Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA
Open accessAbstract
In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.
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dsRNA contaminants can be purified by high-performance liquid chromatography or cellulose chromatography.
“dsRNA contaminants can be purified by high-performance liquid chromatography (HPLC) or cellulose chromatography13,14”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [13]
The Results report that HPLC removed dsRNA contaminants from in vitro-transcribed RNA. Our copy does not report cellulose chromatography as a method for removing them, so it supports only the HPLC part of the cited sentence. (LLM, conf=0.88)
Experimental — accuracy not yet measureddsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.
“This dsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [13]
The Results report that HPLC purification removed dsRNA contaminants, eliminated type I interferon induction by nucleoside-modified mRNA in transfected dendritic cells, and enhanced translation in those cells. But dsRNA depletion alone is not sufficient to explain the interferon claim: HPLC-purified mRNA without nucleoside modification remained a potent IFN-α inducer. (LLM, conf=0.86)
Experimental — accuracy not yet measuredRemoval of dsRNA by HPLC eliminates immune activation and improves translation of linear nucleoside-modified IVT mRNA.
“It has been shown that removal of dsRNA by HPLC eliminates immune activation and improves translation of linear nucleoside-modified IVT mRNA16.”
From Engineering circular RNA for potent and stable translation in eukaryotic cells (2018) · cites this paper as [16]
The lack of biological efficacy of HPLC-purified basic Epo mRNA highlights the importance and potency of sequence-engineering.
“Although high performance liquid chromatography (HPLC) purification depletes contaminants and, thus, contributes to reduced immunostimulation as well as improves mRNA expression to some extent,36,37,38 the lack of biological efficacy of HPLC-purified basic Epo mRNA highlights the importance and potency of sequence-engineering.”
From Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals (2015) · cites this paper as [38]
dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.
“It has been shown that dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.13,14”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [13]
Previous strategies to increase mRNA stability have yielded modest improvements in RNA stability.
“These strategies have yielded modest improvements in RNA stability10, 13–16, and thus alternative approaches to RNA stabilization, such as circularization, are desirable.”
From Engineering circular RNA for potent and stable translation in eukaryotic cells (2018) · cites this paper as [16]
The Introduction and Discussion describe reduced immune activation and improved translation with modified or HPLC-purified mRNA, but do not characterize improvements in RNA stability as modest. Searches for RNA stability, half-life, degradation, and circularization found no passage in our copy that establishes the claimed comparison. (LLM, conf=0.74)
Experimental — accuracy not yet measureddsRNA can activate PRR such as TLR3, whereas ssRNA activates TLR7 and TLR8 to produce IFN-I, thus inducing inflammation in the host and interrupting mRNA translation.
“dsRNA can activate PRR such as TLR3,52,156 whereas ssRNA activates TLR7 and TLR8 to produce IFN-I, thus inducing inflammation in the host and interrupting mRNA translation.157,158”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [156]
HPLC is a gold standard for mRNA purification in a lab setting; however, it presents challenges when scaling up.
“HPLC is a gold standard for mRNA purification in a lab setting;100 however, it presents challenges when scaling up”
From COVID-19 mRNA vaccines: Platforms and current developments (2022) · cites this paper as [100]
Previous studies showed that the protein yield of mRNA purified by reverse phase HPLC increases by 10-1000 times.
“Previous studies showed that the protein yield of mRNA purified by reverse phase HPLC increases by 10–1000 times.156,453”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [156]
Removing double-stranded RNA contaminants further ablates in vitro transcribed RNA's immunostimulatory activity.
“Additional efforts to remove double-stranded (ds)RNA contaminants even further ablated IVT RNA's immunostimulatory activity [8,9]”
From The dawn of mRNA vaccines: the COVID-19 case (2021) · cites this paper as [9]
Chromatographic purification can remove double-stranded RNA in IVT mRNA preparations, diminishing immune activation and increasing translational efficiency.
“Chromatographic purification can remove double-stranded RNA, an analogue of viral genome, in IVT mRNA preparations, diminishing immune activation and increasing translational efficiency2,4,7,183.”
From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [183]
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- Authors
- Karikó, K., Muramatsu, H., Ludwig, J., & Weissman, D.
- Affiliations
- University of Bonn (DE)
- Corresponding author
- Weissman, Drew
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2011-09-02
- Volume
- 39
- Issue
- 21
- Pages
- e142-e142
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC3241667
- OpenAlex
- W2162970812
- Cited by
- 898
- References
- 34
- Citations by year
- 2026: 73 · 2025: 115 · 2024: 133 · 2023: 108 · 2022: 141 · 2021: 115 · 2020: 50 · 2019: 33 · 2018: 40 · 2017: 19 · 2016: 22 · 2015: 19 · 2014: 10 · 2013: 9 · 2012: 11
- FWCI
- 3.90
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- University of Pennsylvania
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R42HL087688, HL87688)
- National Institute of Allergy and Infectious Diseases (R01AI050484, AI050484)
- National Institute of Dental and Craniofacial Research (DE019059, R21DE019059)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Keywords
- mRNA therapeutics, nucleoside-modified mRNA, in vitro transcription, HPLC purification, innate immune activation, type I interferon, inflammatory cytokines, mRNA translation efficiency, gene therapy vectors
- MeSH terms
- Animals, Cells, Cultured, Chromatography, High Pressure Liquid, Cytidine, Humans, Pseudouridine, RNA, Double-Stranded, RNA, Messenger, Transcription, Genetic, Transfection, Protein Biosynthesis, Genetic Therapy, Mice, HEK293 Cells
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