Our team was divided into a dry group, a wet group and an HP group. The dry group completed the web page building and mathematical modeling. The wet group completed the project design and experiments. The hp group completed the education, promotion video production and other human practice activities. IDT and other companies provided the materials and reagents for our experiments. Zhejiang University - University of Edinburgh Institute and Zhejiang University Medical Center provided us with financial and space support. Zhejiang University Institute of Hematology provided technical guidance. For more information, please visit our WIKI.

We are concerned about the aging of the population. Human aging is reflected in organ function decline due to organ fibrosis, etc. The level of fibrosis can be effectively reversed by removing senescent fibroblasts. We are concerned that CAR-T has the potential to be applied. Therefore, we have done so by screening for applicable aging-associated targets and constructing a killing system that removes senescent fibroblasts. In addition, to avoid the severe toxicity exhibited by the side effects of CAR-T therapy. We designed two novel switches and examined their efficiency. One of them is a negative feedback switch that enables automated modulation of T-cell killing, thus enabling the avoidance of side effects. The other one is a caffeine-induced switch. For more information, please visit our WIKI.

We collected and analyzed aging-related single-cell data and compiled some viable targets. We constructed an anti-aging CAR-T killing system, which provides the basis for cell therapy applications in anti-aging. The CAR structures and sequences we provided demonstrated superior killing efficiency in the experiments. We also identified excellent transfection in both Jurkat and Raw 264.7 cell lines. In addition, we constructed negative feedback effector proteins for automated control of T cell function, which is the first application of negative feedback in T cells. We fused caffeine antibody (COSMO) into the structure of CAR, which is also a bold attempt to improve the structure of CAR. For more information, please visit our WIKI.

Our group aimed to develop a method to reverse the senescence. We chose the chimeric antigen receptor (CAR)-T cell therapy, screened out the aging target DPP4, and constructed anti-DPP4 CAR-T cells. To test the killing effect of our CAR-T cells, we co-cultured CAR-T cells (experimental group) and Jurkat (control group) with K562 cells transfected with DPP4 plasmid. Compared to the control group, anti-DPP4 CAR-T cells showed good killing ability, which showed the success of engineering. To prevent cytokine release syndrome during CAR-T therapy, we designed three devices that would inhibit the CAR expression or downregulate the CAR activity in CAR-T cells when IL-6 concentration reached a certain level. We expressed these devices separately with anti-CD19 CAR in Jurkat cells and found that cells transfected with IL-6R-PD-1 showed an obvious reduction of cytotoxicity, suggesting the potential for negative regulation in CAR-T therapy.

With the diversification and deepening of research content, collaboration plays an increasingly important role in the achievement of goals. One explanation for collaboration: to achieve a common goal, the full use of organizational resources, relying on the collective strength of the team to complete a task. This year, ZJUintl-China has cooperated extensively with other iGEM teams(such as NUM-China, NJU-China) and social forces, which is the success of our team and Ambrosia. We agree that it is important to establish broad, long-term, solid collaborative links between different iGEM teams, which will bring great growth potential to each iGEM team and iGEM.

Our teams take full account of the impact on the world from design to implementation in order to engineer a user-friendly, safe and effective product. First of all, we chose anti-aging, which has been pursued by humans for millennia, as the topic of our project. To avoid the potential side effect of CAR-T, after consultation with experts and literature research, we designed a user-friendly negative feedback switch monitoring interleukin-6 and caffine triggered CAR-T. Considering peoples' concerns about CAR-T's cost and long preparation cycle, we designed the engineered erythroid progenitor cells to generate exosomes for CAR-T programming as our future plan. In addition, recognising that not much is known about synthetic biology, we have led and organised six other teams to produce a synthetic biology atlas to promote its popularity. We also go into the mountains of poverty to introduce synthetic biology to the primary school children there.

