As a part of iGEM community, we are aware of our responsibilities. From topic selection to experimental implementation, ZJUintl-China iGEM consistently engaged in the real world and strived to make our project good and responsible for society. From the selection of our project theme, we took full account of the needs of the community. The fastest-growing ageing population is an important problem in China. According to WHO, by 2040, the number of people over 60 will reach 402 million with the development of health care and declining fertility rates. As Roman playwright, Terentius claimed, “senectus ipsa est morbus”, aging and disease are closely related. During aging, various diseases will appear, such as diabetes, hypertension and osteoporosis. These diseases cause great suffering to older people and pose a huge challenge to the government's health care system. The same issue also exists elsewhere in the world. Population aging is becoming an essential global problem. In response to this challenge, our team determined to choose anti-aging as the topic of our project. During our literature research, we noticed that senescent cells are characterized by senescence-associated secretory phenotypes (SASP). They can continuously secrete a variety of inflammatory factors that affect other healthy cells, thereby expanding the scope of senescence. In short, senescent cells themselves are the source of ageing. This reminded us that the elimination of senescent cells can be a potential anti-aging method. Thus, ZJUintl-China iGEM aimed to design one anti-aging CAR-T to reverse physiological ageing.

Anti-aging as an ambitious topic has been pursued by mankind for millennia. In the face of this big challenge, we actively communicate with a wide range of stakeholders with various knowledge backgrounds, such as biomedical scientists and businessmen. After careful consideration of their suggestions, we revisited the team's project and integrated insights from the community into our project, including the optimization of plasmid design and the addition of the subproject. We sincerely believe that by constantly listening to people from all walks of life and iterating on our projects accordingly, we ultimately make our projects accountable and beneficial to the world.

After choosing the elimination of senescent cells as our anti-ageing method, we attempted to find the proper type of targeted senescent cells and identify the senescence marker on the cellular surface. Given that ageing is a complex process that occurs in multiple organs and that the removal of large numbers of senescent cells may affect organ function, the selection for the specific senescent maker and the target organ has troubled us for a long time. Prof. Meng gave us useful advice. He mentioned that the liver has a high regenerative capacity and thus is a good target organ. Even though the anti-aging CAR-T removes numerous senescent cells, the liver's strong self-renewal ability allows these cells to be replenished very quickly. This inspired us quite a lot. After the literature review, we selected senescent fibrotic cells as the target cells for anti-aging CAR-T. Considering Prof. Meng’s advice, except for classic cardiac fibrosis, we also selected liver and lung with self-renewal ability as our target organs. Dipeptidyl Peptidase 4 was chosen as the cellular surface marker of senescent fibroblasts.

Tongrentang Chinese Medicine is a well-known Chinese pharmaceutical seller which has a long history of influence. We were curious as to how sellers of medical and healthcare products, who are closely connected to the frontline market, viewed our project. Therefore, we interviewed Mrs.Yu, the general manager of Tongrentang sales in Hunan Province, China. Mrs.Yu drew on his own extensive experience in selling healthcare supplies and pointed out that consumers prefer products that are easy to use, even in the medical field. This greatly inspired us. Before the interview, we were looking for ways to control the strength of the CAR-T response to prevent cytokine release syndrome, and Mrs.Yu's suggestion prompted us to look for a common household compound that could act as a molecular switch to initiate CAR-T. We eventually adopted caffeine as the switch to initiate CAR expression. We believe that initiating CAR-T killing by drinking coffee when needed can bring great convenience, optimize the user experience and be more user-friendly. Although we later discovered that we could design a negative feedback line for IL-6 to enable fully automated control of CAR-T work, this interview was still very significant. Mrs.Yu's comments prompted us to think from the user's point of view and laid the foundation for further improvements.

Prof. Dongrui Wang is a biomedical scientist and a professional CAR-T researcher. Therefore, we interviewed with him to get professional advice for our project. He mentioned that several immune cells are also suitable to be armed with chimeric antigen receptors (CAR) and have various unique advantages. For example, CAR-macrophage will not cause severe GvHD in an HLA-mismatched scenario. This encouraged us to review the selection of chassis for CAR. Finally, considering the high efficiency of liposome-mediated transfection of macrophages, we decided to first validate the caffeine-triggered CAR expression circuit in macrophages and take this CAR-macrophage as a new subproject.

Listening to people from different educational backgrounds helped us to obtain a holistic view of our project. Thus, we held four mini-chats with the medical student, pharmacy student, business student, and businessman, each of whom provided much interesting advice from their perspective. However, we noticed that all of them, especially the businessmen and business students, mentioned that the high cost of CAR-T might hinder the roll-out of the project and expressed great concern about this. It is our responsibility to take full account of the needs of our users and design products that are truly beneficial to the world. Therefore, we thought about how to reduce the cost of CAR-T. We found that the lack of versatility of CAR-T was a major reason for its high cost. For the traditional process of CAR-T therapy, users’ autologous T are collected and then reprogrammed and transformed into CAR-T at the manufacturing facility. After all these complex time-consuming processes, the CAR-T can be transfused back into the user's body. Thus, we sought a potential solution for universal CAR-T production. Inspired by the work of Jonathan A. Epstein’ team, we designed engineered erythroid progenitor cells to generate special exosomes that contain mRNA and can transform T cells into CAR-T in vivo. Ideally, these programmed erythroid progenitor cells will constantly produce universal and functional exosomes. Users only need to inject exosomes to generate anti-aging CAR-T in vivo and thus maintain their youth and vitality. Due to time limitations, we did not complete this experiment. However, we have designed the relevant plasmids for future plans.

Through human practice, we realized that listening to people from different backgrounds allows us to revisit our projects with a fresh perspective and make further improvements. Prof. Meng's comments inspired us to determine the final target organs and senescence marker on the cellular surface. From the perspective of an experienced salesperson, Mrs. Yu emphasized the importance of user-friendliness. Therefore designed the caffeine-initiated CAR-T. With his expertise in CAR-T, Prof. Wang made us aware of the advantages of macrophages as chassis cells, which led to the creation of the subsequent CAR-Macrophage. In four Mini-chats, three students with different majors and a businessman unanimously made a strong call to reduce the price of CAR-T. This reminded us that the high cost could be the biggest obstacle for our ambrosia-T to obtain widespread acceptance of users and truly improve the quality of life for older people. In response to this challenge, we designed our future plan, an engineered erythroid progenitor, which generates exosomes that can produce CAR-T in vivo. Although our project only focused on the anti-ageing in the liver, heart, and lungs to a certain extent by simply removing senescent fibroblasts. However, we believe that with the support of society and experts, CAR-T will be of great value in the field of anti-ageing. In the future, perhaps immortality will no longer be a crazy fantasy.