Enhancing E. coli Nissle colonization with CRISPR-Cas immunity

Project Summary Inflammatory bowel disease (IBD) prevalence is rising globally and live biotherapeutic products (LBPs) have shown some success in relieving the symptoms of IBD. However, LBP efficacy has been limited in part due to inadequate engraftment and colonization. Novel technologies are needed to identify and address the factors limiting the engraftment and efficacy of LBPs in the clinic. Lytic and lysogenic phage predation has the potential to inhibit the colonization of LBP strains and obstruct their engraftment. Bacterial CRISPR-Cas systems, while extensively used for genome engineering, provide adaptive immunity against phage predation by degrading foreign DNA in a programmable and sequence specific manner. Thus, the addition of a rationally designed CRISPR-Cas system to an LBP can provide phage immunity and support bacterial survival in the presence of phage predation. Escherichia coli Nissle 1917 (EcN) is an LBP with a long history of human use for a variety of applications, including treatment of UC and as a chassis for the delivery of therapeutic payloads. However, like most LBPs, EcN variably engrafts in patients, which can limit its utility in the clinic. We have identified and isolated EcN phages from stool samples of patients with UC, and further validated the existence of these phages in in vitro experiments. This work suggests that these phages could directly prevent the survival and efficacy of this therapeutically significant strain in the clinic. Ancilia Bioscience’s long-term goal is to harness CRISPR’s natural function to develop a new class of LBPs with enhanced engraftment and therapeutic efficacy. The overall objective of this proposal is to improve EcN colonization in the presence of phage. Our central hypothesis is that phages can directly prevent the therapeutic efficacy of LBPs. By strategically engineering CRISPR, we can immunize EcN to robustly colonize the intestinal tract and increase bacterial engraftment. This proposal aims to investigate phage immunity as a method to augment EcN therapeutic efficacy and fitness in the context of IBD. These studies will be executed by Ancilia Biosciences through collaborations with Dr. Rodolphe Barrangou, Todd R. Klaenhammer Distinguished Professor at NC State University, and Dr. Casey Theriot, Associate Professor in Infectious Disease at NC State University. Furthermore, we expect these results to establish the feasibility of engineering CRISPR-Cas immunity for improved bacterial engraftment to enable the therapeutic efficacy and commercialization of LBPs for a range of applications.