Compositions and methods for small circular RNA-based immunotherapies

Abstract

Provided herein are compositions and methods for inducing an immune response using small circular ribonucleic acid (circRNA) molecules. In particular, the compositions comprise a small circRNA encoding at least one antigen or immunogenic peptide fragment, wherein the composition is self-adjuvanted and configured to elicit a T-cell–mediated immune response.

Description

PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601COMPOSITIONS AND METHODS FOR SMALL CIRCULAR RNA-BASED IMMUNOTHERAPIESCROSS-REFERENCE TO RELATED APPLICATIONS|0001] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 704,656, filed October 8, 2024, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The text of the computer readable sequence listing filed herewith, titled “45500- 601_SEQUENCE_LISTING”, created October 8, 2025, having a file size of 32,350 bytes, is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH|0003] This invention was made with government support under grant number AI168684 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.TECHNICAL FIELD]0004] The present disclosure relates to compositions and methods for inducing an immune response using small circular ribonucleic acid (circRNA) molecules. In particular, the compositions comprise a small circRNA encoding at least one antigen or immunogenic peptide fragment, wherein the composition is self-adjuvanted and configured to elicit a T-cell-mediated immune response.BACKGROUND

[0005] Approaches to modulate the immune system to treat or prevent infectious diseases, cancers, and other diseases and conditions have been developed. Yet there remains a need for more efficient, flexible, and effective platforms to prevent and treat such diseases and conditions, including pathogenic viral infections and associated cancers, such as SARS-CoV-2 and human papillomavirus (HPV) infections, and HPV-associated cancer.SUMMARY

[0006] Aspects of the present disclosure relate to compositions and methods for inducing an immune response using small circular ribonucleic acid (circRNA) molecules. In particular, the compositions comprise a small circRNA encoding at least one antigen or immunogenic peptide fragment, wherein the composition is self-adjuvanted and configured to elicit a T-cell-mediated immune response.

[0007] The compositions and methods described herein are designed to promote efficient antigen expression and presentation in immune cells, thereby stimulating a potent and durable adaptive immune response.

[0008] Embodiments of the present disclosure provide a composition for inducing an immune response comprising a small circular ribonucleic acid (circRNA) molecule encoding at least one antigen or anPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 immunogenic peptide fragment thereof, wherein the composition is self-adjuvanted and elicits a T-cell immune response.|0009] In some embodiments, the antigen is selected from a protein, polypeptide, peptide, or fragment thereof derived from a pathogen, tumor-associated antigen, or autoantigen. In some embodiments, the antigen sequence may be codon-optimized for enhanced translation efficiency in host cells. In some embodiments, the immunogenic peptide fragment comprises a T-cell epitope or a multivalent peptide sequence derived from an antigen of a pathogen, tumor-associated antigen, or autoantigen. In some embodiments, the T-cell epitope is optionally an MHC class I- restricted peptide epitope or a MHC class II- restricted peptide epitope.

[0010] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or salt thereof. Suitable carriers include, but are not limited to, lipid nanoparticles, liposomes, emulsions, polymeric nanoparticles, or biodegradable delivery systems configured for systemic or localized administration. The composition may optionally include stabilizers, surfactants, or cryoprotectants to improve storage and formulation stability.

[0011] Also provided herein is a method of modulating an immune response in a subject, comprising administering to the subject a composition comprising a small circular ribonucleic acid (circRNA) molecule encoding an antigen or immunogenic peptide fragment thereof, wherein the circRNA is translated in a host cell to produce multivalent peptide antigens and activate antigen-specific T cells.

[0012] Administration may occur via intramuscular, intradermal, subcutaneous, intravenous, mucosal, or other routes suitable for delivery of nucleic acid-based compositions.

[0013] In some embodiments, the administration of the composition enhances antigen processing and presentation by antigen-presenting cells. In some embodiments, the composition enhances proteolytic degradation of the antigen. In some embodiments, this facilitates generation of antigenic peptides that are efficiently presented to T cells, thereby augmenting the magnitude and extent of the immune response. In some embodiments, the composition enhances adaptive immune responses. In some embodiments, the enhancement of adaptive immune responses, including cellular and humoral immunity, in turn promotes expansion of antigen-specific T cells, generation of memory T cells, and the production of antibodies. In some embodiments, the composition improves immunomodulatory efficacy relative to an unmodified antigen or a linear mRNA vaccine. In some embodiments, the administration of the composition induces a stronger and longer-lasting T-cell immune response relative to a linear RNA or peptide vaccine encoding the same antigen.

[0014] In some embodiments, the composition is a nucleic acid vaccine, optionally delivered as a protein-encoding RNA, or a derivative thereof. In some embodiments, the vaccine is co-formulated with immunostimulatory oligonucleotides, cytokines, or checkpoint inhibitors to further enhance immune activation.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0015] In some embodiments, the composition is administered as a cancer immunotherapy, a prophylactic vaccine, or a therapeutic vaccine. In some embodiments, the composition may be used alone or in combination with other immunotherapeutic agents, such as immune checkpoint inhibitors, cytokines, or chemotherapeutic drugs. In some embodiments, the composition is used for prevention or treatment of a viral infection, microbial infection, cancer, autoimmune disorder, or derivative thereof. In some embodiments, the prophylactic vaccine is for the prevention of a microbial infection, a viral infection, cancer, an autoimmune disease or any derivative thereof. In some embodiments, multiple circRNA molecules encoding distinct antigens may be co-administered to induce a broad protective immune response. In some embodiments, the therapeutic vaccine is for the treatment of a microbial infection, a viral infection, cancer, diabetes, cardiovascular disease, an autoimmune disease, or any derivative thereof. In some embodiments, repeated or booster administrations are employed to sustain antigen-specific T-cell activation and achieve durable therapeutic benefit.

[0016] Current mRNA vaccines use long and linear mRNA that is extensively modified, which incurs complicated, inefficient, and error-prone mRNA manufacturing via in vitro transcription, limited loading capacity in nanocarriers, and moderate biostability and pharmaceutical stability. Currently reported circular RNA therapeutics use very long circular RNA (multiple thousands of kilobases), which have limitations in terms of large scale manufacturing, purification, and quality control. These long circular RNA may also elevated some unwanted RNA-elicited immune side effects. By contrast, embodiments of the present technology provide: (1) circular RNA that is much shorter, and can be readily manufactured at large scales using automatic nucleic acid synthesizers in current oligonucleotide manufacturing cGMP facilities; (2) small circular RNA that also minimize unwanted side effects, if any, that are induced by RNA; (3) small circular RNA is are self-adjuvanted, meaning that as RNA vaccines they do not need additional immunostimulant adjuvants; and (4) inside cells, the small circular RNA continuously synthesizes peptides, resulting in intracellular synthesis of multivalent peptides that are efficient at vaccine immunomodulation, as well as molecular target binding and interaction.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains drawings executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0018] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0019] Figure 1 shows a schematic illustration of highly stable small circRNA vaccines that elicit potent and long-lasting T cell responses for tumor immunotherapy. Small circRNA comprise peptide- antigen-encoding RNA and a short IRES and a Kozak consensus sequence that initiate peptide translation. Small circRNA was synthesized by ligating RNA oligonucleotide precursor(s) using RNA ligase and DNA splint(s). The absence of termini and the minimal sizes of small circRNA enable it toPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 resist exonuclease degradation and minimize endonuclease degradation and hydrolysis, resulting in excellent thermostability and biostability. Nanocarriers delivered small circRNA vaccines to draining lymph nodes and intranodal APCs, in which (1) circRNA activates PRRs to elicit proinflammatory innate immunity with low PKR activation and low cytotoxicity, and (2) circRNA is efficiently translated to antigen peptides for antigen presentation over a prolonged duration. This allows small circRNA vaccines to elicit potent and long-lasting antigen-specific T cell responses. Modular small circRNA vaccines can be easily adjusted to encode various peptide antigens for versatile applications: (1) by encoding MHC-I- and MHC-II-restricted antigens, circRNA elicits CD8+and CD4+T cell responses, respectively; and (2) by encoding tumor-associated antigens, tumor neoantigens or (onco)viral antigens, circRNA finds use for generating off-the-shelf shared vaccines and personalized vaccines. Moreover, small circRNA vaccines reduce the immunosuppression and enhance the infiltration of antitumor immune cells in distant tumors. As a result, small circRNA vaccines, especially when combined with ICB, show robust immunotherapeutic efficacy for multiple types of tumor, including ICB-resistant BrafV600Emelanoma.

[0020] Figure 2 shows highly stable small circRNA vaccines for efficient peptide translation and antigen presentation. (A) Top: schematic illustration of the synthesis of small circRNA and liRNA by ligation of RNA oligonucleotide precursors and DNA splints. Bottom: Sanger sequencing of the cDNA of circRNA-SIINFEKL indicates precise and uniform ligation of RNA precursors into circRNA. The denoted RNA sequence is converted from the Sanger sequencing results of cDNA. (B) crTMV IRES- based circRNA-SIINFEKL elicited efficient antigen presentation and T cell priming. Left: flow cytometric quantification of the mean fluorescence intensity (WTI) of SIINFEKL / H-2Kb complexes on DC2.4 cells treated with circRNA or controls for 24 h. Right: the activities of SIINFEKL-specific B3Z CD8+T cell hybridoma co-incubated with the as-treated DC2.4 cells. B3Z cell activity': absorbance value. (C) The secondary structures of crTMV IRES (left) and crTMV-based circRNA-SIINFEKL (right), as predicted using NUPACK. The blue box denotes IRES. (D) Potent and durable priming of B3Z cells by DC2.4 cells treated with circRNA-SIINFEKL. 5moU-mRNA-OVA: a benchmark proteinencoding 5moU-modified mRNA with CleanCap, 5'- and 3 '-UTRs, and 3' ADO. (E) Confocal fluorescence microscopy images show the efficient production of a model peptide, Flag, from circRNA- Flag in live DC2.4 cells 24 h after transfection. (F-H) circBroccoli was highly stable in live DC2.4 cells as shown by flow cytometry (F,G) and confocal fluorescence microscopy (H) analysis of cell Broccoli fluorescence intensities. Cells were transfected with circBroccoli or linear Broccoli control for 1-168 hours, before adding fluorescence -activating Broccoli cognate, DFHBI-1T. Asterisks in g denote statistical significance of Broccoli MFI AUC of linear Broccoli relative to that of circBroccoli (I) circRNA-seq results showing the sequence integrity of circRNA-SIINFEKL recovered from live DCs after transfection for 24 hours. circRNA-SIINFEKL in PBS was used as a positive control. The authenticity rate of each nucleotide was calculated as the rate of unmutated nucleotides. (J) Percentages of intact circRNA-SIINFEKL, liRNA-SIINFEKL and 5moU-mRNA-OVA after storage in PBS at -20PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601°C, 4 °C or 23 °C for up to 180 days. At -20 °C, 4 °C and 23 °C, the half-lives of circRNA-SIINFEKL are estimated to be 401, 78 and 16 days, respectively; 143, 44 and 6 days, respectively, for 5moU- mRNA-OVA; and 2.6, <0.5 and <0.5 days, respectively, for liRNA-SIINFEKL. Data were quantified from gel electrophoresis using ImageJ. (K) Upon transfection into DC2.4 cells, small circRNA-Flag showed durable Flag expression for at least 7 days, in contrast to fLuc expression for less than 3 days from 5moU-mRNA-fLuc. Intracellular Flag was stained using phycoelythrin (PE)-conjugated anti-Flag antibody, and the WTI of cells was measured by flow cytometry. (L) Dynamic light scattering (left) and cryogenic electron microscopy (cryo-EM) images (right) showing the sizes and morphology of blank and circRNA-loaded ionizable SM-102 LNPs. (M) Percentages of intact circBroccoli loaded in LNPs after storage in PBS at 4 °C or -20 °C (8% cryoprotectant sucrose) for 72 days. Data were quantified from flow cytometric analysis of DC2.4 cells transfected (24 h) with circBroccoli LNPs recovered from storage (paired t-test). (N, O) Representative agarose gel electrophoresis images (N) and the intact circRNA percentages (O) of circRNA-SIINFEKL (1 nmol) and circRNA-RBD (1 nmol) after incubation in a series of diluted FBS in PBS (37 °C, 30 min). Data were quantified from gel electrophoresis by normalizing the band densities of FBS-treated circRNA to that of PBS-treated circRNA (t-test). RNA (100 nM) was transfected using Lipofectamine 3000 unless denoted otherwise. Data represent mean ± s.e.m. *P< 0.05, **P< 0.01, ***p< 0.001, * ** *P< 0.0001, one-way ANOVA with Bonferroni post-test.

