Harnessing Regnase-1 and Roquin-1 activity to modulate T cell function

In the past decade, T cells have served as an important immune cell type in the paradigm shift from molecular to cellular medicine. In particular, chimeric antigen receptor (CAR) T cells have shown promise in the treatment of several hematological malignancies, leading to six U.S. Food and Drug Administration (FDA) approvals to date for indications such as leukemia and multiple myeloma. However, the efficacy of CAR-T cell therapies has been limited in solid tumor indications, posing a need for different strategies to engineer more potent therapeutic cells. Advances in genetic engineering and synthetic biology offer new tools and concepts to potentially design T cells with more effective or complex functions. This thesis explores the development of more potent and dynamic therapeutic T cells using CRISPR-Cas9 genome editing technology and RNA synthetic biology. Specifically, I harness the roles of regulatory RNA binding proteins (RBPs) Regnase-1 and Roquin-1, which restrict inflammatory gene expression in T cells, to increase T cell inflammatory function and to modulate transgene expression in T cells. First, I compare the function and phenotype of Regnase-1 knockout (Reg1-KO), Roquin-1 knockout (Roq1-KO), and Regnase-1 and Roquin-1 double knockout (DKO) human T cells engineered with a clinical-stage CAR targeting mesothelin (M5 CAR) and a clinical-stage T cell receptor (TCR) targeting New York esophageal squamous cell carcinoma 1 (NY-ESO-1, 8F TCR). While genetic disruption of Regnase-1 or Roquin-1 alone increases antitumor function of engineered T cells, dual disruption of both genes results in more potent antitumor function, most strikingly characterized by massive proliferation in vivo that is associated with toxicity in NOD/SCID/IL2Rγ-/- (NSG) mice treated with CAR-T cells. Next, I explore the use of stem loops targeted by Regnase-1 and Roquin-1 to leverage the powerful regulatory function of these RBPs on transgenes of interest in T cells. I demonstrate that stem loops can function in conjunction with another regulatory RNA element and be used to modulate transgene expression in a Regnase-1 and Roquin-1 dependent manner. My hope is that continual advances in genetic engineering, synthetic biology, and cell biology will improve the therapeutic function and scope of cell therapies.