Synthetic circRNA therapeutics: innovations, strategies, and future horizons

Abstract

Small molecule drugs are increasingly emerging as innovative and effective treatments for various diseases, with mRNA therapeutics being a notable representative. The success of COVID-19 vaccines has underscored the transformative potential of mRNA in RNA therapeutics. Within the RNA family, there is another unique type known as circRNA. This single-stranded closed-loop RNA molecule offers notable advantages over mRNA, including enhanced stability and prolonged protein expression, which may significantly impact therapeutic strategies. Furthermore, circRNA plays a pivotal role in the pathogenesis of various diseases, such as cancers, autoimmune disorders, and cardiovascular diseases, making it a promising clinical intervention target. Despite these benefits, the application of circRNA in clinical settings remains underexplored. This review provides a comprehensive overview of the current state of synthetic circRNA therapeutics, focusing on its synthesis, optimization, delivery, and diverse applications. It also addresses the challenges impeding the advancement of circRNA therapeutics from bench to bedside. By summarizing these aspects, the review aims to equip researchers with insights into the ongoing developments and future directions in circRNA therapeutics. Highlighting both the progress and the existing gaps in circRNA research, this review offers valuable perspectives for advancing the field and guiding future investigations.

Keywords

circRNA vaccine / group I intron / internal ribosome entry site / lipid nanoparticle / synthetic circRNA

References

[1] Liu CX, Chen LL. Circular RNAs: characterization, cellular roles, and applications. Cell. 2022; 185(12): 2016-2034.

[2] Sanger HL, Klotz G, Riesner D, Gross HJ, Kleinschmidt AK. Viroids are single-stranded covalently closed circular rna molecules existing as highly base-paired rod-like structures. Proc Natl Acad Sci USA. 1976; 73(11): 3852-3856.

[3] Hsu MT, Cocaprados M. Electron-microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature. 1979; 280(5720): 339-340.

[4] Arnberg AC, Vanommen GJB, Grivell LA, Vanbruggen EFJ, Borst P. Some yeast mitochondrial rnas are circular. Cell. 1980; 19(2): 313-319.

[5] Grabowski PJ, Zaug AJ, Cech TR. The intervening sequence of the ribosomal-RNA precursor is converted to a circular rna in isolated-nuclei of tetrahymena. Cell. 1981; 23(2): 467-476.

[6] Kos A, Dijkema R, Arnberg AC, Vandermeide PH, Schellekens H. The hepatitis delta (delta) virus possesses a circular RNA. Nature. 1986; 323(6088): 558-560.

[7] Cocquerelle C, Daubersies P, Majerus MA, Kerckaert JP, Bailleul B. Splicing with inverted order of exons occurs proximal to large introns. Embo J. 1992; 11(3): 1095-1098.

[8] Gao Y, Wang JF, Zhao FQ. CIRI: an efficient and unbiased algorithm for circular RNA identification. Genome Biol. 2015; 16: 4.

[9] Gao Y, Zhao FQ. Computational strategies for exploring circular RNAs. Trends Genet. 2018; 34(5): 389-400.

[10] Lu TT, Cui LL, Zhou Y, et al. Transcriptome-wide investigation of circular RNAs in rice. RNA. 2015; 21(12): 2076-2087.

[11] Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One. 2012; 7(2): e30733.

[12] Zhang Y, Zhang XO, Chen T, et al. Circular intronic long noncoding RNAs. Mol Cell. 2013; 51(6): 792-806.

[13] Li ZY, Huang C, Bao C, et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol. 2015; 22(3): 256-264.

[14] Guarnerio J, Bezzi M, Jeong JC, et al. Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations. Cell. 2016; 165(2): 289-302.

[15] Muniz L, Nicolas E, Trouche D. RNA polymerase II speed: a key player in controlling and adapting transcriptome composition. EMBO J. 2021; 40(15): e105740.

[16] Li J, Sun D, Pu WC, Wang J, Peng Y. Circular RNAs in cancer: biogenesis, function, and clinical significance. Trends Cancer. 2020; 6(4): 319-336.

[17] Nigro JM, Cho KR, Fearon ER, et al. Scrambled exons. Cell. 1991; 64(3): 607-613.

[18] Capel B, Swain A, Nicolis S, et al. Circular transcripts of the testis-determining gene Sry in adult-mouse testis. Cell. 1993; 73(5): 1019-1030.

[19] Dubin RA, Kazmi MA, Ostrer H. Inverted repeats are necessary for circularization of the mouse testis Sry transcript. Gene. 1995; 167(1-2): 245-248.

[20] Kristensen LS, Andersen MS, Stagsted LVW, Ebbesen KK, Hansen TB, Kjems J. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet. 2019; 20(11): 675-691.

[21] Liang DM, Wilusz JE. Short intronic repeat sequences facilitate circular RNA production. Gene Dev. 2014; 28(20): 2233-2247.

[22] Conn SJ, Pillman KA, Toubia J, et al. The RNA binding protein quaking regulates formation of circRNAs. Cell. 2015; 160(6): 1125-1134.

[23] Kelly S, Greenman C, Cook PR, Papantonis A. Exon skipping is correlated with exon circularization. J Mol Biol. 2015; 427(15): 2414-2417.

[24] Memczak S, Jens M, Elefsinioti A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441): 333-338.

[25] Ashwal-Fluss R, Meyer M, Pamudurti NR, et al. circRNA biogenesis competes with Pre-mRNA splicing. Mol Cell. 2014; 56(1): 55-66.

