Are you familiar with cancer? While the term "cancer" is commonly used to refer to all malignant tumors, medically speaking, cancer strictly refers to malignancies that originate in epithelial tissues. It is the most prevalent type among all malignancies. Colorectal cancer, specifically carcinoma of the colon and rectum, is one of the most common malignancies in the gastrointestinal tract. In its early stages, it often presents with subtle symptoms, but as the tumor grows, it can lead to changes in bowel habits, rectal bleeding, diarrhea, alternating between diarrhea and constipation, localized abdominal pain, and other symptoms. In advanced stages, it may manifest as anemia, significant weight loss, and other systemic symptoms. Its incidence and mortality rates are second only to gastric cancer, esophageal cancer, and primary liver cancer among malignant digestive system tumors.

According to the latest global cancer data from 2020, colorectal cancer ranks third among the top ten cancers worldwide, with 1.93 million cases. It is also the second-leading cause of new cancer cases in China, with 560,000 reported cases. Several factors and precancerous conditions have been associated with its development:Epidemiological studies show that colorectal cancer has a higher incidence in regions such as North America, Western Europe, Australia, and New Zealand, while its incidence is lower in places like Japan, Finland, and Chile. Researchers attribute this geographical variation to dietary habits, with higher rates among those with high-fat diets.The occurrence of colorectal cancer may be influenced by genetic factors, which have gained increasing attention in recent years.

Causes of Colorectal CancerEpidemiological studies show that colorectal cancer has a higher incidence in regions such as North America, Western Europe, Australia, and New Zealand, while its incidence is lower in places like Japan, Finland, and Chile. Researchers attribute this geographical variation to dietary habits, with higher rates among those with high-fat diets.The occurrence of colorectal cancer may be influenced by genetic factors, which have gained increasing attention in recent years.

Based on the information provided in the table, it's evident that current treatments for colorectal cancer can come with various side effects and limitations. Therefore, there is a pressing need for innovative and more effective approaches to treat colorectal cancer.

Efficacy: 5-fluorouracil (5-FU) stands as a cornerstone in colorectal cancer treatment due to its remarkable efficacy. It disrupts cancer cell DNA and RNA synthesis, effectively halting their growth and division.Proven Track Record: Over the years, 5-FU has demonstrated its effectiveness in treating colorectal cancer, often leading to tumor shrinkage and improved patient outcomes.Diverse Treatment Approaches: 5-FU can be administered through various methods, allowing oncologists to tailor treatment strategies to the specific needs of each patient.

While 5-FU offers advantages in colorectal cancer treatment, it also presents serious side effects, including but not limited to:

The Need for Advanced 5-FU-Based Treatment Methods:Despite its efficacy, 5-FU is associated with significant side effects that can impact a patient's quality of life. Therefore, there's a growing need for advanced treatment methods based on 5-FU. Our iGEM project aims to address this challenge by developing a novel treatment method that offers improved outcomes with reduced side effects, bringing hope to colorectal cancer patients worldwide.

Escherichia coli Nissle 1917.Background and Origin: Escherichia coli Nissle 1917 (ECN) was isolated from the feces of a soldier who did not develop diarrhea during a Shigella outbreak. It is the only non-pathogenic strain of E. coli and serves as a probiotic. In clinical practice, it is primarily used to treat inflammatory gastrointestinal disorders such as Crohn's disease and ulcerative colitis. Recent research has revealed that ECN possesses tumor-targeting capabilities and can enhance the efficacy of chemotherapy drugs when used in combination. ECN is the primary component of the pharmaceutical product "Mutaflor," which has been in use in the European market for over half a century.

Advantages: It has a well-defined genetic background, making it convenient for genetic engineering. ECN inhibits the colonization of pathogenic bacteria (such as enterohemorrhagic E. coli) and the production of toxins (such as Shiga toxin-producing E. coli), thereby maintaining the stability of the gut microbiota. It also regulates the secretion of immune factors in the body, enhancing host immune defense capabilities, among other functions.
Our project consists of three main components: the treatment part, the targeting part, and the biosafety part.
As shown in the diagram:

In the targeting therapy module, we employ cell surface display technology to anchor the HIpA protein on the strain's surface using the ice nucleation protein (INP). The HIpA protein binds to the cancer cell's surface HSPG (Heparan Sulfate Proteoglycan), allowing the engineered strain to specifically target and anchor to cancer cells.

Cell Surface Display Technology and Ice Nucleation Protein (INP)Cell Surface Display Technology: Through genetic engineering techniques, the gene sequence of the target protein is fused with the gene sequence of signal peptides or carrier proteins. This fusion gene is then introduced into microorganisms, allowing them to express and localize the protein on the cell surface.Ice Nucleation Protein (INP): INP is a commonly used protein carrier in cell surface display technology. INP protein possesses a large external region that can be utilized to display other proteins on the cell surface.
Binding of HSPG Protein and HIpA Protein:During the carcinogenesis of colorectal cancer cells, HSPG proteins are highly expressed. The affinity of the HIpA fusion protein for human (LoVo and HCT116) and murine (CT26) colorectal cancer cell lines is at the submicromolar level.

Patients are administered 5-fluorocytosine (5-FC). 5-FC is a prodrug, which means it doesn't have therapeutic effects on its own and has no side effects. The strain's expression of CD enzyme allows the conversion of 5-FC into 5-FU, which then attacks cancer cells, resulting in a highly effective treatment.Utilized Medication:5-FU: With a chemical formula of C₄H₃FN₂O₂, 5-FU is a fluorinated pyrimidine and a traditional antineoplastic agent that inhibits both DNA and RNA synthesis. [Mechanism of DNA inhibition: 5-FU is converted in the body to FdUMP, which forms a stable ternary complex with thymidylate synthase and 5,10-methylenetetrahydrofolate, preventing the generation of thymidylate, a crucial substance for DNA replication in cancer cells during the S phase, thus inhibiting cancer cell DNA replication.] [Mechanism of RNA inhibition: It can be incorporated into RNA in place of uracil and misincorporation of fluorouracil into RNA leads to inhibition of RNA synthesis.]

