Synthetic biology refers to designing and constructing new biological components, devices, and systems based on certain rules and existing knowledge, as well as redesigning existing natural biological systems to serve human-specific purposes. Simply put, synthetic biology aims to solve problems related to energy, materials, health, and the environment by artificially designing and constructing biological systems that do not exist in nature.
Figure.1 Synthetic biology.
There are three crucial concepts in synthetic biology: standardization, decoupling, and abstraction. In other words, the biological systems we design should be divided into modules, each of which can perform a certain function independently (cohesion), and the modules can work together to achieve a synergistic effect (coupling).
We divide the designed plasmid and Salmonella into multiple functional modules, each of which has its own corresponding function. These modules can work together to achieve a synergistic effect, which meets the requirements of synthetic biology and the iGEM competition.
Figure.2 An overview to the project design
The shSTAT3/shPD-L1 plasmid is the core of our project design. It is based on the RNAi mechanism to silence the expression of STAT3 and PD-L1 genes, activate the body's own immune response, and inhibit tumor growth.
Our modification of Salmonella involves two modules: knocking out the asnB gene to activate its own immune system and enhance T cell-mediated tumor killing; and inserting the flagellar protein gene flaB to enhance the strain's ability to adhere, invade, and colonize deep tissues.
Next, we will provide a detailed description of the design for each module.
2.1 PD-L1/PD-1
Translation: PD-L1 is a molecule present on the surface of cancer cells that inhibits the immune pathway and acts as a receptor for Programmed Death-1 (PD-1). PD-1 is an important immune checkpoint molecule and a member of the immunoglobulin superfamily, widely expressed on the surface of immune cells [1]. PD-L1 is also expressed on various cell types, including antigen-presenting cells (APCs), T cells, B cells, and actively responds to pro-inflammatory cytokine signaling such as IFN-γ, STAT family, and IFN regulatory factor 1 (IRF1) [2]. The binding of PD-L1 on tumor cells to PD-1 initiates programmed cell death in T cells, leading to immune evasion by tumor cells. However, when PD-L1 expression is too low, it is associated with autoimmune diseases [3].
The expression of PD-L1 is associated with poor prognosis in many cancers. Literature reports suggest that PD-L1 may be involved in cell proliferation and tumor progression, which could be a contributing factor to poor prognosis[4]. As a molecular marker of cancer, PD-L1 plays a significant role in disease diagnosis, treatment, and prognosis.
Figure.3 PD-L1 initiate programmed cell death in T cells[5].
2.2 shRNA silences PD-L1 gene expression
shRNA, short for small hairpin RNA, is an RNA sequence with a tight hairpin turn that is often used for RNA interference to silence the expression of target genes.
Figure.3 PD-L1 initiate programmed cell death in T cells[5].
The plasmid（Gene sequence） we designed can transcribe shRNA targeting PD-L1, controlled by the U6 promoter. Under the control of the U6 promoter, the antisense sequence of PD-L1 is transcribed to generate pre-shRNA. The pre-shRNA needs to be processed by Drosha and its double-stranded RNA binding partner DGCR8 to form shRNA. The shRNA is then cleaved by Dicer and TRBP/PACT enzymes, removing the hairpin structure and generating a double-stranded siRNA with two free bases at each 3' end. The functional strand (guide strand) of these siRNAs is loaded into the RISC complex, which guides it to complementary target sites and catalyzes the cleavage and degradation of the target mRNA. The guide sequence of an shRNA molecule needs to be perfectly complementary to the target sequence.
Figure.5 shRNA target PD-L1.
3.1 STAT3
STAT3 is a member of the signal transducer and activator of transcription (STAT) family, which also includes STAT1, STAT2, STAT4, STAT5a, STAT5b and STAT6 [6]. They are structurally similar and contain an amino terminal for the synergistic binding of STAT proteins with multiple common DNA sites; a coiled helix domain for recruiting STAT3 to the receptor and its subsequent phosphorylation, dimerization and nuclear translocation; an DNA binding domain A SH2 domain is highly conserved and mediates intermolecular connections, and a transcriptional activation domain [7]. STAT3 is involved in biological processes including proliferation, metastasis, angiogenesis, immune response and chemical resistance. Continuous STAT3 activation often occurs in cancer ,while it is related to tumor progression and poor prognosis[8].
3.2 STAT3 and tumor
It is known to all that STAT3 signaling pathway is involved in the occurrence and development of many kinds of tumors. In colorectal cancer, the constitutive activation of STAT3 increases tumor cell viability and promotes tumor cell proliferation by up-regulating the expression of Cyclin D1, Survivin and Myc.
A large number of studies have shown that STAT3 can directly enhance the expression of VEGF to drive tumor angiogenesis and growth[9]. In addition, STAT3 can also reduce the production of ROS by changing the process of oxidative phosphorylation and glycolysis in cancer cells, which is beneficial to the growth of cancer cells in vivo[10]. At the same time, abnormally activated STAT3 can trigger immune escape or inhibit the anti-tumor ability of DC. It has been reported that STAT3 can bind directly to the promoter region of PD-L1, thus promoting the transcription and expression of PD-L1, which suggests that STAT3 may participate in tumor cell immune escape through PD-L1, which provides a feasible idea for follow-up research.
