Abstract
Messenger RNA vaccines have shown strong prophylactic efficacy against viral infections. Here we show that antigen-encoding small circular RNAs (circRNAs) loaded in lipid nanoparticles elicit potent and durable T cell responses for robust tumour immunotherapy after subcutaneous injection in mice, particularly when combined with immune checkpoint inhibition. The small circRNA vaccines are highly stable and show low levels of activation of protein kinase R as well as low cytotoxicity, enabling long-lasting antigen translation (longer than 1 week in cells). Relative to large protein-encoding unmodified or modified mRNAs and circRNAs, small circRNA vaccines elicited up to 10-fold antigen-specific T cells in mice and accounted for 30–75% of the total peripheral CD8+ T cells over 6 months. Small circRNA vaccines encoding tumour-associated antigens, neoantigens and oncoviral or viral antigens elicited substantial CD8+ and CD4+ T cell responses in young adult mice and in immunosenescent aged mice. Combined with immune checkpoint inhibition, monovalent and multivalent circRNA vaccines reduced tumour-induced immunosuppression and inhibited poorly immunogenic mouse tumours, including melanoma resistant to immune checkpoint blockade.
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Data availability
The data supporting the results in this study are available within the paper and its Supplementary Information. Source data are available via figshare at https://doi.org/10.6084/m9.figshare.25919746. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
Change history
12 August 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41551-025-01490-w
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Acknowledgements
G.Z. acknowledges funding support from NIH (R01CA266981, R01AI168684, R35GM143014 and R21NS114455), DoD CDMRP Breast Cancer Breakthrough Award Level II (BC210931/P1), American Cancer Society Research Scholar Grant (RSG-22-055-01-IBCD), University of Michigan Rogel Cancer Center Discovery Award, NIH-NCATS KL2 scholarship (KL2TR002648) via Virginia Commonwealth University (VCU) C. Kenneth and Dianne Wright Center for Clinical and Translational Research (UL1TR002649), VCU Massey Cancer Center Molecules to Medicine pilot grant and VCU Commercialization Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Microscopy was performed at the VCU Microscopy Facility, supported in part by funding from NINDS Center Core Grant 5 P30 NS047463 and, in part, by funding from the NCI Cancer Center Support Grant P30 CA016059. Flow cytometry was performed at the VCU Massey Cancer Center Flow Cytometry Shared Resource, which is supported, in part, with funding from NCI Cancer Center Support Grant P30 CA016059. We thank the University of Michigan Flow Cytometry Core, the ULAM (Unit for Laboratory Animal Medicine) In Vivo Animal Core (IVAC), and the Advanced Genomics Core for service. We thank A. Ribas, J. Moon, C. Wu and NIH Tetramer Core for kindly providing cells and tetramer reagents.
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Contributions
G.Z. conceptualized the project. G.Z. and Y.Z. designed the project. Y.Z., X.L., Q.W., Tinging Shen, S.Z., S.L., S.Y., Ting Su, L.M., L.X., Y.G. and C.G. performed the experiments. Y.Z., X.L., X.-Y.W. and G.Z. analysed the data. Y.Z., B.Z., K.H., K.M.T., X.Q., J.L. and G.Z. analysed the RNA-seq and circRNA-seq data. Y.Z., X.L. and G.Z. drafted and revised the paper, with assistance from K.H., S.Z., K.M.T. and J.L. All authors reviewed the paper.
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G.Z. and Y.Z. are listed as inventors in a related patent application (WO2022173730A1). G.Z. is a co-founder, central scientific officer and equity holder of AmpedRNA Biosciences, LLC. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Liposomes promote circRNA delivery and antigen presentation in DCs.
a, DLS data showing the size distribution of blank liposome and lipo-circRNA with different N/P ratios in PBS (solid) and 1% FBS (dotted). b, Zeta potential of blank liposome and lipo-circRNA. c-d, In vitro cell uptake of circRNA and antigen presentation in DCs mediated by lipo-circRNA. c, Flow cytometry results and d, MFI of the SIINFEKL presentation in SIINFEKL-circRNA-treated DC2.4 cells (24 h). SIINFEKL antigen presentation on DC2.4 cells that were treated with Lipofectamine 3000-transfected circRNA and lipo-circRNA with different N:P ratios, respectively. Ns: non-significant, *p < 0.05, ***p < 0.001, by one-way ANOVA with Bonferroni post-test. e-h, In vitro intracellular delivery of circRNA in DCs by liposome. e, Confocal microscopy images of DC2.4 cells treated with lipo-circRNA for 1 h, 3 h, or 6 h. Blue: nuclei stained with Hoechst33342. Green: endolysosome stained with LysoTracker Green DND-26. Red: Cy5-circRNA. Insets: close-up views of single cells. f, Flow cytometry results of DC2.4 cells incubated with Lipo-circRNA and free circRNA for different times. g, Flow cytometry results showing MFI of DCs incubated with free or Lipo-Cy5-circRNA. ****p < 0.0001, by t-test of the AUC. h, The signal ratio of Cy5-circRNA outside/inside (O/I) the endolysosome.
