After comprehending the purpose of the iGEM competition, the members of JLU-NBBMS diligently ascertained that the problem our team research should be intricately linked to pressing societal and national challenges in order to formally establish it.

The incidence and mortality of colorectal cancer have been steadily increasing in recent years, contrasting with its rare diagnosis decades ago. In 2016, the incidence of colorectal cancer ranked second among women, while its mortality ranked fourth, both showing an upward trend. Among men, the incidence and mortality rates were fourth and second respectively, although not as high as those in women, they too exhibited a gradual rise. According to statistics from the World Health Organization (WHO), colorectal cancer and rectal cancer became the fourth most prevalent malignant tumors in 2020. Global deaths caused by these cancers reached 930,000 that year, accounting for 9.4% of all malignant tumor-related deaths worldwide. Finding effective treatments for colorectal cancer has become an urgent issue.

Colorectal cancer refers to malignancies of the colorectal epithelium, encompassing both colon and rectal cancers. However, current treatment modalities exhibit limited efficacy in terms of cure rate and long-term survival rate. Similar to most cancer treatments, conventional approaches for colorectal cancer primarily involve surgery, radiotherapy, chemotherapy, targeted therapy, and immunization. While these methods undeniably possess certain therapeutic effects against cancerous cells, they also entail toxic side effects such as cardiac cytotoxicity, nephrotoxicity, bone marrow suppression, and neurotoxicity[3].

Furthermore, surgical intervention not only results in a substantial wound area, but also carries postoperative complications like arrhythmia and dyspnea that cannot be overlooked, alongside a high recurrence rate. Additionally, it may induce pathological changes within tissues and organs with inherent cytotoxic properties, leading to adverse reactions akin to vomiting.

The conventional therapy for cancer exhibits some limitations, necessitating additional approaches to achieve a complete cure for colorectal cancer. While surgery currently stands as the primary choice for most patients with colorectal cancer, its major drawback lies in the incomplete removal of tumor cells and their propensity for relapse. Chemotherapy and radiotherapy not only fail to completely eradicate residual tumor cells post-surgery, but also entail significant side effects.

After extensive deliberation spanning over a month, we have established the research center titled "Investigation into Novel Approaches for Colorectal Cancer Treatment", with the aim of complementing traditional therapeutic methods.

When considering novel approaches to combat cancer, we fortuitously came across news, regarding the upcoming 2023 Second Nucleic Acid Drug and Vaccine Innovation Summit in Hangzhou from April 8th to 9th. This conference will primarily focus on the research, development, and industrialization of innovative anti-tumor nucleic acid interference drugs. Consequently, our interest in nucleic acid interference drugs was piqued.

RNA interference (RNAi) is a process that can specifically and selectively destroy the expression of target genes. Some of the small double-stranded RNA (dsRNA) molecules can efficiently and specifically inhibit the transcription of mRNA, promoting the degradation of mRNA to inhibit specific gene expression. RNAi is now considered to be an accurate, efficient and stable gene silencing technology[4]. In 2006, RNAi technology won the Nobel Prize.

Small nucleic acid drugs based on RNA interference technology can silence the expression of oncogenes, with good patentability and both symptoms and causes. Small nucleic acid drugs specifically kill cancer cells and stimulate the triple mechanism of immune response to play an anti-cancer role, with good safety, high targeting, wide anti-cancer spectrum and strong killing effect on cancer cells, which can effectively treat a variety of solid tumors.

The first small interfering RNA (siRNA)-based drug, patisiran, received approval from the US Food and Drug Administration (FDA) in 2018. Givosiran and lumasiran were subsequently approved by the FDA in 2019 and 2020 respectively, for the treatment of acute intermittent porphyria (AHP) and primary hyperuricemia (PH1). Inclisiran obtained approval from the European Medicines Agency (EMA) in 2021 for managing hypercholesterolemia. According to incomplete statistics, nearly 400 novel nucleic acid drugs have entered global clinical pipelines with a total of sixteen nucleic acid drugs being authorized for marketing purposes, including nine antisense oligonucleotide (ASO) drugs, five siRNA drugs, and two mRNA vaccines. The evident technical advantages coupled with their broad application across various therapeutic areas indicate that nucleic acid drugs will spearhead the transformation of the biomedical industry.

