For TNBC, the first thing we need to solve is the problem of identification and initiation, so we designed a gene loop. Currently, targeted therapy drugs for breast cancer are greatly affected by other factors such as environment and tumor status, which may have unstable recognition and reduce its effect. Therefore, we changed the design of targeting from the receptor binding on the cell surface to the specific initiation of the treatment loop inside the cell, that is, a triple-negative breast-cancer-specific promoter was designed in the loop to drive the specific expression of the edited anti-tumor effector in the triple-negative breast cancer cells. However, according to the similarity of the cancer cells and normal tissue or other complex reasons, the promoter specificity is very difficult to reach a higher level, (the experimental data show that the highest can reach 60%, targeted therapy drugs in clinical practice not considering the body complex situations), so we consider to choose a significant differences in tumor tissues and normal tissues of indicators as the premise of loop start, to increase the specificity of the loop. Our team found that three negative breast cancer cells exist in some specific lower expression of microRNAs^[3-8], so we will use these microRNAs as indicators of loop start, i.e. adding the miRNA switch in the loop, to control genes in the loop through differences between normal cells and three negative significant as the amount of the miRNA expression in breast cancer cells, so as to add a bumper to the overall loop.

In order to have a deeper understanding of the treatment status of triple-negative breast cancer and the prospect of our project, we went to Xiangya No. 3 hospital to conduct a survey and interview with professor Yanguang Wen from the department of mammary gland surgery. Through consulting professor Wen on the current treatment status of triple-negative breast cancer, we know that there is still a need to find new treatments for triple-negative breast cancer, and our project has certain prospects. We understand that the current problem of gene therapy killing through the loop faced is how to capture escaped cells^[9], so we improved the loop by using the superposition of Module1 and Module3, which can express anti-tumor effector jointly, to guarantee the killing effect. In terms of the safety of gene therapy, we do not intend to use viral or bacterial vectors, but use safer transferrin combined with liposomes to directly target tumor cells, so that the transduction efficiency is higher and the risk is lower. At the same time, professor Yanguang Wen suggested we pay attention to the advantages and disadvantages of traditional therapy represented by radiotherapy and chemotherapy and our gene therapy, which may provide new ideas for our experiments.

According to the ideas provided by professor Yanguang Wen, in order to further explore this issue and understand the advantages and disadvantages of our gene loop therapy compared with traditional cancer therapy, we invited professional debaters to conduct a fierce debate on the advantages and disadvantages of traditional therapy and gene therapy. Before the debate, we had an in-depth discussion on this issue with debaters from all sides. The debaters asked us in detail about our expertise in gene circuits and traditional therapies, which greatly inspired us to think about gene circuits and traditional therapies from their perspective. They wanted to know how genetic circuits could improve their targeting, that is, how cancer cells could self-destruct without affecting normal cells. Our team members answered this question in detail according to our experiment. In the debate, opponents who support traditional therapy mainly raise doubts about the side effects, development prospect and effectiveness of gene therapy. Are the side effects of gene therapy accurately understood and effectively reduced? What are the prospects for gene therapy? How to ensure the effectiveness of gene therapy? These three questions allow us to reflect on questions about how we can reduce side effects and improve effectiveness in experiments, and provide new ideas for our experiments.

According to the new ideas provided by the debate competition, we discussed with the experimental group timely to improve our project. If we use suicide gene directly as the anti-tumor effect of the content, the existing problem is the expression time is not controllable, thus, as far as we concerned, we should combine our loop with the clinical use of a wide range of chemotherapy drugs to kill, but we won’t add toxic substances directly; instead, based on the loop, under controlled conditions, the premise of a certain amount of non-toxic substance will be transferred into chemotherapy (due to the transformation, the precursor drug imply intracellular killing of cancer cells, and has no amplification effects, which will not affect the next cell), thus to achieve the higher controllability, and security.

After the debate on the advantages and disadvantages of traditional therapy and gene therapy, we improved the project and went to xiangya third hospital to interview professor Boni Ding about the improved project. Through the interview with professor ding, we knew that the most difficult point to treat triple negative breast cancer is its heterogeneity, that is, it is difficult to catch all the cancer cells with different stages of development or different states. So we want to add another Module (namely Module2), in which a specific promoter found in TNBC has can be used as loop start adsorption index of endogenous miRNA sponge structure combination^[10-12], to control the level of the endogenous miRNA, in order to realize motivation of our loop in the condition of different height heterogeneity of cancer cells, to achieve more widespread destruction, and finally solve specificity (targeted) and heterogeneous problem, cooperating with the first Module (Module 1) P1 cell-specific promoters complementary cooperation.

During the experiment, HP activities were closely combined with the experimental progress. Problems and ideas in the experiment were collected through specific investigations and activities for better opinions and feedbacks, which were then discussed and applied in the experiment. The latest experimental results showed that the specificity of direct join of the promoter designed according to the specific expression of transcription factor is high, but its amplification efficiency is very low (compared to a strong promoter CMV). Therefore, the prime questions is that how to combine the advantages of both, makes the promoter can satisfy both strong specificity and enlarge the advantage of high efficiency. After sorting out our questions, we ranged an on-site consulting with the well experienced in the design of the promoter Shuming Sun associate professor, department of molecular biology professor who suggest that we write a question section in the form of a seminar, with the students to discuss the solution at the same time, promoting the knowledge of synthetic biology, so that more college students have a deeper understanding and understanding of synthetic biology after learning about our igem practice. According to the opinions, we conducted a special seminar, with the help of the teacher. First we introduced the project background in class, describes in detail the problems we met, then inspire students' thinking. After discussion, some students mentioned in the synthesis of specific promoter, there are also students coming up with ideas as joining specificity enhancer of combination. With gaining the specific experimental methods to resolve the problem at the same time, college students present in have a specific and vivid understanding synthetic biology and our projects. At the suggestion of many schoolmates, we are preparing for the establishment of synthetic biology community of Central South University, through which allows us to use our power to transmit and publicize synthetic biology, combine experiments to carry out relevant interesting activities and research in a better way.

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