A Novel Engineered Probiotic for the Prevention of Clostridium Difficile Infection

项目摘要 本项目的目标是开发一种新的药物R-4329，用于预防梭菌（C.）菌感染 （土发委会）。R-4329是一种益生菌，经工程改造重组表达和口服递送独特的微生物蛋白 被称为分泌抗原A（佐贺），我们的研究小组发现它可以防止多种肠道感染， 包括C. difficile，通过增强肠膜屏障的完整性/功能1，2。 C.艰难梭菌是一种普遍存在的厌氧革兰氏阳性细菌，其可以形成孢子并变得高度耐药。 环境压力和一线抗生素，如克林霉素7。病原菌株C.难明的秘密 毒素（A和B），破坏肠上皮细胞，导致炎症性结肠炎，严重腹泻， 腹痛、流感样症状和可能的死亡7。C.艰难梭菌感染（CDI）通常发生在 当个体的内源性微生物区系发生改变或严重减少时的驱虫治疗。自2000年以来， CDI的发病率从15万例以下上升到25万例以上，已成为世界上 在美国，治疗最严重的医院获得性感染的经济负担超过10亿美元。 一旦C.艰难梭菌在结肠中建立居所，很难用最后的抗生素根除 （万古霉素），并因此导致CDI和炎性结肠炎的终身复发。有效 治疗CDI的方法是粪便微生物群移植（FMT）疗法，但这种方法 异质性和定义不明确的治疗方法，其安全性仍然是需要FDA特别关注的问题 批准8.迫切需要新的和明确的治疗方法来预防和治疗复发性CDI。 口服益生菌提供了一种令人兴奋的替代方案来管理CDI和乳杆菌菌株（嗜酸乳杆菌，干酪， reuteri，plantarum）来抑制肠道病原体感染并减轻与肠道病原体相关的 夏威夷9.不幸的是，作用机制一直难以理解，在治疗中具有适度的有益效果。 其临床效用的背景10.然而，这些最近的研究表明， 定向和靶向抗感染功能可用于控制CDI。事实上，我们最近的研究结果 发表在《科学》杂志上的一项研究表明，益生菌的工程或“重新编程”， 重组表达的佐贺产生针对包括CDI 2在内的肠道感染的更强的保护功能。 我们的目标是开发一种新的益生菌菌株，以口服形式递送重组佐贺（称为R-4329）。 自然地起作用以诱导保护性调节信号的给药药物。第二阶段SBIR 该应用旨在建立在成功的基础上，并将R-4329推向临床测试。具体目标 包括：1）优化R-4329上游工艺开发并创建GMP主细胞库，2）生产 R-4329并开发GLP分析产品放行/稳定性试验，3）完成R-4329剂量优化 和基于生物标志物的小鼠PD研究，4）在小鼠中进行R-4329 GLP毒理学研究，以支持我们的IND 备案

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.