Aging is manifested as a decline in various body functions, caused by cellular senescence, accompanied by altered gene expression and a senescence-associated secretory phenotype (SASP) (fig.1) (Borghesan et al., 2020). For senescent cells, the immune system recognizes and removes them. However, over-activation of immune cells leads to their exhaustion, which is accompanied by an increase in the rate of senescent cell production as we age, and the senescence of immune cells, or immunosenescence (Santoro et al., 2021). In the heart, for example, myocardial fibrosis is a typical age-related disease that can manifest clinically as arrhythmias or heart failure. Senescent fibroblasts play a key role in myocardial fibrosis. Also, aging fibroblasts promote fibrosis in the liver, lung, kidney, and other organs (fig.2).

In previous studies, removal of senescent cells (Senolytic) has been shown to alleviate some aging phenotypes and reverse aging indicators (Amor et al., 2020; Di Micco et al., 2021). Removal of senescent fibroblasts has been shown to alleviate liver fibrosis. Since the main clinical causes of human death are now due to diseases and organ failure, removal of senescent cells is now an effective way to combat aging.

CAR-T immunotherapy is highly effective by targeting cancer cells, and as research continues, synthetic biological components and lines are being added to CAR-T cells to enhance or achieve their efficacy. Since the killing properties of T cells target all target cells, targeted killing of senescent cells by CAR-T is possible. Previous studies reversed the aging phenotype by killing mouse liver senescent cells with urokinase-type plasminogen activator receptor (uPAR)-targeted CAR-T cells, suggesting that CAR-T has rich promise in the field of anti-aging (Amor et al., 2020). Previous studies have focused on pathological phenotypes that result from aging, such as failure and cancer (Amor et al., 2020; Gasek et al., 2021). We prefer to focus on the damage to the organs of the aging process and target the removal of its harmful components. Therefore, we selected single-cell transcriptomic data from the heart and lung of aging (disease-free) mammals and young animals for differential gene expression analysis to select more promising targets for anti-aging applications.

Inflammatory cytokine release syndrome (CRS, also known as cytokine storm, CS), the most common side effect of CAR-T immunotherapy, occurs when immune cells release many cytokines into the environment (Liu et al., 2020). As for the mechanism of CRS, one potential explanation is that Granzyme B secreted by CAR-T cells activates GSDME in target cells to mediate focal death of target cells. The scorching of target cells stimulates macrophages to release inflammatory cytokines such as IL-6. Limiting the function of CAR-T cells through switches is promising in circumventing side effects. Recent studies have demonstrated a transgenic expression system regulated by resveratrol to control CAR-T cell immunotherapy and improve the safety of tumor immunotherapy by regulating CAR expression (ON) and suppression (OFF) in T cells by resveratrol. Another study controlled the viability of CAR-T somatic cells in solid tumors by focused ultrasound, which could mitigate off-target effects and reduce the damage of CAR-T to all normal structures. Considering that anti-aging therapy does not need to be as fast as anti-cancer therapy, but rather requires continuous stable and gentle treatment, negative feedback loops may have a good application.

The project is to engineer assembled T cells to target and kill senescent fibroblasts to reverse senescence-associated phenotypes, with the following main subprojects:

1) Engineer chimeric antigen receptors (CAR)-T cells to kill senescent cells - 1. Validate the efficiency of our constructed anti-Dipeptidyl Peptidase 4 (DPP4) CAR-T killing system (fig.3); 2. Screen and validate new heart & lung senescence-associated targets.

Our project aims to engineer T cells to remove senescent cells through chimeric antigen receptors. The chimeric antigen receptor itself is not a naturally occurring protein, but a novel protein structure formed by artificial fusion, which satisfies the idea of synthetic biology. At the same time, we have modified the single-chain variable fragment of the antibody of the chimeric antigen receptor to give it the ability to recognize senescent cells. In addition, we innovated the binding of the interleukin 6 receptor to proteins such as Notch to form automated control structures that inhibit the action of T cells. We also fused caffeine antibodies into the structure of chimeric antigen receptors, aiming to develop extremely simplified switches. The application of these switches themselves based on synthetic biology will at the same time contribute to the future application of CAR-T immunotherapy in the clinic.

Our project aims to engineer T cells to remove senescent cells by chimeric antigen receptors, but we also want to avoid side effects through the principle of negative feedback and switching. Our goal is to alleviate the fibrosis problem faced by the elderly and to help them rejuvenate their bodies while reducing their risk of developing disease. Therefore, we first designed several novel protein structures. By transducing the gene sequences corresponding to these protein structures into Jurkat cell lines, successful transduction cell lines were obtained. The effect of our designed components in the successfully transduced cells was verified in the corresponding cellular environment.

We learned about the iGEM competition after seeing the publicity for the NMU-China 2021 team. We saw the previously published paper on Senolytic CAR T cells reverse senescence-associated pathologies and wanted to expand on that idea, so we screened for new targets (more information in "measurement "). Also, during the exchange with the NMU-China 2022 team, we learned that avoiding side effects by means of switches is an effective way, so we designed novel switches with the advice of our PIs.