[0021] Figure 3 shows LNPs efficiently delivered small circRNA to lymph nodes and APCs to elicit T cell responses. (A) Design of nanocarrier screening for small circRNA vaccines, using circRNA- SIINFEKL as a model and 5moU-mRNA-OVA as a control. RNA: 5pg, subcutaneous (s.c.) injection at the tail base of C57BL / 6 mice (n = 5) on days 0 and 14. (B) Tetramer staining on day 21 showed that SM-102 LNPs of circRNA-SIINFEKL elicited the highest frequency of PBMC SIINFEKL+CD8+T cells among all these RNA nanoformulations. (C) Luminex results of normalized serum cytokine and chemokine levels 12 h after booster immunization (day 14). SM-102 LNP-circRNA-SIINFEKL induced relatively low reactogenicity-associated chemokines. Each cytokine or chemokine level (x) was respectively normalized as follows:(D-F) Upon s.c. injection at the foot pad of BALB / c mice (n = 5), SM-102 LNPs efficiently delivered IR800-circRNA-SIINFEKL (0.5 nmol) to draining popliteal lymph nodes (dLNs) (circled in D), as shown by whole-body IVIS imaging (D), quantified IR800 fluorescence intensities of popliteal dLNs (E) and tissue fluorescence intensities quantified from ex vivo IVIS imaging (F). (G) Flow cytometry results showing the Cy5-circRNA± APC subsets among total CD45+cells in draining lymph nodes 24 hours after s.c. injection of free Cy5- circRNA or Cy5-circRNA LNPs, respectively. (H) Confocal microscopy images showing efficient LNP delivery of Cy5-circRNA to DC2.4 cells and rapid endosome escape of Cy5-circRNA, the latter of which was indicated by the cytosolic Cy5-circRNA outside endolysosomes (circRNA, 100 nM; treatment, 0.5 h). Inset: one cell. (I) As quantified from the above confocal microscopy images, the Cy5-circRNA fluorescence signal intensity ratios of outside / inside (O / I) endolysosome indicates thePCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 rapid endosome escape of LNP-circRNA in DC2.4 cells. Liposomal circRNA (lipo-circRNA) served as a control.|0022] Figure 4 shows small circRNA vaccine activated PRRs for intrinsic immunostimulation with low PKR activation. (A-C) Gene transcriptome analysis results from BMDCs LNP-transfected with circRNA-SIINFEKL, 5moU-mRNA-OVA and PBS, respectively (24 hours). The log2-transformed fold change (FC) represents log2(ratio of the mean expression induced by vaccine relative to PBS) (n = 3). (A) Transcription heatmaps of genes involved in inflammation, migration, antigen processing and presentation, TLRs, RLRs, CLRs and miscellaneous immune-related genes. (B) Volcano plot of differentially accessible peaks between blank LNPs and circRNA-SIINFEKL LNPs. (C) log2(FC) of specific genes of interest related to the indicated pathways. (D) PRR reporter cell activities upon treatment with circRNA-SIINFEKL (100 nM, 24 h) and controls. Poly(LC) served as a positive control. (E) RT-PCR results of Ifnb levels in wild-type (WT) RM1 and RM1-IPS1-K0 cells treated with circRNA-SIINFEKL (100 nM, 48 h). (F) Western blot analysis of PKR and pPKR in HEK293T cells after transfection with PBS, small circRNA-SIINFEKL, large circRNA-RBD and poly(LC) positive control (0.5 pg per well), respectively, for 4 hours. (G) Western blot intensity ratio of pPKRto 13-actin in treated HEK293T cells. Relative phosphorylation is indicated, calculated as the band intensity ratio (X) of pPKR to P-actin and then normalized to the relative phosphorylation induced by no RNAtreatment group’(H) HEK293T cell viability transfected with the indicated RNA (0.5 pg per well, 24 hours) or PBS control. Cell viability was normalized to PBS-treated cells. In (D-H), RNA was transfected using Lipofectamine 3000. *P < 0.05, **P< 0.01, ***P< 0.001,0.0001, by one-way ANOVA with Bonferroni post-test. NSm not significant.

[0023] Figure 5 shows small circRNA vaccine outperformed several benchmark modified mRNA and large circRNA vaccines to elicit robust and durable T cell responses with great safety in young adult mice and aged mice. (A-H) Benchmark studies of small circRNA vaccine versus three types of modified mRNA vaccine, an unmodified mRNA vaccine (no nucleoside modification), and a large circRNA vaccine in DCs and young adult mice. (A) Three-day AUC of the H-2Kb-SIINFEKL WTI on DCs LNP- transfected with circRNA-SIINFEKL and three modified mRNAs, respectively. Data were quantified from flow cytometry results. Asterisks: statistical significance relative to circRNA. (B) Timeline of in vivo benchmark study. C57BL / 6 mice (6-8 weeks) were immunized with circRNA-SIINFEKL, as well as three modified mRNAs, unmodified mRNA-OVA and large circRNA-OVA, at escalating doses, respectively. (C-E) Benchmarking circRNA-SIINFEKL against three types of modified mRNA for their ability to elicit T cell responses in mice (n= 9 for PBS, circRNA-SIINFEKL and 5moU-mRNA- OVA; n = 4-5 for the other groups). (C) Tetramer staining results on day 21 indicates that circRNA- SIINFEKL elicited higher fractions of PBMC SIINFEKL-specific T cells than all modified mRNAs. circRNA-SIINFEKL elicited dose-dependent T cell responses, which plateaued at ca. 60% (dose 2 x 30 pg). Asterisks: statistical significance relative to circRNA. (D) circRNA-SIINFEKL caused less mousePCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 body weight drop and more rapid weight recovery than modified mRNAs, as exemplified 1 -2 days after the 2nd 30 pg dose. (E) 180-day kinetics of the PBMC SIINFEKL-specific CD8+T cell percentages in the immunized mice, indicating that circRNA-SIINFEKL elicited more potent and more durable T cell responses than these modified mRNAs, at all corresponding doses before the T cell responses plateaued. Asterisks denote statistical significance of the T cell fraction AUG relative to that for circRNA. (F) Estimated total counts of peripheral and splenic SIINFEKL-specific CD8+T cells per mouse (day 35, day 180). circRNA-SIINFEKL (3 10 pg) elicited SIINFEKL-specific CD8 T cell counts that are >2- fold typical doses of adoptive TCR-T cells (5 x 106, dashed line) used for mouse tumor immunotherapy. (G) Tetramer staining results on day 21 and day 35 indicates that circRNA-SIINFEKL elicited higher fractions of PBMC SIINFEKL-specific T cells than unmodified mRNA-OVA and large circRNA-OVA. Asterisks: statistical significance relative to circRNA. C57BL / 6 mice (6-8 weeks) were subcutaneously immunized with circRNA-SIINFEKL (n = 9), unmodified mRNA-OVA (n = 4) and large circRNA- OVA (n = 4) (dose 3pg and 10 pg, days 0, 14 and 28). (H) Luminex heatmaps showing the serum chemokine and cytokine levels 6 h after the third immunization (day 28). Each cytokine or chemokine level (x) was independently normalized for each dose as follows:Overall, circRNA-SIINFEKL induced the least reactogenicity -associated chemokine and cytokine levels among all vaccines. (I-K) Low-dose circRNA-SIINFEKL (5 pg; days 0, 14 and 28) elicited potent and durable T cell immunity in immunosenescent aged mice (1 year old; n= 5). (I) Tetramer staining showed superior PBMC SIINFEKL-specific CD8+T cell responses elicited by circRNA-SIINFEKL than 5moU-mRNA-OVA or CpG-adjuvanted OVA (day 21) (t-test). (J) Intracellular IFNy and TNF staining results in PBMC CD8+T cells from the above immunized aged mice (day 35). T cells were restimulated with SIINFEKL peptide. (K) circRNA-SIINFEKL vaccine protected immunized aged mice from EG7- OVA tumor cell challenge (1 x 106 cells, s.c. administration on day 70) (asterisks denote statistical significance relative to circRNA). Vaccines were delivered by SM-102 LNPs and subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, by one-way ANOVA with Bonferroni post-test, unless denoted otherwise.