Figure.6 STAT3 family[11].
3.3 RNAi interferes with the silencing STAT3 mechanism
The plasmid we designed can transcribe the shRNA targeting STAT3, which is controlled by H1 promoter. Under the control of the H1 promoter, the antisense sequence of STAT3 will be transcribed to produce pre-shRNA,pre-shRNA, which needs to be processed with Drosha and its double-stranded RNA combined with chaperone DGCR8 to form shRNA,shRNA, which is then digested by Dicer and TRBP/PACT, and the hairpin structure is removed to produce double-stranded siRNA at two 3 'ends. The functional chains of these siRNA (boot chains) are then loaded into the RISC complex, and the RISC complex guides them to the complementary target site and catalyzes the cutting and degradation of the target mRNA. The guide sequence of a shRNA molecule is designed to complement the target sequence perfectly.
Figure.7 shRNA target STAT3.
We plan to express shSTAT3 and shPD-L1 simultaneously in one plasmid, which can inhibit tumor growth by a dual mechanism.
Figure.8 Dual-targeting gene element.
Figure.9 Our plasmid.
In the previous module, we inhibited tumor proliferation through the RNAi mechanism. The next issue we are considering is how to deliver the plasmid into the cells.
After extensive literature review, we have decided to use Salmonella as the vector to deliver the plasmid into tumor cells.
Salmonella is a Gram-negative, facultative anaerobic, non-spore-forming rod-shaped bacterium, covered with multiple flagella. Salmonella possesses intracellular invasiveness, allowing it to survive in both aerobic and anaerobic environments, which is advantageous for targeting tumor cells and eliminating a portion of them[12]. Salmonella can invade through mucosa and persist in host lymphoid tissues, continuously inducing strong mucosal and cellular immune responses in the host[13].
Salmonella, as a vector, not only has high efficiency and safety but also has an affordable price for the general public. Additionally, Salmonella possesses inherent anti-tumor activity, which can be further enhanced by combining it with our plasmids to improve anti-tumor efficacy.
Figure.10 Our Salmonella.
We have selected the delayed lysis strain χ11802 as the carrier for delivering the plasmids.
χ11802 is a delayed lysis strain of Salmonella. The essential genes for the synthesis of the peptidoglycan layer in χ11802 Salmonella, asdA and murA, are controlled by the arabinose promoter araCPBAD. This design prevents the strain from synthesizing crucial components of the cell wall in an environment lacking arabinose, leading to the automatic lysis of the cells and the release of the plasmids. Additionally, this process facilitates the presentation of tumor antigen peptides through increased MHC-I molecules, thereby inducing a stronger CD8+ T cell-mediated anti-tumor immune response.
However, after multiple gene deletions, the χ11802 strain exhibits decreased invasive and colonization abilities in tissues, making it difficult to achieve a sustained and effective therapeutic effect. Therefore, we plan to further modify the attenuated Salmonella χ11802 strain in order to obtain a safe, efficient, and strongly tissue-colonizing attenuated Salmonella engineered bacterium.
Next, we will carry out modifications on multiple modules of χ11802.
The shSTAT3/shPD-L1 recombinant plasmid we designed can partially relieve immunosuppression and promote the body's autoimmunity. We considered to make modifications in Salmonella to further promote the body's autoimmunity.
asnB is a synthesis gene of L-asparaginase Ⅱ, which is highly conserved in Gram-negative bacteria. The enzyme catalyzes the hydrolysis of L-asparagine to aspartate and ammonia. The catabolism of L-asparagine can promote the survival and replication of Salmonella, and contribute to the virulence of Salmonella[14]
Moreover, studies have shown that L-asparaginase II plays an important role in infection and immunity. It is a necessary enzyme to inhibit T cell proliferation, cytokine production and proliferation, and down-regulate T cell receptor expression. Therefore, the knockout of Salmonella asnB gene can also enhance T cell activation and anti-tumor activity[15]. L-asparagine depletion leads to downregulation of TCR-β expression, Interferon-γ (IFN-γ) and IL-2 production, thereby inhibiting T cell proliferation, and Salmonella (STM3106) lacking L-asparaginase II gene shows reduced virulence in vivo. The amount of liver colonization was significantly less than that of the wild type [15]. Thus, the elimination of L-asparaginase Ⅱ synthesis gene asnB can promote autoimmunity and improve the efficiency of tumor cell clearance.
Figure.11 Knocking out asnB
Our first step in modifying Salmonella is to knock out the asnB gene.