Due to the numerous advantages of siRNA drugs and their prominent role in tumor therapy, we have opted to employ RNAi mechanisms for silencing oncogene expression in our approach towards treating tumors.

Coincidentally, our recent lessons on traditional Chinese medicine shed light on certain aspects of this ancient practice. TCM posits that the universe and human body consist of two opposing yet interdependent elements known as "yin" and "yang", with the balance and coordination between these elements being crucial for maintaining bodily health. Many diseases arise from disharmony between "yin" and "yang".

This immediately recalls proto-oncogenes and tumor suppressor genes to us.

The proto-oncogene and tumor suppressor genes are both crucial regulators of cell growth and proliferation. Under normal circumstances, the expression levels of oncogenes in the genome remain low, playing a pivotal role in maintaining normal physiological functions and regulating cell growth and differentiation.

Tumor suppressor genes, present in normal cells, belong to a class of genes that can inhibit cell growth and possess potential tumor-suppressing effects. They play a vital role in negatively regulating cell growth, proliferation, and differentiation. The interplay between proto-oncogene and tumor suppressor genes ensures the relative stability of positive and negative regulatory signals.

Under normal circumstances, the roto-oncogene and tumor suppressor gene function as "yin" and "yang", coordinating and regulating each other to maintain the proper functioning of the human body.

However, mutations or inactivation of these genes can lead to malignant cell transformation and tumor formation.

In certain conditions, abnormal activation of proto-oncogenes may result in their transformation into oncogenes with over-expression while mutation-induced decreased expression of tumor suppressor genes can induce cellular carcinogenesis. This phenomenon is referred to as an imbalance between "yin" and "yang" according to TCM.

From the perspective of TCM, cancer is believed to arise from an imbalance between "yin" and "yang". Therefore, it is crucial to focus on harmonizing "yin" and "yang" in order to inhibit the expression of proto-oncogenes.

In TCM, various methods are employed for disease treatment with the aim of reconciling "yin" and "yang". Over the past few decades, numerous studies have demonstrated that TCM has the potential to activate the immune system for disease management [6].

Drawing inspiration from this concept, we propose a therapeutic approach for cancer that involves activating the immune system while simultaneously inhibiting oncogene expression.

PD-L1 is a molecule present on the surface of cancer cells that inhibits the immune pathway. Binding of PD-L1 to the programmed death receptor 1 (PD-1) on the surface of immune cells can initiate programmed cell death in T cells, inducing immune evasion of tumor cells. Silencing the PD-L1 gene can relieve the inhibition of cancer cells on immune cells, activate the immune system, and inhibit the occurrence and development of tumors[7].

STAT3 is a member of the Signal Transducer and Activator of Transcription (STAT) family. STAT3 is involved in various biological processes including proliferation, metastasis, angiogenesis, immune response, and chemoresistance. Activation of the STAT3 signaling pathway can upregulate the expression of PD-L1. In colorectal cancer, STAT3 increases tumor cell viability and promotes tumor cell proliferation[8].

Silencing the STAT3 gene and the PD-L1 gene can be used to treat colorectal cancer from multiple aspects, such as activating the body's own immune system and inhibiting tumor growth.

We have considered designing a recombinant plasmid containing shSTAT3 and shPD-L1 sequences to inhibit the transcription of mRNA through the RNAi mechanism, leading to mRNA degradation and silencing of the STAT3 and PD-L1 genes for the treatment of colorectal cancer.

5.1 Salmonella as a vector

The shSTAT3/shPD-L1 plasmid can inhibit tumor growth, but how to deliver the plasmid into tumor cells is a new challenge. In the cell experiment stage, we have many methods to introduce the plasmid into tumor cells. However, our project aims to treat colorectal cancer, which is a solid tumor in the human body. We hope that our product can be used in clinical treatment of colorectal cancer in the future. Therefore, our design cannot only stay at the cell experiment stage, and we must consider how to deliver the plasmid into tumor cells in the human body.

