Engineering next generation probiotics for delivery of therapeutics

项目摘要/摘要 多肽和蛋白质药物(PPD)是治疗多种疾病的有效疗法。肠外注射 由于其生物利用度和稳定性较差，给药受到限制。口服PPD面临额外的挑战 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 用于口服针对一些具有挑战性的慢性疾病的治疗性PPD。这批货 系统健壮、高效、性价比高、易于管理、安全。我们最近发表的结果提供了 利用乳杆菌作为运送多肽和蛋白质的活载体的可行性的概念证明 具有增强的组织生物利用度和有效性。然而，我们目前的平台在临床上有几个限制 应用，包括使用抗生素基因作为选择标记，质粒分离不稳定性和 缺乏内置的生物遏制策略，这可能会引起对工程微生物传播的安全担忧 进入到环境中。该项目的目标是开发下一代基于益生菌的平台，用于 提供更稳定和有效的生物遏制的多肽和蛋白质疗法。具体来说，我们将 (1)构建、鉴定和验证一种基于营养缺乏性互补的无抗生素抗性基因 在益生菌菌株中实现治疗性多肽的稳定高效表达的表达系统 (2)开发和验证用于生物遏制的可诱导杀伤开关的有效性和稳定性。 以及(3)验证基因工程益生菌的有效性和稳定性 在动物模型中表达具有内置可诱导杀伤开关的治疗性多肽。如果成功，平台将 在该项目中开发的模块化设计将适用于数百种益生菌乳杆菌 已经显示出特定物种的益处，以及交付其他PPD。这个药物输送系统将是 适用于多种人类疾病--许多慢性和顽固性疾病--来自代谢、心血管和 免疫功能障碍与衰老和神经退行性疾病有关。它还将提供一个强大的研究工具来 调节肠道微生物区系的结构和功能，进一步研究微生物与微生物的相互作用 和宿主-微生物的相互作用，并阐明了促进健康作用的分子机制 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.