A reversible small-molecule-switchable self-amplifying RNA expression platform

RNA-based therapeutics offer versatile strategies for disease prevention and treatment, yet precise control over gene expression remains a major challenge. Self-amplifying RNA (saRNA), derived from the alphavirus genome, could be engineered to contain multiple subgenomic promoters, providing a unique RNA-only architecture for modular and independent regulation of downstream genes. However, the potential of saRNA as a programmable gene circuit enabling small-molecule-controlled on/off regulation of gene expression has remained largely unexplored. In this study, we engineered a series of saRNA constructs incorporating multiple regulatory modules, including a destabilizing domain (DD), the RNA-binding protein L7Ae, a tetracycline-responsive repressor (TetR), and kink-turn (k-turn) RNA motifs. This design allows individual downstream genes driven by distinct subgenomic promoters to be independently and reversibly regulated by the FDA-approved small-molecule ligands trimethoprim (TMP) or doxycycline (Dox). The engineered saRNAs were encapsulated into lipid nanoparticles, and saRNA-mediated expression of luciferase or fluorescent reporter proteins was systematically evaluated both in vitro and in vivo. Our results demonstrate that TMP and Dox function as effective molecular switches to autonomously turn on or off specific gene expression programs encoded within a single saRNA molecule. Collectively, this work establishes saRNA as a programmable RNA gene circuit platform with ligand-responsive, multi-gene regulatory capability, providing a versatile foundation for the development of controllable RNA therapeutics and synthetic biology applications.