There are two stages to deliver the plasmid into solid tumors in the human body: the effective localization of the vector in the target tumor and the effective penetration of the tumor tissue. We must consider how to achieve this.

After extensive literature review, we have decided to use Salmonella as the vector to deliver the plasmid into tumor cells.

Bacteria-mediated cancer therapy (BMCT) offers new possibilities and hope for cancer treatment, as it can highly target tumors, exert strong anti-tumor effects, improve the tumor immune-suppressive environment, effectively inhibit cancer metastasis and recurrence, serve as a drug delivery system, overcome drug resistance, and enhance combination therapy efficacy[10].

Salmonella therapy for cancer has gained significant attention in recent years. 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 [11]. Salmonella can invade through mucosal membranes and persist in host lymphoid tissues, continuously inducing strong mucosal and cellular immune responses in the host [12]. Compared to other bacteria, Salmonella has several advantages, including high tumor specificity, deep tissue penetration, inherent bacterial toxicity, ease of genetic modification, and good safety profile [13]. Furthermore, Salmonella exhibits inherent anti-tumor activity [14].

Salmonella not only has significant advantages as a vector for delivering plasmids into tumor cells, but it can also induce immune responses and exhibit anti-tumor activity, enhancing the efficacy of our plasmid therapy for tumors. Therefore, we have decided to incorporate the plasmid into Salmonella and use Salmonella as the carrier for plasmid delivery.

5.2 Constructing a Salmonella vector

Through genetic engineering techniques, attenuated Salmonella can be obtained for the purpose of carrying exogenous genes, cytokines, and cytotoxic drugs to inhibit tumor growth. Attenuated Salmonella has minimal toxic side effects, can overcome the penetration restrictions of tumor tissues, and can induce immune responses, leading to the production of anti-tumor immunity, including cellular and humoral immunity [12].

ΔPmurA::TTaraCPBADmurAΔasd::TTaraCPBADc2 Δ(wza-wcaM)-8 Δpmi ΔrelA::araCPBADlacITT ΔrecF

We have chosen the delayed lysis strain χ11802 for modification, aiming to obtain a safe, efficient, and highly tissue-colonizing attenuated Salmonella strain. The genotype of χ11802 is as follows:

ΔPmurA::TTaraCPBADmurAΔasd::TTaraCPBADc2 Δ(wza-wcaM)-8 Δpmi ΔrelA::araCPBADlacITT ΔrecF

χ11802 is a delayed lysis strain, in which the essential genes asdA and murA for the synthesis of the peptidoglycan layer in Salmonella are placed under the control of the arabinose promoter araCPBAD. This design prevents the strain from synthesizing crucial components of the cell wall in an environment lacking arabinose in the host, leading to self-lysis of the cells and the release of plasmids. Additionally, it facilitates the presentation of tumor antigen peptides by increasing the expression of MHC-I molecules, thereby inducing a stronger CD8+ T cell-mediated anti-tumor immune response.

However, after multiple gene deletions in the χ11802 strain, its invasive and colonization abilities in tissues are reduced, making it difficult to achieve long-lasting and efficient therapeutic effects. Therefore, we plan to further modify the attenuated Salmonella χ11802 strain in order to obtain a safe, efficient, and highly tissue-colonizing engineered strain.

Therefore, we have decided to make improvements to χ11802 in several aspects:

1. Enhancing the targeting ability and colonization capacity of Salmonella towards tumor cells.

2. Promoting the body's own immune response.

Based on the aforementioned modification ideas and extensive literature research, we have finalized the following directions for modification:

1. Deletion of the L-asparaginase II synthesis gene asnB to activate the host's immune response and enhance the tumor-killing effect of T cells.

2. Insertion of the flagellin protein gene flaB to enhance the strain's ability to adhere, invade, and colonize deep tissues.

We expect to obtain the final ΔasnB::Ptrcfχ11802 Salmonella strain, which can better target tumor tissues and serve as a vector for plasmids. It is designed to kill tumor cells through multiple pathways. We have named this strain SCI, as a tribute to the power of science and the significant contributions of the SCI database to academic research.

