Achievements

	Name	Explanation	Medal
1	Competition Deliverables	1. Wiki 2. Project Promotion Video 3. Team Presentation 4. Judging Form	Bronze
2	Attribution	Our team consists of 25 members in total, including two leaders for our experimental section: Yuchi Li and Qianhui Yang, two leaders for Human Practices: Qiang Liu and Zhiyi Juan, 11 members, 9 advisors, and our PI. With assistance from advisors Nannan Jiang, Ruoyan Zhang, and Chenyu Tao, we finished the design of our project, wet lab experiments, and the arrangement of our Human Practices activities. Our student leaders are involved in all arrangements and work as follows. Molecular biology experiments: Haoyu Sun, Qiongdan Liang. Cell experiments: Hongjun Liu. Plasmid design: Haoyu Sun. Plasmid extraction experiments: Zirun Ying. sEVs extraction: Hanyu Yang. Events planner: Hanyu Yang, Ruining Hu. Interview and communication: Yingjun Peng. Art: Sijie Wei. Modeling: Qiang Liu, Yingjun Peng. Wiki: Qingmiao Zhou, Jiashang Chen, Shengjun Fang (Fellow student from School of Artificial Intelligence, Nanjing University)	Bronze
3	Project Description	Alopecia is a skin disorder that occurs in a wide population, often causing damaging psychological consequences. The NJU-China iGEM team chose to focus on androgenetic alopecia (AGA), which exhibits the highest prevalence. Current drugs on the market for treating AGA produce unstable results and cause various side effects. Our team began with a comprehensive study of its molecular mechanism, from which we selected several genetic targets. Our goal was to develop a drug with minimal side effects, stable results, and ease of use. To achieve this goal, we applied RNAi technique and mRNA embedding elements to regulate gene expression particularly. Since the delivery system is crucial to the effect of RNA therapy, we used small extracellular vesicles (sEV) to carry and deliver siRNA and mRNA to follicle cells. We also compared different protocols for sEV preparation and chose tangential flow filtration (TFF) and bind-elute size exclusion chromatography (BE-SEC) as the optimal solution. Furthermore, apart from AGA, our research may also pave the way for an sEV-mediated systemic delivery platform of therapeutic cargo for other dermatologic diseases.	Bronze
4	Contribution	We designed six siRNAs that can down-regulate 5α Reductase and six siRNAs that down-regulate the expression of the stress-activated ion channel protein Piezo1. After a series of validation experiments, we screened the most effective siRNAs, 5αR-siRNA-1 (BBa_K4173009) and Piezo1-siRNA-5 (BBa_K4173019). We also designed a plasmid that expresses β-catenin, a protein that enhances the Wnt/β-catenin pathway and thus promotes cell proliferation. And the plasmid was experimentally validated to express the mRNA of β-catenin in vitro.	Bronze
5	Engineering Success	After designing the siRNAs, we enabled them to be wrapped into sEVs by adding components to both ends of the siRNA sequence and inserting it into the plasmid expression vector pcDNA3.1-mCherry (BBa_K4173048 and BBa_K4173058), and we also verified the validity of the sequence by experiment. We modified the mRNA of β-catenin by adding a C/D box RNA at one end (BBa_K4173045) and designed specific CD63-L7Ae fusion protein (BBa_K4173046). CD63 is one of the most widely used exosome markers with n-terminal and c-terminal intracellular structural domains. L7Ae, the 60S large ribosomal subunit of the archaeal ribosomal protein 7Ae. The target protein can be captured and kept in close contact with L7Ae through the interaction of L7Ae with the C/D box RNA structure added at the UTR of the reporter gene. When L7Ae binds to the c-terminus of CD63, the target protein can be immobilized on the membrane of exosomes. Finally, CD63-L7Ae can serve as an active packaging device for specific RNA entry into sEVs. We have also demonstrated experimentally that β-catenin can be encapsulated into sEVs.	Silver
6	Collaboration	We held the second Nanjing iGEM Association (NIA) meetup this year and invited 7 iGEM teams in total to communicate with each other and exchange our design of the project in the first meetup. Additionally, we simulated our final presentation and provided comments to each other during the second meetup. Moreover, we participated in many online and offline meetups with other teams and received many questions and suggestions about our project, which we carefully considered for further integration into our project. Team Nanjing-China and our team held a debate competition to deepen the audience's understanding of biological technology and related moral issues, which also enhance our own knowledge and promote friendship.	Silver
7	Human Practices	Through literature research, epidemiological analysis, and other methods to understand the severe background of alopecia, we set out to conduct a survey and get to know the public's demand for better drugs (effectiveness, side effects, etc.). Then we interviewed doctors at Xi 'an Jiaotong University and learned about the current situation of alopecia treatment and the deficiencies of current drugs (long treatment cycle, side effects, etc.). After designing our drug delivery system, we introduced our project to two companies known for small extracellular vesicles and alopecia studies and received their comments and suggestions. To receive feedback from the public, we distributed the questionnaire again after estimating our product price, and we obtained their expected range of drug price and their views on gene therapy drugs.	Silver
