Piro, Francesca (2024) Investigation of post-transcriptional regulators for long-term efficacy of T cell-based immunotherapies. [Tesi di dottorato]

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Abstract

CAR-T therapy, the immunotherapy based on the selective engineering of T cells with chimeric antigen receptors (CAR) has obtained tremendous success in treatment of liquid malignancies, yet it results quite less effective for solid-tumor therapy. There are several reasons to this poor outcome, with main challenges that urge to be addressed. These include low persistence and functionality of T cells within the tumor microenviroment (TME), and life-threatening side effects caused by an excess of CAR-T activation and cytokine release. In particular, a main cause of loss of functionality of engineered T cells is the chronic antigen exposure in the TME, a condition better known as exhaustion. This is a dysfunctional state promoted by tumor and inhibitory immune cells in which CD8+ T cells loose their avidity, proliferative and cytotoxic capacity. Improving the long-term functionality of effector T cells is critical to overcome inhibitory factors within the TME and elicit tumor regression. In this picture, synthetic biology is becoming a new key player in engineering cell-based immunotherapies. With specific genetic circuits which sense environment inputs and trigger controlled outputs, synthetic biology aims at reprogramming cell fate and functions in a spatial-temporal fashion. In this work, post-transcriptional regulators that leverage on RNA interference (RNAi), are used to augment the efficacy of the immunotherapy by specific targeting key regulatory genes of T cell functionality and dysfunction. Our objective is to engineer CD8+ T cells or CAR-T cells with a controllable ON-OFF switch by incorporating a sensing module. This module consists of a synthetic promoter that activates in response to exhaustion, triggering the expression of a post-transcriptional RNAi actuator module as the output. In specific, we aim to silence hyporesponsive-associated proteins CBL-b, CISH, SOCS1 downstream the TCR signalling and exhausted driver proteins DNMT3α, TOX, TOX2. The silencing is achieved through the design of DNA-encoded miRNA-based shRNAs (shmiR). In my PhD work, the design of shmiR and downregulation of exhaustion targets was evaluated in HEK293 and CD8+ T cells. In the latter, downregulation of proteins CBL-b, CISH, SOCS1 improves Granzyme B secretion while downregulation of exhausted-related proteins decreases expression of TOX, CTLA-4 or PD-1. The shmiR cassette were subsequentially cloned under different RNA pol II promoters and integrated in Jurkat and T cells. In particular, shEF1α and synthetic promoter inducible promoter responding to NR4A2 exhaustion transcription factor, induce the expression of shmiR. In summary, we developed a potential versatile strategy that operates locally in the TME and induces a genetic switch from ON to OFF, improving T cell functionality overcoming exhaustion. Our constructs pave the way for the development of novel immunotherapy that could be used to potentially overcome current limitation in the treatment of solid tumor.

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