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: 9761466
• 负责人: Gautam Dantas
• 金额: $ 70.48万
• 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/9761466
• 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 工程益生菌代表了一种强大的工具，可以使用它“敲入”基因功能和通向途径 肠道微生物组，更改肠道微生物组的结构以检验有关社区体系结构的假设 和疾病，并提供治疗。但是，已知的益生菌由于定殖而无法持续存在 肠道菌群的抵抗力限制了其作为研究或治疗工具的价值。此外，受控 通过工程益生菌提供治疗剂和其他基因产品受到缺乏健壮和的限制 复杂体内环境的可调合成生物学工具。拟议研究的理由是 益生菌疗法的承诺目前受到持久性差和缺乏强大的工程的限制 工具。该建议的主要动机是了解宿主和微生物机制 益生菌整合和开发工具以设计益生菌疗法。在强大的初步数据的指导下， 跨学科的建议将追求三个具体目标：至1）确定益生菌定居的决定者 肠道，2）设计和优化与肠道相关的生物传感器和表达电路，3）证明了效率 通过增强的益生菌结肠剂的体内苯酮尿症（PKU）疗法的体内递送的。 该提案的第一个目的是优化和确定益生菌定植的机制，测试 假设益生菌肠道定植是通过调节表达来增强和调整的 从粪便宏基因组中选择的定植因子。特别是，我们将选择耐用的殖民者 从表达由体内驱动的定植因子的详尽组合文库的益生菌 特征启动子。第二个目的是开发可调基因表达的合成生物学工具 控制和生物植物的工程益生菌，检验了将传感器结合的假设 温度，pH，胆汁酸和短链脂肪酸将使基因表达的空间控制 沿着胃肠道。第三个目的是证明工程化的益生菌底盘可以 可靠地向肠道提供治疗剂，检验我们工程益生菌可以保持的假设 PKU的鼠模型中的递送苯丙氨酸 - 氨基酸酯（PAL2），以减少血清苯丙氨酸。 该建议具有创新性，因为我们的整合和完整的研究团队将有所改善 了解基础科学和翻译水平的益生菌疗法。提出的实验 意义重大，因为它们会1）提高我们对遗传因素和微生物相互作用的理解 管理胃肠道殖民化，2）生成和优化用于体内电路控制的合成生物学工具 这将是模块化的，并且广泛适用于益生菌工程，3）探索替代方案的效率， PKU的连续交付疗法。拟议的研究具有影响力，因为它将1）发展可靠 益生菌殖民者，2）更新体内合成生物学的工具箱，3）建立工程设计 益生菌作为持续治疗交付或对肠道社区体系结构的控制调制的车辆。