Multi-tiered containment system for engineered microbes

使用合成微生物提供治疗药物提供了极好的潜力来管理这两种疾病 农业和人类医学。然而，转基因生物(GMO)的使用立即带来了 对他们的牵制感到担忧。因此，必须制定战略，防止永久性的 转基因生物在寄主和环境中的定殖和积累。此外，关闭开关必须是 有效地根除转基因生物。特别是在人类医学中，需要一个有效的杀死开关来 克服因免疫反应而导致并发症时的安全顾虑 治疗分子。直到我们克服了这些根本的技术挑战，临床应用 微生物疗法的研究将是人类医学所无法企及的。我们的长期目标是开发益生菌 细菌作为治疗载体，改善人类健康。此应用程序的总体目标是 建立遏制和安全策略，我们设想这些策略将广泛适用于革兰氏阳性和 和革兰氏阴性细菌。我们的中心假设是a。)编码粘附素的基因的缺失将减少 生物体与宿主细胞相互作用的能力；b)胸腺嘧啶核苷营养不良会限制环境中的生长； C.)通过重组噬菌体递送CRISPR可以特异性地从 环境复杂。我们的假设是基于我们已发表的发现而提出的。我们演示了 鲁氏乳杆菌粘附素是黏附上皮细胞的关键，缺乏ThyA的细胞依赖于 外源添加胸腺嘧啶核苷，在体内编码具有生物活性的噬菌体，以及 CRISPR-Cas在罗氏乳杆菌中具有功能。拟议研究的理由是成功地完成了 这项工作有望生产出一种可用于安全原位递送的鲁氏乳杆菌原型。 使用嵌入式生物遏制系统治疗一系列疾病的治疗技术 适用于革兰氏阴性菌和革兰氏阳性菌。我们计划在以下时间完成总体目标 完成三个具体目标。在目标1中，我们将开发一种减少殖民潜力的运载工具。 在目标2中，我们将开发一种基于胸苷营养缺乏症的环境遏制策略。在目标#3中，我们 将开发重组噬菌体，通过CRISPR-Cas实现对路支杆菌的菌株特异性杀灭。路氏乳杆菌噬菌体 将被设计成编码针对工程L.reuri的CRISPR阵列。将实施战略，以 减少‘逃逸者’，体内的有效性和特异性将被确定。当成功时，我们的工作将有一个 积极的影响，因为我们将开发出一种合成微生物安全策略的功能原型， 我们希望这将成为一种新的研究工具，以提供现场治疗。进化守恒论 Thya结合丰富的噬菌体和CRISPR-Cas系统，使我们的平台得到了广泛的应用 对革兰氏阳性菌和革兰氏阴性菌均适用。

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.