Engineering Reciprocal Gene Circuits to Direct T-Cell Shuttling in Cancer Immunotherapy

Abstract

T cell exhaustion remains a major challenge in cancer immunotherapy, limiting the efficacy of adoptive T cell therapies. Chronic antigen stimulation within the tumour microenvironment (TME) drives T cells into a dysfunctional state, reducing their ability to mount a sustained anti-tumour response. Building on the work ‘Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodelling’ by Weber et al., we designed two complementary gene circuits to dynamically shuttle T cells between the TME and the draining lymph nodes (dLNs), where they can periodically recover before re-engaging tumours. Our strategy employs complementary gene circuits regulating the expression of chemokine receptors CCR7 and CXCR3 in response to chemokine signals. This allows T cells to migrate adaptively, balancing tumour infiltration with phases of rest and activation. To enhance circuit precision, we incorporate microRNA (miRNA)-based regulation to ensure mutually exclusive receptor expression, preventing functional interference. Additionally, we introduce inducible periodic receptor expression to fine-tune T cell trafficking dynamics. Using a microfluidic in vitro platform, we assess the ability of engineered T cells to respond to chemokine gradients, validating their capacity for controlled migration and functional recovery. Our findings demonstrate that synthetic gene circuits can provide a novel, programmable means of overcoming exhaustion by optimising T cell distribution between suppressive and supportive niches. This work presents a promising framework for improving T cell-based cancer immunotherapies, offering a strategy to enhance persistence and efficacy through engineered periodic migration.