1. Overview

According to the World Health Organization (WHO), the number of newly diagnosed cancer cases worldwide in 2020 was approximately 19.29 million, with the mortality rate increasing year by year. As traditional therapies, surgery and radiotherapy are not ideal for advanced malignant tumors while chemotherapy suffers from resistance during the treatment process, increasing the risk of recurrence and serious side effects due to the poor selectivity of chemotherapeutic agents. To increase the sensitivity of tumor cells to chemotherapy, RNA interference (RNAi) technology is adapted to down-regulate resistance-causing genes. Attenuated Salmonella presents several advantages, including its tumor targeting ability, intratumoral penetration, native bacterial cytotoxicity, low cost and its capability of immunostimulation, making it an ideal delivery vector. In addition, by genetically engineering the bacteria, it is possible to endow them with more beneficial properties. Meanwhile, loading the chemotherapeutic drug into nanoparticles (NPs) can enhance effective therapeutic responses and reduce adverse side effects.

We therefore designed a combination therapy that integrates bacteriotherapy of engineered bacteria delivering shRNA with nanoparticle-mediated chemotherapy to reduce tumor chemotherapy resistance and improve targeting. We named our drug BIOTARGET.

2. Chassis Organism

Depending on our needs, our chassis must meet the following criteria:

High invasiveness

Attenuated Salmonella is an intracellular invasive Gram-negative parthenogenetic anaerobic bacterium which has a tumor lysing effect by directly attacking tumor cells and leading to their death through its killing effects, activating the host immune system through its immunogenicity to enhance the therapeutic effects and competing with tumor cells for nutrients and growth factors, thus inhibiting tumor growth.

Tumor targeting ability

The concentration of attenuated Salmonella at tumor sites is 1000 times higher than in normal tissues.

Expression and secretion of proteins

Attenuated Salmonella can express and secrete proteins efficiently.

Induction of gene silencing in host cells

Engineered Salmonella is able to invade tumor cells and produces shRNAs targeting chemoresistance genes. Then shRNA is released into the cytoplasm with the help of listeriolysin O, encoded by the hlyA gene, which ultimately activates the RNAi pathway to induce gene silencing.

Adaptation to different types of tumors

In our experiments, our attenuated Salmonella increased its targeting to breast and ovarian cancer cells by expressing RGD peptide. By replacing the RGD peptide with other tumor-targeting peptides or antibody fragments and the shRNA sequence on the plasmid with various resistance genes corresponding to different tumor, the tumor targeting of the therapeutic system can be further improved and the therapeutic needs of different types of tumors can be achieved.

Safety

Attenuated Salmonella has been safely used as a live attenuated vaccine in humans for more than 20 years. Additionally, we have modified its genome to further reduce its virulence.

Adaptation to the tumor environment

Due to the vigorous metabolism, rich nutrients, rapid growth of the tumor and relatively insufficient blood supply, tumor sites present hypoxic and necrotic zones, which are favourable for the accumulation and multiplying of Salmonella as it tends to have low oxygen metabolism.

Our project can be divided into four modules:

- Targeting Module

- Gene Regulation Module

- Safety Module

- Drug Delivery Module

3. Targeting module

We screened tumor markers and found that in most tumors, integrin expression is abnormally elevated. Therefore, we would like to target tumors using RGD peptides. Despite various functional modified types of RGD (e.g., c(RGDfK), iRGD), their structural stability and binding ability to integrins remained unknown when both modified and ordinary RGD peptides are loaded onto bacterial extra-membrane presentation system. Therefore, we wanted to determine the stability through structural simulations, as well as to compare the binding ability of the regular RGD peptide and the modified RGD peptide to integrins through molecular docking. In this process, we found that the affinity could not be fully judged by molecular docking alone. Therefore, we further used molecular dynamics simulations of different types of RGD peptides binding to integrins to select RGD peptides. Finally, we concluded that ordinary RGD peptides are sufficient for targeting in terms of affinity and stability.

We used the Lpp-OmpA structure to demonstrate RGD peptides on the outer membrane of Salmonella to enhance tumor-specific targeting of bacteria.

To enhance the therapeutic system's tumor targeting capabilities and to adapt it for various cancer types, the RGD peptide can be substituted with alternative tumor-homing peptides or antibody fragments.