[26] Du WW, Yang WN, Liu E, Yang ZG, Dhaliwal P, Yang BB. Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res. 2016; 44(6): 2846-2858.

[27] Li X, Liu CX, Xue W, et al. Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection. Mol Cell. 2017; 67(2): 214.

[28] Liu CX, Li X, Nan F, et al. Structure and degradation of circular RNAs regulate PKR activation in innate immunity. Cell. 2019; 177(4): 865.

[29] Xia PY, Wang S, Ye BQ, et al. A circular RNA protects dormant hematopoietic stem cells from DNA sensor cGAS-mediated exhaustion. Immunity. 2018; 48(4): 688.

[30] Hansen TB, Jensen TI, Clausen BH, et al. Natural RNA circles function as efficient microRNA sponges. Nature. 2013; 495(7441): 384-388.

[31] Qiu M, Xia W, Chen R, et al. The circular RNA circPRKCI promotes tumor growth in lung adenocarcinoma. Cancer Res. 2018; 78(11): 2839-2851.

[32] Chen X, Mao R, Su W, et al. Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKalpha signaling in STK11 mutant lung cancer. Autophagy. 2020; 16(4): 659-671.

[33] Huang Q, Chu Z, Wang Z, et al. circCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models. Nat Commun. 2024; 15(1): 3904.

[34] He Y, Jiang L, Liu H, et al. Hippocampal circAnk3 deficiency causes anxiety-like behaviors and social deficits by regulating the miR-7080-3p/IQGAP1 pathway in mice. Biol Psychiatry. 2024; 95(9): 896-908.

[35] Li B, Bai WW, Guo T, et al. Statins improve cardiac endothelial function to prevent heart failure with preserved ejection fraction through upregulating circRNA-RBCK1. Nat Commun. 2024; 15(1): 2953.

[36] Chen CY, Sarnow P. Initiation of protein-synthesis by the eukaryotic translational apparatus on circular RNAs. Science. 1995; 268(5209): 415-417.

[37] Yang Y, Fan XJ, Mao MW, et al. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res. 2017; 27(5): 626-641.

[38] Meyer KD, Patil DP, Zhou J, et al. 5’ UTR m A promotes cap-independent translation. Cell. 2015; 163(4): 999-1010.

[39] Li Y, Chen B, Zhao JJ, et al. HNRNPL circularizes ARHGAP35 to produce an oncogenic protein. Adv Sci. 2021; 8(13): 2001701.

[40] Gao XY, Xia X, Li FY, et al. Circular RNA-encoded oncogenic E-cadherin variant promotes glioblastoma tumorigenicity through activation of EGFR-STAT3 signalling. Nat Cell Biol. 2021; 23(3): 278.

[41] Lu DC, Chatterjee S, Xiao K, et al. A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity. Eur Heart J. 2022; 43(42): 4496.

[42] Chen CK, Cheng R, Demeter J, et al. Structured elements drive extensive circular RNA translation. Mol Cell. 2021; 81(20): 4300-4318. e13.

[43] Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018; 9(1): 2629.

[44] Dousis A, Ravichandran K, Hobert EM, Moore MJ, Rabideau AE. An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nat Biotechnol. 2023; 41(4): 560.

[45] Baden LR, El Sahly HM, Essink B, et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021; 384(5): 403-416.

[46] Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020; 383(27): 2603-2615.

[47] Yan Y, Liu XY, Lu A, Wang XY, Jiang LX, Wang JC. Non-viral vectors for RNA delivery. J Control Release. 2022; 342: 241-279.

[48] Green MR, Sambrook J. How to win the battle with RNase. Cold Spring Harb Protoc. 2019; 2019(2).

[49] Foster JB, Choudhari N, Perazzelli J, et al. Purification of mRNA encoding chimeric antigen receptor is critical for generation of a robust T-cell response. Hum Gene Ther. 2019; 30(2): 168-178.

[50] Liu X, Barrett DM, Jiang S, et al. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice. Blood Cancer J. 2016; 6: e430.

[51] Maus MV, Haas AR, Beatty GL, et al. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013; 1(1): 26-31.

[52] Wesselhoeft RA, Kowalski PS, Parker-Hale FC, Huang Y, Bisaria N, Anderson DG. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol Cell. 2019; 74(3): 508-520. e4.

[53] Wang C, Liu H. Factors influencing degradation kinetics of mRNAs and half-lives of microRNAs, circRNAs, lncRNAs in blood in vitro using quantitative PCR. Sci Rep. 2022; 12(1): 7259.

[54] Niu D, Wu YR, Lian JQ. Circular RNA vaccine in disease prevention and treatment. Signal Transduct Target Ther. 2023; 8(1): 341.

[55] Li HL. circRNA: a promising all-around star in the future. Epigenomics UK. 2023; 15(12): 677-685.

[56] Han YJ, Shin SH, Lim CG, Heo YH, Choi IY, Kim H. Synthetic RNA therapeutics in cancer. J Pharmacol Exp Ther. 2023; 386(2): 212-223.

[57] Muller S, Appel B. In vitro circularization of RNA. RNA Biol. 2017; 14(8): 1018-1027.

[58] Obi P, Chen YG. The design and synthesis of circular RNAs. Methods. 2021; 196: 85-103.

[59] Lee CH, Han SR, Lee SW. Group I intron-based therapeutics through-splicing reaction. Prog Mol Biol Transl. 2018; 159: 79-100.