[0024] Figure 6 shows the broad application of small circRNA vaccines to elicit T cell responses against various peptide antigens. (A) Design of T cell response study for small circRNAs encoding various types of peptide antigen in C57BL / 6 mice (6-8 weeks; n= 5). RNA, 5 pg per RNA; CpG, 5 pg; peptides, 10 pg; s.c. injection at tail base. Vaccines were delivered by LNPs. T cells were restimulated with OVA for intracellular cytokine staining. (B) Intracellular IFNy and TNF staining showed that MHC-II-restricted circRNA-ISQ elicited effector CD4+T cells in PBMCs (day 21). 5moU-mRNA- OVA served as a benchmark. (C) Intracellular IFNy and TNF staining showed that MHC-II-restricted circRNA-ISQ enhanced the ability of MHCI- restricted circRNA-SIINFEKL to elicit SIINFEKL- specific CD8+T cell response (day 21). Both monovalent and bivalent circRNAs outperformed 5moU- mRNA-OVA to elicit the corresponding antigen-specific CD4+or CD8+T cell responses. (D-K) Small circRNA vaccines elicited T cell responses against various cancer and viral peptide antigens. (D)PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601Tetramer staining results showed the fractions of ADPGK ±CD8+T cells among all live PBMC CD8+T cells, indicating that circRNA-ADPGK neoantigen vaccine elicited robust T cell responses. (E) circRNA- ADPGK elicited T cell memory (day 70). (F) circRNA-ADPGK elevated PD-1 expression on CD8+T cells (day 21). (G) Intracellular IFNy and TNF staining of PBMC CD8+T cells (day 21) from C57BL / 6 mice immunized with circRNA-TRP2 / gplOO or CpG-adjuvanted peptide vaccines (days 0 and 14). (H) The frequencies of PBMC E7±CD8+T cells over 70 days post priming from C57BL / 6 mice immunized with circRNAE743_62 or CpG-adjuvanted peptide vaccines (days 0, 14 and 28). (I) Intracellular IFNy and TNF staining of PBMC CD8+T cells (day 21) from the above immunized mice. (J) Tetramer staining results showing the fractions of RBD440_459±CD8+T cells among all live PBMC CD8+T cells from as-immunized C57BL / 6 mice (days 0, 14 and 28), indicating that circRNA-RBD440 459 elicited potent RBD440_459-specific CD8+T cell responses. (K) Intracellular IFNy and TNF staining of PBMC CD8+T cells (day 21) from the above immunized mice (asterisks in D, H and J: statistical significance relative to circRNA). Vaccines: subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. (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.10025] Figure 7 shows that the small circRNA neoantigen vaccine reduced tumor immunosuppression for potent tumor immunotherapy. (A) Study design of TME immune analysis and tumor immunotherapy in mice. MC38 tumor cells were subcutaneously inoculated in the flank of C57BL / 6 mice, and treatment started when tumors reached around 60 mm3 on day 6. (B-D) MC38 tumor immune microenvironment analysis (day 15) upon treatment with circRNA-ADPGK, alone or combined with anti-PD-1 (n= 6-8). 5moU-mRNA-ADPGK served as a control. (B) The percentage of different immune cells among CD45+cells in TME after the indicated treatment. (C) Tetramer staining results showed the fractions of ADPGIK±CD8+T cells among total live CD8+T cells in TME, indicating that circRNA-ADPGK vaccine combined with anti-PD-1 enhanced antigen-specific cytotoxic T cells within the TME. (D) The ratio of CD8+T cells to Treg cells within the TME. e-g, RNA-seq transcriptome analysis of MC38 tumors (day 15) after the above treatment (n = 3). (E) Transcription heatmaps of selected genes related to immune modulation. (F) The log-transformed mean expression ratio (log2(FC) of pathway -related genes in immunotherapy -treated tumors compared with that in PBS-treated tumors. (G) Triwise radar plots depicting Gene Ontology enrichment analysis of T cell priming and DC activation pathways (left) and regulation of T cell proliferation (right). Black dots, all genes; red dots, genes related to the corresponding immune functions. (H) Low-dose circRNA-ADPGK (5 pg) for potent combination immunotherapy of MC38 tumor, as shown by MC38 tumor growth and Kaplan-Meier mouse survival curves. liRNA and 5moU-mRNA encoding the same peptide antigen were used as controls (n = 14 for PBS, anti-PD-1, circRNA-ADPGK and circRNA-ADPGK+anti-PD-l; n = 7 for the other groups). Asterisks denote statistical significance between the AUG of circRNA-ADPGK tumor growth and that of circRNA-ADPGK+anti-PD-l. P= 0.0480: statistical analysis between the AUC of circRNA-ADPGK tumor growth and that of mRNA-ADPGK. (I) MC38 tumor volumes after lymphocyte depletion usingPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 anti-CD8, anti-CD4 or anti-NKl. l antibodies. RNA vaccines were delivered by SM-102 LNPs and subcutaneously injected at tail base. RNA, 5 pg; antibodies, 100 pg, intraperitoneal (i.p.) injection. Data represent mean ± s.e.m. *P< 0.05, **P< 0.01, ***P< 0.001, 0.0001, by one-way ANOVA withBonferroni post-test unless denoted otherwise.

[0026] Figure 8 shows monovalent or multivalent small circRNA vaccines for robust combination immunotherapy of multiple types of tumor. (A) Design of tumor immunotherapy studies in mice. Tumor cells were inoculated subcutaneously in mouse flank, and treatment started when tumor volumes were around 50 mm3. Vaccines were loaded in LNPs and subcutaneously injected at tail base; antibodies were intraperitoneally injected. (B) Average TC-1 tumor volumes after treatment with circRNA-E?43 62+anti-PD-l and controls (n = 6-7). (C) Spider plots of individual TC-1 tumor growth curves and complete regression (CR) rates after the above treatment. (B , C) vaccine, 5 pg RNA, 5pg CpG, 10 pg E?43 62 peptide antigens; anti-PD-1, 200 pg. Asterisks: statistical significance relative to circRNA ICB. (D) Average volumes of B16F10 melanoma treated with MHC-I / II-restricted tetravalent circRNA- T2 / g / Tl / Tl vaccine, alone or combined with anti-PD-1+anti-CTLA-4. Asterisks denote statistical significance relative to circRNA-T2 / g / Tl / Tl. (E) B16F10 melanoma tumor growth after circRNA- T2 / g / Tl / Tl vaccine treatment and lymphocyte depletion. Asterisks: statistical significance relative to circRNA-T2 / g / Tl / Tl. (F) Spider plots of individual B16F10 melanoma tumor growth curves and CR rates. (G) Kaplan-Meier survival curves of the as-treated B16F10 melanoma-bearing mice. Asterisks: statistical significance relative to circRNA+ICB. (D-G) circRNA, 10 pg; antibodies, 100 pg per ICB antibody, 200 pg anti-CD8, anti-CD4 or anti-NKl. l. (H) Average (left) and individual (right) tumor growth curves of Braf600ESMI melanoma treated with circRNA-T2 / g / Tl / Tl combined with anti-PD- 1+anti-CTLA-4, as well as controls. Asterisks: statistical significance relative to circRNA-T2 / g / Tl / Tl. (I) Kaplan-Meier survival curves of Braf60® SMI melanoma-bearing mice treated as above. Asterisks: statistical significance relative to circRNA+ICB. (H,I) circRNA, 30 pg; ICB antibodies, 200 pg each. Vaccines: loaded in SM-102 LNPs and subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. (n= 6-8). *P< 0.05, **P< 0.01, ***P< 0.001, ****P<0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.

[0027] Figure 9 shows 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 lipocircRNA. (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 hours). 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 hour, 3 hours, or 6 hours. Blue: nuclei stained with Hoechst33342. Green: endolysosomePCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 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 < o.OOOl, by t-test of the AUC. (H) The signal ratio of Cy5-circRNA outside / inside (OA) the endolysosome.

[0028] Figure 10 shows 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 hours. (D) Radiance efficiency of major organs and inguinal lymph nodes after s.c. injection of IR800-circRNA at tail base for 24 hours. He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; and LN: inguinal lymph node. (E) The frequency of circRNA eDCs and macrophages in inguinal lymph node tissues 24 hours after s.c. injection at tail base.

[0029] Figure 11 shows 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 day 21 - day 70 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 upregulated PD-1 expression on SIINFEKL-specific CD8+T cells on day21.(D) circRNA-SIINFEKL elicited SIINFEKL specific T cell memory (day 70). Tern: effector memory T cell; Tcm: central memory T cell. (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 x 105 EG7.OVA tumor challenge 70 days post-vaccination. Vaccine delivery by liposome, s.c. injected at tail base, 5 pg RNA, 2 nmole CpG, 20 pg OVA. *: relative to circRNA. (H) Mouse body weights after tumor 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 oneway ANOVA with Bonferroni post-test unless denoted otherwise.

[0030] Figure 12 shows 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 3pg and 10 pg doses, respectively. (B) 120-day kinetics of the PBMC SIINFEKL-specific CD8+T cell percentages in the above immunized mice, indicating that circRNA-SIINFEKL elicited overall strongerPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 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 (Tern+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.

[0031] Figure 13 shows monovalent small circRNA vs modified mRNA or large circRNA vaccines for robust tumor immunotherapy. (A) For immunotherapy studies in mouse models of subcutaneous EG7.OVA (B-D), and TC-1 (E), tumors were inoculated into the right flank of C57B1 / 6 mice. Vaccine: 5 pg RNA or 5 g CpG+10 pg protein or peptide antigens, s.c. injected in liposome at mouse tail base; antibodies: 200 pg, i.p. (B-D) EG7.OVA tumor growth (B) and Kaplan-Meier survival curves (C) of EG7.OVA tumor-bearing mice treated with circRNA-SIINFEKL, 5moUmRNA- OVA, and CpG- adjuvanted OVA, respectively. aCD8, aCD4, and aNKl.l were injected intraperitoneally (i.p..) for lymphocyte depletion. (D) Body weights of EG7.OVA tumor-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 aPD-1 (i.p..) alone or combined with circRNA-SIINFEKL. RNA: 30 pg, s.c. injection at tail base. CR: complete regression rate. (F) TC-1 tumor volumes after lymphocyte depletion using aCD8, aCD4 or aNKl.l. 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).

[0032] Figure 14 shows validation of small circRNA synthesis. (A) Schematic illustration of small circRNA synthesis. (B) HPLC and gel electrophoresis verified the purity of circRNA, which showed a different retention time than that of linear RNA. The corresponding fractions of HPLC samples were collected and subject to agarose gel electrophoresis. (C) Agarose gel electrophoresis validated the synthesis of small circRNA. Even though ligation-based circRNA synthesis could result in multimeric circRNA, such byproducts were not detectable under this experiment condition. This is likely due to the relatively low concentration and the favored intramolecular ligation relative to intermolecular ligation, the latter forming multimeric circRNA.

[0033] Figure 15 shows In vitro antigen presentation by DCs treated with small circRNA vaccine. (A) The secondary structures (predicted by NUPACK) of IRES LINE1 and Rbm3 (left) and the corresponding circRNA-SIINFEKL (right). Blue boxes indicate IRES domains. (B) Flow cytometric analysis of SIINFEKL / H- 2Kb complex level on DC2.4 cells. DC2.4 cells were transfected with various SIINFEKL or OVA vaccines for 24 hours, and were then stained with an APC -conjugated H- 2Kb / SIINFEKL antibody followed by flow cytometric analysis. *p< 0.05, **p< 0.01, ***p<0.001, by one-way ANOVA with Bonferroni post-test.

