Stem Loop Mediated Transgene Modulation in Human T Cells

Controlling gene expression is useful for many applications, but current methods often require external user inputs, such as the addition of a drug. We present an alternative approach using cell-autonomous triggers based on RNA stem loop structures in the 3' untranslated regions (UTRs) of mRNA. These stem loops are targeted by the RNA binding proteins Regnase-1 and Roquin-1, allowing us to program stimulation-induced transgene regulation in primary human T cells. By incorporating engineered stem loops into the 3' UTRs of transgenes, we achieved transgene repression through Regnase-1 and Roquin-1 activity, dynamic upregulation upon stimulation, and orthogonal tunability. To demonstrate the utility of this system, we employed it to modulate payloads in CAR-T cells. Our findings highlight the potential of leveraging endogenous regulatory machinery in T cells for transgene regulation and suggest RNA structure as a valuable layer for regulatory modulation.