Stem Loop Mediated Transgene Modulation in Human T\nCells

Controlling gene\nexpression is useful for many applications, but\ncurrent methods often require external user inputs, such as the addition\nof a drug. We present an alternative approach using cell-autonomous\ntriggers based on RNA stem loop structures in the 3′ untranslated\nregions (UTRs) of mRNA. These stem loops are targeted by the RNA binding\nproteins Regnase-1 and Roquin-1, allowing us to program stimulation-induced\ntransgene regulation in primary human T cells. By incorporating engineered\nstem loops into the 3′ UTRs of transgenes, we achieved transgene\nrepression through Regnase-1 and Roquin-1 activity, dynamic upregulation\nupon stimulation, and orthogonal tunability. To demonstrate the utility\nof this system, we employed it to modulate payloads in CAR-T cells.\nOur findings highlight the potential of leveraging endogenous regulatory\nmachinery in T cells for transgene regulation and suggest RNA structure\nas a valuable layer for regulatory modulation.