[60] Belfort M, Lambowitz AM. Group II intron RNPs and reverse transcriptases: from retroelements to research tools. Csh Perspect Biol. 2019; 11(4): a032375.

[61] Liu X, Zhang Y, Zhou SR, Dain LR, Mei L, Zhu GZ. Circular RNA: an emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 2022; 348: 84-94.

[62] Sharma NK, Dwivedi P, Bhushan R, Maurya PK, Kumar A, Dakal TC. Engineering circular RNA for molecular and metabolic reprogramming. Funct Integr Genomics. 2024; 24(4): 117.

[63] Kim YS, Kim DH, An D, et al. The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis. Anim Cells Syst (Seoul). 2023; 27(1): 208-218.

[64] Puttaraju M, Been MD. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Res. 1992; 20(20): 5357-5364.

[65] Lee KH, Kim S, Lee SW. Pros and cons of in vitro methods for circular RNA preparation. Int J Mol Sci. 2022; 23(21): 13247.

[66] Loan Young T, Chang Wang K, James Varley A, Li B. Clinical delivery of circular RNA: lessons learned from RNA drug development. Adv Drug Deliv Rev. 2023; 197: 114826.

[67] Petkovic S, Muller S. RNA circularization strategies in vivo and in vitro. Nucleic Acids Res. 2015; 43(4): 2454-2465.

[68] Qiu Z, Hou Q, Zhao Y, et al. Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression. bioRxiv. June 23 June 22, 2022. Available from: doi:10.1101/2022.06.20.496777

[69] Frommer J, Hieronymus R, Arunachalam TS, et al. Preparation of modified long-mer RNAs and analysis of FMN binding to the aptamer from. RNA Biol. 2014; 11(5): 609-623.

[70] Sturm MB, Roday S, Schramm VL. Circular DNA and DNA/RNA hybrid molecules as scaffolds for ricin inhibitor design. J Am Chem Soc. 2007; 129(17): 5544-5550.

[71] Kumar R, El-Sagheer A, Tumpane J, Lincoln P, Wilhelmsson LM, Brown T. Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry. J Am Chem Soc. 2007; 129(21): 6859-6864.

[72] Kato D, Oishi M. Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles. Acs Nano. 2014; 8(10): 9988-9997.

[73] Abe H, Kimura Y. Chemical ligation reactions of oligonucleotides for biological and medicinal applications. Chem Pharm Bull. 2018; 66(2): 117-122.

[74] Paredes E, Evans M, Das SR. RNA labeling, conjugation and ligation. Methods. 2011; 54(2): 251-259.

[75] Ho CK, Wang LK, Lima CD, Shuman S. Structure and mechanism of RNA ligase. Structure. 2004; 12(2): 327-339.

[76] Wilson GG, Murray NE. Molecular cloning of the DNA-ligase gene from bacteriophage-T4 .1. Characterization of the recombinants. J Mol Biol. 1979; 132(3): 471-491.

[77] Kurschat WC, Müller J, Wombacher R, Helm M. Optimizing splinted ligation of highly structured small RNAs. RNA. 2005; 11(12): 1909-1914.

[78] Gaglione M, Di Fabio G, Messere A. Current methods in synthesis of cyclic oligonucleotides and analogues. Curr Org Chem. 2012; 16(11): 1371-1389.

[79] Moore MJ, Sharp PA. Site-specific modification of pre-messenger-RNA - the 2’-hydroxyl groups at the splice sites. Science. 1992; 256(5059): 992-997.

[80] Park W-S, Miyano-Kurosaki N, Abe T, Takai K, Yamamoto N, Takaku H. Inhibition of HIV-1 replication by a new type of circular dumbbell RNA/DNA chimeric oligonucleotides. Biochem Biophys Res Commun. 2000; 270(3): 953-960.

[81] Middleton T, Herlihy WC, Schimmel PR, Munro HN. Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography. Anal Biochem. 1985; 144(1): 110-117.

[82] Steger J, Graber D, Moroder H, Geiermann AS, Aigner M, Micura R. Efficient access to nonhydrolyzable initiator tRNA based on the synthesis of 3’-azido-3’-deoxyadenosine RNA. Angew Chem Int Edit. 2010; 49(41): 7470-7472.

[83] Kim YS, Kim D, An D, et al. The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis. Anim Cells Syst. 2023; 27(1): 208-218.

[84] Liu CX, Guo SK, Nan F, Xu YF, Yang L, Chen LL. RNA circles with minimized immunogenicity as potent PKR inhibitors. Mol Cell. 2022; 82(2): 420-434. e6.

[85] Ho CK, Shuman S. Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc Natl Acad Sci USA. 2002; 99(20): 12709-12714.

[86] Wood ZA, Sabatini RS, Hajduk SL. RNA ligase: picking up the pieces. Mol Cell. 2004; 13(4): 455-456.

[87] Wang LK, Ho CK, Pei Y, Shuman S. Mutational analysis of bacteriophage T4 RNA ligase 1 - Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction. J Biol Chem. 2003; 278(32): 29454-29462.

[88] Yin SM, Ho CK, Shuman S. Structure-function analysis of T4 RNA ligase 2. J Biol Chem. 2003; 278(20): 17601-17608.

[89] Cech TR. Self-splicing of the ribosomal-rna precursor of tetrahymena. Fed Proc. 1985; 44(3): R10-R10.

