Engineering drug-responsive replication machinery for precise control of self-amplifying RNA

Abstract Precise, reversible control of gene expression from self‑amplifying RNA (saRNA) remains difficult, limiting the therapeutic flexibility of this otherwise potent platform. Although alphavirus‑derived saRNAs encode non‑structural proteins that drive RNA replication and offer an intrinsic regulatory point, no existing approach has enabled direct, drug‑dependent control of this machinery for high‑fidelity modulation of expression. Here we engineer saRNA constructs whose replication is activated by the approved small‑molecule drug trimethoprim, using drug‑responsive degradation domains fused to individual non‑structural proteins to regulate self‑amplification. As each replication protein contributes differently to RNA copying, we systematically screened fusion configurations and identified an optimal design combining modified replication proteins with a regulated payload. This construct achieved more than a 10 4 ‑fold difference between on and off states with negligible background expression. In mice, oral trimethoprim enabled tunable, reversible and temporally programmed expression patterns. When encoding a human immunodeficiency virus antigen, an escalating trimethoprim regimen enhanced germinal centre responses, a key determinant of antibody affinity maturation. This drug‑regulated saRNA platform provides a controllable and clinically compatible strategy for vaccines, immunotherapies and gene therapies.