Ambrosia-T is a modified CAR-T cell therapy that can effectively alleviate physiological aging, and improve cardiac, lung, and liver fibrosis in the elderly with fewer side effects. The treatment provides a plasmid that contains anti-DPP4 and IL-6 negative feedback switch functions, making it simple and safe to use. Operators only need to electrotransfer the plasmid into autologous T cells and inject them into the user's body once achieving the appropriate killing effect. Operators were required to wear appropriate personal protective tools and complete all experiments in biosafety level 2 facilities. Experimental wastes should be collected and treated specifically in accordance with relevant laws. If electrotransfection efficiency is low during the experiment, the operator can try lentivirus transfection if safety allows. ZJUintl-China is attempting to file for Ambrosia-T's patent with the Chinese Intellectual Property Office to implement it as quickly as possible.

We actively communicate with society and integrate insights from the community into our project. Here are major reasons.

1. To provide a better user experience, we interviewed the regional sales director of Tongrentang Chinese Medicine. She suggested that user-friendly products are always preferred. So we designed the CAR-T triggered by common caffeine.

2. CAR-T expert introduced us to the benefits of CAR-Macrophage. Considering the advantages of macrophages in cell culture and liposome transfection, we decided to first validate the caffeine-initiated CAR expression circuit in CAR-Macrophage

3. We conducted 4 mini-chat with students and businessmen. Most of them are concerned about the high price and longer preparation of CAR-T. As a future plan, we designed engineered erythroid progenitor cells to generate exosomes encapsulated with specific mRNAs, which can program T cells into CAR-T. This cost-effective and immediately available product allows the generation of CAR-T in vivo with the injection of exosomes.

We developed two models, a model in which CAR-T kills target cells and an intracellular model in which CAR-T cells express CAR-T proteins. Based on our experiment results and data from previous studies about targeted CAR-T cell transfer that have been applied in clinical trials, like anti-CD19 CAR-T cell therapy, a computational model was built for predicting the therapeutic effect of the DDP4-targeted CAR-T cell. For the Intracellular CAR-T protein expression model, it was developed with parameters and models from previous studies to check whether the CAR-T gene we transfected was effective. All in all, the simulation of the modelling made up the in vivo experiment that has not been conducted, which serves as a good prediction for the effectiveness of the DPP4-targeted CAR-T cell and transcribed CAR-expressing genes, though the quantitative results provide limited reference since it’s a conceptual model.

Our design is divided into two parts, killing and controllable elements, which jointly prove the feasibility of our design. First, we designed an anti-aging CAR-T killing system. CAR-DPP4 demonstrated superior killing efficiency in removing senescent cells in our experiments. For the controllable element, we added caffeine antibody (COSMO) into the structure of CAR to modulate killing intensity. Furthermore, we constructed negative feedback loops to moderate cytokine release syndrome (CRS) during CAR-T therapy. When IL-6 concentration, an important marker for CRS, reached a certain high level, what we designed would trigger the inhibition of cellular CAR expression or disruption of downstream signalling pathways. In our experiments, cells transfected with IL-6R-PD-1 plasmid showed a reduction of cytotoxicity, which proves that our negative feedback element regulation is effective. For more information, please visit our WIKI.

Goethe once said, "Conformity is always the highest need of all good-minded people." Collaboration is also one of the principles that iGEM participants follow carefully, and it has deeply influenced the exploration path of successive iGEMers. Collaboration with other iGEM teams can simplify complex problems and enable the development of collective wisdom to find the best solution, which is the embodiment of the efficiency, vitality, and innovation of an iGEM team. This year, we have established a solid collaborative partnership with NMU-China, and the two teams are working hand in hand to create a new chapter in problem solving, innovation and development.

ZJUintl-China 2022 strives to popularize synthetic biology knowledge to people of all ages and fields through engagement with the general public and other iGEM teams, while also improving Ambrosia-T. For people of different ages and professional backgrounds, we have carried out various offline synthetic biology activities: volunteer teaching in poor areas, vivid animation screenings, biological experiment courses, popular science lectures, and seminars. To increase the impact of our actions, we have opened the “ZJUintl iGEM” WeChat official account, offering rich synthetic biology materials online and offline: interesting videos and slides, books, and comprehensive synthetic biology maps in collaboration with 5 other iGEM teams. Additionally, to continuously improve Ambrosia-T, we exchange experimental progress and problems with experts weekly, and host national and regional iGEMer meetings to hear their opinions. We also interviewed people from all walks of life and improved the cost and concept of AmbrosiA-T based on this.