[90] Doudna JA, Cech TR. The chemical repertoire of natural ribozymes. Nature. 2002; 418(6894): 222-228.

[91] Puttaraju M, Been MD. Group-I permuted intron exon (Pie) sequences self-splice to produce circular exons. Nucleic Acids Res. 1992; 20(20): 5357-5364.

[92] Ford E, Ares M. Synthesis of circular RNA in bacteria and yeast using rna cyclase ribozymes derived from a group-I intron of phage-T4. Proc Natl Acad Sci USA. 1994; 91(8): 3117-3121.

[93] Puttaraju M, Perrotta AT, Been MD. A circular trans-acting hepatitis-delta virus ribozyme. Nucleic Acids Res. 1993; 21(18): 4253-4258.

[94] Perriman R, Ares M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo. RNA. 1998; 4(9): 1047-1054.

[95] Bohjanen PR, Colvin RA, Puttaraju M, Been MD, GarciaBlanco MA. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription. Nucleic Acids Res. 1996; 24(19): 3733-3738.

[96] Rausch JW, Heinz WF, Payea MJ, Sherpa C, Gorospe M, Le Grice SFJ. Characterizing and circumventing sequence restrictions for synthesis of circular RNA. Nucleic Acids Res. 2021; 49(6): e35.

[97] Lee KH, Kim S, Song J, Han SR, Kim JH, Lee S-W. Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme. Mol Ther Nucleic Acids. 2023; 33: 587-598.

[98] Cui JY, Zhang LX, Zhang ZF, et al. A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila. Nucleic Acids Res. 2023; 51(14): e78.

[99] Diegelman AM, Kool ET. Generation of circular RNAs and-cleaving catalytic RNAs by rolling transcription of circular DNA oligonucleotides encoding hairpin ribozymes. Nucleic Acids Res. 1998; 26(13): 3235-3241.

[100] Branch AD, Robertson HD. A replication cycle for viroids and other small infectious RNAs. Science. 1984; 223(4635): 450-455.

[101] Mikheeva S, Hakim-Zargar M, Carlson D, Jarrell K. Use of an engineered ribozyme to produce a circular human exon. Nucleic Acids Res. 1997; 25(24): 5085-5094.

[102] Chen C, Wei H, Zhang K, et al. A flexible, efficient, and scalable platform to produce circular RNAs as new therapeutics [Preprint]. BioRxiv, 2022. 2022-6-1. doi:10.1101/2022.05.31.494115

[103] Petkovic S, Müller S. RNA self-processing: Formation of cyclic species and concatemers from a small engineered RNA. Febs Lett. 2013; 587(15): 2435-2440.

[104] Dallas A, Balatskaya SV, Kuo TC, et al. Hairpin ribozyme-antisense RNA constructs can act as molecular lassos. Nucleic Acids Res. 2008; 36(21): 6752-6766.

[105] Kazakov SA, Balatskaya SV, Johnston BH. Ligation of the hairpin ribozyme in cis induced by freezing and dehydration. RNA. 2006; 12(3): 446-456.

[106] Lu ZP, Filonov GS, Noto JJ, et al. Metazoan tRNA introns generate stable circular RNAs in vivo. RNA. 2015; 21(9): 1554-1565.

[107] Litke JL, Jaffrey SR. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol. 2019; 37(6): 667.

[108] Unti MJ, Jaffrey SR. Highly efficient cellular expression of circular mRNA enables prolonged protein expression. Cell Chem Biol. 2023; 31(1): 163-176. e5.

[109] Fitzgerald KD, Semler BL. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochim Biophys Acta. 2009; 1789(9-10): 518-528.

[110] Hellen CUT, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Gene Dev. 2001; 15(13): 1593-1612.

[111] Mokrejs M, Vopálensky V, Kolenaty O, et al. IRESite:: the database of experimentally verified IRES structures (https://www.iresite.org). Nucleic Acids Res. 2006; 34: D125-D130.

[112] Shen L, Yang J, Zuo C, et al. Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection. Mol Ther. 2023; 32(1): 168-184.

[113] Amaya L, Grigoryan L, Li Z, et al. Circular RNA vaccine induces potent T cell responses. Proc Natl Acad Sci USA. 2023; 120(20): e2302191120.

[114] Yang JL, Zhu JF, Sun JJ, et al. Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy. Mol Ther Nucl Acids. 2022; 30: 184-197.

[115] Chen R, Wang SK, Belk JA, et al. Engineering circular RNA for enhanced protein production. Nat Biotechnol. 2022; 41(2): 262-272.

[116] Mabry R, Becker A, Wesselhoeft A, et al. 1222 In situCAR therapy using oRNA™ lipid nanoparticles regresses tumors in mice. presented at: Regular and Young Investigator Award Abstracts; 2022.

[117] Zhou YX, Wu JC, Yao SH, et al. DeepCIP: A multimodal deep learning method for the prediction of internal ribosome entry sites of circRNAs. Comput Biol Med. 2023; 164: 107288.

[118] Xu C, Zhang L, Wang W, et al. Improving the circularization efficiency, stability and translatability of circular RNA by circDesign. bioRxiv Oct 29 2023. 2023;

[119] Zhang H, Zhang L, Lin A, et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature. 2023; 621(7978): 396-403.

[120] Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015; 217: 337-344.

[121] Jackson RJ, Hellen CUT, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Bio. 2010; 11(2): 113-127.

