Multi-tiered containment system for engineered microbes

使用合成微生物来提供治疗药物，为管理这两种疾病提供了极好的潜力。 农业和人类医学。然而，使用转基因生物（GMOs） 对他们的包容性表示担忧。因此，必须制定防止永久性 转基因生物在宿主和环境中的定殖和积累。此外，开关必须 以有效地根除转基因生物。特别是在人类医学中，需要一种有效的开关， 克服由于免疫反应而发生并发症时的安全性问题， 治疗分子。在我们克服这些基本的技术挑战之前， 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 细菌作为治疗递送载体来改善人类健康。本申请的总体目标是 建立我们设想将广泛适用于革兰氏阳性 和革兰氏阴性菌。我们的主要假设是a）。删除编码粘附素的基因 生物体与宿主细胞相互作用的能力; B.）胸苷营养缺陷型会限制环境中的生长; c.）重组噬菌体的CRISPR递送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。我们证明 那个L。罗伊氏粘附素对于粘附上皮细胞至关重要，缺乏thyA的细胞依赖于该细胞 外源添加胸苷，L. reuteri在体内编码具有生物活性的噬菌体， CRISPR-Cas在L. reuteri。拟议研究的理由是， 这项工作有望产生一种罗伊氏乳杆菌的原型，可用于安全的原位递送 使用嵌入式生物遏制系统治疗一系列疾病的治疗方法， 适用于革兰氏阴性和革兰氏阳性微生物。我们计划透过以下方法，达到整体目标 实现三个具体目标。在目标#1中，我们将开发一种具有降低定殖潜力的运载工具。 在目标#2中，我们将开发基于胸苷营养缺陷型的环境遏制策略。在目标#3中，我们 将开发重组大肠杆菌以实现菌株特异性杀灭L. reuteri via CRISPR-Cas. L.罗伊氏噬菌体 将被工程化以编码靶向工程化L的CRISPR阵列。reuteri。将实施战略， 减少“逃逸”，并确定体内功效和特异性。一旦成功，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， 我们希望它能作为一种新的研究工具，在原位提供治疗。进化保守论 结合大量的噬菌体和CRISPR-Cas系统， 适用于革兰氏阳性菌和革兰氏阴性菌。

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.