In every phase of our project, from design to execution and final implementation, Integrated Human Practices served as a pivotal element. By incorporating input from experts, fellow iGEM participants, and the wider public, we've been able to effectively steer our project in the desired direction and set the stage for achieving our anticipated results.

Our Own Story

In 2021, Litong Wu and Yanheng Zhu frequently played basketball games together. Litong Wu observed that his injuries, particularly to his joints, took longer to heal compared to Yanheng Zhu. Seeking the underlying reason, Litong realized his Body Mass Index (BMI)—a measure of body fat based on height and weight, where a value below 23.9 is considered normal—surpassed that of Yanheng's. More significantly, Litong was classified as obese, with a BMI exceeding 32. This revelation led to a question: is obesity negatively impacting his knee joints?

Fig.1 Our student leader and student member played basketball together

Yingcai Program in China

Yingcai program is a national program to select a group of high school students with excellent character and learning ability to enter the university, participating in scientific research under the guidance of famous scientists.

In 2022, Wu Litong was selected for the Yingcai Program. With Professor Hua Zichun's team from Nanjing University, he researched the impact of a high-fat diet-induced obesity and adipose exosomes on post-traumatic osteoarthritis in young mice, particularly from DMM (destabilization of the medial meniscus) surgery. They also examined how obesity exacerbates the condition. Their findings suggested that obesity induced by a high-fat diet can worsen post-traumatic osteoarthritis resulting from DMM surgery in young mice. Furthermore, it was determined that obese adipose exosomes primarily impact the surface of the knee's articular cartilage.

In light of the previous research, we've embarked on an iGEM project with a new, exciting objective - how can we employ synthetic biology methods to devise innovative therapy forms that safeguard our joints from damage caused by obese adipocyte exosomes?

Fig.2 The certification and the Poster in 2022 Yingcai biology program

Consult Public Health Experts on Obesity & Osteoarthritis Solutions

In the early stages of our project, we faced difficulties pinpointing a specific issue within Osteoarthritis to tackle and figuring out a manageable approach for addressing it. To overcome this hurdle, we sought advice from Prof. Guo Baosheng, who is affiliated with the Medical School of Nanjing University.

In our initial interview with Professor Guo, we gained understanding that knee osteoarthritis is primarily triggered by the degeneration of articular cartilage, or more specifically, its attenuation. Being overweight significantly amplifies this problem as it increases the friction on the joint, thereby accelerating the abrasion.

He also introduced us to Professor. Zhao, who specialized in obesity. She showed us her lab's previous work in sequencing obese adipocyte exosomes.

Understanding our goals and time limits for this iGEM season, we read a lot of research and literature using PubMed. A key finding in Professor. Zhao's previous research was that miRNA-3074-5p in obese adipocyte exosomes played an important role in the regulation of joint. We wanted to understand what this could mean for the health of their joints.

After several discussions, we showed Prof. Wei Wei and Prof. Li Huang our first project design. With their help, we planned out the experiments needed at each step of our project.

Please click the link to see the video interview in Bilibili:

• Video: Interview with Professor Guo about osteoarthritis.
• Video: Interview with Professor Zhao about obese adipocyte exosomes.

Learn Obesity Conditions at National Metabolic Management Center

Living in Nanjing, we've seen firsthand how obesity is becoming an increasing problem in our society. While initially designing our project, our team visited the National Metabolic Management Center to get a deeper understanding of the issue of obesity.

In our discussions with Dr. Feng, who serves as the Department chair of the Obesity Multidisciplinary Clinic, we learned about the ramifications and treatment options related to Obesity. Obesity significantly enhances the chances of developing various long-term diseases and health conditions. These include cardiovascular diseases, type 2 diabetes, hypertension, certain cancers, sleep apnea, and Osteoarthritis.

Dr. Feng highly praised our project, she pointed out that if a drug could simultaneously tackle obesity and protect joint health, it would serve as an ideal treatment for obese patients with osteoarthritis. This makes our project highly relevant.