[122] Chen YG, Kim MV, Chen X, et al. Sensing self and foreign circular RNAs by intron identity. Mol Cell. 2017; 67(2): 228-238. e5.

[123] Chen YG, Chen R, Ahmad S, et al. N6-methyladenosine modification controls circular RNA immunity. Mol Cell. 2019; 76(1): 96-109. e9.

[124] Bishop DHL, Claybroo JR, Spiegelm S. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J Mol Biol. 1967; 26(3): 373.

[125] Cheng ZF, Deutscher MP. An important role for RNase R in mRNA decay. Mol Cell. 2005; 17(2): 313-318.

[126] Gabriel TF, Michalewsky JE. Oligonucleotide separations by high-pressure liquid chromatography on a weak anion-exchanger. J Chromatogr. 1973; 80(2): 263-265.

[127] Minkner R, Boonyakida J, Park EY, Wätzig H. Oligonucleotide separation techniques for purification and analysis: what can we learn for today’s tasks? Electrophoresis. 2022; 43(23-24): 2402-2427.

[128] Karikó K, Muramatsu H, Ludwig J, Weissman D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 2011; 39(21): e142.

[129] Durbin AF, 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): e00833.

[130] Park OH, Ha H, Lee Y, et al. Endoribonucleolytic cleavage of m(6)A-containing RNAs by RNase P/MRP complex. Mol Cell. 2019; 74(3): 494-507. e8.

[131] Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004; 432(7014): 173-178.

[132] High KA, Roncarolo MG. Gene therapy. N Engl J Med. 2019; 381(5): 455-464.

[133] Young TL, Wang KC, Varley AJ, Li BW. Clinical delivery of circular RNA: lessons learned from RNA drug development. Adv Drug Deliv Rev. 2023; 197: 114826.

[134] Stewart MP, Sharei A, Ding X, Sahay G, Langer R, Jensen KF. In vitro and ex vivo strategies for intracellular delivery. Nature. 2016; 538(7624): 183-192.

[135] Shih FH, Chang HH, Wang YC. Utilizing adeno-associated virus as a vector in treating genetic disorders or human cancers. IUBMB Life. 2024.

[136] Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 2003; 4(5): 346-358.

[137] Raguram A, Banskota S, Liu DR. Therapeutic in vivo delivery of gene editing agents. Cell. 2022; 185(15): 2806-2827.

[138] Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet. 2014; 15(8): 541-555.

[139] Lin MT, Pulkkinen L, Uitto J, Yoon K. The gene gun: current applications in cutaneous gene therapy. Int J Dermatol. 2000; 39(3): 161-170.

[140] Mahvi DM, Sheehy MJ, Yang NS. DNA cancer vaccines: a gene gun approach. Immunol Cell Biol. 1997; 75(5): 456-460.

[141] An J, Zhang CP, Qiu HY, et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway. Nat Biomed Eng. 2024; 8(2): 149-164.

[142] Jayasooriya V, Ringwelski B, Dorsam G, Nawarathna D. mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells. Lab Chip. 2021; 21(19): 3748-3761.

[143] Zhao YB, Moon E, Carpenito C, et al. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res. 2010; 70(22): 9053-9061.

[144] Bouakaz A, Michel Escoffre J. From concept to early clinical trials: 30 years of microbubble-based ultrasound-mediated drug delivery research. Adv Drug Deliv Rev. 2024; 206: 115199.

[145] Snipstad S, Sulheim E, de Lange Davies C, et al. Sonopermeation to improve drug delivery to tumors: from fundamental understanding to clinical translation. Expert Opin Drug Deliv. 2018; 15(12): 1249-1261.

[146] Zhang C, Ma YF, Zhang JJ, et al. Modification of lipid-based nanoparticles: an efficient delivery system for nucleic acid-based immunotherapy. Molecules. 2022; 27(6): 1943.

[147] Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev Genet. 2022; 23(5): 265-280.

[148] Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021; 6(12): 1078-1094.

[149] Zong Y, Lin Y, Wei T, Cheng Q. Lipid nanoparticle (LNP) enables mRNA delivery for cancer therapy. Adv Mater. 2023; 35(51): e2303261.

[150] Jia Y, Wang X, Li L, Li F, Zhang J, Liang XJ. Lipid nanoparticles optimized for targeting and release of nucleic acid. Adv Mater. 2024; 36(4): e2305300.

[151] Olden BR, Cheng Y, Yu JL, Pun SH. Cationic polymers for non-viral gene delivery to human T cells. J Control Release. 2018; 282: 140-147.

[152] Fidan Y, Muçaj S, Timur SS, Gürsoy RN. Recent advances in liposome-based targeted cancer therapy. J Liposome Res. 2023: 1-19.

[153] Chackerian B. Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines. 2007; 6(3): 381-390.

[154] Ludwig C, Wagner R. Virus-like particles-universal molecular toolboxes. Curr Opin Biotechnol. 2007; 18(6): 537-545.

[155] Ong HK, Tan WS, Ho KL. Virus like particles as a platform for cancer vaccine development. PeerJ. 2017; 5: e4053.

[156] Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM. Virus-like particles in vaccine development. Expert Rev Vaccines. 2010; 9(10): 1149-1176.

[157] Rocha JVR, Krause RF, Ribeiro CE, et al. Near infrared biomimetic hybrid magnetic nanocarrier for MRI-guided thermal therapy. ACS Appl Mater Interfaces. 2024.

