A Novel Engineered Probiotic for the Prevention of Clostridium Difficile Infection

项目摘要 该项目的目标是开发一种预防梭状芽孢杆菌(C.)的新药R-4329。艰难梭菌感染 (CDI)。R-4329是一种经过改造的益生菌，可重组表达和口头传递一种独特的微生物蛋白 被称为分泌型抗原A(SAGA)，我们的团队证明它可以预防多种肠道感染， 包括艰难梭菌，通过增强肠道膜屏障1、2的完整性/功能性。 艰难梭菌是一种普遍存在的厌氧革兰氏阳性细菌，它能产生孢子并对 环境压力和一线抗生素，如克林霉素7。艰难梭菌致病菌株的秘密 毒素(A和B)破坏肠道上皮细胞，导致炎性结肠炎、严重腹泻、 腹痛，流感样症状，以及可能的死亡7。艰难梭菌感染(CDI)通常发生在 抗生素-当个体的内源性微生物区系改变或严重减少时的治疗。自2000年以来， CDI发生率从不到15万例增加到25万例以上，已成为世界上最大的 在美国，最重要的医院获得性感染需要治疗的经济负担超过10亿美元。 艰难梭菌一旦在结肠中定居，就很难用最后的抗生素根除。 (万古霉素)，从而导致CDI和炎症性结肠炎的终身复发。卓有成效的 治疗CDI的方法一直是粪便微生物区系移植(FMT)疗法，但这种方法 异质性和不明确的治疗方法，其安全性仍然是一个令人担忧的问题，需要专门的FDA 批准8.迫切需要新的和定义明确的疗法来预防和治疗复发的CDI。 口服益生菌提供了一种令人兴奋的替代方案来管理CDI和乳杆菌(嗜酸菌、干酪、 植物)已被探索用来抑制肠道病原体感染和减轻抗生素相关 腹泻。不幸的是，作用机制一直很难理解，只有适度的有益影响。 它们的临床用途的背景10。然而，这些最近的研究表明，改良的益生菌与 定向和定向的抗感染功能在控制CDI方面可能是有用的。事实上，我们最近的结果 发表在《科学》杂志上的文章表明，对益生菌进行工程设计或重新编程可以 重组表达的SAGA产生了对肠道感染更具保护性的功能，包括CDI2。 我们的目标是开发一种新的益生菌菌株来传递重组SAGA(称为R-4329)作为口服 使用自然起作用以诱导保护性调节信号的药物。此第二阶段SBIR 应用的目的是在成功的基础上再接再厉，将R-4329推向临床测试。具体目标 1)优化R-4329上游工艺开发，创建GMP主细胞库；2)制造 R-4329和开发GLP分析产品释放/稳定性分析，3)完成R-4329的剂量优化 和基于生物标记物的小鼠PD研究，4)在小鼠身上进行R-4329 GLP毒理学研究，以支持我们的IND 归档。

Project Summary The goal of this project is to develop a novel drug, R-4329, for prevention of Clostridium (C.) difficile infection (CDI). R-4329 is a probiotic engineered to recombinantly express and orally deliver a unique microbial protein known as secreted antigen A (SagA) which our team showed can protect against multiple enteric infections, including C. difficile, by enhancing the integrity/functionality of the gut membrane barrier1, 2. C. difficile is a ubiquitous anaerobic Gram-positive bacterium that can sporulate and become highly resistant to environment stresses and frontline antibiotics such as clindamycin7. Pathogenic strains of C. difficile secret toxins (A and B) that damage intestinal epithelial cells, resulting in inflammatory colitis, severe diarrhea, abdominal pain, flu-like symptoms, and possible death7. C. difficile infection (CDI) often occurs following antibiotic-treatment when the endogenous microflora of individuals is altered or severely reduced. Since 2000, the rate of CDI has increased from below 150,000 to over 250,000 cases and has been become one of the most significant hospital-acquired infections with an economic burden of over $1 billion to treat in the U.S.7. Once C. difficile establishes residence in the colon, it is difficult to eradicate with last resort antibiotics (vancomycin) and consequently results in lifelong relapses of CDI and inflammatory colitis. An effective approach to treating CDI has been fecal microbiota transplant (FMT) therapy but this approach is heterogeneous and a poorly defined therapeutic, for which its safety is still a concern requiring special FDA approval8. New and well-defined therapeutics are desperately needed to prevent and treat recurrent CDI. Oral probiotics offer an exciting alternative to manage CDI and strains of Lactobacillus (acidophilus, casei, reuteri, plantarum) have been explored to inhibit enteric pathogen infection and mitigate antibiotic-associated diarrhea9. Unfortunately, mechanism of action has been difficult to understand with modest beneficial effects in the context of their clinical utility10. However, these recent studies suggest that improved probiotics with directed and targeted anti-infective functionality may be useful at controlling CDI. Indeed, our recent results published in the journal Science demonstrate that engineering or ‘reprogramming’ of probiotics to recombinantly express SagA yields a more protective functionality against enteric infections, including CDI2. Our goal is to develop a novel probiotic strain to deliver recombinant SagA (referred to as R-4329) as an orally administered drug that functions naturally to induce protective regulatory signals. This Phase II SBIR application is intended to build upon success and advance R-4329 towards clinical testing. The specific aims are: 1) optimize R-4329 upstream process development and create a GMP master cell bank, 2) manufacture R-4329 and develop GLP analytical product release/stability assays, 3) complete R-4329 dose optimization and biomarker-based PD studies in mice, 4) perform R-4329 GLP toxicology studies in mice to support our IND filing.