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Peer-reviewedCC BYNature Biomedical Engineering

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

  • Parisa Yousefpour1
  • Justin R. Gregory1
  • Kristen Si2,1
  • Jan Lonzarić2
  • Yingzhong Li2,1
  • Junmin Wang3
  • Kashif Qureshi1
  • Amir Ledbetter1
  • Mariane B. Melo4,5,1
  • Alexander Hostetler2,1
  • Ashley A. Lemnios4,1
  • Jonathan Dye1
9 more authors
  • Linette Rodriguez4,1
  • Tanaka K. Remba1
  • Rachel Yeung1
  • Melissa Güereca6
  • Yuebao Zhang7
  • Shengwei Wu1
  • Yizhou Dong8
  • Ron Weiss9,10
  • Darrell J. Irvine11,12,13,14,15
15 affiliations
  1. 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
  2. 2Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
  3. 3Bioinformatics Graduate Program, Boston University, Boston, MA, USA
  4. 4Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
  5. 5Howard Hughes Medical Institute, Chevy Chase, MD, USA
  6. 6Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
  7. 7Division of Pharmaceutics and Pharmacology, College of Pharmacy, Ohio State University, Columbus, OH, USA
  8. 8Icahn Genomics Institute, Precision Immunology Institute, Department of Immunology and Immunotherapy, Department of Oncological Sciences, Tisch Cancer Institute, Friedman Brain Institute, Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
  9. 9Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected]
  10. 10Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected]
  11. 11Consortium for HIV/AIDS Vaccine Development, The Scripps Research Institute, La Jolla, CA, USA. [email protected]
  12. 12Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected]
  13. 13Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. [email protected]
  14. 14Howard Hughes Medical Institute, Chevy Chase, MD, USA. [email protected]
  15. 15Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected]
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Abstract

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug-regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

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Cite

BibTeXRIS
BibTeX
@article{yousefpour2026engineering,
  title = {{Engineering drug-responsive replication machinery for precise control of self-amplifying RNA}},
  author = {Parisa Yousefpour and Justin R. Gregory and Kristen Si and Jan Lonzarić and Yingzhong Li and Junmin Wang and Kashif Qureshi and Amir Ledbetter and Mariane B. Melo and Alexander Hostetler and Ashley A. Lemnios and Jonathan Dye and Linette Rodriguez and Tanaka K. Remba and Rachel Yeung and Melissa Güereca and Yuebao Zhang and Shengwei Wu and Yizhou Dong and Ron Weiss and Darrell J. Irvine},
  journal = {Nature Biomedical Engineering},
  year = {2026},
  month = jun,
  doi = {10.1038/s41551-026-01723-6},
  url = {https://doi.org/10.1038/s41551-026-01723-6},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Engineering drug-responsive replication machinery for precise control of self-amplifying RNA
AU  - Parisa Yousefpour
AU  - Justin R. Gregory
AU  - Kristen Si
AU  - Jan Lonzarić
AU  - Yingzhong Li
AU  - Junmin Wang
AU  - Kashif Qureshi
AU  - Amir Ledbetter
AU  - Mariane B. Melo
AU  - Alexander Hostetler
AU  - Ashley A. Lemnios
AU  - Jonathan Dye
AU  - Linette Rodriguez
AU  - Tanaka K. Remba
AU  - Rachel Yeung
AU  - Melissa Güereca
AU  - Yuebao Zhang
AU  - Shengwei Wu
AU  - Yizhou Dong
AU  - Ron Weiss
AU  - Darrell J. Irvine
PY  - 2026
DA  - 2026/06/23
JO  - Nature Biomedical Engineering
DO  - 10.1038/s41551-026-01723-6
UR  - https://doi.org/10.1038/s41551-026-01723-6
AB  - The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug-regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.
SN  - 2157-846X
ER  -