[158] Safdar A, Wang P, Muhaymin A, Nie G, Li S. From bench to bedside: Platelet biomimetic nanoparticles as a promising carriers for personalized drug delivery. J Control Release. 2024; 373: 128-144.

[159] Chen HR, Yao HB, Chi JX, et al. Engineered exosomes as drug and RNA co-delivery system: new hope for enhanced therapeutics? Front Bioeng Biotechnol. 2023; 11: 1254356.

[160] Iqbal Z, Rehman K, Mahmood A, et al. Exosome for mRNA delivery: strategies and therapeutic applications. J Nanobiotechnology. 2024; 22(1): 395.

[161] Luo Z, Cheng X, Feng B, et al. Engineering versatile bacteria-derived outer membrane vesicles: an adaptable platform for advancing cancer immunotherapy. Adv Sci (Weinh). 2024:e2400049.

[162] Ji P, Wu P, Wang L, et al. Lysosome-targeting bacterial outer membrane vesicles for tumor specific degradation of PD-L1. Small. 2024:e2400770.

[163] Yang X, Chen M, Weng C, et al. Red blood cell membrane-coated nanoparticles enable incompatible blood transfusions. Adv Sci (Weinh). 2024; 11(29): e2310230.

[164] Ye J, Yu Y, Li Y, et al. Nanoparticles encapsulated in red blood cell membranes for near-infrared second window imaging-guided photothermal-enhanced immunotherapy on tumors. ACS Appl Mater Interfaces. 2024; 16(27): 34607-34619.

[165] Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand. 2003; 177(4): 437-447.

[166] Liu F, Su R, Jiang X, Wang S, Mu W, Chang L. Advanced micro/nano-electroporation for gene therapy: recent advances and future outlook. Nanoscale. 2024; 16(22): 10500-10521.

[167] Meganck RM, Borchardt EK, Castellanos Rivera RM, et al. Tissue-dependent expression and translation of circular RNAs with recombinant AAV vectors in vivo. Mol Ther Nucleic Acids. 2018; 13: 89-98.

[168] Meganck RM, Liu J, Hale AE, et al. Engineering highly efficient backsplicing and translation of synthetic circRNAs. Mol Ther Nucleic Acids. 2021; 23: 821-834.

[169] Lavenniah A, Luu TDA, Li YQP, et al. Engineered circular RNA sponges act as miRNA inhibitors to attenuate pressure overload-induced cardiac hypertrophy. Mol Ther. 2020; 28(6): 1506-1517.

[170] Yi Z, Qu L, Tang H, et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat Biotechnol. 2022; 40(6): 946-955.

[171] Katrekar D, Yen J, Xiang YC, et al. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs. Nat Biotechnol. 2022; 40(6): 938.

[172] Yang Z, Chang L, Chiang CL, Lee LJ. Micro-/nano-electroporation for active gene delivery. Curr Pharm Des. 2015; 21(42): 6081-6088.

[173] Yang N, Zhang C, Zhang Y, et al. CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia. J Transl Med. 2024; 22(1): 274.

[174] Shah PD, Huang AC, Xu XW, et al. Phase I trial of autologous RNA-electroporated cMET-directed CAR T cells administered intravenously in patients with melanoma and breast carcinoma. Cancer Res Commun. 2023; 3(5): 821-829.

[175] Campillo-Davo D, De Laere M, Roex G, et al. The ins and outs of messenger RNA electroporation for physical gene delivery in immune cell-based therapy. Pharmaceutics. 2021; 13(3): 396.

[176] Van Driessche A, Ponsaerts P, Van Bockstaele DR, Van Tendeloo VF, Berneman ZN. Messenger RNA electroporation: an efficient tool in immunotherapy and stem cell research. Folia Histochem Cytobiol. 2005; 43(4): 213-216.

[177] Fan L, Yao L, Li Z, et al. Exosome-based mitochondrial delivery of circRNA mSCAR alleviates sepsis by orchestrating macrophage activation. Adv Sci (Weinh). 2023; 10(14): e2205692.

[178] Liu J, Jiang J, Deng C, et al. Nanochannel electro-injection as a versatile platform for efficient RNA/DNA programming on dendritic cells. Small. 2023; 19(43): e2303088.

[179] Huang XG, Kong N, Zhang XC, Cao YH, Langer R, Tao W. The landscape of mRNA nanomedicine. Nat Med. 2022; 28(11): 2273-2287.

[180] Akinc A, Maier MA, Manoharan M, et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol. 2019; 14(12): 1084-1087.

[181] Samaridou E, Heyes J, Lutwyche P. Lipid nanoparticles for nucleic acid delivery: current perspectives. Adv Drug Deliv Rev. 2020; 154: 37-63.

[182] Kim J, Eygeris Y, Gupta M, Sahay G. Self-assembled mRNA vaccines. Adv Drug Deliv Rev. 2021; 170: 83-112.

[183] Patel S, Ashwanikumar N, Robinson E, et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat Commun. 2020; 11(1): 983.

[184] Patel SK, Billingsley MM, Frazee C, et al. Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells. J Control Release. 2022; 347: 521-532.

[185] Large DE, Abdelmessih RG, Fink EA, Auguste DT. Liposome composition in drug delivery design, synthesis, characterization, and clinical application. Adv Drug Deliv Rev. 2021; 176: 113851.

[186] Xu XY, Ho W, Zhang XQ, Bertrand N, Farokhzad O. Cancer nanomedicine: from targeted delivery to combination therapy. Trends Mol Med. 2015; 21(4): 223-232.