In summary, we have designed and constructed a novel approach based on RNAi and bacterial vectors for the treatment of colorectal cancer, providing a new option for the management of this disease.

[1] Zheng, R., Zhang, S., Zeng, H., Wang, S., Sun, K., Chen, R., Li, L., Wei, W., & He, J. (2022). Cancer incidence and mortality in China, 2016. Journal of the National Cancer Center, 2(1), 1-9.
[2] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021 May;71(3):209-249. doi: 10.3322/caac.21660. Epub 2021 Feb 4. PMID: 33538338.
[3] Liu YQ, Wang XL, He DH, Cheng YX. Protection against chemotherapy- and radiotherapy-induced side effects: A review based on the mechanisms and therapeutic opportunities of phytochemicals. Phytomedicine. 2021 Jan;80:153402. doi: 10.1016/j.phymed.2020.153402. Epub 2020 Oct 31. PMID: 33203590.
[4] Saurabh, S., Vidyarthi, A.S. & Prasad, D. RNA interference: concept to reality in crop improvement. Planta 239, 543–564 (2014). https://doi.org/10.1007/s00425-013-2019-5.
[5] Alshaer W, Zureigat H, Al Karaki A, Al-Kadash A, Gharaibeh L, Hatmal MM, Aljabali AAA, Awidi A. siRNA: Mechanism of action, challenges, and therapeutic approaches. Eur J Pharmacol. 2021 Aug 15;905:174178. doi: 10.1016/j.ejphar.2021.174178. Epub 2021 May 24. Erratum in: Eur J Pharmacol. 2022 Feb 5;916:174741. PMID: 34044011.
[6] Su XL, Wang JW, Che H, Wang CF, Jiang H, Lei X, Zhao W, Kuang HX, Wang QH. Clinical application and mechanism of traditional Chinese medicine in treatment of lung cancer. Chin Med J (Engl). 2020 Oct 15;133(24):2987-2997. doi: 10.1097/CM9.0000000000001141. PMID: 33065603; PMCID: PMC7752681.
[7] KEIR M E, BUTTE M J, FREEMAN G J, et al. PD-1 and its ligands in tolerance and immunity [J]. Annu Rev Immunol, 2008, 26: 677-704.
[8] DONG J, CHENG X-D, ZHANG W-D, et al. Recent Update on Development of Small-Molecule STAT3 Inhibitors for Cancer Therapy: From Phosphorylation Inhibition to Protein Degradation [J]. J Med Chem, 2021, 64(13): 8884-915.
[9] Abroun S, Saki N, Ahmadvand M, Asghari F, Salari F, Rahim F. STATs: An Old Story, Yet Mesmerizing. Cell J. 2015 Fall;17(3):395-411. doi: 10.22074/cellj.2015.1. Epub 2015 Oct 7. PMID: 26464811; PMCID: PMC4601860.
[10] Badie F, Ghandali M, Tabatabaei S A, et al. Use of Salmonella Bacteria in Cancer therapy: direct, drug delivery and combination approaches[J]. Frontiers in Oncology, 2021, 11: 624759.
[11] Nakashima C, Yamamoto K, Kishi S, et al. Clostridium perfringens enterotoxin induces claudin-4 to activate YAP in oral squamous cell carcinomas[J]. Oncotarget, 2020, 11(4): 309.
[12] Kim V M, Blair A B, Lauer P, et al. Anti-pancreatic tumor efficacy of a Listeria-based, Annexin A2-targeting immunotherapy in combination with anti-PD-1 antibodies[J]. Journal for immunotherapy of cancer, 2019, 7(1): 1-132.
[13] PANGILINAN C R, LEE C-H. -Based Targeted Cancer Therapy: Updates on A Promising and Innovative Tumor Immunotherapeutic Strategy [J]. Biomedicines, 2019, 7(2).
[14] LEE T-H, LIN G-Y, YANG M-H, et al. reduces tumor metastasis by downregulation C-X-C chemokine receptor type 4 [J]. Int J Med Sci, 2021, 18(13): 2835-41.