4. Gene regulation module

We used engineered Salmonella to mediate trans-kingdom RNAi, inhibiting the expression of tumor chemoresistance genes. Briefly, engineered Salmonella is able to invade tumor cells by producing shRNAs targeting chemoresistance genes via the T7 expression cassette. The shRNA is released into the cytoplasm with the help of listeriolysin O, a product encoded by the hlyA gene. Anaerobic-induced gene expression and tumor-targeting of Salmonella could confine this process to tumor tissues to enhance the efficacy and safety of treatment.

The environment of tumor tissues is more hypoxic compared to normal tissues. In this way it is possible to restrict hlyA expression to tumor tissues also to restrict trans-kingdom RNAi to tumor tissues. This is tumor-specific trans-kingdom RNAi.

In fact, the function of this module is much more than gene regulation. We can use the T7 expression cassette to express a variety of other gene products, such as cytotoxic proteins or anti-cancer peptides, in order to expand the application of the system, not only to silence drug-resistant genes, but also to directly kill tumor cells or enhance the immune response.

5. Safety module

To further enhance the safety and efficacy of Salmonella, we employed the delayed lysis strain χ11802. In this strain, the asd and murA genes, which are crucial for bacterial cell wall synthesis, were knocked out. These two genes were added to the plasmid vector, and regulated by an arabinose promoter araC PBAD, allowing for controlled survival of Salmonella in vivo and improving its safety profile. Additionally, the msbB gene in χ11802 was deleted, and the gene encoding T7 RNA polymerase was inserted at the same locus. The msbB gene encodes myristoyl transferase, an enzyme responsible for adding myristic acid to the C3' position of lipid A in Escherichia coli (E. coli) BL21(DE3), a critical step in the lipopolysaccharide (LPS) biosynthesis pathway. This modification successfully attenuated the virulence of Salmonella while simultaneously enhancing the efficiency of shRNA expression.

For the safety switch, firstly, we want to simulate the changes in the human immune system (especially the immune changes in the peripheral blood of the human body) after a Salmonella injection. The variables are mainly the amount or concentration of Salmonella injected, and we hope to simulate changes in the amount of immune cells, or changes in immune substances (cytokines, etc.) in plasma/tissue fluids. Further, we wish to simulate the process of transcriptional regulation by hypoxic promoters, among this, the heart of the problem is the changes in gene transcription by oxygen binding to regulatory proteins. For the delayed lysis system, the main focus is to simulate the regulation of bacterial lysis by arabinose. The concentration range of arabinose stored within Salmonella after injection may exhibit nonlinear changes, which could affect the delayed lysis of Salmonella. Combining these three models, we simulate the safety situation during the actual implementation of our project.

6. Drug delivery module

One of the key challenges in chemotherapy, aside from the development of tumor drug resistance, is the side effects caused by the non-specific distribution of chemotherapeutic agents via the bloodstream, which affects healthy tissues. To address this, we aim to further refine the targeted delivery of chemotherapeutic agents, reducing off-target toxicity and minimizing the damage to normal tissues.

Chemotherapeutic drug was encapsulated within nanoparticles, and attenuated Salmonella was utilized as a carrier for the precise delivery of these nanoparticles to tumor tissues. Traditional approaches using biotin and electrostatic attraction did not yield stable attachment; hence, we employed mPEG-PLGA-PLL, which forms a chemical bond with the cell wall of the attenuated Salmonella while simultaneously encapsulating chemotherapeutic drug.

We selected mPEG-PLGA-PLL nanoparticles for chemotherapeutic drug delivery by attaching them to Salmonella. The poly (ethylene glycol) monomethyl ether (mPEG) component facilitates evasion from the reticuloendothelial system, while PLGA degrades slowly in vivo, enabling the sustained release of chemotherapeutic drug. The cationic polymer, poly(L-lysine) (PLL), provides excellent biocompatibility and tunability, aiding in the targeted and controlled release of chemotherapeutic drug. PLL is positively charged, with a flexible and stable structure that allows for easy adjustment of molecular weight. Furthermore, by introducing side chains and specific targeting groups, the polymer backbone can be modified to enhance carrier performance and ensure the slow release of chemotherapeutic drug. Customizing PLL side chains also enables targeted delivery to different sites and allows for more personalized functionalities.