Team:CKWA-China/Human Practices

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Inspiration

1. A major problem in cancer.

As our project design and laboratory part progressed, we encountered a very large number of practical difficulties, including three main areas: schematic design, experimental implementation and clinical application. For this purpose, we have interviewed three different groups, including: experts in proteomics to help us with design aspects; pharmaceutical companies of recombinant proteases and protein production centers to help us with difficulties encountered in experimental production; and experts in clinical aspects to help us with difficulties that we may encounter when the product is put into use. To achieve this goal, we communicate and interact with different stakeholders through seminars, expert interviews, and public questionnaires. We believe that our solution will not only bring significant benefits to the community, but will also address the issues prevalent in our final product development phase. 1. Pre-project research Cancer, has always been a major challenge on the road to human survival. This includes cure and prognosis, both of which are very difficult to work with. In particular, many in situ cancers also have complicated prognostic difficulties after resection and treatment, resulting in low 5-year as well as 10-year survival cycles, to the extent that it makes the quality of survival of patients severely reduced. Among others, we observed in our reading of the literature that overexpression of the c-myc gene, which is associated with a variety of cancers, has a strong impact on the recurrence aspect of prognosis in particular. For example, in the literature: c-myc gene amplification in breast, bladder and kidney cancer tissues, it was mentioned that the c-myc gene is very strongly associated with the amplification of breast, bladder and kidney cancers. In a 2012 CELL review, "MYC on the Path to Cancer", a systematic explanation of the molecular functions, signaling pathways and roles of MYC in cancer development is provided. The MYC gene is one of the most widely studied intranuclear oncogenes, and the MYC gene family consists of three major members, C-Myc, N-Myc and L-Myc, of which C-Myc was first identified as the cellular homolog of the avian myeloma virus transformation sequence. The role of L-Myc is still poorly understood, and the expression of N-Myc is tissue-specific and can replace c-Myc in mouse development. proto-oncogene MYC is a crossover point for a number of growth-promoting signaling pathways and an immediate early response gene downstream of many ligand-membrane receptor complexes (Figure 1A). MYC expression is highly controlled and its expression levels are tightly regulated by a number of transcriptional regulatory modality-related mechanisms within the proximal promoter region. Early studies of oncogenic retroviruses causing fulminant chicken tumors paved the way for the discovery of MYC, which led to the identification of v-myc oncogenes causing myelomonocytosis. The homologous sequence of v-myc in the host genome is c-myc. Although the search for similar human retroviruses could not generalize retroviral oncogenes in human cancers, it was also found that human MYC is usually altered in Burkitt lymphoma (Burkitt lymphoma) due to balanced chromosomal translocations and is therefore a true human oncogene. MYC is frequently found in MYC is frequently translocated in multiple myeloma and is also one of the most highly amplified oncogenes in a variety of different human cancers. It has been shown that defective Wnt-APC signaling in human colon cancer promotes transcriptional activation of MYC by TCF, and in T-cell leukemia MYC is downstream of the uncontrolled Notch signaling pathway. Thus alterations in MYC are most commonly seen on the pathway to cancer. In addition to its role in tumor formation, MYC can also work with Sox2, Oct4 and KLF4 to reprogram fibroblasts to a pluripotent stem cell state. Based on its central role in cell growth, proliferation, tumor formation and stem cells, author Chi V. Dang states in the article that he takes these key questions such as what is the molecular function of Myc, the protein product of MYC, how does MYC contribute to tumor formation, the differences between the MYC proto-oncogene and the runaway forms found in various human cancers, and whether MYC or Myc's Can target genes be targets for cancer therapy? Summary: c-Myc and Cancer Dysregulation of the proto-oncogene MYC has a key role in human tumorigenesis. Unlike other proto-oncogenes that are active due to mutation or truncation, MYC is dysregulated due to loss of transcriptional control, resulting in protein overexpression. For example, in human Burkitt's lymphoma, translocation of the MYC gene to the immunoglobulin heavy or light chain site results in c-Myc overexpression. Amplification of the MYC gene was also found in several cases of human epithelial cell tumors and diffuse large B-cell lymphomas, directly correlating with poor prognosis of the disease. At this point, we also found the desired research direction, i.e., can target genes of MYC or Myc become targets for cancer therapy? With this question in mind we sought to learn more about the literature, among which we found Professor Gerard Evan from the University of Cambridge, whose seminal contributions include the discovery of Myc's involvement in apoptosis and the development of early tools for studying Myc, such as the 9E10 antibody against the Myc tag. In one of his interviews, he mentioned that "the commonality of cancer is an important clue that people avoid ...... "...... [We need] to come up with a cure, no matter who you are or what you have. Now, a lot of people think this is science fiction, but I think all the data clearly shows it's not . " ...... This is one of the great mysteries in cancer biology, and no one knows why it is. No one knows why tumors are susceptible to disruption of these signals and die." https://share.vidyard.com/watch/qwjWihLudbTT6hkY2f13jJ? In our next literature study, we found a paper published in Nature Biotechnology by researchers from Lund University in Sweden entitled "A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer. The researchers found that children with acute pyelonephritis (APN) had significantly lower MYC expression levels during the acute infection period. Through in vitro infection experiments, it was confirmed that infection with uropathogenic Escherichia coli (UPEC), such as E. coli CFT073 and E. coli 536, rapidly reduced c-MYC protein expression levels. To investigate the mechanism of c-MYC degradation, the investigators screened the E. coli 536 mutant for activity, identified the region responsible for c-MYC inhibition located on pathogenicity islands (PAIs) PAI I, and found that c-MYC was inhibited by the supernatant of E. coli 536 cultures. Proteomic analysis of the bacterial culture supernatants of wild-type E. coli 536 and PAI I deletion mutant strain PAI I536 identified the MYC inhibitor as the protease Lon. Lon protease is an ATP-dependent serine protease that interacts with c-MYC by directly cleaving the serine-rich repeat sequence of c-MYC, depriving c-MYC of essential functional domains, such as, DNA binding site and C-terminal MAX binding site. At