Fig.3 Visiting National metabolic management center and interviewing with Dr. Feng, who serves as the Department chair of the Obesity Multidisciplinary Clinic

Investigate Public Knowledge of Obesity and Osteoarthritis

We conducted a survey on public knowledge of obesity, knee osteoarthritis, and their relationship to learn about public acceptance and guide educational activities. By analyzing the answers, we found that:

1. About 75% of the respondents have considered that there is a relationship between obesity and knee osteoarthritis, while 58% believe obesity increases the risk of developing knee osteoarthritis.
2. Among all the respondents who are overweight (43%), 80% have attempted weight loss, while only 21% had great results. Additionally, only 21% consider biological therapy an effective way for weight loss, which indicates the need for increased education regarding the necessity and efficacy of biological therapy among individuals who are overweight.
3. When asked which measures can reduce the impact of obesity on knee osteoarthritis, 92% of our respondents mentioned weight reduction, while only about half of that, 47%, chose seeking medical assistance.

Based on the results above, we concluded that although people may have general knowledge of obesity and osteoarthritis, they are not provided with sufficient and detailed knowledge to further deal with this problem, which means need more education and publicity is of the most important. We were further addressed in our Edu & Communication page.

Nanjing-NFLS Alliance Meetup

We invited several PhD students from Nanjing University, University College London, University of Queensland, National University of Singapore, and University of California, San Francisco, and held a meeting online, in which we discussed our project and lab security.

Fig.4 Nanjing-NFLS alliance Meetup with aboard universities

We received feedback about potential improvements and were alerted to possible risks associated with our product. Following this, The PhD students introduced the safety regulations governing lab operations to us. This comprehensive feedback and briefing greatly helped us refine our approach for future experiments.

Fig.5 PhD students from aboard universities give us many useful advices on our project

Fig.6 Nanjing-NFLS team exchanged safety and security rules with PhD students from aboard universities

The Attitude of Stakeholder in Pharmaceuticals

Novozymes Pharmaceuticals, established in 2012, is a global supplier of bioactive compounds and widely contributed in other fields as well. The company has been particularly noticeable for its indelible contribution to global efforts combating the new coronavirus, through the development and production of nucleic acids. Recently, we have had the chance to interview the Vice President and Technical Director of Novizen Pharmaceuticals, Mr. Feng Su, as well as Mr. Tang Bo, the Deputy General Manager.

Micro-RNA technology expertise: Our discussion primarily centered around the biosafety of micro-RNA drugs. We also addressed various technical difficulties associated with these drugs. Mr. Feng underscored the vital importance of the delivery system in this context. The fundamental features necessary for any effective delivery system include:

1. Shielding the anti-miR/miRNA mimics from premature degradation once they enter the bloodstream.
2. Positioning the anti-miR/miRNA mimics in close proximity to the target cells.
3. Promoting cellular uptake of the anti-miR/miRNA mimics.
4. Ensuring that the system does not trigger any immunogenic response.
5. Incorporating materials that are both biocompatible and biodegradable.

Commercial aspects of micro-RNA drugs: The Vice President of Novozymes discussed the potential of micro-RNA drugs in the market and their operation. He urged us to focus on technology and R&D, and suggested collaboration with established pharmaceutical enterprises.

Fig.7 visiting Novozymes Pharmaceuticals

Public Knowledge of New Biological Therapy (the safety of micro-RNA drugs)

We surveyed public attitude towards using new biological therapy on obesity and osteoarthritis, which means public attitude on the safety of micro-RNA drugs. By analyzing the survey result, we found that:

1. Safety is the main concern that the respondents of the second survey have on the new therapy, with 80% mentioning it. We need pay more attention on ensuring and advocating the safety of our therapy.
2. About 30% of our respondents in the second survey approve using synthetic biology methods to alleviate the impact of obesity on knee osteoarthritis, and 38% need more information before making a judgment. We hope to provide quality information to them and help them make an informed decision.

In conclusion, our analysis reveals that while a substantial portion of the public recognizes the connection between obesity and knee osteoarthritis, there is a clear need for enhanced education on the efficacy of weight management strategies, especially biological therapy. Addressing concerns related to safety and access to treatments is crucial for wider acceptance and utilization. Additionally, providing comprehensive information on synthetic biology methods can empower individuals to make well-informed decisions regarding their health. These insights will inform our educational initiatives, which can be read in our education page.

