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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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Published on: December 29, 2021

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

Parisa Yousefpour1, Justin R Gregory1, Kristen Si1,2

  • 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Nature Biomedical Engineering
|June 23, 2026
PubMed
Summary

Researchers developed a new self-amplifying RNA (saRNA) system controlled by the drug trimethoprim. This breakthrough allows precise, on-demand gene expression for advanced vaccines and therapies.

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Area of Science:

  • Molecular Biology
  • RNA Therapeutics
  • Drug Discovery

Background:

  • Self-amplifying RNA (saRNA) offers a potent platform for gene expression but lacks precise, reversible control.
  • Existing saRNA systems, including those derived from alphaviruses, have limitations in direct, drug-dependent modulation of their replication machinery.
  • Achieving high-fidelity control over saRNA-driven gene expression is crucial for therapeutic applications.

Purpose of the Study:

  • To engineer a novel saRNA construct with drug-dependent, reversible control over gene expression.
  • To establish a system for precise modulation of saRNA replication using an approved small-molecule drug.
  • To evaluate the therapeutic potential of this controllable saRNA platform in preclinical models.

Main Methods:

  • Engineered saRNA constructs by fusing drug-responsive degradation domains to non-structural replication proteins.
  • Systematically screened different fusion configurations to optimize regulation of self-amplification.
  • Utilized trimethoprim as the activating small molecule to control saRNA replication and payload expression.
  • Validated the system's performance in vitro and in vivo using a human immunodeficiency virus antigen model in mice.

Main Results:

  • Developed saRNA constructs activated by trimethoprim, achieving over 10^4-fold difference between on and off states with minimal background expression.
  • Demonstrated tunable, reversible, and temporally programmed gene expression in mice following oral trimethoprim administration.
  • Showed that an escalating trimethoprim regimen enhanced germinal center responses when saRNA encoded a human immunodeficiency virus antigen.

Conclusions:

  • The engineered drug-regulated saRNA platform provides unprecedented control over gene expression.
  • This system offers a clinically compatible strategy for developing advanced vaccines, immunotherapies, and gene therapies.
  • The trimethoprim-inducible saRNA technology represents a significant advancement in programmable RNA therapeutics.