Designer Probiotics for the treatment of intestinal infection and inflammation

项目摘要 新的药物输送平台对于将高特异性治疗药物定向输送到靶点是至关重要的 以最大限度地提高治疗效果，同时限制非目标副作用。大多数努力 目前正在进行的这种靶向给药系统的开发主要集中在 开发合成纳米颗粒，这是一种生产、储存和分销成本高昂的材料。在这里，我们 建议开发具有成本效益、自我复制和灵活、可编程的设计者益生菌 有针对性地将治疗药物直接输送到疾病部位。我们建议使用一种合成生物学方法 通过基因工程获得广泛安全使用的益生菌--大肠杆菌Nissle 1917，以表达一种 可以将治疗有效载荷分泌到肠道环境中的纳米机器。这些设计好的细菌将会是 配备了一种经过改造的3型分泌系统，可以将蛋白质分泌到肠腔中，而不是将其 先天靶标，哺乳动物细胞的胞浆。作为概念的证明，并朝着发展这些 设计者益生菌作为治疗剂，我们将工程这些设计者益生菌来分泌一类新的井- 有文献记载的治疗性生物分子具有极高的特异性，单域抗体，也称为 VHH。我们将专注于VHH多聚体的交付，这些多聚体具有深刻的中和活性，以VHH为基础 中和剂(VNAs)，可抑制基本细菌毒素或促炎细胞因子的活性。 此外，我们将研究这些菌株作为新的治疗范例的潜力。 肠道感染和炎症性疾病。具体地说，我们将调查这些措施的效果 用于预防治疗艰难梭菌感染(CDI)、溶血性尿毒症综合征(HUS)的菌株 和炎症性肠病(IBD)。我们理解，可能会有一些关于 将转基因细菌作为治疗药物进行管理。然而，随着我们进入‘时代’ 微生物组，这种干预措施似乎极有可能成为 用于治疗感染和炎症性疾病的医疗器械，特别是那些根植于 胃肠道，特别是因为这类药物很有可能克服许多问题 与抗生素和全身性免疫抑制剂的广泛使用有关。虽然努力 这里专门致力于开发这些用于治疗CDI的设计型益生菌， HUS和IBD，一旦被确立为治疗范例，这个设计的益生菌平台可以扩展 治疗各种肠道疾病。例如，设计者益生菌可以被编程为 提供各种基于蛋白质的治疗有效载荷，包括抑制细胞因子，如IL-10 肠炎症或针对肠道细菌主要暴露的毒力蛋白的VNAs 病原体，例如粘附素或毒力因子递送系统的基本成分。

Project Summary New drug delivery platforms are vitally needed for the targeted delivery of high-specificity therapeutics to sites of disease in order to maximize therapeutic efficacy while limiting off-target side effects. The majority of efforts currently underway for the development of such targeted drug delivery systems are focused on the development of synthetic nanoparticles, materials which are costly to produce, store, and distribute. Here, we propose to develop cost-effective, self-replicating and flexible, programmable designer probiotics for the targeted delivery of therapeutics directly to sites of disease. We propose to utilize a synthetic biology approach to genetically engineer a widely and safely administered probiotic, Escherichia coli Nissle 1917, to express a nanomachine that can secrete therapeutic payloads into the intestinal milieu. These designer bacteria will be equipped with a type 3 secretion system modified to secrete proteins into the intestinal lumen rather than their innate target, the cytosol of mammalian cells. As proof of concept and towards the development of these designer probiotics as therapeutics, we will engineer these designer probiotics to secrete a new class of well- documented therapeutic biomolecules of exquisite specificity, single domain antibodies, also referred to as VHH. We will focus on the delivery of VHH multimers that exhibit profound neutralizing activity, VHH-based neutralizing agents (VNAs), which inhibit the activity of essential bacterial toxins or proinflammatory cytokines. Furthermore, we will investigate the potential of these strains as novel therapeutic paradigms for the treatment of both intestinal infections and inflammation disorders. Specifically, we will investigate the efficacy of these strains in the prevention of treatment of Clostridium difficile infections (CDI), hemolytic uremic syndrome (HUS) and inflammatory bowel disease (IBD). We appreciate that there might be some concern regarding the administration of genetically modified bacteria as therapeutics. However, as we enter the `era of the microbiome,' it seems extremely likely that such interventions are to become an integral component of the armamentarium utilized to treat infections and inflammatory disorders, particularly those rooted in the gastrointestinal tract, especially because of the high likelihood that such agents can overcome many issues associated with the wide-spread usages of antibiotics and systemic immunosuppressive agents. While efforts here are specifically devoted towards the development of these designer probiotics for the treatment of CDI, HUS, and IBD, once established as a therapeutic paradigm, this designer probiotic platform can be extended to treat a variety of intestinal based diseases. For example, the designer probiotics could be programmed to deliver a variety of protein-based therapeutic payloads, including cytokines, such as IL-10, that suppress intestinal inflammation or VNAs designed to target essential exposed virulence proteins of enteric bacterial pathogens, e.g., adhesins or essential components of virulence factor delivery systems.