Discuss Follow-up Experiments at CCiC

We took part in the 10th Conference of China iGEMer Community (CCiC). During the conference, we discussed our project concept, engineering design, and integrated human practices through a poster and online presentation. Experienced high school and Collegiate iGEM teams, familiar with miRNA, assisted us in transforming our follow-up experiments into protocol outlines. Notably, they guided us on how to identify the target gene of miRNA. We're thankful for the collaborative platform of CCiC that allowed us to interact with multiple teams!

Fig.8 The booklet to introduce our project in CCiC

Fig.9 The poster displayed in CCiC

Consulting Osteoarthritis and Obesity Experts on Implementation

During the second interview with Professor Guo, we gained a much deeper understanding on biological factors related to knee osteoarthritis. Professor Guo suggested doing an overlap between target gene that potentially have effects on osteoarthritis and gene that control knee cartilage to ensure the target gene that can cause knee osteoarthritis. If found, cultivate them in the cell test solution to verify. If it really has impacts on the cartilage, we need to first identify whether it inhibits or promotes the formation of cartilage. If it inhibits the formation, we can use an inhibitor to inhibit the RNA; on the contrary, if it promotes the formation, we can use a mimic to promote it. Both needs do cell experiments. In the future, direct intraarticular injection of miRNA can be tested by animal experiments and clinical trials.

Since the only method to cure osteoarthritis in obese subjects available now is to lose weight, the medicine has a much easier way, which means it will have a grand market. Despite this, the medicine still has some limits. First of all, inhibitor and mimic are synthetic drugs, which means they may have side effects. Second, micro-RNA is targeted at a specific gene, not a body part. So, it doesn't have a strong pertinence on knee and the cartilage. Which is where we need further research and improvement.

Fig.10 Interviewing with Professor Zhao on the experiment implementation.

Please click the link to see the video interview in BiliBili:
Video: Interview with Professor Guo about osteoarthritis (2).

When queried about the process of confirming miR-3074's target genes in joint action, Professor Zhao explained the meticulous methodology. She described a multi-step approach involving miRNA profiling and cross-referencing with databases like TargetScan and Miranda. By identifying common target genes, such as SMAD4, associated with arthritis-related mechanisms like cartilage development, researchers can begin to uncover the gene interactions underlying joint health.

Regarding strategies to inhibit miR-3074, Professor Zhao outlined an experimental pathway. The verification process entails fluorescent labeling of extracellular vesicles and miRNAs to ascertain their joint entry capabilities. Utilizing dual luciferase assay with distinct fluorescence assists in verifying miR-3074's presence in joints and establishing specificity.

Professor Zhao emphasized the burgeoning potential of bioengineered therapies involving miRNAs. Drawing parallels with successful applications in countering drug addiction, she underscored the transformative potential of engineered miRNAs in treating conditions like osteoarthritis.

In terms of safety, ethical concerns and potential risks were discussed. While patient-derived extracellular vesicles seem promising due to their autologous nature, therapies using engineered miRNAs from external sources necessitate careful scrutiny for compatibility and adverse effects. Ongoing international research focuses on assessing the safety of these innovative approaches. In summary, the interview highlighted Professor Zhao Yue's pioneering work in deciphering miRNA targets and exploring their therapeutic utility in addressing obesity and osteoarthritis. The potential of bioengineered miRNA therapies offers hope for novel treatments, yet the safety and ethical dimensions remain integral to their future implementation.

Fig.10 Interviewing with Professor Guo on the experiment implementation

Please click the link to see the video interview in BiliBili:
Video: Interview with Professor Zhao about ovese adipocyte exosomes (2).

Proposed Patients and Customers

Department of Health

The Department of Health's mission is advancing the health and well-being of all people by transforming the public health system through unique partnerships. They also take the lead in osteoarthritis treatment. When the department is informed of such a safe and efficient device, it will utilize such a product that can help its residents from the pain of OA.