Cytosine Deaminase (CD)CD catalyzes the deamination of non-toxic 5-fluorocytosine (5-FC) at the carbon 4 position (as shown in the blue structure), converting it into the therapeutic drug 5-fluorouracil (5-FU) (as depicted in the diagram below).

In consideration of potential biosafety concerns associated with our project, we have designed a suicide system.

In the suicide system, we have selected pTcl42 as the promoter, which allows the engineered strain to express MazF. After the engineered strain has completed its therapeutic role, patients can introduce a heating suppository. When it is necessary to completely eliminate the engineered strain, the suppository activates its heating function to trigger the bacterial suicide system.
Temperature-Controlled Promoter Tcl42Temperature-inducible promoters are a class of promoter sequences that regulate gene transcription activity within specific temperature ranges. They are widely used in the fields of synthetic biology and genetic engineering to achieve temperature-sensitive gene expression control.The mechanism of temperature-controlled promoters relies on temperature fluctuations to alter promoter activity, thereby regulating gene transcription levels. At low temperatures, these promoters are inhibited, resulting in low transcriptional activity. In contrast, at higher temperatures, they become activated, leading to increased gene transcription. This temperature sensitivity is achieved through the interaction of specific regulatory elements within the promoter sequence and temperature-sensing factors.We have chosen the pTcl42 promoter because it has an ideal activation temperature threshold of 42°C, with low expression levels at 37°C. In its natural host, Salmonella enterica serovar Typhimurium, Tlp is presumed to regulate virulence genes upon entry into a warm host organism. Additionally, Tcl is a temperature-sensitive mutant of the bacteriophage lambda's "cl" gene. In its natural environment, cl serves as a transcriptional repressor, allowing the phage to establish and maintain latency.

Mechanism of Action of MazFMazF belongs to the toxin-antitoxin (TA) systems, which are extensively studied and have well-defined mechanisms. The mazF gene is downstream of the TA system mazEF, encoding a stable toxin protein. MazF is an mRNA interferase (ribonuclease) that acts independently of ribosomes. It can specifically cleave single-stranded mRNA at certain sequence sites and is highly conserved in most microorganisms and some archaea.In Escherichia coli, MazF recognizes ACA sequences and hydrolyzes the phosphodiester bond at the first A position of either the 5' or 3' end, leading to ribosome release from cleaved mRNA and the inhibition of protein synthesis. Consequently, aberrantly encoded polypeptides are released and degraded by intracellular proteases, ultimately resulting in cell death.

Our project combines three key systems: the chassis microorganism, targeting technology, and the suicide system to create an innovative treatment approach for colorectal cancer. We have selected ECN as our chassis microorganism due to its safety and ease of genetic engineering. In the targeting module, we utilize cell surface display technology to anchor the HIpA protein on the ECN strain's surface using the ice nucleation protein (INP). This allows the engineered strain to precisely locate and identify cancer cells. HIpA protein binds to the cancer cell surface's HSPG protein, ensuring accurate targeting. For the treatment part, patients are administered 5-fluorocytosine (5-FC), a prodrug that remains inactive on its own. The engineered ECN strain expresses CD enzyme, which converts 5-FC into the active drug 5-fluorouracil (5-FU). This 5-FU then selectively attacks cancer cells, achieving an effective therapeutic outcome. Additionally, we've implemented a suicide system as a safety measure. When needed, patients can use a heating suppository containing the thermosensitive pTcl42 promoter to induce the expression of MazF toxin in the engineered strain, ensuring its complete elimination.

Target Audience:The target audience for our project is the colorectal cancer patient community. We have engaged in communication and collaboration with a team of experts in the field and with patients and their families. This has helped us confirm the positioning of our drug as an adjuvant treatment designed to relatively effectively alleviate the side effects for patients in the early, middle, and late stages of colorectal cancer treatment.

Product Design:Distinguishing ourselves from conventional oral methods, based on our research, we have designed two suppositories. The first suppository contains 5-FC, engineered bacterial lyophilized powder, and the necessary accompanying excipients. The second suppository includes a heating function.

Production Process:Engineered bacteria are cultivated and fermented in a biofactory, and their lyophilized powder is produced using freeze-drying techniques. Lyophilized powder containing engineered E. coli ensures a shelf life of at least two years. Subsequently, in a pharmaceutical facility, suppositories are produced and packaged using 5-FC and the lyophilized powder.
Consumer Usage:The first suppository is inserted into the rectum. At this point, the suppository gradually dissolves, releasing the engineered bacteria and 5-FC. The engineered bacteria adhere to cancer cells and convert 5-FC into 5-FU, which is effective in killing cancer cells. When treatment is completed, and it is necessary to ensure the death of the bacterial strain to prevent genetic leakage, the second suppository with a heating function is inserted into the rectum. This activates the heating function, leading to the death of the engineered bacteria.

Our project integrates these three systems to create a novel, precise, and safe treatment method for colorectal cancer.ECN, guided by targeting technology, delivers the therapeutic payload of 5-FU to cancer cells, while the suicide system provides a fail-safe mechanism for the controlled elimination of the engineered strain when necessary. This comprehensive approach aims to improve treatment effectiveness while maintaining biosafety.

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