Internal Toxin Neutralizer for Treating STEC-infection

摘要 产滋贺毒素E.大肠杆菌（STEC）是出血性腹泻的最常见原因， 据估计，美国每年有73，000人，造成严重的发病率。最新和最大的STEC 德国于2011年爆发，影响超过3，800人，包括54人死亡。目前没有 有效治疗STEC感染。STEC感染的病理学来源于两种外毒素--滋贺 毒素1（Stx 1）和滋贺毒素2（Stx 2）-由肠内STEC分泌。虽然抗生素治疗可以 减少STEC的负荷，它也增加了滋贺毒素的释放，导致发展更多的风险增加。 严重的溶血性尿毒综合征（HUS）和肾衰竭（高达25%）。CDC建议 抗生素不用于STEC患者，仅支持性治疗（例如口服和静脉注射液体、疼痛 控制）使用。虽然抗毒素抗体已被确定，抗体不能穿过细胞， 膜使它们对已经被宿主细胞吸收的毒素无能为力，限制了它们的临床应用。 应用程序.我们假设，一种胞质可及的抗毒素应该能够中和细胞外的 和细胞内的滋贺毒素，从而导致更长的治疗窗和更好的治疗效果。 本研究的总体目标是设计一组针对滋贺毒素的细胞内毒素中和剂（ITNs 2（Stx2）。作为所提出的ITN的支架，我们将使用设计的锚蛋白重复蛋白（DARPin）。DARPins 代表了一种多功能的结合蛋白，其已被工程化以结合不同的靶标， 具有皮摩尔亲和力和低免疫原性。在这个项目中，我们将首先隔离绑定和 中和Stx 2（目标1）。同时，我们将筛选一组细胞穿透肽（CPP）， 将ITNs运输到细胞中（目标2）。在目标3中，我们将使用最好的抗Stx 2 DARPin组装抗Stx 2 ITNs 和CPP，并评估这些抗Stx 2 ITNs在体外和体内的治疗潜力。的临近 使用经杀虫剂处理过的蚊帐来对抗循环中的毒素，为治疗STEC和非STEC提供了一种新的范例 细菌感染

Abstract The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.