Rise Therapeutics, LLC SBIR Phase I Award, July 2022

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.