性测定。目前使用的活小鼠毒性测定是不道德的，由于动物窒息死亡的缓慢模式，有一个长期的需要，最近政府的压力，取代活小鼠毒性测定与肉毒杆菌活性的动物检测。该项目旨在开发基于细胞的检测方法，以准确测量BoNT A型和C型的活性。这个机会来自于我们最近获得的BoNT作用的分子机制的详细知识，以及我们对一种新的连续细胞系的鉴定，该细胞系表现出对BoNT的稳健结合。使用合成生物学方法，我们将工程化该细胞系，以在简单的多孔发光测定中报告BoNT A型和C型的活性。该项目的成功实施将有助于通过更容易地生产兽药和人用药物来改善动物健康，将数十万只小鼠从不道德的测试中拯救出来，并在养殖动物和野生环境中的肉毒杆菌中毒爆发期间提供长期寻求的BoNT存在测试。