ZJUintl-China concurs that education ought to be a lifetime pursuit for all people. Our educational initiatives are primarily organized into three categories: public education, education for elementary school pupils in poor areas where lack educational resources, and education for students studying biology who are roughly the same age. We built a WeChat public platform, which serves as a forum for public education on synthetic biology and for showcasing the work of our team. We produced an accessible atlas of synthetic biology knowledge for the general public to read. Some of our team members went to low-income and poor-education regions to give lectures and hold seminars to introduce basic knowledge of synthetic biology, the human immune system, cellular immunotherapy, and chimeric antigen receptor-T cell therapy. We also inspired biology major students. We believe this will pique more people's interest in synthetic biology, encouraging them to learn more about it and make contributions to this area.

ZJUintl-China is an iGEM 2022 team that values diversity and inclusivity. We worked hard to identify and address inequalities within and outside the team. Within the team, we did well in recruiting team members, team cooperation, and team financial management. The selection of our team members was fair and unbiased, regardless of gender, religion, and speciality. The task and funding allocation depended on the needs and realities. We maximized the effectiveness of everyone's time, energy, and resources. Outside the team, we showed our inclusivity when doing the publicity, mini-chat, and team website design. We introduced the knowledge of synthetic biology and anti-aging immunotherapy to all kinds of people. We provided public science lectures in underprivileged and educationally underdeveloped areas. We gained insight from people of various identities through interviews to help advance the study of synthetic biology and anti-aging immunotherapy. Our wiki website is friendly to those with a physical disability which makes it difficult for them to read.

We actively communicate with society and integrate insights from the community into our project. Here are major reasons.

1. To provide a better user experience, we interviewed the regional sales director of Tongrentang Chinese Medicine. He suggested that user-friendly products are always preferred. So we designed the CAR-T triggered by common caffeine.

2. CAR-T expert introduced us to the benefits of CAR-Macrophage. Considering the advantages of macrophages in cell culture and liposome transfection, we decided to first validate the caffeine-initiated CAR expression circuit in CAR-Macrophage

3. We conducted 4 mini-chat with students and businessmen. Most of them are concerned about the high price and longer preparation of CAR-T. As a future plan, we designed engineered erythroid progenitor cells to generate exosomes encapsulated with specific mRNAs, which can program T cells into CAR-T. This cost-effective and immediately available product allows the generation of CAR-T in vivo with the injection of exosomes.

Four heart-related datasets of mouse, rat and monkey were used for marker selection. Limma and DEGSeq2 were used for differential gene expression analysis. Genes significantly (p < 0.05) upregulated genes (LogFC > 0) in senescent samples were considered as potential markers. A Python-based web crawler was used to automate the filtering process of membrane protein based on UniProt database. A total of 63 genes (12, 51 upregulated in 4 and 3 datasets respectively) entered the follow-up screening. Expression of the potential markers should be relatively tissue-specific and low in T cells. Using UCSC, HumanProteinAtlas and Mouse Cell Atlas of Zhejiang University databases, combined with results of literature analysis and other previous data set analysis, the final 6 potential targets were identified (Anxa3, Icam1, Vsir, Tspan8, Cyba, Fxyd5). Cell subset analysis was performed on the four datasets using Seurat 4.1.1 to obtain the expression level of each target in the cell subset.

We developed two models, a model in which CAR-T kills target cells and an intracellular model in which CAR-T cells express CAR-T proteins. Based on our experiment results and data from previous studies about targeted CAR-T cell transfer that have been applied in clinical trials, like anti-CD19 CAR-T cell therapy, a computational model was built for predicting the therapeutic effect of the DDP4-targeted CAR-T cell. For the Intracellular CAR-T protein expression model, it was developed with parameters and models from previous studies to check whether the CAR-T gene we transfected was effective. All in all, the simulation of the modelling made up the in vivo experiment that has not been conducted, which serves as a good prediction for the effectiveness of the DPP4-targeted CAR-T cell and transcribed CAR-expressing genes, though the quantitative results provide limited reference since it’s a conceptual model.