[0034] Figure 16 shows the high stability of small circRNA relative to long modified mRNA and large circRNA. (A) confocal microscopy images (green: Broccoli-DFHBI-IT; blue: nuclei) of DC2.4 cellsPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 treated with linear or circular Broccoli for 1-168 h, prior to adding DFHBI-1T before analysis. (B) Representative agarose gel electrophoresis images of small circRNA-SIINFEKL, liRNA-SIINFEKL, and 5moU-modified CleanCap® mRNA-OVA after storage in PBS for the indicated number of days.

[0035] Figure 17 shows the validation of circRNA-RBD synthesis. (A) HPLC chromatogram of circRNA RBD product after column purification. (B) an agarose gel electrophoresis image showing the successful synthesis of circRNA-RBD (marked by arrow). circRNA-RBD product was treated with RNase R and column purified before gel electrophoresis. The >3k-nt band is presumably dimeric circRNA. (C) Sanger sequencing of circRNA-RBD Cdna (SEQ ID NO: 29) indicates the precise circularization circRNA-RBD.

[0036] Figure 18 shows the serum concentrations of chemokines 12 hours after administration of 5 pg circRNA-SIINFEKL and 5moU-mRNA-OVA, each of which was formulated in liposome, SM-102 LNPs, MC3 LNPs, and KC2 LNPs, respectively (n = 5). Ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Bonferroni post-test.

[0037] Figure 19 shows LNPs promoted the intracellular delivery of circRNA in DCs. (A) Confocal microscopy images showing efficient LNP-circRNA-Cy delivery to DC2.4 cells (100 nM circRNA). Cy5 signal from circRNA not colocalized with Ly sotracker indicates circRNA endosome escape. Blue: nuclei stained with Hoechst33342. Green: endolysosome stained with LysoTracker Green DND-26. Red: Cy5- circRNA. (B) Flow cytometric analysis (MFI) of DC2.4 cells incubated with free Cy5- circRNA or LNP-Cy5- circRNA showed that LNPs enhance circRNA delivery to DCs. LNPs were prepared using SM-102 as the ionizable lipid. ****p < 0.0001, by t-test of the AUC.

[0038] Figure 20 shows circRNA elevated the expression of DC co-stimulatory factors and reduced PKR activation. DC2.4 cells were treated with PBS, CpG+OVA, 5moU-mRNA-OVA, and circRNASIINFEKL (24 hours), respectively, followed by flow cytometric analysis. RNA was transfected with lipofectamine 3000.

[0039] Figure 21 shows circRNA-OVA synthesis and PKR activation by 5moU-mRNA-OVA. (A-B) An agarose gel electrophoresis image (A) and their Sanger sequencing results of the cDNA of circRNA- OVA (B) showing its successful synthesis. (C) Western blot analysis of PKR and pPKR, and gel intensity ratio of pPKR over [3-actin in DC2.4 cells LNP-transfected with circRNA-SIINFEKL or 5moU-mRNA-OVA (100 nM, 24 hours). *p < 0.05, by paired t-test. (D) Representative complete gel images.

[0040] Figure 22 shows flow cytometric results of SIINFEKL / H-2Kb complex levels on vaccine treated DC2.4 cells for benchmarking small circRNA-SIINFEKL vaccine against multiple modified mRNA-SIINFEKL and modified mRNA-OVA vaccines. circRNA-SIINFEKL, 5moU mRNASIINFEKL, W-mRNA-SIINFEKL, and 5moU-mRNA-OVA were compared and all formulated in SM-102 LNPs, for in vitro antigen presentation at the corresponding escalating concentrations. All these mRNAs have 573' UTRs, 3' ADO, and CleanCap®. DC2.4 cells were transfected with vaccinesPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 for 1-3 days, followed by flow cytometric analysis of H-2Kb / SIINFEKL levels. Ns: non-significant, *p< 0.05, **p< 0.01, ***p< 0.001, ****p < 0.0001, by one-way ANOVA with Bonferroni post-test.|0041] Figure 23 shows mass dose and molar dose based comparison of small circRNA vaccines vs three modified mRNA vaccines for PBMC SIINFEKL-specific T cells elicited in mice. (A) Study design. C57BL / 6 mice (n = 4-8; 6-8 weeks) were immunized with circRNA SIINFEKL and three modified mRNAs at escalating doses, respectively. (B) Tetramer staining results on day 21 indicates that circRNA-SIINFEKL elicited higher fractions of PBMC SIINFEKL-specific T cells than modified mRNAs at the corresponding doses. (C) Molar dose-based comparison of small circRNA vaccines vs three modified mRNA vaccines for PBMC SIINFEKL-specific T cells elicited in mice. Data were converted from Figure B). Asterisks: mass statistical significance of the T cell fraction AUC relative to that for circRNA.

[0042] Figure 24 shows that the mouse body weight changes after administration with small circRNA- SIINFEKL as well as modified mRNA-SIINFEKL and modified mRNA-OVA vaccines at escalating doses. (A) Study design, All RNAs, including circRNA-SIINFEKL, 5moU-mRNA-SIINFEKL, W- mRNA-SIINFEKL, and 5moU-mRNA-OVA, all separately formulated in SM-102 LNPs. All these mRNAs have 573' UTRs, 3' A120, and CleanCap®. C57BI / c mice (n=4-5; 6-8 weeks) were immunized with SM-102 LNPs of circRNA-SIINFEKL and three benchmarking modified mRNA at escalating doses, respectively. (B) circRNA-SIINFEKL caused less mouse body weight loss than benchmark modified mRNAs at 100 pg dose, as exemplified 1-2 days after the 2nd dose. Data represent mean ± s.e.m. (n = 3-5). c, circRNA-SIINFEKL caused less mouse body weight loss and more rapid recovery than benchmark modified mRNAs except for the doses of 100 pg, as exemplified 0.5-3 days after the 3rd dose. Data represent mean ± s.e.m. (n = 5, except for 100 pg-dose groups in which n = 3). Asterisks: statistical significance relative to circRNA. Ns: nonsignificant, *p< 0.05, **p< 0.01, by one-way ANOVA with Bonferroni post-test.

[0043] Figure 25 shows the representative flow cytometry plots for the analysis of PBMC SIINFEKL±CD8+T cells on day 35 after three doses of circRNA-SIINFEKL and benchmark modified mRNA at escalating doses (Fig. 5), as stained using PE-labeled H-2Kb-SIINFEKL tetramer.

[0044] Figure 26 shows the representative flow cytometry plots for the analysis of PBMC CD8+T cell memory elicited by RNA vaccines at escalating doses (day 180) in mice. The CD8+T cell fractions of effector memory T cells (Tem, CD62L-CD44+), central memory T cells (Tcm, CD62LhlghCD44+), and naive T cells (CD62L+CD44-) were quantified.

[0045] Figure 27 shows the kinetics of PD-1 expression levels on total PBMC CD8+T cells in mice immunized with circRNA-SIINFEKL and modified mRNA at escalating doses (Fig. 5). The results indicate that dose-dependent PD-1 upregulation shortly after immunization. The PD-1 expression levels were significantly reduced starting from at most 21 days after the last immunization (day 49).PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0046] Figure 28 shows the Luminex results of the serum cytokines and chemokines from mice immunized with circRNA-SIINFEKL and benchmark RNA vaccines at a dose of 10 pg (Fig. 5). Serum concentrations of chemokines (Eotaxin, RANTES, MCP-1. MIP-la. MIP-1[3, and KC) and cytokines (IL-3, IL-1 [3, TNF- a , IL-9, IL-6, IFN-I3, IL-2, IL-13, IL-5, IL-la, IL-10, IL-12p40, G-CSF, and GM- CSF) were measured 6 hours after the third dose of escalating doses of circRNA-SIINFEKL, mRNA- OVA, 5moU-mRNA-OVA, and circRNA-OVA formulated in SM-102 LNPs (n = 4). Mice and ministered with PBS or blank LNPs were used as controls. These data were summarized in Fig. 5h. Data: mean ± standard error of the mean (s.e.m.); Asterisks indicate statistically significant differences between circRNA and other groups. **p < 0.01, ***p < 0.001, by one-way ANOVA with Bonferroni post-test. *p< 0.05; **p< 0.01; ***p< 0.001; *** *p < 0.0001, one-way ANOVA with Bonferroni posttest.

[0047] Figure 29 shows the representative flow cytometry plots showing that circRNA-SIINFEKL (3 x 5 pg on days 0, 14, 28) elicited CD8+T cell memory (day35) in aged mice (1 year) (n = 5).

[0048] Figure 30 shows small circRNA-Adpgk elicited potent neoantigen Adpgk-specific T cell responses. C57BL / 6 mice (n = 5) were vaccinated on day 0, day 14, and day 28 and PBMCs were analyzed [vaccines were s.c. administered at mouse tail base; circRNA-Adpgk, liRNA-Adpgk (5 pg), and (CpG+Adpgk) (5 pg CpG+10 pg Adpgk)]. (A) Tetramer staining results showed the fractions of Adpgk+CD8+T cells among all live PBMC CD8+T cells, indicating that circRNA-Adpgk neoantigen vaccine elicited robust and dose-dependent T cell responses. (B) MFI of PD-1 expression on Adpgk+CD8+T cells than that on total CD8+T cells in peripheral blood on day 21 (two-tailed paired t test). (C) Representative flow cytometry dot plots of effector emory T cells (Tern, CD62L-CD44+), central memory T cells (Tern, CD62LhlghCD44+), and naive T cells (CD62L+CD44-) in peripheral blood on day 70, showing circRNA-induced T cell memory. (D) The effector memory T cells on Adpgk+CD8+T cells than that on total CD8+T cells in peripheral blood on day 70. Data represent mean ± s.e.m. (n = 5). Asterisks in (A) indicate statistically significant differences between circRNA and other groups. **p < 0.01, ***p < 0.001, by one-way ANOVA with Bonferroni post-test.

[0049] Figure 31 shows small circRNA-E7 elicited potent HPV E7-specific T cell responses. C57BL / 6 mice (n = 5) were vaccinated on day 0, day 14, and day 28, followed by PBMC CD8+T cell analysis [vaccines were s.c. administered in liposome at mouse tail base; circRNA-E7 (5 pg), HRNA-E7 (5 pg), and (CpG+E743 -62) (5 CpG+10 pg E743-62)]. (A-B) Representative flow cytometry plots (s) and frequency (B) of PBMC E7+CD8+T cells on day 21, as stained using PE-labeled H-2Db-RAHYNIVTF tetramer. *p < 0.05, **p < 0.01, by one-way ANOVA with Bonferroni post-test. (C) Representative flow cytometry results of PBMC Tem, Tcm, and naive T cells on day 70. (D) Elevated PD-1 expression on E7+CD8+T cells relative to that on total PBMC CD8+T cells on day 21 (**p < 0.01, two-tailed paired t test).PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0050] Figure 32 shows representative gating tree used in flow cytometric analysis of tetramer staining of PBMC CD8+T cells.