Extended Data Fig. 2 Liposomes promoted the delivery of circRNA to draining lymph nodes and to key intranodal APC subsets in mice.
a, Liposomes promoted the delivery of IR800-circRNA to draining popliteal lymph nodes (circled) in BALB/c mice (0.2 nmol, s.c. injected at foot pad). b, AUC of the radiance efficiency. **p < 0.01, by t-test of the AUC. c, Ex vivo fluorescence images of BALB/c mice after s.c. injected at tail base for 24 h. d, Radiance efficiency of major organs and inguinal lymph nodes after s.c. injection of IR800-circRNA at tail base for 24 h. He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; and LN: inguinal lymph node. e, The frequency of circRNA+ cDCs and macrophages in inguinal lymph node tissues 24 h after s.c. injection at tail base.
Extended Data Fig. 3 Small circRNA-SIINFEKL elicited potent T-cell responses in young adult mice.
a, Study design. C57BL/6 mice (n = 5) were vaccinated on day 0 and day 14, and PBMC T cell responses were analyzed starting from day 21. b, Tetramer staining on day21 – day70 showed that circRNA-SIINFEKL elicited potent SIINFEKL-specific CD8+ T cell response in mice (n = 5) that outperformed current benchmarks 5moU-modified CleanCap mRNA-OVA and CpG-adjuvanted OVA. c, circRNA-SIINFEKL elicited SIINFEKL-specific T cell memory (day 70). Tem: effector memory T cell; Tcm: central memory T cell. d, circRNA-SIINFEKL upregulated PD-1 expression on SIINFEKL-specific CD8+ T cells on day21. e, PD-1 MFI on live PBMC CD8+ T cells. f, circRNA-SIINFEKL enhanced PD-1 expression levels (MFI) and frequencies on SIINFEKL+CD8+ T cells than that on total CD8+ T cells in peripheral blood on day 21, indicating immune exhaustion often resulting from chronic immunostimulation and providing an opportunity to combine circRNA vaccines with immune checkpoint blockade for optimal T cell responses (Two-tailed paired t test). g, circRNA-SIINFEKL enabled mice to resist 3×105 EG7.OVA tumour challenge 70 days post-vaccination. Vaccine delivery by liposome, s.c. injected at tail base, 5 μg RNA, 2 nmole CpG, 20 μg OVA. *: relative to circRNA. h, Mouse body weights after tumour challenge. Data represent mean ± SD (b-e) and mean ± s.e.m. in other figure panels (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.
Extended Data Fig. 4 Peptide-encoding small circRNA vaccine elicit stronger and more durable T-cell responses than protein-encoding unmodified mRNA and large circRNA vaccines in young adult mice.
a, Design of T cell response study in mice. C57BL/6 mice (n = 4-5; 6-8 weeks) were immunized with circRNA-SIINFEKL, unmodified mRNA-OVA, and large circRNA code OVA protein at 3 μg and 10 μg doses, respectively. b, 120-day kinetics of the PBMC SIINFEKL-specific CD8+ T cell percentages in the above immunized mice, suggesting that circRNA-SIINFEKL elicited overall stronger and more durable T cell responses than OVA-encoding unmodified mRNA and large circRNA vaccines. Asterisks: statistical significance of the T cell fraction AUC relative to that for circRNA. c, circRNA-SIINFEKL elicited larger fractions of memory T cells (Tem + Tcm) than unmodified mRNAs (day 90), indicating great T cell memory elicited by small circRNA vaccine. Data were quantified from CD44 and CD62L staining of PBMC CD8+ T cells.
Extended Data Fig. 5 Monovalent small circRNA vs modified mRNA or large circRNA vaccines for robust tumour immunotherapy.
a, For immunotherapy studies in mouse models of subcutaneous EG7.OVA (b-d), and TC-1 (e), tumours were inoculated into the right flank of C57Bl/6 mice. Vaccine: 5 μg RNA or 5 μg CpG + 10 μg protein or peptide antigens, s.c. injected in liposome at mouse tail base; antibodies: 200 μg, i.p. b-d, EG7.OVA tumour growth (b) and Kaplan-Meier survival curves (c) of EG7.OVA tumour-bearing mice treated with circRNA-SIINFEKL, 5moU-mRNA-OVA, and CpG-adjuvanted OVA, respectively. αCD8, αCD4, and αNK1.1 were injected intraperitoneally (i.p.) for lymphocyte depletion. d, Body weights of EG7.OVA tumour-bearing mice treated with circRNA-SIINFEKL vs. controls. e, Individual (upper panel) and average (lower panel) EG7.OVA tumor growth curves in C57BL/6 mice treated with the indicated circRNA-SIINFEKL or large circRNA-OVA, as well as αPD-1 (i.p.) alone or combined with circRNA-SIINFEKL. RNA: 30 μg, s.c. injection at tail base. CR: complete regression rate. f, TC-1 tumour volumes after lymphocyte depletion using αCD8, αCD4 or αNK1.1. Data represent mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001, by one-way ANOVA with a Bonferroni post-test (n = 6-8).
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Zhang, Y., Liu, X., Shen, T. et al. Small circular RNAs as vaccines for cancer immunotherapy. Nat. Biomed. Eng 9, 249–267 (2025). https://doi.org/10.1038/s41551-025-01344-5
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DOI: https://doi.org/10.1038/s41551-025-01344-5
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