the same time, the investigators further demonstrated the mechanism by which pathogenic E. coli 536 regulates the expression level of c-MYC through the pore-forming toxin (PFT) α-hly. α-hly induced Ca2+ flux activates casein kinase 1 alpha 1 (CK1α1), and the activated CK1α1 is reported to be associated with c-MYC. CK1α1 binds to c-MYC, phosphorylates c-MYC, and is degraded by proteases. cK1α1 is also involved in the degradation of the c-MYC transcriptional enhancer c-MYB, which presumably further attenuates MYC expression. In addition, it was found that pathogenic E. coli 536 targets the transcriptional regulator of c-MYC: CCAAT enhancer-binding protein delta (CEBPD), which in turn accelerates the degradation of c-MYC. In vivo experiments with Lon, an α-hly-independent inhibitor that acts directly and alone on c-MYC, delivered to mice either intravesically or orally, found that Lon protease delayed tumor development in MYC-dependent bladder and colon cancers and improved survival in cancer model mice, respectively. These results suggest that bacteria have evolved strategies to control c-MYC levels in the host and that Lon proteases hold promise as therapeutic cancer drugs targeting c-MYC. This has greatly stimulated our interest, and we believe that Lon proteins may become potentially excellent targeting agents for c-MYC that could effectively improve the survival of patients with cancers due to c-MYC overexpression, including bladder and kidney cancers. So we planned to use experimental techniques to produce Lon protease, to judge the use and application value of Lon protease, and to be able to package it as a product with the standard of a biological drug and to improve this product according to the needs of actual stakeholders. 2.Interview 2.1 Project design phase. In the project design phase, since our project today is a protease-based anti-cancer drug, we interviewed proteomics experts and clinical experts for this purpose to get a preliminary understanding of the product that will be used in the future and to have a clear goal of how the product will be used. My design goal is to design a drug that can be used as an adjunct to surgical treatment of bladder cancer with TUR-BT, which can be followed by adjuvant perfusion therapy to achieve a reduction in recurrence of bladder cancer after surgery. 2.1.1 Proteomics Expert Since our goal this year is to make a drug that can protein, we interviewed Lotro Technologies, for which James Dewey Watson is the general scientific advisor, and Lotro Technologies was happy to accept our interview, arranging for Dr. Yanjing Su and his scientific team to be interviewed by us. Jeffrey Su, B.Sc., M.Sc., Nankai University, Ph.D., Carleton University, Canada, and Postdoctoral Fellow, National Protein Engineering Center (PENCE), Canada. In the first interview, we introduced our project background and experimental design to Dr. Su, who gave positive comments on our project design idea and thought that our design idea was very clear. At the same time, Dr. Su gave his opinion that the molecular weight of rLon protein was 89Kda, which is a very large amount of protein and may be a challenge for protein drugs, and he gave his opinion as a proteomics expert to try to further optimize the molecular structure of this protein to see if some key structures can be found to optimize a large protein into a smaller peptide structure. . At the same time, we talked about an idea that if we can validate the protein potency, then can we try to refer to the way of mRNA drugs, and make the mRNA corresponding to the protein combined with LNP and other carriers to make targeted drugs to deliver to the cancer lesions, one can increase the potency, and the other can reduce the possible side effects. 2.1.2 Clinical experts Meanwhile, at the beginning of the project design, in order to get medical clinical information related to bladder cancer triggered by C-myc gene overexpression, we interviewed Dr. Mi Zhou from Beijing Chaoyang Hospital, who graduated from Peking University School of Medicine and is also a senior who participated in the iGEM competition. In the interview, Dr. Zhou introduced us to the current clinical treatment of bladder cancer. There are two common types of bladder cancer at present, treatment of non-muscle-invasive bladder cancer and surgical treatment of muscle-invasive bladder cancer, of which non-muscle-invasive bladder cancer (non muscle-invasive bladder cancer) or superficial bladder cancer (superficial bladder cancer) accounts for 75% to 85% of all bladder tumors. Certain factors are closely associated with the prognosis of non-muscle-invasive bladder cancer. Among these factors, the number of tumors, frequency of tumor recurrence, especially at 3 months postoperatively, tumor size, and tumor grade are strongly associated with recurrence. The factors most associated with tumor progression include pathologic grading of the tumor and tumor stage. The prognosis for tumors at the bladder neck is poor. The main treatment options are surgical resection and postoperative adjuvant therapy. The surgical resection treatment is based on an assessment of the nature of the tumor, the depth of infiltration, and the choice of conservative transurethral resection of bladder tumor (TUR-BT) with postoperative radiation and chemotherapy, and close postoperative follow-up is required. An alternative, more radical surgical treatment is radical cystectomy, in which the patient's entire bladder is directly removed. Recurrence occurs in 10% to 67% of patients within 12 months after TUR-BT and 24% to 84% of patients within 5 years after surgery. There are two peak periods of recurrence after TUR-BT for non-muscle invasive bladder cancer, 100 to 200 days after surgery and 600 days after surgery. Because of the concern about recurrence, more and more patients are choosing the radical treatment of radical cystectomy. However, from Dr. Zhou's conversation with us, we learned that although recurrence is lower with this type of resection, the quality of patient's survival is namely lower, such as the need for abdominal wall stoma, lifelong wearing of urinary collection bags, urinary incontinence and other inconveniences. Clinically speaking, in order to reduce the problem of high recurrence and progression after surgery, adjuvant bladder perfusion therapy is generally used for bladder cancer patients after surgery. The perfusion drugs include chemotherapeutic drugs such as epirubicin or mitomycin, etc. It has been clinically proven that bladder perfusion chemotherapy can reduce the tumor recurrence rate by 40%. Another is postoperative bladder perfusion immunotherapy with BCG, for example, for intermediate-risk non-muscle-infiltrating bladder uroepithelial carcinoma, the probability of tumor recurrence after surgery is 45% and the probability of progression is 1.8% with BCG perfusion. However, BCG instillation has more side effects and needs to be used with caution. At the time of the interview, we had a very good idea to use Lon protein as a perfusion drug to assist postoperative treatment of TUR-BT surgery, which is used to reduce the risk of cancer recurrence. Also this approach would circumvent one of the initial considerations in our design project of injecting protease into the body and worrying about generating free

