After obtaining strong experimental evidence and clinical trials, ZJUintl-China 2022 focuses on the application and transformation of knowledge to facilitate the systematic absorption of Ambrosia-T research results into routine clinical practice and help more people delay aging and alleviate aging-related diseases. Ambrosia-T is an innovative product for the treatment of pathologic aging-related diseases and the pursuit of longevity. It is a modification of traditional CAR-T cell therapy. After analyzing the market and industry background of Ambrosia-T, to understand the audience's willingness to accept cell therapy and further refine our Ambrosia-T, we contacted patients and physicians in Human Practice. We believe Ambrosia-T has the potential to be a future first-line treatment to extend human life and achieve healthy aging, so it is our responsibility to propose real-world implementation and guide others to use it.

To put Ambrosia-T into the market as soon as possible, ZJUintl-China analyzed the current industry status and market potential of Ambrosia-T in the field of anti-aging, analyzed the cost of Ambrosia-T, and pointed out its advantages and innovation. To ensure Ambrosia-T can be managed correctly, ZJUintl-China wrote the implementation method and possible problems of the negative feedback switch in detail. At the same time, we propose possible future directions for Ambrosia-T to aid in continuous product improvement.

Driven by declining national fertility rates and a marked increase in life expectancy, the World Health Organization expects the number of people aged 65 years and older to grow from an estimated 524 million in 2010 to nearly 1.5 billion by 2050, with most of the growth in developing countries.[1] This greatly affects the normal operation of individuals, families, and society, and increases the burden of the state on medical care, social security, work and retirement, medical care, and sickness subsidies. Aging is the result of the general aging of the cells composing the individual, which is the comprehensive performance of the decline and disorder of the physiological function of the organ. Aging is a complex physiological and pathological process, accompanied by a significant increase in the incidence of cardiovascular diseases, cancer, diabetes, and neurodegenerative diseases. [2] Therefore, anti-aging and improving aging-related diseases have become the research hotspots of researchers around the world.

Since the accumulation of senescent cells can form tumors and induce other diseases, Senolytic drugs aim to target senescent cells and destroy them by their action. [3] According to the research of Sajeev and Sumant, the global longevity and anti-aging therapeutics market was valued at $25.1 billion in 2020 and is expected to reach $44.2 billion by 2030. [4] According to the treatment, anti-aging therapy can be divided into drug therapy, gene therapy, and immunotherapy. "Since drug therapy can target multiple biological pathways in a variety of tissues, side effects, such as neutropenia and thrombocytopenia in the case of navitoclax, are more intense, making it difficult to describe the underlying mechanism of any potential therapeutic effect." [5]Therefore, the demand for therapies that selectively, effectively, and safely target senescent cells has led to the rapid rise of CAR-T cell-based anti-aging methods and become a new star in the field of anti-aging. In 2020, the CAR-T cell therapy targeting upar protein that is specifically highly expressed in senescent cells was published in Nature, which successfully alleviated liver fibrosis and early-stage liver cancer in the elderly, and opened up the application of CAR-T in the field of aging. [6]

Ambrosia-T is a CAR-T cell therapy designed to target the highly expressed protein DPP4 on the surface of senescent fibroblasts. Different from traditional CAR-T cell therapy, Ambrosia-T also introduces a concentration monitoring switch of inflammatory factor IL-6 on the surface of T cells, so that anti-DPP4 CAR-T cells can not express at high concentration of IL-6, thereby greatly alleviating the cytokine storm caused by traditional CAR-T cells. In addition, this therapy is simpler and safer than other CAR-T cells. We provide a plasmid with both anti-DPP4 and IL-6 negative feedback switch functions. After isolating and extracting T cells, the operator only needs to electrotransfer the plasmid into T cells to obtain the Ambrosia-T product.

The expression of DPP4 protein in liver, lung, and heart fibroblasts has been confirmed to cause liver, lung, and heart fibrosis[7][8]. its selective expression in senescent cells also confirmed its high specificity[9]. inhibition of DPP4 protein expression has also been shown to alleviate liver, lung, and heart fibrosis as well as diabetic symptoms[10][11]. Therefore, Ambrosia-T can not only be applied to alleviate aging, but also help to treat a variety of aging-related diseases such as fibrosis of the liver, lung, heart, and other organs.

The end users of Ambrosia-T are large hospitals, elderly people who aspire to live longer, patients with fibrosis of the heart, liver, and lung organs, and even large research institutes with innovative, smart, and practical ideas. Their scientific ideas or products can be combined with Ambrosia-T to create better CAR-T anti-aging products. Ambrosia-T can be used in many applications, as supported by our product design. With this, our goal is to bring synthetic biology to a wider range of applications and closer to realization. We believe that the full implementation and implementation of Ambrosia-T will be able to facilitate further development in the anti-aging field in a safe manner.

Patients who approved for Ambrosia-T infusion need undergo the following treatment process:

Collection: The peripheral blood mononuclear cells are firstly extracted from patients via leukapheresis, and T lymphocytes are further purified by magnetic beads.

Activation: T lymphocytes are activated by monoclonal antibodies, coated magnetic beads or artificial antigen presenting cells.

Modification: The plasmids encoding the anti-DPP4 CAR gene and IL-6-based feedback loops gene are co-transfected into the T cells through electrotransfection to produce the Ambrosia-T.

