In vitro evolution of enhanced RNA replicons for immunotherapy
Open accessAbstract
Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo . To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo . In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8 T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.
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Previous efforts to decrease immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle.
“Previous efforts to decrease the immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle16–18.”
From Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency (2025) · cites this paper as [16]
The Introduction and Results report in vitro evolution of saRNA replicon sequences and identify mutations that reduced interferon responses in some variants. The paper also describes nanoparticle delivery, but does not present delivery-vehicle optimization as a way to decrease immunogenicity or describe co-expression of viral inhibitory proteins. No page numbers are available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measuredA pro-inflammatory tumour microenvironment can be induced by lipid nanoparticle-mRNA formulations delivering cytokines or co-stimulatory molecules.
“Alternatively to vaccination, a pro-inflammatory tumour microenvironment can be induced by lipid nanoparticle–mRNA formulations delivering cytokines or co-stimulatory molecules33,106,161,163,214 (Table 2).”
From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [161]
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References 45 · Cited by 71
- Authors
- Li, Y., Teague, B., Zhang, Y., Su, Z., Porter, E., Dobosh, B., Wagner, T., Irvine, D. J., & Weiss, R.
- Affiliations
- Koch Institute for Integrative Cancer Research At MIT (US)
- Massachusetts Institute of Technology (US)
- University of Rhode Island (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Irvine, Darrell J.
- Weiss, Ron
- ORCID
- Venue
- Scientific Reports
- Published
- 2019-05-06
- Volume
- 9
- Issue
- 1
- Pages
- 6932-6932
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC6502795
- OpenAlex
- W2943015962
- Cited by
- 71
- References
- 45
- Citations by year
- 2026: 7 · 2025: 18 · 2024: 9 · 2023: 13 · 2022: 13 · 2021: 8 · 2020: 3
- FWCI
- 1.99
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Funders
- U.S. Department of Health & Human Services | NIH | National Cancer Institute (CA206218, P30-CA14051)
- Howard Hughes Medical Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Keywords
- self-replicating RNA, RNA replicon, in vitro evolution, Venezuelan equine encephalitis virus, non-structural proteins, NSP2, NSP3, transgene expression, interleukin-2, cancer immunotherapy, B16F10 melanoma, CD8+ T cells, RNA therapeutics, gene therapy
- MeSH terms
- Alleles, Animals, Genetic Vectors, Humans, Immunity, Immunotherapy, Melanoma, Experimental, Mutation, Neoplasms, Promoter Regions, Genetic, Replicon, RNA, Transcription, Genetic, Genetic Therapy, Gene Expression, Gene Order, Cell Line, Tumor, Mice
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