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

该项目建议建立检测A型和C型肉毒神经毒素(BoNTs)的无动物定量检测方法。肉毒中毒是一种严重的瘫痪疾病，对人类和动物都有影响。麻风性肉毒杆菌神经毒素A-G型是由土壤中以及腐烂的物质和烹调不当的食物中经常存在的厌氧细菌产生的。与其他肉毒杆菌血清型不同，A型和C型BoNTs专门针对神经元内蛋白SNAP25，并导致持续长达6个月的肌肉瘫痪。A型肉毒中毒最初是在人类身上发现的，但也会困扰动物，而C型肉毒中毒是一种动物疾病。长期以来，对受影响的动物和环境样本中的有毒物质进行灵敏检测的需求由来已久。为了防止肉毒杆菌中毒的爆发，受管理的动物接种了含有灭活的BoNTs的肉毒杆菌疫苗。在活体小鼠毒性试验中，正在测试这些物质是否缺乏活性有毒物质。此外，用于人类和兽医的抗毒素也在活体小鼠毒性测试中进行了效力测试。最后，由于A型BONT是持续时间最长的肌肉麻痹，因此在医学上被广泛用于局部肌肉治疗和美容，而在反复使用BONT/A导致免疫抵抗的情况下，BONT/C正在被作为一种额外的医学选择进行研究。不幸的是，药物波恩特制剂的制造也依赖于活体小鼠毒性测试。由于动物窒息死亡的缓慢模式，目前使用的活体小鼠毒性检测是不道德的。长期以来，人们一直需要用肉毒杆菌活性的无动物检测取代活体小鼠毒性检测，最近也受到了政府的压力。该项目旨在开发基于细胞的分析，以准确测量BoNT类型A和C的活性。这一机会来自我们最近对BONT作用的分子机制的详细了解，以及我们发现了一种新的连续细胞系，该细胞系显示出与BoNT的牢固结合。利用合成生物学方法，我们将设计这种细胞系，在一个简单的多孔光发射实验中报告A型和C型BoNT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类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，并为在养殖动物和野生环境中爆发肉毒杆菌中毒期间提供一种长期受欢迎的测试方法。

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.