Engineered probiotic bacteria for colorectal cancer immunoprevention and interception

PROJECT SUMMARY Synthetic biology is driving a new era of medicine through the genetic programming of living cells. One particular focus has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo. Since colorectal cancer (CRC) initiation and progression are significantly influenced by interactions between intestinal microbes and the mucosal immune system, these interactions can be specifically modulated with engineered probiotics for immoprevention and interception. Our laboratories have demonstrated that a probiotic bacteria, E. coli Nissle 1917 (EcN), when orally administered, selectively colonizes colorectal polyps and adenomas versus normal tissue in murine models, and colonizes tumors in CRC patients. We additionally demonstrated that engineered EcN can produce diagnostic or therapeutic moleclues in a model of early stage CRC. This UG3/UH3 proposal aims to expand upon this approach by engineering EcN to identify precancerous lesions, characterize novel stage-specific immune targets, and rationally design immunomodulatory strains for CRC prevention and interception. In the UG3 Phase, we will employ a tamoxifen-inducible model of biallelic adenomatous polyposis coli (APC) gene deletion (ApcFl/FlCdx2-CreERT2) to characterize the dynamics of CRC stage-specific EcN colonization and immune cell infiltration/activation. We will construct a library of violacein-producing EcN to identify multiple stages of CRC, from precancerous adenoma to invasive carcinoma. We will then determine the kinetics of adenoma progression following biallelic deletion of Apc and assess the ability of EcN to colonize precancerous and cancerous lesions. Additionally, we will characterize the immune cell repertoire of EcN-colonized precancerous lesions of varying stages using spatial transcriptomics. In the UH3 Phase, we will design and engineer immunotherapeutic bacteria for CRC prevention and interception. We will create EcN strains expressing inflammatory payloads (e.g., GM-CSF and IFNg) and deliver them to mice prior to Apc deletion to determine if EcN-based immunotherapy can prevent the development of neoplastic lesions. Furthermore, to intercept CRC progression, we will design EcN strains expressing immunomodulatory payloads (e.g., GM-CSF and anti-CTLA-4 and anti-PD-L1 blocking nanobodies) and deliver them to mice after verifying the presence of precancerous polyps. Finally, we will examine the durability of the immune response elicited by engineered bacteria by challenging mice with organoids reflective of advancing stages of carcinogenesis. The overall goal of this proposal is to develop effective probiotic-based immunotherapies that prevent and halt CRC progression by harnessing the power of engineered EcN strains. These novel approaches have the potential to significantly improve CRC outcomes and provide new avenues for cancer prevention and interception.