8	Proposed Implementation	Our team has applied for patents on the technical route of tangential flow filtration and bind-elute size exclusion chromatography to extract small extracellular vesicles in large quantities and applied for patents on the sEV-wrapped mRNA drug system. We plan to use microneedles as the drug delivery mechanism to first make sEV drugs for androgen alopecia, and after success, use this mature drug system to make sEV drugs for other skin diseases.	Silver
9	Integrated Human Practices	After designing the mRNA system, we introduced our project to professors studying small extracellular vesicles, and they recommended that we should add an experimental component to confirm the packaginge of mRNA and the location of the mRNA because L7-ae is a membrane system, the mRNA could be inside or outside the sEV. So, we designed an experiment, culturing RNA enzymes and sEVs together to see the mRNA expression. Moreover, we interviewed professors who study alopecia and learned about the selection of common targets for androgenetic alopecia. Finally, we chose to down-regulate 5aR to inhibit androgenetic degeneration, down-regulate piezo1 to reduce apoptosis and use β-catenin mRNA to promote hair growth. In terms of experimental design, we got to know the public's concern about side effects, so we designed siRNA and verified its low off-target effect by BLAST against the human microbiome, showing the low risk of side effects.	Gold
10	Project Modeling	To gain an great insight into the patients’ views and needs among AGA, and demonstrate the effectiveness of our project, we have conducted 4 models: 1. Epidemiology analysis of androgenetic alopecia We evaluated more than 3000 AGA patients of different age, gender, nationality, and obtained the epidemiologic characteristics of AGA. 2. Questionnaire analysis We distributed a questionnaire to learn the public’s views and needs among AGA, and did a comprehensive analysis with R. We concluded that AGA can be a problem disturbing more and more people, and the public’s preference for AGA treatment. 3. China market analysis We did a market analysis of alopecia treatment in China, finding that a better treatment is urgently needed. The results give a theoretical support to our entrepreneurship. 4. sEVs extraction technique model We evaluated the result of our sEVs extraction method. With this model and several tests, we determined the appropriate conditions to extract the most sEVs without breaking them.	Gold
11	Proof of Concept	Androgenetic alopecia has been a long-existing problem all over the world and current drugs are reported to have some disadvantages. Therefore, we must guarantee the feasibility of every part of our project. 5α-reductase (5αR) inhibition is one of the effective methods to suppress androgenetic alopecia. Moreover, Piezo1 causes apoptosis of hair follicle stem cells (HFSC), leading to alopecia, and the Wnt/β-catenin signaling pathway is associated with hair regeneration. After designing plasmids, we need to prove our products wouldn’t lead to side effects and have been improved in some aspects. In this part, we show some outcomes of Proof of Concept based on the experimental results, purchasing power analysis of different countries, and our interview and survey of industrialization. The viability shown by these parts ensure the feasibility of our project.	Gold
12	Partnership	We developed partnership with two other teams throughout the year: 1. With BUCT Our project corresponds with BUCT's on the topic of hair loss, so in the partnership we exchange information and data, and raise suggestions to help polish each other's project. At the early stage, we had meetings to discuss each other's project, both of us contribute some insights based on which we modified our project. In the HP, BUCT helped us modify and distribute our questionnaire, and in return we shared our results with them. As we both need to consult doctors and experts professional in skin diseases, we helped each other to raise questions during the interview and exchange our feedback. Together we conducted the market research, and organized several education and science communication activities both on and offline, including a offline popularization on the World No Hair Loss Day. 2. With SZU-China Our projects share similarity on applying RNAi technique, so we helped each other overcome difficulties on the experiment design. In the information exchange, SZU-China gave us some suggestions about siRNA selection, and we shared with them our sequence editing tool. In the HP, SZU-China assisted us to scheme a science popularization, and in return we helped them in a RNAi introduction activity. Together we conducted some other activities to promote synthetic biology, including an offline lecture in our school. On October, we also helped each other with the presentation of our project.	Gold
13	Education and Communication	Our team conducted educational activities for about 300 people in different age groups. For children aged 7~10, we held an activity in the Nanjing Science and Technology Museum, taught them basic knowledge about biology, and led them to conduct safe biological experiments. For high school students, we held a lecture and introduced the mechanism of alopecia and the history and current progress of synthetic biology. For students majoring in biology in college, we held a lecture and introduced the background of the RNAi technique, small extracellular vesicles, synthetic biology, and the iGEM competition. For the general public, we held an online broadcast and introduced the cause and effect of alopecia, existing treatments, and our project's design. Through education activities targeting different groups of people, we got to know the common thoughts of the public about alopecia, such as its harm and treatments, and synthetic biology, such as its definition and purpose.	Gold

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