We are working to develop a safer and more efficient way to eliminate solid tumors with engineered Salmonella VNP20009. We added recognition system, delivery system (for plasmids), and killing system to this attenuated Salmonella strain, and established a model to predict the population change of the engineered Salmonella VNP20009 after injected into human body.

This system includes a surface display system for Gram-negative bacteria like Salmonella, and a Single-chain variable fragment (scFv). The surface display system can display scFv at the surface of Salmonella so that scFv can recognize the specific antigen on the surface of cancer cells.

Single-chain variable fragments (scFv) are small antibody fragments that consist of the variable regions of the heavy and light chains, connected by a flexible peptide linker. These fragments retain the antigen-binding ability of full-sized antibodies while being smaller and more versatile for therapeutic applications. To enhance Salmonella’s targeting efficacy, we expressed a HER2 scFv(BBa_K5363017) on the surface of VNP20009 to target breast cancer cells. (Bereta et al., 2007)

As for the surface display system, we chose Lpp-OmpA (BBa_K103006), which is a signal peptide can be used as an outer-membrane-targeting anchor in E. coli and Salmonella. And we compare the display efficiency of it with Lpp-OmpA-1(BBa_K5363014) mentioned in (Francisco et al., 1992).

Moreover, we tried Non-OmpA(BBa_K5363016) as surface display system, which consists of OmpA (amino acids 46–159) devoid of Lpp. (Jeiranikhameneh et al., 2017)

Upon the scFv binding antigens on the surface of cancer cells, VNP20009 invades the tumor cells, transitioning to live inside vesicles in the invaded cell. To ensure it delivers its therapeutic payload, we engineered a self-lysing system triggered by intracellular signals. Specifically, we identified a SPI2 promoter that activates only within the intracellular environment. We then coupled the PsseJ promoter(BBa_K5363013) (a promoter for SPI2-associated genes) with Lysis gene E (LyseE)(BBa_K5363015) from bacteriophage φX174, inducing rapid bacterial lysis upon intracellular activation. (Raman et al., 2021) Then the plasmids we added into Salmonella can be released to the cytosol of cancer cells.

After self-lysis, VNP20009 releases its therapeutic payload into the cancer cell’s cytoplasm. This payload comprises three plasmids, one of which contains the BAX gene(BBa_K5363000). Once translated, BAX protein localizes to the mitochondrial outer membrane, where it forms pores that release cytochrome c and other apoptotic factors, ultimately leading to cancer cell apoptosis. Since BAX protein has risks to induce apoptosis in normal cells as well, we utilized STIFs to manipulate the mRNA circularization process, to achieve efficient translational control over the expression of the effector gene (BAX)(BBa_K5363000) in human cells, following the "closed-loop" model of translation. (Shao et al., 2024)

An expression vector was designed to produce a synthetic mRNA transcript containing a control region with RBP-specific aptamers in the 3'-UTR, specifically MS2-box motifs(BBa_K5363001). To minimize background activity from BAX mRNA translation independent of STIFs, trans-acting shRNA-binding sites were replaced with cis-acting hammerhead ribozyme (HHR) motifs(BBa_K5363002), which induce spontaneous self-excision of the natural poly(A) signal. An antibody linked to rotaviral non-structural protein 3 (NSP3)(BBa_K5363007), an eIF4F-binding protein (eIFBP), recognizes the intracellular signal, while another antibody recognizing the same signal is linked to bacteriophage-derived MS2 coat protein (MCP)(BBa_K5363003), which binds to tandem repeats of cognate MS2-box motifs(BBa_K5363001). These plasmids form a killing system activated by STIF. If the two antibodies recognize the specific signal, forming a loop with BAX mRNA connected to NSP3-antibody-signal-antibody-MCP, translation of BAX protein will occur to induce cell apoptosis of cancer cells.