[0051] Figure 33 shows representative gating tree used in flow cytometric analysis of (A) CD8+T cells and Adpgk-specific CD8+T cells, (B) CD4+T cells and Treg cells, and (B) NK cells in the TME of MC38 tumors.|0052] Figure 34 shows representative gating tree used in flow cytometric analysis of (A) DC cells, (B) macrophages, (C) and (C) MDSC cells in the TME of MC38 tumors.10053] Figure 35 shows representative gating tree used in flow cytometric analysis of circRNA± CD8+eDCs in mouse draining lymph nodes.10054] Figure 36 shows representative gating tree used in flow cytometric analysis of circRNA± CD4+Tcell-priming CD4+eDCs in draining lymph nodes|0055] Figure 37 shows representative gating tree used in flow cytometric analysis of circRNA+migratory CD103+eDCs in draining lymph nodes.|0056] Figure 38 shows representative gating tree used in flow cytometric analysis of circRNA± macrophage in draining lymph nodes.|0057] Figure 39 shows the pulmonary delivery of small circRNA vaccines for flu prophylaxis. (A) Ex vivo fluorescence images of Balb / c mice 24 hours, 48 hours and 72 hours after intratracheal (i.t.) administration of free IR800-circRNA or IR800-circRNA LNPs (0.5 nmole circRNA). (B) Radiant efficiency of major organs from the above mice 48 hours after i.t. administration of circRNA. (C) Lung radiant efficiency 24 hours, 48 hours and 72 hours after i.t. circRNA administration. (D) The frequency of circRNA+pulmonary eDCs and macrophages 24 hours after i.t. circRNA administration. (E) Tetramer staining showed that, by pulmonary delivery using LNPs, circRNA-SIINFEKL elicited superior PBMC SIINFEKL-specific CD8+T cell responses than 5moU-mRNA-OVA or CpG- adjuvanted OVA in C57BL / 6 mice (n = 5) (day 21) ( / -test). (F) Study design of T cell response in Balb / c mice immunized with circRNA-M2e vaccines. RNA: 5 pg; CpG: 5 pg; M2e peptide: 10 pg). Mice were i.t. immunized three times at 2-week intervals. Four weeks after the final immunization, mice were challenged with a lethal dose of PR8 H1N1 influenza virus. (G) Tetramer staining showed robust PBMC M2e-specific CD4+T cell response elicited by circRNA in mice (n = 5) (day 35). (H) Intracellular IFN- y and TNF-a staining in CD4+T cells from as-immunized mice (day 35). (I) Tetramer staining showed that circRNA elicited significant PBMC M2e-specific CD4+T cell response in aged mice (1 year). Male mice showed more robust circRNA-elicited T cell responses than female mice (n = 4 per sex per group) (day 35) ( / -test). (J-K) Luminex results of the BAL levels (J) and the serum levels (K) of a panel of cytokines and chemokines, as measured 12 hours (BAL and serum) or 24 hours (BAL) after the third dose. Heatmaps in (J) shows fold changes relative to that in PBS-treated mice. (1) Survival curves of the above mice immunized with circRNA-M2e vaccines or controls after lethal challenge with 4 x LD50 ofHINl influenza PR8 virus. (M) Body weight changes ofPR8 influenza virus-challenged mice. DataPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 represent mean ± s.e.m. (n = 5-10). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.|0058] Figure 40 shows the design and characterization of circRNA-M2e and its LNPs. (A) Predicted secondary structure of circRNA-M2e. (B) circRNA-M2e was successfully synthesized via ligation of oligo RNA precursors, as verified by agarose gel electrophoresis. (C,D) Size characterization of circRNA-M2e LNPs by DLS (C) and cryo-EM (D).

[0059] Figure 41 shows a comparison of the structures of circRNA-SIINFEKL, circRNA-OVA, and three modified mRNA encoding OVA or SIINFEKL.DEFINITIONS

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0061] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0062] For the recitation of nmneric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0063] The term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present disclosure) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the mouth (oral), nose (nasal), lungs (e.g., inhalant, intratracheal), skin (transdermal), oral mucosa (buccal), by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0064] The term “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acidPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.|0065] The terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). "Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.

[0066] The terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention.

[0067] The term "pharmaceutical composition" as used herein means a product comprising an active, compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a person. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation.

[0068] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as antibiotics, antifungals, antimicrobials, can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (2006); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 11th Ed., The McGraw-Hill Companies.10069] The term “subject” as used herein refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, mice, rats, etc.). As used herein, the term “patient” typically refers to a human subject that is being treated for a disease or condition or prophy tactically.

[0070] The term “therapeutically effective amount” refers to the amount of a composition of the present invention that is capable of achieving a therapeutic effect in a subject in need thereof. For example, aPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 therapeutically effective amount of a composition of the present invention can be the amount that is capable of preventing or relieving one or more symptoms associated with a disease or disorder.|0071] An "antigen" refers to a molecule, such as a polypeptide, containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to generate a humoral and / or cellular antigen-specific response. The term is used interchangeably with the term "immunogen." Similarly, an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in nucleic acid immunization applications, is also included in the definition of antigen herein. For purposes of the present disclosure, immunogens can be derived from any organism for which an immune response is desired, including immunogens derived from viruses, bacteria, fungi, parasites and the like.

[0072] The term “vaccine,” as used herein, refers to any pharmaceutical composition containing at least one antigenic peptide or other immunogen or at least one nucleic acid encoding at least one antigenic peptide or other immunogen, which can be used to prevent or treat a disease or condition in a subject.

[0073] By "fragment" is intended a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and / or an internal deletion of the native polypeptide.

[0074] By "immunogenic fragment" is meant a fragment of the reference polypeptide that includes one or more epitopes and thus elicits one or more immunological responses. An “immunogenic fragment” of a particular protein will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full- length molecule, that define an epitope, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains the ability to elicit an immunological response as defined herein.

[0075] The term "epitope" as used herein refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 500 amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, elicits an immunological response in the subject to which it is administered. Often, an epitope will bind to an antibody generated in response to such sequence. There is no critical upper limit to the length of the epitope, which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes from the molecule in question. An epitope for use in the subject disclosure is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived. For example, viral genomes are in a state of constant flux and contain several variable domains which exhibit relatively high degrees of variability between isolates. The term "epitope" encompasses sequences identical to thePCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature).|0076] An "immunological response" or "immune response" to an antigen is the development in a subject of a humoral and / or a cellular immune response to an antigen present in a composition of interest. For purposes of the present disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T- lymphocytes and / or other white blood cells.

[0077] An "immunogenic composition" is a composition that comprises an antigenic molecule, where administration of the composition to a subject results in the development in the subject of a humoral and / or a cellular immune response to the antigenic molecule of interest.

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.DETAILED DESCRIPTION|0079] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limitingCompositions10080] Provided herein are immunogenic compositions (e.g., vaccine compositions). In certain aspects, the compositions comprise a nucleic acid (e.g., circular RNA) encoding at least one antigen or immunogenic peptide fragment. In some embodiments, the composition is self-adjuvanted and configured to elicit a T-cell-mediated immune response.

[0081] Embodiments of the present disclosure provide nucleic acids encoding antigenic polypeptides. In some embodiments, the nucleic acid is a circular RNA. The present disclosure is not limited to particular constructs. Circular RNAs can be produced from linear RNAs in a number of ways. Methods for circularizing RNA are described, for example, in Obi et al., Methods. 2021 Mar 2; 196:85— 103; herein incorporated by reference in its entirety.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0082] In some embodiments, the circular RNA comprises at least one chemical modification or chemically modified base or nucleoside. The chemical modifications may comprise any modification which is not naturally present in the RNA or any naturally-occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single mRNA may include both naturally-occurring and non-naturally -occurring modifications. Chemical modifications may be located in any portion of the mRNA molecule and the mRNA molecule may contain any percentage of modified nucleosides (1-100%). In some embodiments, every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine) In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl- pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (mods.ma.albany.edu / home). Modified residues may be included in transcription reactions to generate modified RNA.

[0083] In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary nucleosides having a modified uracil include pseudouridine fP), pyridin-4-one ribonucleoside, 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5 -iodo-uridine or 5 -bromo-uridine), 3-methyl-uridine, 5-methyl-uridine, 5 -methoxy -uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5 -carboxy hydroxymethyl-uridine, 5 -carboxy hydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-inctlioxycarbonylmclhyl-2-thio-uridinc. 5- aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5- methy laminomethyl-2-seleno-uridine, 5 -carbamoy Imethy 1-uridine, 5 -carboxymethylaminomethyl- uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5 -propyny 1-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine, 5-methyl-2-thio-uridine, l-methyl-4- thio-pseudouridine, 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio- 1-methylpseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1-methylpseudouridine, 3-(3-amino-3- carboxypropyl)uridine, l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine , 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, a-thio-uridine, 2’-O- methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl-pseudouridine, 2-thio-2’-O-methyl-uridine, 5- methoxycarbonyhnethyl-2’-O-methyl-uridine, 5 -carbamoy lmethyl-2’-O-methy 1-uridine, 5- carboxymethylaminomethyl-2’-O-methyl-uridine, 3,2’-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2’-O-methyl-uridine, 1 -thio-uridine, deoxythymidine, 2’-F-ara-uridine, 2’- F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601In some embodiments, the modified uridine residue is selected from the group consisting of: pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5- methyluridine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5 -azauridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza- uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.

[0084] In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5- methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza-pseudoisocytidine, 1- methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-mcllioxy-5-mclhyl-cylidinc. 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O-methyl- cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O-dimethyl-cytidine, 5- formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1 -thio-cytidine, 2’-F-aracytidine, 2’-F- cytidine, and 2’-OH-aracytidine. In some embodiments, the at least one chemical modification comprises 5 -methylcytosine (base) or 5 -methylcytidine (nucleoside). In some embodiments, the at least one chemical modification comprises 5 -methylcytosine.

[0085] In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl- adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6- isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl-adenosine, N6,2’-O-dimethyl-adenosine, N6,N6,2’-O-trimethyl-adenosine, l,2’-O-dimethyl-adenosine, 2’-O-ribosyladenosine (phosphate), 2- amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2 ’ -OH-ara-adenosine, and N 6-( 19-amino-pentaoxanonadecyl)-adenosine.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0086] In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7- aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl- guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8- oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio- guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2- dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2,7-dimethyl-2’-O-methyl- guanosine, 2’-O-methyl-inosine, l,2’-O-dimethyl-inosine, and 2’-O-ribosylguanosine (phosphate).Lipid nanoparticles

[0087] In some embodiments, the nucleic acid compositions are provided in or formulated with lipid nanoparticles. The present disclosure is not limited to particular lipids for use in lipid nanoparticles.

