RNA Modifications Modulate Activation of Innate Toll-Like Receptors

Freund, Isabel; Eigenbrod, Tatjana; Helm, Mark; Dalpke, Alexander H.

doi:10.3390/genes10020092

Cited by 92 publications

(73 citation statements)

References 125 publications

(148 reference statements)

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“…Thus, the precise requirements for the second binding pocket and the true nature of any minimal TLR8 ligand remain unclear. Moreover, given the broad number of conceivable di- or trinucleotide ligands for the second binding pocket, it seems unlikely that such motifs are not contained in self RNA, or that they could all be rendered inactive by modifications such as pseudouridine or 2’O-methylation ( Freund et al., 2019 ). In addition, it is unclear which inhibitory modifications affect TLR8 activation and which inhibit upstream RNases.…”

Section: Main Textmentioning

confidence: 99%

“…In addition, it is unclear which inhibitory modifications affect TLR8 activation and which inhibit upstream RNases. 2’O-methylation is a known inhibitor of TLR8 (and TLR7) activity ( Freund et al., 2019 ) but also of upstream RNaseT2 and RNase2 ( Ostendorf et al., 2020 ). Conceivably, self RNA does not avoid immunorecognition in the classical sense but rather contains inhibitory motifs perhaps similar to those synthetically designed for TLR7 and TLR8 ( Schmitt et al., 2017 ) using 2’O-methylation, but further studies will be necessary to determine if these sequences have natural counterparts in our self RNA.…”

Section: Main Textmentioning

confidence: 99%

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Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids

2020

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All lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (NA). In vertebrates, NA-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. Effector mechanisms include i) immune signaling, ii) restriction of NA functions such as inhibition of mRNA translation, and iii) cell death pathways. An appropriate effector response is necessary for host defense, but dysregulated NA-sensing can lead to devastating autoimmune and autoinflammatory disease. Their inherent biochemical similarity renders the reliable distinction between self NA under homeostatic conditions and altered or exogenous NA particularly challenging. In this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity.

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Section: Main Textmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids

2020

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“…The technology used for the synthesis of these in vitro transcribed RNAs-predominantly using phage RNA polymerases (RNAPs)-is robust and well established for the large-scale production of synthetic RNA. However, it is also known that introduction of synthetic in vitro transcribed mRNAs into cells or animal models results in an immune response against the synthetic molecules (Weissman et al 2000;Karikó et al 2004;Akira et al 2006;Sahin et al 2014;Freund et al 2019). Such outcomes are undesirable in therapeutic applications in which an immune response is detrimental or unnecessary (for example, protein-replacement therapies).…”

Section: Introductionmentioning

confidence: 99%

“…Such outcomes are undesirable in therapeutic applications in which an immune response is detrimental or unnecessary (for example, protein-replacement therapies). Incorporation of modified nucleosides into synthetic mRNA mitigates the immune response to some extent by mimicking endogenous mRNAs (Karikó et al 2005;Durbin et al 2016;Pardi et al 2017;Richner et al 2017;Freund et al 2019). Another major stimulant of the immune response comes from contaminants present in the IVT reactions.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Asahara

Tzertzinis

et al. 2020

RNA

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The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3 ′ ′ ′ ′ ′ extension of the runoff product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3 ′ ′ ′ ′ ′-extension of the runoff product. We show that template-encoded poly(A) tailing does not affect 3 ′ ′ ′ ′ ′-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.

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“…Such outcomes are undesirable in therapeutic applications in which an immune response is detrimental or unnecessary (for example, protein-replacement therapies). Incorporation of modified nucleosides into synthetic mRNA mitigates the immune response to some extent by mimicking endogenous mRNAs [4,[6][7][8][9]. Another major stimulant of the immune response comes from contaminants present in the in vitro transcription reactions.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Asahara

Tzertzinis

et al. 2019

Preprint

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The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed mRNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo, that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-self extension of the run-off product. We show that template-encoded poly-A tailing does not affect 3'-self extension but reduces the formation of the antisense RNA by-products and that combining high-temperature IVT with templateencoded poly-A tailing prevents formation of both kinds of by-products.

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RNA Modifications Modulate Activation of Innate Toll-Like Receptors

Cited by 92 publications

References 125 publications

Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids

Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

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