Expression of Eukaryotic Payloads in Cancer Associated F. Nucleatum

Fusobacterium nucleatum (FN) species are enriched in human colorectal tumors and their presence is correlated with poor prognosis. Bacteriophage ("phage") have high intrinsic safety and specificity and offer a promising treatment strategy to target FN associated with colorectal cancer (CRC). Phage engineering allows specific delivery of anti-tumor immune stimulating payloads. A major obstacle in phage engineering is to express eukaryotic payloads in unconventional prokaryotic bacterial hosts, such as FN, while ensuring high expression and activity.

To address these challenges, a de novo molecular system was established. Initially, a shuttle vector was generated using a native FN plasmid. Two promoters that drive strong bacterial gene expression in FN were identified using both bioinformatic tools and mass spectrometry and their activity was validated using a luciferase-based detection tag.

Payload protein expression was further improved using various algorithms for exposing the ribosome binding site and optimizing codon usage and ribosome elongation speed. Biological activity of the eukaryotic payload was confirmed in vitro, validating that the FN host can express an active payload.

After determining the optimal conditions for payload expression via a plasmid, a FN phage was engineered to carry the payload within its genome. Following phage infection, payload expression by the bacterial host was observed.

Our results demonstrate the capability to develop molecular expression systems for unconventional bacterial species and to engineer phages to deliver and express eukaryotic payload proteins in their hosts. Engineered phages may be used as a novel therapeutic modality for CRC and potentially for other diseases and conditions.