Oral delivery of protein-based drugs ("biologics') would vastly improve and simplify our approach to treat andprevent disease1-4. However, biologics suffer from low stability and short half-life when administered in oralform and are sensitive to the caustic conditions of the intestinal tract5-7. Oral delivery of biologics via bacteriacan circumvent these hurdles. Compared to conventional drug delivery systems, bacteria exhibit theadvantages of in situ production of biologicals, targeting ability, and amenability to different payloads. Byleveraging gut microbiome-host immune cell intertwining, bacteria delivery of therapeutics can also lead tomodulation of the host immune response8-10. Rise Therapeutics has pioneered the developed of novelrecombinant strains of the probiotic Lactococcus lactis demonstrating tremendous proof-of-concept for theapproach in over 15 animal models. In these studies, oral delivery of our recombinant probiotics enablestargeted 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 enteringhuman 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 ofbiologics, 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 thegold standard, Gram positive bacteria, like L. lactis are gaining traction due to much simpler purificationprocesses 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 farbelow levels require for some commercial purposes. This limitation increases cost of goods, prevents use ofhigher doses in humans, and curb its use for industrial protein expression applications. Achieving high yielddensities of pure and stable recombinant bacteria is essential to enabling commercially viable and successfulproduct development.To solve this challenge, we will use synthetic biology approaches to modify key growth regulatory pathways toimprove manufacturability of our promising L. lactis platform. L. lactis adapts to environmental circumstances tosurvive. In a bioreactor setting, L. lactis has a controlled metabolism that permits the utilization of certain typeof sugars. When under stress, and event that typically occurs at late stage of fermentation, L. lactis switches toan aerobic metabolism, which is detrimental to the bacteria survival. In this application, we proposed toengineer a new L. lactis chassis with improved metabolic and respiration capacities to augment and improvebacterial fermentation and cellular biomass.
Public Health Relevance Statement: Project Narrative
Probiotic bacteria represent an ideal platform for delivering therapeutic agents orally. In particular, Lactococcus
(L.) lactis has been extensively used to express and deliver therapeutic proteins in humans with no overt
toxicity ever observed. Despite the promising data with this recombinant probiotic delivery approach,
manufacturing limitations hinder a wider adoption of this delivery platform for drug application, and for potential
industrial uses. Here, we proposed to generate a novel L. lactis chassis with improved manufacturability and
protein expression properties to enable development of a broader range of new drugs.
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