[0088] In some embodiments, lipid nanoparticles include one or more (e.g., 2, 3, 4, or more) different lipids (e.g., of the same or different classes). For example, in some embodiments, the lipid nanoparticle comprises an ionizable (e.g., cationic) lipid, a structural or helper phospholipid, cholesterol, and a PEG lipid.|0089] In some embodiments, the cationic lipid is 5 -(dimethylamino) -pentanoic acid, (6Z)-l,2-di- (4Z)-4-decen-l-yl-6-dodecen-l-yl ester (CL1), (CKK-E12), (TCL053), bis((Z)-12-(((non-2-yn-l- yloxy)carbonyl)oxy)octadec-9-en-l-yl) 3,3’-((2-(diethylamino)ethyl)azanediyl)dipropionate (RCB4- 8), 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate (LP-01), [(4-Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate) (ALC-0315), O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (DLIN-MC3-DMA), [(Z)-non-2-enyl] 8-[2-(dimethylamino)ethylsulfanylcarbonyl-[8- [(Z)-non-2-enoxy]-8-oxooctyl]amino]octanoate (ATX-002), Di((Z)-non-2-en-l-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), l,l'-((2-(4-(2-((2-(Bis(2- hydroxydodecyl)amino)ethyl)(2 -hydroxy dodecyl) amino)ethyl)piperazin- 1 - yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), or analogs or combinations thereof.

[0090] Such lipids are commercially available (e.g., from Broad Pharma, San Diego, CA or Sigma- Aldrich, St. Louis, MO). Additional lipids are described, for example, in Tenchov et al., ACS Nano 2021, 15, 11, 16982-17015; herein incorporated by reference in its entirety.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0091] In certain embodiments, lipid nanoparticles further comprise a pulmonary surfactant. Experiments described herein determined that the use of a pulmonary surfactant (e.g., coating the lipid layer or mixed with the lipid) improved the performance of vaccine compositions.|0092] The present disclosure is not limited to particular pulmonary surfactants. Examples of additional pulmonary surfactants include but are not limited to, poractant alfa, colfosceril palmitate, a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents; pumactant, a mixture of DPPC and PG; KL-4, composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B; venticute, composed of DPPC, PG, palmitic acid and recombinant SP-C; lucinactant, composed of DPPC, POPG, and palmitic acid; beractant; alveofact, extracted from cow lung lavage fluid; survanta extracted from minced cow lung with additional DPPC, palmitic acid, and tripalmitin; beraksurf, extracted from minced calf lung with additional DPPC, palmitic acid, and tripalmitin; calfactant, extracted from calf lung lavage fluid; and ovinactant, extracted from material derived from minced sheep lung.

[0093] In some embodiments, the average LNP diameter of the LNP composition may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 120 mm.|0094] Nucleic acid (e.g., circular RNA) are encapsulated or otherwise combined with lipid nanoparticles using any suitable method. The efficiency of encapsulation of a cargo, describes the amount of nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%; e.g., greater than 90%; greater than 95%). For the lipid nanoparticles described herein, the encapsulation efficiency of a nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.Antigenic DolvDepiides10095] The present disclosure is not limited to a particular antigenic polypeptide.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0096] In some embodiments, the antigenic polypeptide is a SARS-CoV-2 structural protein or fragment thereof (e g., spike protein (S), envelope protein (E), nucleocapsid protein (N), or membrane protein (M)).|0097] Non-limiting examples of viral pathogens that affect humans and / or nonhuman vertebrates from which immunogens can be derived, include retroviruses, RNA viruses and DNA viruses. The group of retroviruses includes both simple retroviruses and complex retroviruses. The simple retroviruses include the subgroups of B-type retroviruses, C-type retroviruses and D-type retroviruses. An example of a B- type retrovirus is mouse mammary tumor virus (MMTV). The C-type retroviruses include subgroups C-type group A (including Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-type group B (including murine leukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The complex retroviruses include the subgroups of lentiviruses, T-cell leukemia viruses and the foamy viruses. Lentiviruses include HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II, simian Tcell leukemia virus (STLV), and bovine leukemia virus (BLV). The foamy viruses include human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).

[0098] Examples of other RNA viruses from which immunogens can be derived include, but are not limited to, the following: members of the family Reoviridae, including the genus Orthoreovirus (multiple serotypes of both mammalian and avian retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, African horse sickness virus, and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus); the family Picomaviridae, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcine enteroviruses , the genus Cardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses including at least 113 subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); the family Calciviridae, including Vesicular exanthema of swine virus, San Miguel sea lion virus, Feline picornavirus and Norwalk virus; the family Togaviridae, including the genus Alphavirus (Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus, Chikungunya virus, O'NyongSI 6- Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, OmskPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Buny virus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza ty pe B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Buny virus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (CrimeanS 16- Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza ty pe B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); the family Rhabdoviridae, including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses (Marburg virus and Ebola virus); the family Arenaviridae, including Lymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, and Lassa virus; the family Coronoaviridae, including the SARS virus, Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus, Human enteric corona virus, and Feline infectious peritonitis (Feline coronavirus).

[0099] Illustrative DNA viruses from which immunogens can be derived include, but are not limited to: the family Poxviridae, including the genus Orthopoxvirus (Variola major, Variola minor, MonkeyPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine papular stomatitis virus); the family Iridoviridae (African swine fever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the family Herpesviridae, including the alphaherpesviruses (Herpes Simplex virus Types 1 and 2, Varicella-Zoster, Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine keratoconjunctivitis virus, infectious bovine rhinotracheitis virus, feline rhinotracheitis virus, infectious laryngotracheitis virus) the beta- herpesvirises (Human cytomegalovirus and cytomegaloviruses of swine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pig herpes virus, Lucke tumor virus); the family Adenoviridae, including the genus Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simian adenoviruses (at least 23 serotypes), infectious canine hepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many other species, the genus Aviadenovirus (Avian adenoviruses); and non-cultivatable adenoviruses; the family Papoviridae, including the genus Papillomavirus (Human papilloma viruses, bovine papilloma viruses, Shope rabbit papilloma virus, and various pathogenic papilloma viruses of other species), the genus Polyomavirus (polyomavirus, Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus, and other primate polyoma viruses such as Lymphotrophic papilloma virus); the family Parvoviridae including the genus Adeno-associated viruses, the genus Parvovirus (Feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc). Finally, DNA viruses may include viruses which do not fit into the above families such as Kuru and Creutzfeldt-Jacob disease viruses and chronic infectious neuropathic agents (CHINA virus).

[0100] Non-limiting examples of bacterial pathogens from which immunogens can be derived include both gram negative and gram positive bacteria. Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus infuenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., FusobacteriumPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii.|0101] Examples of infectious fungi from which immunogens can be derived include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Examples of infectious parasites include Plasmodium such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Other infectious organisms (i.e. protists) include Toxoplasma gondii.

[0102] In some embodiments, the antigenic polypeptide is a cancer antigen. Examples include but are not limited to HPV proteins (e.g., E7), EGF, The C-T antigens (MAGE-1, MAGE-3, BAGE, BAGE, GAGE, KK-LC-1, and NY-ESO-1), GD-3, hyaluronic acid-mediated motility (RHAMM), and carboanhydrase IX (G250 / CAIX).Vaccine Compositions

[0103] Embodiments of the disclosure provide vaccine or pharmaceutical compositions comprising the circular RNA compositions described herein.

[0104] For example, in some embodiments, provided herein is a formulation or delivery system (e.g., lipid nanoparticle) comprising a circular RNA. Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of undesired pyrogens, as well as other impurities that could be harmful to humans or animals.

[0105] The compositions described herein may be formulated for any suitable delivery method. In certain embodiments, the vaccine compositions are delivered intramuscularly or via intranasal or pulmonary administration, although other delivery methods are specifically contemplated.

[0106] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought aboutPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.|0107] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0108] Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant. If the compounds or pharmaceutical compositions comprising them are administered as aerosols, for example as nasal aerosols or by inhalation, this can be carried out, for example, using a spray, a nebulizer, a pump nebulizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler. Pharmaceutical forms for administration of the compounds as an aerosol can be prepared by processes well known to the person skilled in the art. For their preparation, for example, solutions or dispersions of the compounds in water, water / alcohol mixtures or suitable saline solutions can be employed using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others and, if appropriate, customary propellants, for example include carbon dioxide, CFCs, such as, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane; and the like. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.

[0109] In formulations intended for administration to the respiratory tract, including intranasal formulations, die compound will generally have a small particle size for example of the order of 50 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.

[0110] Alternatively the active ingredients may be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).

[0111] The compositions of the present disclosure generally may be formulated in a neutral or salt form. Pharmaceutically -acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the freePCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).

[0112] In some embodiments, pharmaceutical compositions (e.g. compositions formulated for pulmonary delivery) further comprise an emulsifier. Examples of emulsifiers include but are not limited to, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, alkyl polyoxyethylene sulfates, sodium dodecyl sulfate, dioctyl sodium sulfosuccinate, quaternary ammonium compounds, cetyltrimethyllammonium bromide, lauryldimethylbenzylammonium chloride, polyoxyethylene fatty acid derivatives of the sorbitan esters (e.g., Tween series), polyoxyethylene fatty alcohol ethers Sorbitan fatty acid esters polyoxyethylene alkyl ethers (macrogols) polyoxyethylene sorbitan fatty acid esters (including polysorbate 80), polyoxyethylene polyoxypropylene block copolymers (poloxamers), polyethylene glycol 400 monostearate, lanolin alcohols, and ethoxylated lanolin.

[0113] In some embodiments, the composition comprises polysorbate 80 at a concentration of approximately 0.1%.

[0114] The pharmaceutical preparations herein can also be housed in a syringe, a delivery device, or the like, depending upon the intended mode of delivery and use. In certain embodiments, the compositions, are in unit dosage form, meaning an amount of a composition appropriate for a single dose, in a premeasured or pre-packaged form.

[0115] The compositions herein may optionally include one or more additional agents, such as other drugs for treating infection by a pathogen. For example, compositions including antiseptics, antibiotics, antifungal agents, antiviral agents, antiparasitic agents; and additional vaccines may be added to pharmaceutical compositions of the disclosure.

[0116] Alternatively, such agents may be contained in a separate composition from the composition comprising the RNAs and co-administered concurrently, before, or after the composition comprising the RNAs.Kits and devices

[0117] Embodiments of the disclosure provide kits and devices for utilizing the compositions described herein. In certain embodiments, provided herein are delivery devices for delivering vaccine compositions comprising circular RNA, for example, to a mucus membrane of a subject. In particular, provide herein are devices for intranasal and pulmonary delivery.

