Tunable therapeutic modulation of the gut microbiome by engineered probiotics

通过工程益生菌对肠道微生物组进行可调节的治疗调节

• 批准号: 9977923
• 负责人: Gautam Dantas
• 金额: $ 69.87万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-10 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977923
• 关键词: Affect; American; Architecture; Basic Science; Bile Acids; Biochemical; Biosensor; Cecum; Classical phenylketonuria; Colon; Communities; Complex; Data; Development; Disease; Elements; Engineered Probiotics; Engineering; Environment; Enzymes; Feedback; Gastrointestinal Diseases; Gastrointestinal tract structure; Gene Expression; Genes; Goals; Health; Human; Immune System Diseases; Immunology; In Situ; Knock-in; Libraries; Measures; Metabolic Diseases; Metagenomics; Microbe; Microbiology; Motivation; Mus; Nutrient; Pathway interactions; Patients; Phenylalanine; Phenylalanine Ammonia-Lyase; Phenylalanine Hydroxylase; Phenylketonurias; Probiotics; Production; Research; Serum; Small Intestines; Structure; Technology; Temperature; Testing; Therapeutic; Treatment Efficacy; Update; Urine; Volatile Fatty Acids; Work; colonization resistance; combinatorial; design; efficacy testing; enhancing factor; exhaustion; experimental study; gastrointestinal; gene function; gene product; genetic element; gut colonization; gut metagenome; gut microbiome; gut microbiota; host-microbe interactions; improved; in vivo; innovation; metabolic engineering; metagenome; microbial; microbial host; microbiota; microorganism; microorganism interaction; mouse model; neurotoxic; probiotic therapy; promoter; remediation; sensor; sex; synthetic biology; targeted delivery; therapeutic gene; tool

ABSTRACT Engineered probiotics represent a powerful tool with which to ‘knock-in’ gene functions and pathways into the gut microbiome, alter the structure of the gut microbiome to test hypotheses regarding community architecture and disease, and to deliver therapeutics. However, known probiotics fail to persist in the gut due to colonization resistance by the gut microbiota, limiting their value as either research or therapeutic tools. Further, controlled delivery of therapeutics and other gene products by engineered probiotics is limited by a lack of robust and tunable synthetic biology tools for the complex in vivo environment. The rationale for the proposed research is that the promise of probiotic therapies is currently limited by poor persistence and a lack of robust engineering tools. The central motivation for this proposal is to understand the host and microbial mechanisms governing probiotic integration and develop tools to engineer probiotic therapies. Guided by strong preliminary data, this interdisciplinary proposal will pursue three specific aims: to 1) identify determinants of probiotic colonization in the gut, 2) design and optimize gut-relevant biosensor and expression circuits, and 3) demonstrate the efficacy of in vivo delivery of a phenylketonuria (PKU) therapeutic by an enhanced probiotic colonizer. The first aim of this proposal is to optimize and identify mechanisms of probiotic colonization, testing the hypothesis that probiotic gut colonization is enhanced and tunable by modulating expression of colonization factors selected from fecal metagenomes. In particular, we will select for durable colonizers from probiotics expressing an exhaustive combinatorial library of colonization factors driven by in vivo characterized promoters. The second aim is to develop synthetic biology tools for tunable gene expression control and biocontainment of engineered probiotics, testing the hypothesis that combining sensors for temperature, pH, bile acids, and short chain fatty acids will enable spatial control over gene expression along the gastrointestinal tract. The third aim is to demonstrate that the engineered probiotic chassis can reliably deliver therapeutics to the gut, testing the hypothesis that our engineered probiotic can stably deliver phenylalanine-ammonia lyase (Pal2) in a murine model of PKU to decrease serum phenylalanine. This proposal is innovative because our integrated and complementary research team will improve understanding of probiotic therapies at both basic science and translational levels. The proposed experiments are significant in that they will 1) improve our understanding of the genetic elements and microbial interactions governing gastrointestinal colonization, 2) generate and optimize synthetic biology tools for in vivo circuit control that will be modular and widely applicable to probiotic engineering, and 3) explore the efficacy of an alternative, continual-delivery therapeutic for PKU. The proposed research is impactful because it will 1) develop reliable probiotic colonizers, 2) update the toolbox for synthetic biology in vivo applications, and 3) establish engineered probiotics as vehicles for sustained therapeutic delivery or controlled modulation of gut community architectures.

抽象的 工程益生菌是一种强大的工具，可以将基因功能和途径“敲入” 肠道微生物组，改变肠道微生物组的结构以测试有关群落结构的假设 和疾病，并提供治疗。然而，已知的益生菌由于定植而无法在肠道中持续存在 肠道微生物群的抵抗力限制了它​​们作为研究或治疗工具的价值。进一步，控制 通过工程益生菌传递治疗药物和其他基因产品受到缺乏稳健和可靠的限制。 适用于复杂体内环境的可调节合成生物学工具。拟议研究的理由是 益生菌疗法的前景目前因持久性差和缺乏稳健的工程技术而受到限制 工具。该提案的核心动机是了解宿主和微生物的控制机制 益生菌整合并开发设计益生菌疗法的工具。在强有力的初步数据的指导下， 跨学科提案将追求三个具体目标：1）确定益生菌定植的决定因素 肠道，2) 设计和优化肠道相关生物传感器和表达电路，3) 证明功效 通过增强的益生菌定殖剂在体内递送苯丙酮尿症（PKU）治疗剂。 该提案的首要目标是优化和确定益生菌定植机制，测试 假设益生菌肠道定植可以通过调节益生菌的表达来增强和调节 从粪便宏基因组中选择的定植因子。特别是，我们将选择持久的殖民者 来自表达体内驱动的详尽定植因子组合库的益生菌 特征性的启动子。第二个目标是开发用于可调基因表达的合成生物学工具 工程益生菌的控制和生物防护，测试结合传感器的假设 温度、pH、胆汁酸和短链脂肪酸将使基因表达的空间控制成为可能 沿着胃肠道。第三个目标是证明工程益生菌底盘可以 可靠地将治疗药物输送到肠道，测试我们的工程益生菌可以稳定地传递这一假设 在 PKU 小鼠模型中输送苯丙氨酸氨裂解酶 (Pal2)，以降低血清苯丙氨酸。 这个提议是创新的，因为我们的综合性和互补性研究团队将改进 在基础科学和转化层面对益生菌疗法的理解。拟议的实验 意义重大，因为它们将 1) 提高我们对遗传因素和微生物相互作用的理解 控制胃肠道定植，2) 生成和优化用于体内回路控制的合成生物学工具 这将是模块化的并广泛适用于益生菌工程，3）探索替代方案的功效， PKU 持续给药治疗。拟议的研究具有影响力，因为它将 1）开发可靠的 益生菌殖民者，2) 更新体内合成生物学应用的工具箱，以及 3) 建立工程化 益生菌作为持续治疗传递或肠道群落结构受控调节的载体。