Tunable gene control via RNA splicing with a clinically approved small molecule.

Abstract

Precise transgene regulation is crucial for safe and effective gene and cell therapies. Current inducible systems often rely on immunogenic exogenous proteins or non-clinically approved inducers, hindering clinical translation. Here we present RisdiON, a compact inducible system controlled by risdiplam, a clinically approved oral drug that acts via splicing modulation. RisdiON utilizes risdiplam-responsive sequences for precise transgene control via endogenous splicing machinery, bypassing exogenous protein regulators, while its split-ATG architecture ensures the expression of native, tag-free proteins. This approach provides robust, dose-dependent induction with minimal leakiness. We demonstrate that RisdiON controls various transgenes in immortalized cell lines and hiPSCs, enables inducible CAR expression in primary T cells, and regulates Cas9 for precise gene editing. Additionally, we achieve reversible transgene expression in vivo using adeno-associated virus (AAV) delivery. The platform is modular and functional across diverse promoters, offering a safe, titratable, and reversible tool when coupled with an orally bioavailable drug to advance next-generation therapies.