Acquisition of a parallel stirred-tank bioreactor system to accelerate and advance the development of next-generation probiotics

收购并行搅拌罐生物反应器系统，以加速和推进下一代益生菌的开发

• 批准号: 10389127
• 负责人: Jan-Peter van Pijkeren
• 金额: $ 11.92万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389127
• 关键词: Adhesions; Advanced Development; Aftercare; Agriculture; Animal Model; Bacteria; Bacterial Adhesins; Bacteriophages; Biological; Biological Containment; Bioreactors; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Containment; Cytolysis; DNA; Development; Disease; Disease Management; Engineering; Environment; Epithelial Cells; Frequencies; Gastrointestinal tract structure; Gene Deletion; Genes; Genetically Modified Organisms; Goals; Gram-Negative Bacteria; Growth; Health; Human; In Situ; Lactobacillus reuteri; Measures; Mediating; Medicine; Microbe; Mission; Mucous body substance; Organism; Probiotics; Proteins; Publishing; Recombinants; Research; Safety; Sampling; Specificity; Surface; System; Therapeutic; Thymidine; Thymidylate Synthase; Time; United States National Institutes of Health; Validation; Work; auxotrophy; base; clinical application; gastrointestinal; immunoreaction; improved; in vivo; microbial; microbial community; microbiota; next generation; novel; prevent; prototype; tool

The use of synthetic microbes to deliver therapeutics offers excellent potential to manage diseases in both agriculture and human medicine. However, use of genetically modified organisms (GMOs) raises immediate concerns with regards to their containment. Therefore, strategies must be developed that prevents permanent colonization and accumulation of the GMO in the host and environment, respectively. Also, a kill-switch must be in-place to eradicate the GMO efficiently. Especially in human medicine, an efficient kill-switch is needed to overcome safety concerns when complications would occur as a consequence of an immune reaction against the therapeutic molecule. Until we have overcome these fundamental technical challenges, clinical applications of microbial therapeutics will be beyond reach in human medicine. Our long-term goal is to develop probiotic bacteria as therapeutic delivery vehicles to improve human health. The overall objective of this application is to establish containment and safety strategies which we envision will be broadly applicable in both gram-positive and gram-negative bacteria. Our central hypotheses are a.) that deletion of genes encoding adhesins will reduce the organism's ability to interact with host cells; b.) that thymidine auxotrophy will limit growth in the environment; c.) that CRISPR-delivery by recombinant bacteriophages can specifically eradicate the target organism from a complex environment. Our hypotheses have been formulated based on our published findings. We demonstrated that L. reuteri adhesins are critical to adhere to epithelial cells, that cells lacking thyA are dependent on exogenously added thymidine, that L. reuteri encodes biologically active bacteriophages in vivo, and that CRISPR-Cas is functional in L. reuteri. The rationale for the proposed research is that successful completion of this work is expected to yield a prototype of Lactobacillus reuteri that can be used for safe in situ delivery of therapeutics to treat an array of diseases with an embedded biological containment system that is broadly applicable in both Gram-negative and Gram-positive microbes. We plan to accomplish the overall objective by completing three specific aims. In Aim #1 we will develop a delivery vehicle with reduced colonization potential. In Aim #2 we will develop an environmental containment strategy based on thymidine auxotrophy. In Aim #3 we will develop recombinant phages to achieve strain-specific killing of L. reuteri via CRISPR-Cas. L. reuteri phages will be engineered to encode CRISPR arrays targeting engineered L. reuteri. Strategies will be implemented to reduce `escapers', and in vivo efficacy and specificity will be determined. When successful, our work will have a positive impact as we will have developed a functioning prototype of a safety strategy for a synthetic microbe, which we expect will serve as a novel research tool to deliver therapeutics in situ. The evolutionary conservation of thyA combined with the abundance of bacteriophages and CRISPR-Cas systems make our platform broadly applicable to both gram-positive and gram-negative bacteria.

使用合成微生物提供治疗为控制这两种疾病提供了巨大的潜力 农业和人类医学。然而，转基因生物（GMO）的使用立即引起了人们的关注。 对它们的遏制表示担忧。因此，必须制定战略来防止永久性的 转基因生物分别在宿主和环境中定植和积累。此外，必须有一个终止开关 有效根除转基因生物。特别是在人类医学中，需要一个有效的终止开关 克服因免疫反应而发生并​​发症时的安全问题 治疗分子。在我们克服这些基本技术挑战之前，临床应用 微生物疗法在人类医学中将是遥不可及的。我们的长期目标是开发益生菌 细菌作为治疗载体来改善人类健康。该应用程序的总体目标是 建立遏制和安全策略，我们认为这些策略将广泛适用于革兰氏阳性菌 和革兰氏阴性菌。我们的中心假设是 a.) 编码粘附素的基因的缺失会减少 生物体与宿主细胞相互作用的能力； b.) 胸苷营养缺陷会限制环境中的生长； c.) 重组噬菌体的 CRISPR 传递可以特异性地根除目标生物体 复杂的环境。我们的假设是根据我们发表的研究结果制定的。我们展示了 罗伊氏乳杆菌粘附素对于粘附上皮细胞至关重要，缺乏 thyA 的细胞依赖于 外源添加胸苷，罗伊氏乳杆菌在体内编码具有生物活性的噬菌体，并且 CRISPR-Cas 在罗伊氏乳杆菌中发挥作用。拟议研究的理由是成功完成 这项工作预计将产生罗伊氏乳杆菌的原型，可用于安全地原位递送 通过广泛应用的嵌入式生物遏制系统来治疗一系列疾病的疗法 适用于革兰氏阴性和革兰氏阳性微生物。我们计划通过以下方式实现总体目标 完成三个具体目标。在目标#1中，我们将开发一种降低定植潜力的运载工具。 在目标#2 中，我们将制定基于胸苷营养缺陷型的环境遏制策略。在目标#3中，我们 将开发重组噬菌体，通过 CRISPR-Cas 实现对罗伊氏乳杆菌的菌株特异性杀伤。罗伊氏乳杆菌噬菌体 将被设计为编码针对工程化罗伊氏乳杆菌的 CRISPR 阵列。将实施策略 减少“逃逸者”，并将确定体内功效和特异性。一旦成功，我们的工作将会有一个 积极影响，因为我们将开发出合成微生物安全策略的功能原型， 我们期望它将作为一种新颖的研究工具来提供原位治疗。进化保守 thyA 与丰富的噬菌体和 CRISPR-Cas 系统相结合，使我们的平台具有广泛的应用 适用于革兰氏阳性菌和革兰氏阴性菌。