[0118] Devices may be provided pre-loaded with one or more doses of a vaccine composition of the present disclosure. Alternately, devices may be provided separately from the vaccine composition (e.g., as a component of a kit for vaccination of a subject).PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0119] Examples of delivery devices for pulmonary (e.g., intranasal or directly to the lung) delivery include but are not limited to, a metered dose inhaler, a dry powder inhaler, a metered dose spray pump, a soft mist inhaler, or a nebulizer.|0120] Metered-dose spray pumps and nebulizers are the most frequently used devices for nasal vaccine delivery. Metered-dose spray pmnps can deliver a precise and constant volume of drug solution or suspension with adjustable particle sizes and geometries (Djupesland PG. Nasal drug delivery devices: characteristics and performance in a clinical perspective-a review. Drug Deliv Transl Res 2013;3:42e62) and provide the advantages of low manufacturing costs, portability, simplicity in applications, and safety. Single-dose metered-dose spray pumps are available, for example, from BD (Accuspray™ device).

[0121] Medical nebulizers like vibrating mesh, air-jet, and ultrasonic nebulizers can also be utilized for intranasal administration. OptiNose is a bi-directional nasal nebulizer that includes a flexible mouthpiece and a sealing nosepiece (Djupesland PG, Skretting A, Winderen M, Holand T. Breath actuated device improves delivery to target sites beyond the nasal valve. Laryngoscope 2006; 116:466e72). The device isolates the nose from the mouth and lungs through automatic closure of the soft palate during oral exhalation and helps the vaccine to deposit in high / deep sites in the nasal passages.

[0122] For pulmonary delivery directly to the lung, exemplary devices include pressurized metered- dose inhalers (pMDIs), dry powder inhalers (DPIs), and nebulizers (Rau JL. Design principles of liquid nebulization devices currently in use. Respir Care 2002;47:1257e78).

[0123] Pressurized metered dose inhalers (pMDIs) use pressurized propellant to deliver a fixed dose of aerosol through a nebulizer nozzle. The canister, metering valve, actuator, and dose counter are the major components of pMDIs. The canister contains a mixture of propellant, drugs, and excipients.

[0124] Soft mist inhalers (SMIs) are propellent-free metered-dose inhalers activated by breathing (Tamura G. Comparison of the aerosol velocity of Respimat soft mist inhaler and seven pressurized metered dose inhalers. Allergol Int 2015;64:390e2; Brand P, Hederer B, Austen G, Dewberry H, Meyer T. Higher lung deposition with Respimat Soft MistTM Inhaler than HFA-MDI in COPD patients with poor technique. Int J Chron Obstruct Pulmon Dis 2008;3:763e70). These deliver aqueous formulations through micro holes, forming soft fog for deep lung deposition. The aerosol generated with SMIs generally has a high fine particle fraction (65%e80%), a low velocity, and a more sustained duration than that of pMDIs (Dalby R, Spallek M, Voshaar T. A review of the development of Respimat soft MistTM inhaler. Int J Pharm 2004;283: le9. 277. Hochrainer D, Ho "lz H, Kreher C, Scaffidi L, Spallek M, Wachtel H. Comparison of the aerosol velocity and spray duration of Respimat Soft MistTM Inhaler and pressurized metered dose inhalers. J Aerosol Med 2005; 18:273e82).|0125] Unlike pMDIs, DPIs are breath-actuated devices, that can be used for the respiratory administration of either single or multidose dry powders. Examples include, for example, BD Solovent,PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601Aktiv-Diy, Aerolizer, Diskus, Flexhaler, Handihaler, Neohaler, Pressair, Rotahaler, Turbohaler, and Twisthaler. Other dry powder inhalers which can be used are described in US patent 6,766,799, US patent 7,278,425 and US patent 8,496,002, each of which are hereby incorporated by reference in their entireties.

[0126] Nebulizers like vibrating mesh, surface acoustic wave (SAW), air-jet, and ultrasonic nebulizers generate large volumes of inhalable aerosols, where the loaded formulations do not require propellant as in pMDIs or drying processes as in DPIs. Air-jet nebulizers employ high-velocity gas to pass through a venturi nozzle and convert liquids into a mist. The liquid is drawn from the nebulizer reservoir up to a feed tube and emerges as fine filaments that collapse into aerosol droplets. Unlike jet nebulizers, ultrasonic nebulizers exploit a piezoelectric crystal vibrating at high frequency to produce a fountain at the liquid-air interface, which forms droplets that can be aerosolized and exit the nebulizer through ventilation (Flament MP, Leterme P, Gayot A. Study of the technological parameters of ultrasonic nebulization. Drug Dev Ind Pharm 2001;27:643e9).

[0127] Similar to ultrasonic nebulizers, mesh nebulizers are more portable and efficient than jet nebulizers, but they are less effective with viscous solutions and suspensions due to their ability to clog the pores. Vibrating mesh nebulizers are commonly used to deliver respiratory -delivered vaccines.

[0128] Any of the compositions described herein may be included in a kit. For example, vaccine compositions, delivery devices, delivery devices loaded with vaccine compositions, and the like.

[0129] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. The kit may comprise one or more containers holding the vaccine compositions, and other agents. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0130] The kit can further comprise a container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or the pulmonary delivery devices described herein. The delivery device may be pre-filled with the compositions.Methods

[0131] In some embodiments, provide herein are methods of inducing an immune response (e.g., vaccination methods). The present disclosure provides methods for treating or preventing a disease or disorder in a subject in need thereof by administering one or more compositions described herein to the subject.|0132] In some embodiments, compositions are administered intramuscularly, orally, intranasally, via pulmonary administration, or a combination thereof. In the present disclosure, the terms “intranasalPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 delivery” “intratracheal delivery” and “pulmonary delivery” are used interchangeably to refer to delivery of compositions to the respiratory tract of a subject.|0133] In some embodiments, one or more booster or subsequent doses of a circular RNA vaccine composition are administered to a subject. The present disclosure is not limited to a particular interval between doses. In some embodiments, doses are administered one or more months or years apart.

[0134] In some embodiments, the circular RNA compositions described herein find use in the treatment of infectious diseases or cancers. In some embodiments, the compositions and administered in combination with an additional therapy for cancer (e.g., chemotherapy, cancer vaccine, or cancer immunotherapy). In some embodiments, the circular RNA encodes a tumor antigen or other antigenic polypeptide. In some embodiments, the circular RNA does not encode an antigenic polypeptide.

[0135] Compositions comprising circular RNAs or mRNAs can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the circular RNAs or mRNAs can be provided in the same or in a different composition. Thus, RNAs and one or more other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising an RNA and a dose of a pharmaceutical composition comprising at least one other agent, such as a drug for treating an infection, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, an RNA and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (e.g., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.EXAMPLES

[0136] The present disclosure has multiple aspects, illustrated by the non-limiting examples as described herein.

[0137] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.Example 1 : Materials and Methods

[0138] circRNA synthesis. All oligonucleotides (sequences in Table 1) were purchased from Integrated DNA Technologies. Small circRNAs were synthesized using T4 RNA ligase 1 or 2 (NewPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601England Biolabs) to ligate linear 51 -phosphorylated RNA precursors (5 pM each) in the presence of 30-mer DNA splints (15 M each). The products were treated with DNase I and exonuclease T (New England Biolabs) or RNase R (Abeam) to remove DNA and linear or lariat RNA. circRNA-RBD and circRNA-OVA (sequence in Table 1) were synthesized in two steps as reported previously44: (1) IVT using AmpliScribe T7 High Yield Transcription Kit (Biosearch Technologies, AS3107) and (2) group I intron-mediated autocatalytic circularization. In brief, Ipg DNA template was used per 20 plIVT reaction (37 °C, 2 hours). Then, DNA templates were degraded with DNase I (37 °C, 15 minutes). The remaining RNA was column purified. Next, GTP (2 mM) was added to the resulting RNA to allow autocatalytic RNA cyclization (55 °C, 15 minutes). circRNA was column purified using an RNA Clean & Concentrator kit (Zymo Research) or HPLC to remove DNA or RNA fragments, nucleotides and enzymes. circRNA products were verified using gel electrophoresis, HPLC and Sanger sequencing of complementary DNA reverse transcribed from circRNA using primers flanking the circularization sites. Primers for circRNA-SIINFEKL reverse transcription: F, 5’-TTCGTTTGCTTTTTGTAGTATAATT- 3' (SEQ ID NO: 30);R,5'-AGTTTTTCAAAGTTGATTATACTCTCC-3' (SEQ ID NO:1). Primers for circRNA-RBD reverse transcription: F, 5 -AAGCGGCTACATCCCAGAAG-3' (SEQ ID NO:2); R, 5’-GGCGCACAAAGGTACCGTGA-3’ (SEQ ID NO:3).

[0139] Table 1. RNA SequencesPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0140] In vitro circRNA stability. To study thermostability, small circRNA, liRNA and 5moU- mRNA-OVA (Trilink BioTechnologies, LLC) were stored in PBS at 4 °C, 25 °C and -20 °C for a series of times. Remaining RNAs were resolved by agarose gel electrophoresis and imaged using a ChemiDoc imaging system (Bio-Rad Laboratories). Intact RNA was quantified using ImageJ software (NIH). To study the in vitro biostability of large circRNA and small circRNA, RNA was incubated in freshly prepared diluted FBS (37 °C, 30 minutes) and was then briefly denatured in RNA loading buffer before gel electrophoresis.

[0141] RNA LNP preparation. An ethanol phase containing all lipids and an aqueous phase of RNA were mixed to synthesize LNPs. The ethanol phase contained ionizable lipids, DSPC, DMG-PEG2000 and cholesterol at a molar ratio of 50:10:1.5:38.5. The aqueous phase contained circRNA or mRNA in 10 mM citrate buffer. The two phases were mixed at a flow rate of 1.8 ml min'1and 0.6 ml min'1(3:1), respectively, using Pump33DS syringe pumps. LNPs were dialysed in 1 x PBS in a microdialysis cassette (20,000 molecular weight cut-off (MWCO), Thermo Fisher Scientific) at 4 °C for 12 hours. LNP diameters and polydispersity index were measured on Zetasizer Nano (Malvern Instruments). RNA concentration and encapsulation efficiency in LNPs were measured using a modified Quant-iT RiboGreen (ThermoFisher) assay, and the circRNA copy numbers per LNP were estimated as before45. For cryo-EM, LNPs were concentrated after dialysis to -90 mg ml'1of lipid using Amicon ultracentrifugation filters before cryo-EM observation at the Molecular Electron Microscopy Core of University of Virginia.|0142] circRNA LNP stability. Circular or linear Broccoli RNA aptamer loaded in LNPs was stored in PBS at 4 °C and -20 °C (supplemented with sucrose) for a series of durations. Then, recovered LNPs were transfected into DC2.4 cells for 1 hour and were then resuspended and further incubated in PBS with 5% FBS and 200 pM DFHBI-1T (Sigma-Aldrich) for 45 minutes. Using untransfected cells as a control, the Broccoli fluorescence intensity (excitation, 488 nm; emission, 525 nm) of cells was analyzed by flow cytometry.|0143] Liposome encapsulation of circRNA. DOTAP, cholesterol and DSPE-PEG2000 (Avanti Polar Lipids) were mixed at molar ratios of 1: 1:0.1 of DOTAP / cholesterol / DSPE-PEG2ooo. Lipid mixtures were dried under a nitrogen stream in glass test tubes. The resulting lipid fdms were placed in a desiccation system overnight. Dry films were hydrated in a solution of circRNA or mRNA in PBS with six cycles of vortexing for 30 seconds every 5 minutes. Samples were then freeze-thawed six times between liquid nitrogen and a 37 °C water bath and extruded using 21 passes in a mini -extruder (Avanti) through a 0.2 pm pore size polycarbonate filter (Whatman). Samples were placed in 10 K MWCO dialysis cartridges (Thermo Scientific) and dialysed against PBS for 6 hours before use.

