A Novel Probiotic Drug Delivery Platform for Oral Administration of Protein-Based Therapies

项目摘要 口服以蛋白质为基础的药物(生物制剂)将极大地改进和简化我们治疗和 预防疾病1-4。然而，生物制剂在口服时稳定性低，半衰期短。 形成并对肠道的腐蚀性条件敏感。通过细菌口服生物制剂 可以绕过这些障碍。与传统的药物传递系统相比，细菌表现出 就地生产生物制剂的优势、靶向能力和对不同有效载荷的适应性。通过 利用肠道微生物群-宿主免疫细胞的相互作用，治疗药物的细菌输送也可以导致 调节宿主免疫反应8-10。Rise Treeutics开创了小说发展的先河 乳酸益生菌重组菌株展示了对 在15个以上的动物模型上进行了实验。在这些研究中，口服我们的重组益生菌能够 有针对性地参与免疫系统的特定受体，以重新平衡宿主免疫系统。 在我们内部GMP制造基础设施的支持下，这些重组菌株现在正在进入 人体临床试验。 乳球菌(L.)乳酸是最有前途的生物制剂输送细菌平台之一。乳酸乳杆菌是一种非 致病的，GRAS指定的，革兰氏阳性细菌，对人类具有非凡的安全性， 包括以转基因形式使用来输送治疗性蛋白质。除了口服药外 生物制品、微生物系统是重组蛋白工业化生产的标志11。工程菌L. 乳酸也被用来表达工业应用中的异源蛋白。而大肠埃希菌是 金标准，革兰氏阳性细菌，如乳酸杆菌，由于更简单的纯化而获得了吸引力 革兰氏阴性菌12、13不支持的过程和重要蛋白质的生化途径。 然而，乳酸乳杆菌平台受到制造限制，细胞生物反应器密度远远超过 低于水平需要用于某些商业目的。这一限制增加了商品的成本，阻止了使用 对人体有更高的剂量，并限制其在工业蛋白质表达应用中的使用。实现高产 纯净和稳定的重组菌密度是商业上可行和成功的关键 产品开发。 为了解决这一挑战，我们将使用合成生物学方法来修改关键的生长调控途径，以 改善我们前景看好的乳酸菌平台的可制造性。乳酸乳杆菌适应环境环境 活下去。在生物反应器中，乳酸乳杆菌具有受控的新陈代谢，允许利用某些类型的 糖分。当受到压力时，乳酸乳杆菌在发酵后期通常会切换到 有氧代谢，不利于细菌的生存。在此应用程序中，我们建议 设计一种新的乳酸乳杆菌底盘，具有更好的代谢和呼吸能力，以增强和改进 细菌发酵和细胞生物量。

Project Summary Oral delivery of protein-based drugs (‘biologics’) would vastly improve and simplify our approach to treat and prevent disease1-4. However, biologics suffer from low stability and short half-life when administered in oral form and are sensitive to the caustic conditions of the intestinal tract5-7. Oral delivery of biologics via bacteria can circumvent these hurdles. Compared to conventional drug delivery systems, bacteria exhibit the advantages of in situ production of biologicals, targeting ability, and amenability to different payloads. By leveraging gut microbiome-host immune cell intertwining, bacteria delivery of therapeutics can also lead to modulation of the host immune response8-10. Rise Therapeutics has pioneered the developed of novel recombinant strains of the probiotic Lactococcus lactis demonstrating tremendous proof-of-concept for the approach in over 15 animal models. In these studies, oral delivery of our recombinant probiotics enables targeted engagement of specific receptors of the immune system to rebalance the host immune repertoire. Enabled by our in-house GMP manufacturing infrastructure, these recombinant strains are now entering human clinical testing. Lactococcus (L.) lactis is one of the most promising bacterial platforms for biologics delivery. L. lactis is a non- pathogenic, GRAS-designated, Gram-positive bacterium with an extraordinary safety profile in humans, including use in genetically modified forms to delivery therapeutic protein. In addition to oral delivery of biologics, microbial systems are a hallmark of industrial production of recombinant proteins11. Engineered L. lactis has also been employed to express heterologous proteins for industrial applications. While E. coli is the gold standard, Gram positive bacteria, like L. lactis are gaining traction due to much simpler purification processes and important proteins` biochemical pathways not supported by Gram negative strains 12, 13. However, L. lactis platform suffers from manufacturing limitations, where cellular bioreactor densities top off far below levels require for some commercial purposes. This limitation increases cost of goods, prevents use of higher doses in humans, and curb its use for industrial protein expression applications. Achieving high yield densities of pure and stable recombinant bacteria is essential to enabling commercially viable and successful product development. To solve this challenge, we will use synthetic biology approaches to modify key growth regulatory pathways to improve manufacturability of our promising L. lactis platform. L. lactis adapts to environmental circumstances to survive. In a bioreactor setting, L. lactis has a controlled metabolism that permits the utilization of certain type of sugars. When under stress, and event that typically occurs at late stage of fermentation, L. lactis switches to an aerobic metabolism, which is detrimental to the bacteria survival. In this application, we proposed to engineer a new L. lactis chassis with improved metabolic and respiration capacities to augment and improve bacterial fermentation and cellular biomass.