Targeted Delivery of Therapeutics into Motor Neurons for Post-exposure Treatment of Botulism

项目摘要 肉毒杆菌神经毒素（Botulinum neurotoxins，BoNTs）是一种细菌毒素家族，由芽孢杆菌产生， 形成革兰氏阳性梭菌属细菌，是已知的最有效的毒素。它们使得 人和动物的致命麻痹性疾病肉毒杆菌中毒。这些毒素是世界上 危险的潜在生物恐怖主义制剂（A类和1级）。它们的目标是运动神经元 具有极端特异性，进入神经元的胞质溶胶，并阻断神经元活动， 肌肉麻痹BONTs威胁的一个主要因素是它们极长的半衰期 在运动神经元的胞质溶胶中-毒素在人体内存在4-6个月 导致持续瘫痪数月到目前为止，没有治疗剂可以抑制毒素内， 神经元的胞质溶胶。 在这里，我们建议为这一最优先事项创建一个有效的暴露后治疗方法。 生物恐怖剂和致命细菌毒素。这种治疗方法是基于最近修改的 发现了新的细菌毒素BoNT/X，并利用这种工程蛋白质提供融合的 抗BoNT中和抗体进入运动神经元的胞质溶胶。我们广泛 初步的研究提供了工作原型，它完全拯救了老鼠， 多个暴露后模型中BoNT的全身毒性。在此，我们提出三个目标， 进一步验证我们的假设，通过（1）完善嵌合毒素平台;（2）优化 针对BoNT的纳米抗体货物;和（3）建立药代动力学参数， 在啮齿动物和豚鼠模型中验证治疗功效。 这些目标的成功将创造一个有效的暴露后治疗的首要任务 生物恐怖剂，并开发一种新的蛋白质为基础的输送平台，针对电机 神经元，从而提供了新的工具，针对细胞溶质的过程和“undruggable”蛋白质， 神经元

Project Summary The family of bacterial toxins, botulinum neurotoxins (BoNTs), produced by spore- forming Gram-positive Clostridium bacteria, are the most potent toxins known. They cause the deadly paralytic disease botulism in humans and animals. These toxins are one of the six most dangerous potential bioterrorism agents (Category A and Tier 1). They target motor neurons with extreme specificity, enter the cytosol of neurons, and block neuronal activity, causing muscle paralysis. A major contributor to the threat of BoNTs is their extremely long half-life within the cytosol of motor neurons – the toxin resides in the cytosol for 4-6 months in humans and causes persistent paralysis for months. To date, no therapeutics can inhibit toxins within the cytosol of neurons. Here we propose to create an effective post-exposure treatment for this top-priority bioterrorism agent and deadly bacterial toxins. This treatment is based on modifying a recently discovered new bacterial toxin BoNT/X and utilizing this engineered protein to deliver a fused neutralizing antibody against BoNTs into the cytosol of motor neurons. Our extensive preliminary studies have provided working prototypes, which completely rescue mice from systemic toxicity of BoNTs in multiple post-exposure models. Here we propose three aims to further validate our hypothesis by (1) refining the chimeric toxin platform; (2) optimizing the nanobody cargo against BoNTs; and (3) establishing pharmacokinetic parameters and validating therapeutic efficacy in both rodent and guinea pig models. Success of these aims will create an effective post-exposure treatment for a top-priority bioterrorism agent, and also develop a novel protein-based delivery platform for targeting motor neurons, thus offering new tools for targeting cytosolic processes and “undruggable” proteins in neurons.