Nature biomedical engineeringJun 23, 2026

Designing drug-controlled machinery for precise self-amplifying RNA replication

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 24, 2026

Abstract

A drug-regulated RNA construct achieved more than a 10-fold difference between active and inactive states.

Self-amplifying RNA constructs were engineered to be activated by trimethoprim, a small-molecule drug.
Drug-responsive degradation domains were used to control the replication of individual non-structural proteins.
Systematic screening of fusion configurations led to the identification of an optimal design for RNA replication.
The system demonstrated negligible background expression, allowing precise control over gene expression levels.
In mice, oral administration of trimethoprim enabled tunable and reversible expression patterns.
An increasing dose of trimethoprim improved immune responses when the construct encoded a human immunodeficiency virus antigen.

Simplified

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 10⁴‑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.

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Funding

Competing interests

Competing interests: P.Y., J.L., Y.L., R.W. and D.J.I. are inventors on a patent filed by MIT related to this work (patent no. 12139721). D.J.I. and R.W. are co-founders and equity holders in Strand Therapeutics. Y.D. is an inventor on a patent filed by The Ohio State University related to the TT3 lipid used in this work (patent no. PCT/US2016/033514). The other authors declare no competing interests.

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Designing drug-controlled machinery for precise self-amplifying RNA replication

Abstract

Full Text

Funding

Competing interests

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