RNA aptamers as novel antidotes against botulism

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，会导致致命的神经麻痹疾病，肉毒杆菌中毒。肉毒杆菌中毒导致的松弛麻痹持续了几个月。BoNTs可能被用作潜在的生物恐怖主义毒剂，因此对国土安全和公共卫生构成极大威胁。目前，肉毒杆菌中毒的预防和治疗尚无有效、安全的解毒剂。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，治疗窗口非常短。此外，马的抗毒素可能会引起严重的副作用，不包括它们作为有效的预防药物。对于患者来说，唯一的选择仍然是在重症监护下进行人工呼吸机几个月。在这项R21探索性研究拨款中，我们提出了一种新一代的肉毒杆菌中毒解毒剂-适体。适配子是一种独特的寡核苷酸，对从小分子到蛋白质的靶标具有高亲和力。适体具有低毒、无免疫原性、制造成本低、储存稳定性好等特点。这些特性使适体成为对抗肉毒杆菌中毒的理想解毒剂。这项研究的目标是开发适配子作为肉毒杆菌中毒的解毒剂。建议的解毒剂既有可能成为有效的预防性治疗，也有可能成为治疗有症状的肉毒杆菌中毒患者的有效治疗药物。未来两年要追求的具体目标是：(1)针对BONT的适体的选择和鉴定。我们将使用SELEX筛选随机RNA文库，分离针对BONT的适配子，并根据与BONT的结合亲和力确定适配子的优先顺序。(2)适配子抗BONT活性的验证。我们将使用体外和基于细胞的分析方法，确定其对内肽酶活性和毒素与其受体结合的抑制作用。因此，从这项研究中确定的适体将被用作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。这种研究方法可以很容易地适用于所有七种血清型的BONT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。 由于生物恐怖主义在全球范围内造成的健康风险不断增加，给科学界带来了巨大的负担，需要迅速开发针对生物和化学毒素的安全预防和治疗方法。这项任务一直是公共卫生的高度优先事项。肉毒杆菌中毒和大多数其他生物毒素(列入NIAID优先病原体名单)缺乏治疗和预防药物。我们的项目将导致基于适体的抗肉毒杆菌中毒药物候选，这是一种新型的肉毒杆菌中毒解毒剂，与现有的抗毒素相比具有许多优势。

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.