RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2019 Mar 24

PMID 30902547

Citations 181

Authors

R Alexander Wesselhoeft

Piotr S Kowalski

Frances C Parker-Hale

Yuxuan Huang

Namita Bisaria

Daniel G Anderson

Affiliations

Soon will be listed here.

Abstract

Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

Citing Articles

A versatile toolbox for determining IRES activity in cells and embryonic tissues.

Koch P, Zhang Z, Genuth N, Susanto T, Haimann M, Khmelinskaia A EMBO J. 2025; .

PMID: 40082722 DOI: 10.1038/s44318-025-00404-5.

Engineered circular RNA-based DLL3-targeted CAR-T therapy for small cell lung cancer.

Cai J, Liu Z, Chen S, Zhang J, Li H, Wang X Exp Hematol Oncol. 2025; 14(1):35.

PMID: 40075480 PMC: 11905684. DOI: 10.1186/s40164-025-00625-8.

Unleashing the potential of mRNA: Overcoming delivery challenges with nanoparticles.

Chen Q, Huo K, Ji S, Pang S, Sun T, Niu Y Bioeng Transl Med. 2025; 10(2):e10713.

PMID: 40060761 PMC: 11883111. DOI: 10.1002/btm2.10713.

Recent advances and perspectives on the development of circular RNA cancer vaccines.

Gong Z, Hu W, Zhou C, Guo J, Yang L, Wang B NPJ Vaccines. 2025; 10(1):41.

PMID: 40025038 PMC: 11873252. DOI: 10.1038/s41541-025-01097-x.

Internal cap-initiated translation for efficient protein production from circular mRNA.

Fukuchi K, Nakashima Y, Abe N, Kimura S, Hashiya F, Shichino Y Nat Biotechnol. 2025; .

PMID: 39972222 DOI: 10.1038/s41587-025-02561-8.

References

Li Z, Huang C, Bao C, Chen L, Lin M, Wang X . Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol. 2015; 22(3):256-64. DOI: 10.1038/nsmb.2959. View

Wang Y, Wang Z . Efficient backsplicing produces translatable circular mRNAs. RNA. 2014; 21(2):172-9. PMC: 4338345. DOI: 10.1261/rna.048272.114. View

Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O . Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis. Mol Cell. 2017; 66(1):22-37.e9. PMC: 5387670. DOI: 10.1016/j.molcel.2017.02.017. View

Dong Y, Love K, Dorkin J, Sirirungruang S, Zhang Y, Chen D . Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. Proc Natl Acad Sci U S A. 2014; 111(11):3955-60. PMC: 3964096. DOI: 10.1073/pnas.1322937111. View

Kawasaki T, Kawai T . Toll-like receptor signaling pathways. Front Immunol. 2014; 5:461. PMC: 4174766. DOI: 10.3389/fimmu.2014.00461. View

Zhang Z, Ohto U, Shibata T, Krayukhina E, Taoka M, Yamauchi Y . Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA. Immunity. 2016; 45(4):737-748. DOI: 10.1016/j.immuni.2016.09.011. View

Kaczmarek J, Kowalski P, Anderson D . Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. 2017; 9(1):60. PMC: 5485616. DOI: 10.1186/s13073-017-0450-0. View

Wesselhoeft R, Kowalski P, Anderson D . Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018; 9(1):2629. PMC: 6035260. DOI: 10.1038/s41467-018-05096-6. View

Suzuki H, Zuo Y, Wang J, Zhang M, Malhotra A, Mayeda A . Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. 2006; 34(8):e63. PMC: 1458517. DOI: 10.1093/nar/gkl151. View

10.

Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y . Extensive translation of circular RNAs driven by N-methyladenosine. Cell Res. 2017; 27(5):626-641. PMC: 5520850. DOI: 10.1038/cr.2017.31. View

11.

Yanez Arteta M, Kjellman T, Bartesaghi S, Wallin S, Wu X, Kvist A . Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles. Proc Natl Acad Sci U S A. 2018; 115(15):E3351-E3360. PMC: 5899464. DOI: 10.1073/pnas.1720542115. View

12.

Davis D . Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 1995; 23(24):5020-6. PMC: 307508. DOI: 10.1093/nar/23.24.5020. View

13.

Oberli M, Reichmuth A, Dorkin J, Mitchell M, Fenton O, Jaklenec A . Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy. Nano Lett. 2017; 17(3):1326-1335. PMC: 5523404. DOI: 10.1021/acs.nanolett.6b03329. View

14.

Petkovic S, Muller S . RNA circularization strategies in vivo and in vitro. Nucleic Acids Res. 2015; 43(4):2454-65. PMC: 4344496. DOI: 10.1093/nar/gkv045. View

15.

Durbin A, Wang C, Marcotrigiano J, Gehrke L . RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling. mBio. 2016; 7(5). PMC: 5030355. DOI: 10.1128/mBio.00833-16. View

16.

Hansen T, Jensen T, Clausen B, Bramsen J, Finsen B, Damgaard C . Natural RNA circles function as efficient microRNA sponges. Nature. 2013; 495(7441):384-8. DOI: 10.1038/nature11993. View

17.

Lin S, Choe J, Du P, Triboulet R, Gregory R . The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell. 2016; 62(3):335-345. PMC: 4860043. DOI: 10.1016/j.molcel.2016.03.021. View

18.

Roers A, Hiller B, Hornung V . Recognition of Endogenous Nucleic Acids by the Innate Immune System. Immunity. 2016; 44(4):739-54. DOI: 10.1016/j.immuni.2016.04.002. View

19.

Kariko K, Buckstein M, Ni H, Weissman D . Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005; 23(2):165-75. DOI: 10.1016/j.immuni.2005.06.008. View

20.

Puttaraju M, Been M . Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Res. 1992; 20(20):5357-64. PMC: 334342. DOI: 10.1093/nar/20.20.5357. View