Engineering next generation probiotics for delivery of therapeutics

项目总结/摘要 肽和蛋白质药物（PPD）是治疗广谱疾病的有效治疗剂。肠胃外 由于它们的生物利用度和稳定性差，给药受到限制。口服PPD面临额外的 胃肠道的障碍。我们开发了一种创新的、基于益生菌的平台技术， 用于口服治疗性PPD，靶向许多具有挑战性的慢性疾病。该递送 该系统稳健、高效、成本效益高、易于管理且安全。我们最近公布的结果提供了 使用乳酸杆菌作为递送肽和蛋白质的活载体的可行性的概念验证 具有增强的组织生物利用度和功效。然而，我们目前的平台在临床上有一些局限性。 本发明还涉及一种应用，其包括使用抗生素基因作为选择标记、质粒分离不稳定性和 缺乏内置的生物防护策略，这可能会引起对工程微生物传播的安全问题 到环境中。该项目的目标是开发下一代基于益生菌的平台， 以更稳定和有效的生物防护递送肽和蛋白质治疗剂。具体来说，我们将 (1)构建、表征和验证一种无抗疟抗性基因的、基于营养缺陷型互补的 表达系统，以实现治疗肽在益生菌菌株中的稳定和高水平表达。 L.（2）开发和验证用于生物遏制的诱导型开关的有效性和稳定性 转基因L.验证工程益生菌的功效和稳定性 在动物模型中表达具有内置诱导型β-开关的治疗肽。如果成功，平台 在这个项目中开发的模块化设计将适用于数百种益生乳酸菌 已经显示出特定物种的好处，以及提供其他PPD。这种药物输送系统将是 适用于许多人类疾病-许多慢性和慢性-从代谢，心血管和 免疫功能障碍导致衰老和神经退行性疾病。它还将提供一个强大的研究工具， 调节肠道微生物群的结构和功能，以进一步研究微生物-微生物之间的相互作用 和宿主-微生物相互作用，并阐明参与健康促进作用的分子机制 这些益生菌。

PROJECT SUMMARY / ABSTRACT Peptide and protein drugs (PPDs) are potent therapeutics to treat a broad spectrum of diseases. Parenteral administration is limited due to their poor bioavailability and stability. Oral delivery of PPDs faces additional obstacles in the gastrointestinal tract. We have developed an innovative, probiotics-based platform technology for the oral delivery of therapeutic PPDs that targets a number of challenging chronic diseases. This delivery system is robust, highly efficient, cost effective, easy to administer and safe. Our recent published results provide proof-of concept for the feasibility of using Lactobacillus as a live vector for the delivery of peptide and protein with enhanced tissue bioavailability and efficacy. However, our current platform has several limitations for clinical application, which include the use of an antibiotic gene as selection marker, plasmid segregation instability and lack of built-in biocontainment strategy, which may raise safety concern with engineered microbes disseminated into the environment. The goal of this project is to develop a next generation probiotics-based platform for the delivery of peptide and protein therapeutics with more stable and effective biocontainment. Specifically, we will (1) construct, characterize and validate an antibiotic-resistance-gene free, auxotrophic complementation-based expression system to achieve stable and high-level expression of the therapeutic peptide in a probiotic strain of L. paracasei; (2) develop and validate the efficacy and stability of inducible kill-switches for the biocontainment of genetically engineered L. paracasei; and (3) validate the efficacy and stability of engineered probiotics expressing the therapeutic peptide with a built-in inducible kill-switch in animal models. If successful, the platform developed in this project with its modular design will be applicable to hundreds of probiotic Lactobacillus species that have shown species-specific benefits, as well as to deliver other PPDs. This drug delivery system will be applicable to numerous human diseases – many chronic and recalcitrant - from metabolic, cardiovascular and immune dysfunction to aging and neurodegenerative diseases. It will also provide a powerful research tool to modulate the structure and function of the gut microbiota, to further investigate the interplay of microbe-microbe and host-microbe interactions, and elucidate the molecular mechanisms involved in the health-promoting effects of these probiotics.