7.1 The movement of bacteria
Like most bacteria,Salmonella rely on flagella for locomotion. Flagella emerge from the bacterial cell and extend beyond its surface. Most Salmonella bacteria possess flagella of a basal body motor embedded in the bacterial membrane, a connecting structure known as the flagellar hook, and a filament composed of multiple flagellin proteins. The flagellar motor is widely regarded as one of nature's most sophisticated molecular engines, comprising various components such as rods, outer membrane rings, periplasmic rings, MS rings, and an export apparatus, which is responsible for transporting hydrogen ions via proton pumps to facilitate rotation. This elongated coil assumes a spiral shape that imparts rotational force enabling cellular movement[16].
Figure.12 A picture of Flagella. Picture comes from the website of Xinlang.
Figure.13 Flagella and their structure.[16]
7.2 flaB
After reducing the toxicity of Salmonella, its impact on normal cells is diminished, thereby enhancing its ability to kill tumor cells. However, due to weak colonization capacity, the efficacy of killing may not be efficient. The flaB gene from Vibrio vulnificus (a Gram-negative bacterium) produces a more potent flagellin[17]. Six flagellin genes (fla B, C, D, E and F) jointly regulate bacterial adhesion and toxicity . Mutations in these genes significantly reduce bacterial motility, adhesion and cytotoxicity with flaB and flaD having the greatest influence leading to a marked reduction in adhesion and cytotoxicity towards Hela cells (a malignant tumor cell line)[18]. FlaB enhances biofilm generation ability indicating that it can enhance the ability of the strain to invade the tumor cells.
Figure.14 Lack of flaB leads to reduced motility[16]
Moreover, studies have shown that L-asparaginase II plays an important role in infection and immunity. It is a necessary enzyme to inhibit T cell proliferation, cytokine production and proliferation, and down-regulate T cell receptor expression. Therefore, the knockout of Salmonella asnB gene can also enhance T cell activation and anti-tumor activity[15]. L-asparagine depletion leads to downregulation of TCR-β expression, Interferon-γ (IFN-γ) and IL-2 production, thereby inhibiting T cell proliferation, and Salmonella (STM3106) lacking L-asparaginase II gene shows reduced virulence in vivo. The amount of liver colonization was significantly less than that of the wild type [15]. Thus, the elimination of L-asparaginase Ⅱ synthesis gene asnB can promote autoimmunity and improve the efficiency of tumor cell clearance.
7.3 Inserting flaB
Salmonella primarily relies on flagella for movement. The flagellar matrix possesses a highly intricate structure, multiple rings providing the necessary kinetic energy for flagellar rotation plays a crucial role in promoting bacterial adhesion and invasion of host cells, thereby enhancing the strain's ability to colonize tumor cells. Additionally, it has been observed that flagellin can elicit an immune response, and in certain cases, injecting specific bacteria into tumors can stimulate inflammation and trigger an anti-tumor immune response, leading to tumor eradication. During the initial stages of bacterial colonization and proliferation within the tumor microenvironment, bacterial ligands also act as immune adjuvants by activating neutrophils and other components of innate immunity, contributing to the clearance of tumor-targeted bacteria. By incorporating flaB gene encoding Vibrio vulnificus' flagellin expression into attenuated Salmonella typhimur-like receptors (TLR4 and TLR5) are more effectively able to recognize bacterial lipopolysaccharide (LPS), a component of Gram-negative cell walls. This synergistic activation stimulates innate immune responses; TLR4 recognizes LPS from Salmonella Typhimurium while TLR5 recognizes secreted flaB protein. Consequently, macrophages and neutrophils infiltrate significantly within the tumor microenvironment resulting in more precise killing of tumor cells [17][19]. Therefore, insertion of flaB gene into Salmonella enhances its invasive potential towards tumor cells.
Figure.15 flaB activates the immune response[17].
Figure.16 Insertion of the flaB gene.
Now we have designed a comprehensive solution to address the challenges that hinder current colorectal cancer treatment, including ineffective targeting of the tumor and inadequate penetration of tumor tissues.
We have designed shSTAT3/shPD-L1 plasmids to inhibit the expression of STAT3 and PD-L1 genes by degrading their mRNA, thereby promoting immune response and killing tumor cells.
We have also chosen Salmonella as a vector and made some significant improvements, including knocking out the asnB gene to activate self-immunity and enhance T cell-mediated tumor killing. We have also inserted the flagellin protein gene flaB to enhance the strain's ability to adhere, invade, and colonize deep tissues.
Therefore, our "SCI" has now been created. After knocking out the genes planned for deletion, inserting the genes, and importing the designed plasmids into Salmonella, a strain of Salmonella that can specifically kill colorectal cancer cells has been born.
A large number of studies have shown that STAT3 can directly enhance the expression of VEGF to drive tumor angiogenesis and growth[9]. In addition, STAT3 can also reduce the production of ROS by changing the process of oxidative phosphorylation and glycolysis in cancer cells, which is beneficial to the growth of cancer cells in vivo[10]. At the same time, abnormally activated STAT3 can trigger immune escape or inhibit the anti-tumor ability of DC. It has been reported that STAT3 can bind directly to the promoter region of PD-L1, thus promoting the transcription and expression of PD-L1, which suggests that STAT3 may participate in tumor cell immune escape through PD-L1, which provides a feasible idea for follow-up research.
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