Designer Probiotics for the treatment of intestinal infection and inflammation

项目摘要 新的药物递送平台对于将高特异性治疗剂靶向递送到位点是至关重要的 以最大限度地提高治疗效果，同时限制脱靶副作用。大多数努力 目前正在进行的这种靶向药物递送系统的开发集中在 合成纳米颗粒的开发，这些材料的生产、储存和分配成本很高。这里我们 建议开发具有成本效益，自我复制和灵活，可编程的设计师益生菌， 将治疗剂直接靶向递送至疾病部位。我们建议利用合成生物学方法 为了对广泛且安全施用的益生菌大肠杆菌Nissle 1917进行基因工程改造，以表达 纳米机器，可以分泌治疗有效载荷到肠道环境中。这些设计师细菌 配备有3型分泌系统，该系统被修改为将蛋白质分泌到肠腔中，而不是它们的 先天靶，哺乳动物细胞的胞质溶胶。作为概念的证明， 设计益生菌作为治疗，我们将设计这些设计益生菌分泌一类新的良好的， 有文献记载的具有精确特异性的治疗性生物分子，单结构域抗体，也被称为 VHH。我们将专注于VHH多聚体的递送，所述VHH多聚体表现出深刻的中和活性，基于VHH的中和活性，以及基于VHH的中和活性。 中和剂（VNA），其抑制必需的细菌毒素或促炎细胞因子的活性。 此外，我们还将研究这些菌株作为新的治疗范例的潜力。 肠道感染和炎症紊乱。具体来说，我们将研究这些药物的疗效。 预防治疗艰难梭菌感染（CDI）、溶血性尿毒综合征（HUS）的菌株 和炎症性肠病（IBD）。我们理解，可能会有一些关注， 施用遗传修饰的细菌作为治疗剂。然而，当我们进入“时代”时， 微生物组，“似乎极有可能，这种干预措施将成为一个不可分割的组成部分， 用于治疗感染和炎症性疾病的医疗设备，特别是那些植根于 胃肠道，特别是因为这种药物很可能可以克服许多问题 与抗生素和全身免疫抑制剂的广泛使用有关。尽管工作 这里特别致力于开发这些设计的益生菌用于治疗CDI， HUS和IBD，一旦作为治疗范例建立，这种设计的益生菌平台可以扩展 治疗各种肠道疾病。例如，设计师益生菌可以被编程为 递送多种基于蛋白质的治疗有效载荷，包括细胞因子，如IL-10，其抑制 设计成靶向肠道细菌的必需暴露毒力蛋白的肠炎或VNA 病原体，例如，粘附素或毒力因子递送系统的必需组分。

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.