Obese and OA Individuals

The primary target audience would be individuals who are already obese or overweight. These are at a higher risk of developing knee arthritis due to the excess stress on their joints. They may be seeking ways to manage their weight and reduce the impact on their knee joints. Obesity itself is an important risk factor for osteoarthritis disease, and weight loss can lead to improvements in other risk factors such as blood pressure and cholesterol levels. There's more to them than simply weight loss.

Business Plan

Publicizing our project at iG 20

iG 20 was hosted by team LZU-CHINA, with participation from over 28 teams. We joined iG 20 to expose more people to our project. We produced four videos, which included an introduction to our school, an overview of our project, a promotion of our team, and a segment about synthetic biology. Through iG 20, we were able to attract more attention to our project and raise awareness about the impact of obesity on our joints.

Learn from Other High School iGEM Team on Integrated Human Practice

The high school online conference was hosted by BGI-mommath, Nanjing-Bioxstem, BS-United China, Squirrel, and the Nanjing-NFLS team. This conference focused on the integrated human practice, with the key emphasis being on "Integration". Each team provided an introduction to their project and their human practice, while iGEMer human practice instructor, Mr. Bao Yuhan, gave detailed instructions to enhance the effectiveness of our human practice efforts. The closing loop of “Background-----Public Attitude-----Implementation-----Publicity to make impact” is critical for every team and is also a benchmark goal of iGEM. It's essential to address the problems we encounter during our project and employ human practice activities to solve these issues. While education, inclusivity, and outreach are important tasks, they are not typically categorized under human practice work.

Fig.12 High school meetup to exchange intergrated human practice

Learn from Similar Projects @ CRISPR team meetup

The CRISPR online conference was jointly hosted by Tongji-Software, WHU-China, AFMU, SMBU, CJUH-CLU-China, and the Nanjing-NFLS team. We were the only high school team to participate in this meeting. It was a fantastic opportunity for us to listen to and learn from the experiences of the other teams.

The CRISPR online conference was jointly hosted by Tongji-Software, WHU-China, AFMU, SMBU, CJUH-CLU-China, and the Nanjing-NFLS team. We were the only high school team to participate in this meeting. It was a fantastic opportunity for us to listen to and learn from the experiences of the other teams.

Fig.13 Similar project team meetup to exchange

Nanjing-China is a collegiate team aiming to use microbial cultivation technology to produce new dyed textile fibers as alternatives to traditional materials. Our shared residence in Nanjing brought us together, and our initial encounter at Nanjing University marked the inception of a productive collaboration. This partnership extended across various domains, encompassing the wet lab, modeling, and integrated human practices (IHP) facets of our respective projects. Throughout the year, our collaboration with Nanjing-China remained steadfast, driven by our common goal of optimizing the luciferase experiment, target gene searching modeling, western blot techniques, and cell transfection procedures that both of our teams were utilizing. Additionally, our joint efforts aimed to promote awareness about synthetic biology. In the realm of modeling, a significant exchange of knowledge occurred, as we shared insights into target gene searching modeling software with their dry lab team, while reciprocally gaining valuable insights from their modeling expertise (View detailed things in partnership page).

Fig.14 The meetup with Nanjing-China team

Timeline of Our Integrated Human Practice

The Last but Not Least

In fact, our student leader, Litong Wu, has personally benefited from this project. Through regular exercise and conscientious diet controls, he successfully reduced his BMI from 32 to 28. Beyond personal impact, we aim to utilize our project as a platform to amplify awareness about the implications of obesity and Osteoarthritis amongst our local community, and eventually, society at large.

Reference

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2. Chakraborty C, Sharma AR, Sharma G, Doss CGP, Lee SS. Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine. Mol Ther Nucleic Acids. 2017 Sep 15; 8:132-143.
3. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S. The Market of Biopharmaceutical Medicines: A Snapshot of a Diverse Industrial Landscape. Front Pharmacol. 2017 Jun 8; 8:314.
4. Bajan S, Hutvagner G. RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs. Cells. 2020 Jan 7;9(1):137.