RNA aptamers as novel antidotes against botulism

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强的物质，可引起致命的神经麻痹性疾病肉毒中毒。肉毒杆菌中毒引起的弛缓性麻痹持续数月。bont可作为潜在的生物恐怖主义制剂，对国土安全和公众健康构成巨大威胁。目前还没有有效和安全的解毒剂用于肉毒中毒的预防和治疗。唯一被批准的解毒剂是马抗毒素，它只能中和血液循环中的毒素，治疗窗口期很短。此外，马抗毒素会引起严重的副作用，因此不能作为有效的预防药物。患者唯一的选择仍然是在重症监护下进行人工呼吸几个月。在这个R21探索性研究资助中，我们提出了一种新一代的肉毒杆菌解毒剂，适体。适配体是一种独特的寡核苷酸，对其靶标具有高亲和力，范围从小分子到蛋白质。适体具有低毒性、无免疫原性、低制造成本和良好的储存稳定性。这些特性使适配体成为对抗肉毒杆菌中毒的理想解毒剂。本研究的目的是开发适合体作为肉毒杆菌中毒的解毒剂。提出的解毒剂有潜力成为有效的预防性治疗和有效的治疗药物来治疗有症状的肉毒杆菌中毒患者。未来两年的具体目标是：(1)选择和鉴定抗BoNT的适体。我们将使用SELEX筛选随机RNA文库，分离针对BoNT的适配体，并根据与BoNT的结合亲和力对适配体进行优先排序。(2)适配体抗bont活性的验证。我们将通过体外和细胞实验来确定对内啡肽酶活性和毒素与其受体结合的抑制作用。因此，从本研究中鉴定的适体将作为设计更有效的肉毒杆菌解毒剂的先导化合物。这项研究将导致一类新的肉毒杆菌中毒解毒剂的产生。该研究方法可以很容易地适用于BoNT的所有7种血清型和生物防御议程上的其他优先病原体。此外，本文提出的研究将对生物防御剂的快速诊断产生重大影响。

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are the most toxic substances known to man, and cause the deadly neuroparalytic disease, botulism. The flaccid paralysis caused by botulism lasts for months. BoNTs can be used as potential bioterrorism agents, and therefore, pose great threat to homeland security and public health. Currently no effective and safe antidote exists for prophylactic and therapeutic treatment for botulism. The only approved antidotes are equine antitoxins, which can only neutralize the toxin in the blood circulation, and have a very short treatment window. In addition, the equine antitoxins can cause severe side effects, excluding them as effective prophylactics. The only alternative for patients remains to be put on artificial ventilation under intensive care for several months. In this R21 exploratory research grant, we propose a new generation of antidotes against botulism, aptamers. Aptamers are unique oligonucleotides that have high affinity for their targets, ranging from small molecules to proteins. Aptamers have low toxicity, no reported immunogenicity, low cost to manufacture, and excellent storage stability. Those characteristics make aptamers ideal as antidotes against botulism. The goal of this research is to develop aptamers as antidotes against botulism. The proposed antidotes have potential to be both effective prophylactic treatment, and effective therapeutic drugs to treat botulism patients with symptoms. The specific aims to be pursued in the next two years are: (1) Selection and characterization of aptamers against BoNT. We will use SELEX to screen random RNA libraries, isolate the aptamers against BoNT, and prioritize aptamers based on binding affinity to BoNT. (2) Validation of anti-BoNT activity of aptamers. We will determine the inhibitory effects on both endopeptidase activity and toxin binding to its receptors, using both in vitro and cell-based assays. The aptamers identified from this study, therefore, will be used as lead compounds for the designing of more potent antidotes against botulism. This study will lead to a new class of antidotes against botulism. The research approach can be readily applicable to all seven serotypes of BoNT and other priority pathogens on biodefense agenda. In addition, the research proposed here will have significant impact on the rapid diagnostics of biodefense agents. The ever increasing health risks due to bioterrorism worldwide have placed a large burden on the scientific community to rapidly develop safe prophylactic and therapeutic treatments for biological and chemical toxins. This task has been a high priority for public health. There is lack of therapeutic and prophylactic drugs for botulism and most of the other biological toxins (as listed in NIAID priority pathogen list). Our project will result in aptamer-based drug candidates against botulism, a new class of antidotes against botulism, with many advantages over currently available antitoxin.