Recurrence of cancer after treatment is a major problem facing humans today, and cancer recurrence is a great threat for cancer patients. Among them, overexpression of c-myc protein is closely related to this process, and patients with overactivation of c-myc gene in cancer cells tend to have poor prognosis.

1.1 What are myc genes?

myc genes have been shown to be a class of regulatory genes that are transcription factors that can encode myc-like proteins to become transcription factors transcription factors. they are essential for cell growth, metabolism and tissue development.

1.2 Impact of myc in cancer

They are essential for cell growth, metabolism and tissue development. Also, MYC is an important group of oncogenes. c-MYC is aberrantly expressed in about 30-50% of human malignancies and plays an extremely important role in affecting cell growth, proliferation, differentiation, apoptosis, as well as cell metabolism and malignant transformation. aberrantly high expression of c-MYC is associated with poor prognosis in many human cancers. Translated with www.DeepL.com/Translator (free version)

1.3 C-myc genes

The Myc family consists of three related human genes: - c-myc (MYC), the first gene to be discovered in this family - l-myc (MYCL) - n-myc (MYCN)

We then recognized an absence in both detection and treatment of Soft Rot Disease. Therefore, we understood the importance of a bio-control solution that is not only effective but also environmentally-friendly. Based on knowledge of the field of synthetic biology, we started to come up with different approaches to soft rot detection and treatment.

Interview（Potato Center Asia-Pacific Headquarter）

We met with senior staffs of the potato center Asia-Pacific Headquarter to learn about the current detection and treatments of potato diseases. We then visited their workplace, which accounted for a large proportion of the current potato-agricultural development. We presented our initial concept to the experts (including the chairman of the potato center, Mr. Lu) and audited a group meeting on agricultural pharmacology. Through the discussions with them, we became more aware of the consequences of Soft Rot disease and the importance of creating an effective bio-control solution.

Experts

Our team had the pleasure of meeting with Dr. Flemming Bensenbacher on 5th July 2019. Dr. Bensenbacer is an academician of the Royal Danish academy of Sciences and President of Carlsberg Foundation. He shared his journey in scientific research with us and the problems he encountered and tackled. We presented our project to him and hosted an interview with questions relating to the application of synthetic biology. This opportunity was crucial to us as we gained from it a valuable depth of knowledge in the potential future pathways of our product development.

I could write some video notes here, a little bit more introduction.

I could write some video notes here, a little bit more introduction.

I could write some video notes here, a little bit more introduction.