RNA circles with minimized immunogenicity as potent PKR inhibitors

Liu, Chu-Xiao; Guo, Si-Kun; Fang, Nan; Xu, Yaohui; Yang, Li; Chen, Lingling

doi:10.1016/j.molcel.2021.11.019

Cited by 86 publications

(91 citation statements)

References 44 publications

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“…revealed similar findings to Breuer et al. 2 The authors revealed that circRNAs synthesized by permuted td introns from T4 bacteriophage or by pre-tRNA group I introns could induce an immune response because of the extraneous fragments that can distort circRNAs folding. In contrast, circRNAs synthesized by T4 RNA ligase (containing short double-stranded RNA [dsRNA] regions) without extraneous fragments exhibited minimal immunogenicity.…”

supporting

confidence: 83%

“…One of the important points that needs to be addressed here is whether the same phenomena will be observed for longer circRNAs, as T4 RNA ligase has limited circularization capacity when the RNA is longer. 2 …”

mentioning

confidence: 99%

“…included production of circRNAs with permuted Anabaena pre-tRNA group I introns, circRNAs approximately 1,200 nt in length, modifications done (m1ψ), cell types used for transfection (HEK293, A549, HeLa, and RAW264.7), transfection reagent used (Lipofectamine MessengerMAX), amount of circRNA transfected (40–200 ng), phosphatase treatment, RNase R digestion, and HPLC to purify circRNAs, dose of circRNA (350 ng), and route of inoculation (visceral fat); 4 the study design of Liu et al. included production of circRNAs with permuted group I introns from phage T4 td gene, Anabaena pre-tRNA group I introns as well as with T4 RNA ligase, circRNAs approximately 336, 410, and 522 nt in length, modifications done (none), cell types used for transfection (A549, HeLa, HEK293, and 293FT), transfection reagent used (Lipofectamine MessengerMAX reagent), amount of circRNA transfected (200 ng), and denaturing PAGE to purify circRNAs; 2 and the study design of Breuer et al. included production of circRNAs with T4 RNA ligase I, circRNAs approximately 200 nt in length, modifications done (none), A549 cells were used for transfection, transfection reagent used (Lipofectamine 2000), amount of circRNA transfected (250 ng), and polyacrylamide-urea gel extraction to purify circRNAs.…”

mentioning

confidence: 99%

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The HOPE for Pandora’s Box of artificial circular RNA immunogenicity

Awan

2022

Molecular Therapy - Nucleic Acids

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supporting

confidence: 83%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The HOPE for Pandora’s Box of artificial circular RNA immunogenicity

Awan

2022

Molecular Therapy - Nucleic Acids

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“…It has been a concerning issue whether the circRNAs produced via in vitro transcription induce strong innate immune responses 38,39,87 . Our vaccination study in rhesus macaques demonstrated that circRNA vaccine could indeed provide safe protection against SARS-CoV-2 infection without causing severe clinical signs of illness ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

Shen

et al. 2021

Preprint

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SARS-CoV-2 has caused a worldwide pandemic. The emerging variants B.1.1.7 in the UK, B.1.351 in South Africa, and P.1 in Brazil have recently spread rapidly, arousing concerns about the efficacy of the current vaccines and antibody therapies. Therefore, there is still a high demand for alternative vaccines with great efficacy, high design flexibility, and fast manufacturing speed. Here, we reported a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 spike protein. Being a circularized RNA molecule, circRNARBD could be rapidly produced via in vitro transcription and is highly stable without nucleotide modification. Lipid-nanoparticle-encapsulated circRNARBD elicited potent and sustained neutralizing antibodies, as well as Th1-biased T cell responses in mice. Notably, antibodies from mice immunized with circRNA encoding RBD variant (K417N-E484K-501Y) effectively neutralized B.1.351 variant. Moreover, we developed therapeutic circRNAs, encoding SARS-CoV-2 neutralizing nanobodies or hACE2 decoys, which could effectively neutralize SARS-CoV-2 pseudovirus. Our study suggests that circular RNA holds the potential to become a novel vaccine and therapeutic platform.

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“…Wesselhoeft RA et al found that the spacer-spacer complementarity and the pairing of homologous arms formed a sheltered splicing bubble potentiates the catalytic intron splicing.Circular RNA produced via Clean PIE(ORF) shows thorough minimalized immunogenicityPrevious studies have reported that circular RNAs made by different in vitro circularization strategies can induce distinct innate immune responses12 . As concealing the splicing site into the ORF, circRNAs that are much more precise are produced, and extraneous RNA sequences were not involved in.…”

mentioning

confidence: 99%

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

Qiu¹,

Zhao²,

Hou³

et al. 2022

Preprint

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Translatable circular RNAs (circRNAs) are emerging as a crucial molecular format for transient protein expression, with high potential to be an alternative for linear mRNA to reshape the landscape of mRNA pharmaceutical industry. Canonical Anabaena permuted intron-exon (Ana PIE) format that developed by ORNA is an efficient method for RNA circularization, and the engineered circRNAs direct supreme protein expression in eukaryotic cells. However, recent studies revealed that this method may unavoidably result in a remain of immunogenicity in the circRNA products, albeit after thorough RNA purification. In the current study, we develop a novel strategy for efficient generation of circRNA, via the permuted T4Td introns mediated autocatalytically ribozymatic reaction mediated ligation of the flanking segment sequences that concealing in ORF or translation initiation sequence (normally equal to IRES). This strategy universally realizes around 90% circularization effectivity, and the circRNA products can be purified to around 90% purity by our new purification method via HPLC, and presented thorough minimized immunogenicity, thus is termed Clean-PIE. The purified circRNAs are found to direct potent and durable expression of various proteins in vitro and in vivo. The partly purified Fluc circRNA by HPLC-SEC was found to direct Fluc expression in muscle for no less than 20 days. The highly purified circRNA exhibits much stronger protein expression in vitro and in vivo, and presumed a longer duration. Additionally, the scale-up of RNA circularization with the RNA precursors from 1 L transcription revealed high circularization effectivity (around 90%) and a high productivity of high-purity final circRNA products. Collectively, Clean PIE is a novel circRNA platform that possesses high circularization effectivity, enabled high RNA purity and thorough minimized immunogenicity, as well as scaling-up accessibility and directing extreme durability of protein expression, thus has the potential to develop advanced RNA vaccines and therapeutics in pharmaceutical industrial scale.

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RNA circles with minimized immunogenicity as potent PKR inhibitors

Cited by 86 publications

References 44 publications

The HOPE for Pandora’s Box of artificial circular RNA immunogenicity

The HOPE for Pandora’s Box of artificial circular RNA immunogenicity

Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

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