[187] Fang Y, Xue JX, Gao S, et al. Cleavable PEGylation: a strategy for overcoming the “PEG dilemma” in efficient drug delivery. Drug Deliv. 2017; 24(2): 22-32.

[188] Mui BL, Tam YK, Jayaraman M, et al. Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Mol Ther Nucl Acids. 2013; 2: e139.

[189] Oberli MA, Reichmuth AM, Dorkin JR, et al. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy. Nano Lett. 2017; 17(3): 1326-1335.

[190] Maeki M, Uno S, Niwa A, Okada Y, Tokeshi M. Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J Control Release. 2022; 344: 80-96.

[191] Pidgeon C, Mcneely S, Schmidt T, Johnson JE. Multilayered vesicles prepared by reverse-phase evaporation - liposome structure and optimum solute entrapment. Biochemistry US. 1987; 26(1): 17-29.

[192] Bangham A. The 1st description of liposomes - a citation classic commentary on diffusion of univalent ions across the lamellae of swollen phospholipids by Bangham, A.D., Standish, M.M., and Watkins, J.C. Cc/Life Sci. 1989;(13): 14-14.

[193] Gouda A, Sakr OS, Nasr M, Sammour O. Ethanol injection technique for liposomes formulation: An insight into development, influencing factors, challenges and applications. J Drug Deliv Sci Technol. 2021; 61: 102174.

[194] Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D. Nanomaterial delivery systems for mRNA vaccines. Vaccines Basel. 2021; 9(1): 65.

[195] Sakurai Y, Abe N, Yoshikawa K, et al. Targeted delivery of lipid nanoparticle to lymphatic endothelial cells via anti-podoplanin antibody. J Control Release. 2022; 349: 379-387.

[196] Herrera-Barrera M, Ryals RC, Gautam M, et al. Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Sci Adv. 2023; 9(2): eadd4623.

[197] Qu L, Yi ZY, Shen Y, et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell. 2022; 185(10): 1728.

[198] Li H, Peng K, Yang K, et al. Circular RNA cancer vaccines drive immunity in hard-to-treat malignancies. Theranostics. 2022; 12(14): 6422-6436.

[199] Zhou J, Ye T, Yang Y, et al. Circular RNA vaccines against monkeypox virus provide potent protection against vaccinia virus infection in mice. Mol Ther. 2024; 32(6): 1779-1789.

[200] Xu S, Xu Y, Solek NC, et al. Tumor-tailored ionizable lipid nanoparticles facilitate IL-12 circular RNA delivery for enhanced lung cancer immunotherapy. Adv Mater. 2024:e2400307.

[201] Huang K, Liu X, Qin H, et al. FGF18 encoding circular mRNA-LNP based on glycerolipid engineering of mesenchymal stem cells for efficient amelioration of osteoarthritis. Biomater Sci. 2024; 12(17): 4427-4439.

[202] Cullis PR, Felgner PL. The 60-year evolution of lipid nanoparticles for nucleic acid delivery. Nat Rev Drug Discov. 2024.

[203] Gurung S, Perocheau D, Touramanidou L, Baruteau J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun Signal. 2021; 19(1): 47.

[204] Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013; 200(4): 373-383.

[205] Liu S, Wu X, Chandra S, et al. Extracellular vesicles: Emerging tools as therapeutic agent carriers. Acta Pharm Sin B. 2022; 12(10): 3822-3842.

[206] Kang W, Xu Z, Lu H, et al. Advances in biomimetic nanomaterial delivery systems: harnessing nature’s inspiration for targeted drug delivery. J Mater Chem B. 2024; 12(29): 7001-7019.

[207] Han Y, Jones TW, Dutta S, et al. Overview and update on methods for cargo loading into extracellular vesicles. Processes (Basel). 2021; 9(2): 356.

[208] Johnsen KB, Gudbergsson JM, Skov MN, et al. Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes. Cytotechnology. 2016; 68(5): 2125-38.

[209] Yang L, Han B, Zhang Z, et al. Extracellular vesicle-mediated delivery of circular RNA SCMH1 promotes functional recovery in rodent and nonhuman primate ischemic stroke models. Circulation. 2020; 142(6): 556-574.

[210] Yu X, Bai Y, Han B, et al. Extracellular vesicle-mediated delivery of circDYM alleviates CUS-induced depressive-like behaviours. J Extracell Vesicles. 2022; 11(1): e12185.

[211] Huang G, Lin G, Zhu Y, Duan W, Jin D. Emerging technologies for profiling extracellular vesicle heterogeneity. Lab Chip. 2020; 20(14): 2423-2437.

[212] Ayala-Mar S, Donoso-Quezada J, Gallo-Villanueva RC, Perez-Gonzalez VH, Gonzalez-Valdez J. Recent advances and challenges in the recovery and purification of cellular exosomes. Electrophoresis. 2019; 40(23-24): 3036-3049.

[213] Hood JL. Post isolation modification of exosomes for nanomedicine applications. Nanomedicine (Lond). 2016; 11(13): 1745-1756.

[214] Jeyaram A, Jay SM. Preservation and storage stability of extracellular vesicles for therapeutic applications. AAPS J. 2017; 20(1): 1.

[215] Colao IL, Corteling R, Bracewell D, Wall I. Manufacturing exosomes: a promising therapeutic platform. Trends Mol Med. 2018; 24(3): 242-256.