Expansion: CAR T cells were expanded to the desired number of cells by static or dynamic culture vessels or devices.

Security test: After expansion and culture, CAR-T cells should be tested for activity and for microbial and endotoxin contamination.

Infusion: The Ambrosia-T are infused back into the patients’ bloodstream in a single infusion.

Recovery: After the infusion, patients should be monitored for the side effects and treatment response.

*Tips:

1. Ambrosia-T for self-use only.

2. Plasmid stock should be kept at -20°C freezer during transportation and storage to ensure its genetic stability until expiration date.

3. After transfection, Ambrosia-T should be stored and transported at liquid nitrogen. Please remelt the product in a 37 ° C water bath with special medium (RPMI-1640+10%FBS) to the titer before using.

4. Avoid Ambrosia-T exposure to extreme conditions including unsuitable pH, temperature, chelating reagents, and chemicals.

5. Avoid repeated freezing and thawing to prevent expiration.

6. This product is expected to slowly clear senescent cells with high expression of DPP4 in human body after injection to achieve the effect of reversing senescence. And because of the unique negative feedback regulation design, there is almost no severe cytokine release syndrome. But medical monitoring is still needed for a period after the injection to ensure safety. If respiratory symptoms, hypotension, anoxia and related symptoms occur after introducing Ambrosia-T, please contact your GP or go to the local hospital immediately.

Although we have initially demonstrated that Ambrosia-T has specific killing activity against DPP4-overexpressing cells at the in vitro level, there are still many challenges for future clinical application scenarios.

The survival rate and cell activity of Ambrosia-T were lower after electrotransfection. If possible, transfection of plasmids by lentivirus can be considered when laboratory safety level is achieved.

We need to ensure that CAR gene expression and the expression of IL-6 based feedback loop are in balance to ensure that our Ambrosia-T can function as intended. This requires modeling of the expression of these two genes and screening of promoters for optimization.

In addition, we need to obtain long-term evidence on the functionality and safety of Ambrosia-T. This requires the development of specific testing procedures to ensure that Ambrosia-T does not cause serious side effects while performing its normal function of clearing senescent cells

Moreover, since in our transfection method, the gene is integrated into random sites in the T-cell genome, this could potentially lead to the evolution of CAR-T cells into cancer cells. In the future, CRISPR-Cas9 could be used to insert genes into specific sites in the genome.

Finally, while gaining the approval of regulators, we need to make Ambrosia-T more acceptable to the public and reassure them. Therefore, we need to educate the public about the anti-aging principles of our CAR-T cells and gain their trust.

1. Cellular experiments can only be conducted in biosafety level 2 facilities (BL-2 level).

2. Please equip with appropriate with PPE including lab coat, face shield, and gloves, no skin should be exposed during experiment and your operation.

3. Associated waste materials contaminated by Ambrosia-TT need to be specially collected and processed according to your regional medical waste disposal law.

4. Hand hygiene and cleaning should be carefully followed.

The production of chimeric antigen receptor T (CAR-T) mainly consists of six steps, including leukapheresis, cell selection, T cell activation, genetic modification, T cell expansion, and harvest. To perform the cost budget of ambrosia-T, this business plan mainly focuses on the variable costs and ignores the annual fixed costs, with reference to Ran’s work. Table 1 illustrates the variable cost of each production. The leukapheresis requires 20000 RMB. For all consumables of all steps, such as media and cytokines, about 120000 are needed. The core plasmid which contains the DNA sequence of modified interleukin-6 receptor and anti-DPP4 CAR and the electrotransfection will cost 1500 and 250 RMB separately. To examine the efficiency and perform the sterile control, 3000 RMB are required. Thus, the cost of ambrosia-T per production is about 144750 RMB[12].

Adoptive transfer of autologous, genetically modified chimeric antigen receptor (CAR) T cells has shown impressive efficacy as a treatment that has emerged from oncology and is also gaining traction in the aging field. However, there are many obstacles to the widespread adoption of CAR T cells, including high cost, inefficient in vitro transfection, long time to manufacture cells, off-target, adverse events after infusion such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human cars. In the face of these problems, various approaches have been proposed, such as adjusting scfv affinity to reduce side effects, universal or allogeneic donor "off-the-shelf" CAR (UCAR) T cells to reduce cost, and solving the long in vitro manufacturing time[13] . Among them, a novel in vivo approach proposed by the cancer field caught our attention - exosome therapy for cancer. Exosomes are small membrane vesicles (30-150nm) containing complex RNAs and proteins that can be secreted by most cell types. Dendritic cell-derived exosomes can prime naive T cells and activate NK cells to shrink tumors[14] . This inspired us to use exosomes to generate CAR-T in vivo and also helped us to solve the problems of Ambrosia-T's high cost and long in vitro cell production time. Therefore, in the future, we propose a method to modify red blood cells to generate exosomes carrying target scfv to activate naive T cells to form CAR-T cells in vivo.

5th Floor, Zhejiang University Medical Center, Wenyi West Road, Cangqian Street, Yuhang District, Hangzhou City, Zhejiang Province, China

For more information about Ambrosia-T, please visit: https://2022.igem.org/Team:ZJUintl-China

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