[216] Xue C, Li G, Zheng Q, et al. The functional roles of the circRNA/Wnt axis in cancer. Mol Cancer. 2022; 21(1): 108.

[217] Kristensen LS, Jakobsen T, Hager H, Kjems J. The emerging roles of circRNAs in cancer and oncology. Nat Rev Clin Oncol. 2022; 19(3): 188-206.

[218] Lin J, Lyu Z, Feng H, et al. CircPDIA3/miR-449a/XBP1 feedback loop curbs pyroptosis by inhibiting palmitoylation of the GSDME-C domain to induce chemoresistance of colorectal cancer. Drug Resist Updat. 2024; 76: 101097.

[219] Ji Y, Ni C, Shen Y, et al. ESRP1-mediated biogenesis of circPTPN12 inhibits hepatocellular carcinoma progression by PDLIM2/NF-kappaB pathway. Mol Cancer. 2024; 23(1): 143.

[220] Song R, Guo P, Ren X, et al. A novel polypeptide CAPG-171aa encoded by circCAPG plays a critical role in triple-negative breast cancer. Mol Cancer. 2023; 22(1): 104.

[221] Wu N, Qadir J, Yang BBB. CircRNA perspective: new strategies for RNA therapy. Trends Mol Med. 2022; 28(4): 343-344.

[222] Seephetdee C, Bhukhai K, Buasri N, et al. A circular mRNA vaccine prototype producing VFLIP-X spike confers a broad neutralization of SARS-CoV-2 variants by mouse sera. Antiviral Res. 2022; 204: 105370.

[223] Wan JW, Wang ZM, Wang LL, et al. Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses. mBio. 2023; 15(1): e0177523.

[224] Wang Y, Lin L, Wang X, et al. Synergically enhanced anti-tumor immunity of in vivo CAR by circRNA vaccine boosting. bioRxiv. 2024. Available from: doi:10.1101/2024.07.05.600312

[225] Wang S, Yang J, Wei L, et al. An exhaustive pre-clinical study of a circRNA drug candidate (cmRNA1210) encoding IL-12sc for anti-tumor therapeutics. J Clin Oncol. 2024; 42(16_suppl): e14569-e14569.

[226] Yang J, Sun J, Zhu J, et al. Circular mRNA encoded PROTAC (RiboPROTAC) as a new platform for the degradation of intracellular therapeutic targets. bioRxiv. 2022. Available from: doi:10.1101/2022.04.22.489232

[227] Zhang LL, Liang DW, Chen CM, et al. Circular siRNAs for reducing off-target effects and enhancing long-term gene silencing in cells and mice. Mol Ther Nucl Acids. 2018; 10: 237-244.

[228] Liu X, Abraham JM, Cheng YL, et al. Synthetic circular RNA functions as a miR-21 sponge to suppress gastric carcinoma cell proliferation. Mol Ther Nucl Acids. 2018; 13: 312-321.

[229] Wang Z, Ma K, Cheng YL, et al. Synthetic circular multi-miR sponge simultaneously inhibits miR-21 and miR-93 in esophageal carcinoma. Lab Invest. 2019; 99(10): 1442-1453.

[230] Schreiner S, Didio A, Hung LH, Bindereif A. Design and application of circular RNAs with protein-sponge function. Nucleic Acids Res. 2020; 48(21): 12326-12335.

[231] Guo SK, Liu CX, Xu YF, et al. Therapeutic application of circular RNA aptamers in a mouse model of psoriasis. Nat Biotechnol. 2024.

[232] Irvine DJ, Maus MV, Mooney DJ, Wong WW. The future of engineered immune cell therapies. Science. 2022; 378(6622): 853.

[233] Xin T, Cheng L, Zhou C, Zhao Y, Hu Z, Wu X. In-vivo induced CAR-T cell for the potential breakthrough to overcome the barriers of current CAR-T cell therapy. Front Oncol. 2022; 12: 809754.

[234] Mabry R, Becker A, Wesselhoeft A, et al. 1222 In situ CAR therapy using oRNATM lipid nanoparticles regresses tumors in mice. J Immunother Cancer. 2022; 10; A1265-A1265.

[235] Ma L, Hostetler A, Morgan DM, et al. Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity. Cell. 2023; 186(15): 3148-3165. e20.

[236] Gulley JL, Madan RA, Pachynski R, et al. Role of antigen spread and distinctive characteristics of immunotherapy in cancer treatment. J Natl Cancer Inst. 2017; 109(4): djw261.

[237] Mackensen A, Haanen J, Koenecke C, et al. CLDN6-specific CAR-T cells plus amplifying RNA vaccine in relapsed or refractory solid tumors: the phase 1 BNT211-01 trial. Nat Med. 2023; 29(11): 2844-2853.

[238] Liu L, Wang PJ, Zhao DD, et al. Engineering circularized mRNAs for the production of spider silk proteins. Appl Environ Microb. 2022; 88(8): e0002822.

[239] He M, Cao C, Ni Z, et al. PROTACs: great opportunities for academia and industry (an update from 2020 to 2021). Signal Transduct Target Ther. 2022; 7(1): 181.

[240] Raab M, Kostova I, Pena-Llopis S, et al. Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer. Cancer Commun. 2024; 44(1): 101-126.

[241] Gurung S, Timmermand OV, Perocheau D, et al. mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria. Sci Transl Med. 2024; 16(729): eadh1334.

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