[0144] Cell culture. EG7.OVA cells were cultured in RPMI-1640 medium with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U ml1penicillin, 100 pg ml1streptomycin and 0.4 mg ml1G418. DC2.4, TC-1, SM-1 and B3Z cells were cultured in RPMI-1640 medium (Gibco) supplemented withPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.60110% FBS, 100 U ml'1penicillin, 100 pg ml'1streptomycin, 2 mM L-glutamine, 50 pM 2- mercaptoethanol, 1 x non-essential amino acids and 10 mM HEPES. DC2.4 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U ml'1penicillin and 100 pg ml1streptomycin. B16F10, MC38 and HEK293T cells were cultured in DMEM supplemented with 10% FBS, 100 U ml'1penicillin and 100 pg mF1streptomycin. All cells were cultured at 37 °C with 5% CO2.

[0145] BMDC isolation and culture. Mouse BMDCs were isolated from C57BL / 6 mice (6-8 weeks). All femurs and tibia were collected and cut at the epiphysis level, and bone marrows were flushed out with 10 ml RPMI-1640 medium (Gibco) 3 times. The resulting cell suspensions were filtered through cell strainers (70 pm cut-off, BD Falcon), centrifuged and then lysed by ACK buffer (Gibco). To culture BMDCs, RPMI-1640 medium supplemented with 10% FBS and 20 ng ml1GM-CSF was added to resuspended cell pellets to obtain at a density of 2 x 106viable cells per 75 mm Petri dish. Three days later, an additional 10 ml cell culture medium was added. Six days later, non-adherent and loosely adherent cells were collected by gentle washing with PBS and then pooled for further studies.

[0146] Cell viability assay. The cytotoxicity of DC2.4 cells (2 x 105cells were seeded per well in 24- well plates) treated with small or large circRNAs was assessed using a ONE-Glo Tox kit (Promega, E7110). RNAs were transfected with Lipofectamine 3000 using serum-free medium 24 hours after cell seeding. After 6 hours, the medium was replaced by complete culture medium, and the cells were incubated for another 18 hours, before adding CellTiter-Fluor reagent. After incubation for 0.5 hours, the fluorescence of cells was measured using a plate reader (Agilent BioTek) (excitation, 400 nm; emission, 505 nm).

[0147] Immunostaining of circRNA translation products in live DCs. Flag-encoding liRNA or circRNA (1 pg) was transfected into pre-seeded DC2.4 cells using Lipofectamine 3000 (Invitrogen). After 24 hours, cells were washed and fixed using 4% paraformaldehyde in PBS at room temperature for 30 minutes and then incubated with 0.3% Triton X-100 in PBS. After blocking with 1% bovine serum albumin in PBS, cells were incubated with 2pg ml1of anti-Flag M2 monoclonal antibody (Sigma-Aldrich) in PBS containing 1% bovine serum albumin for 1 hour. Cells were washed twice with PBS containing 0.3% Triton X-100 and then incubated with a mixture of Alexa Fluor 488-labelled antimouse IgG antibody (Invitrogen) and 4 ',6-diamidino-2-phenylindole (DAPI; 250 ng ml1) for 30 minutes. The cells were mounted on a glass slide using PermaFluor aqueous mounting medium (Thermo Fisher Scientific) for observation by confocal laser scanning microscopy on a Zeiss LSM 780 confocal microscope.

[0148] Intracellular translation kinetics of peptide and protein from mRNA and circRNA. DC2.4 cells were seeded into 24-well plates at a density of 2 x 105cells per well and cultured at 37 °C for 24 hours. Then cells were transfected with 5moU-mRNA-fFuc or circRNA-Flag (1 pg ml1) using Lipofectamine 3000. For 5moU-mRNA-fFuc, after 24 hours, 48 hours and 72 hours, the cell culture supernatants were replaced with a ONE-Glo Luciferase Assay System according to the manufacturer'sPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 instructions, and the bioluminescence was measured using a microplate reader. For circRNA-Flag, the cells were collected, permeated and intracellularly stained with PE-conjugated anti-Flag antibody (BioLegend, 637309), followed by flow cytometric analysis of cell fluorescence intensity.

[0149] PKR activation. PKR activation was studied in mouse DC2.4 cells and human HEK293 cells. DC2.4 cells seeded in 24-well plates were treated with LNPs of circRNA-SIINFEKL or 5moU- mRNAOVA (100 nM) for 24 horns at 37 °C. Then, cells were collected and lysed with cell lysis buffer (Cell Signaling Technology) containing 100 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). Cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4 °C and the supernatants were eluted with 1 x SDS loading buffer and resolved by 10% SDS-PAGE, followed by western blotting using anti-PKR and anti-pPKR antibodies (Thermo Fisher Scientific). HEK293T cells were used to study PKR activation by small circRNA and large circRNA, with PBS and poly(I:C) as controls. Following RNA transfection, cells were lysed for 4 hours, and proteins were resolved by SDS-PAGE, followed by western blotting to detect PKR and p-PKR using the above antibodies. Relative PKR phosphorylation was calculated as the gel density ratio of pPKR / p-actin, which was then normalized to the pPKR / p-actin gel density ratio in PBS-treated cells.

[0150] Flow cytometry. Flow cytometry was used to analyze immunostained cultured cells, mouse blood cells, as well as tumor tissue and lymph node homogenate single cells. Antibodies used for immunostaining are listed in Table 2. Cultured cells were analyzed on a BD Beckman Coulter flow cytometer. Blood and tissue single cells were analyzed on LSRFortessa X-50 (BD Biosciences). Flow cytometry results were analyzed using FlowJo V10 software. Representative flow cytometric analysis gating trees are shown in Fig. 32, Fig. 33, Fig. 34, Fig. 35, Fig. 36, Fig. 37 and Fig. 38.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0151] Table 2. AntibodiesTargets Fluorochromes Clones Vendors Catalogue #

[0152] Antigen presentation on cultured DCs. DC2.4 cells or BMDCs (2 x 105) seeded in 24-well plates were treated with circRNA SIINFEKL or controls for 24 hours. Cells were then collected and stained with APC-labelled anti-mouse H-2Kb / SIINFEKL complex antibody (BioLegend). Cells were then washed and analyzed by flow cytometry.

[0153] B3Z cell activation. Upon recognition of the H-2Kb / SIINFEKL complex, B3Z cells (engineered SIINFEKL specific CD8+T cells) are activated and produce P-galactosidase. The activation level of the B3Z cells can be measured by the absorption of B3Z cell solution upon P-galactosidase treatment. Specifically, DCs were treated with SIINFEKL-specific vaccines for 16 hours (RNA vaccine (1 pg) was transfected using Lipofectamine 3000). Cells were subsequently cocultured with B3Z cells for another 24 hours and were then lysed for 4 hours at 37 °C with a lysis buffer (PBS with 100 mM 2- mercaptoethanol, 9 mM MgC L. 0.2% Triton X-100 and 0.15 mM chlorophenol red-(3-D- galactopyranoside). The reaction was stopped using a stop buffer (1 M sodium carbonate). B3Z T cell activation was quantified by measuring the absorbance at 570 nm with 635 nm as a reference wavelength. The B3Z cell activation is shown as the normalized optical density (OD) relative to the untreated cell control.

[0154] ELISA. DCs were plated at densities of 5 x 105cells per well in 6-well plates. Cells were treated with the indicated formulations for 24 hours. RNA (1 pg) was transfected into the cells usingPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601Lipofectamine 3000. Secreted cytokines (such as IFN(3, IL-6 and IL-12) in cell culture medium were quantified using ELISA kits (R&D Systems).10155] PRR activity studies using reporter cells. HEK-Lucia RIG-I cells, HEK-Blue hTLR3 cells or HEK-Blue hTLR7 cells were respectively seeded into a 96-well plate at a density of 3 x 104 cells per well and cultured at 37 °C for 24 hours. Then cells were transfected with RNAs (1 pg ml-1) using Lipofectamine 3000. After 24 hours, cell culture supernatants were collected and treated with either QUANTIBlue Solution (InvivoGen, rep-qbs) or QUANTI-Luc 4 Lucia (InvivoGen, rep-qlc41gl) according to the manufacturer's instructions. The bioluminescence or OD at 630 nm was measured using a microplate reader.

[0156] In vitro cell uptake of circRNA. In vitro cell uptake of dye-labelled circRNA was examined using confocal laser scanning microscopy and measured by flow cytometry. Specifically, circRNA was labelled with Cy5 via hybridization with a Cy5-modified 30-mer cDNA. Cy5-circRNA loaded in LNPs or liposomes were incubated with DC2.4 cells for a series of durations and stained with LysoTracker Green DND-26 (Life Technologies) and 10 pg ml-1 Hoechst 33342 (Life Technologies) for 0.5 hours. Cells were then washed with DPBS three times before confocal microscopy observation on a Zeiss LSM 780 confocal microscope. Alternatively, DC2.4 cells seeded in 24-well plates were treated with Cy5-labelled circRNA formulations as above, followed by flow cytometric analysis.

[0157] DC co-stimulation. Co-stimulatory factors CD80 and CD86 were stained and measured on vaccine-treated DC2.4 cells by flow cytometry. In brief, DC2.4 cells were treated with circRNA vaccine or controls, dissociated using non-enzymatic dissociation buffer, washed with PBS and resuspended in PBS with dye-labelled anti-mouse CD80 and CD86 antibodies at 4 °C for 30 minutes. Cells were then washed with PBS before flow cytometric analysis.

[0158] RNA-seq. Total RNA was isolated using AllPrep DNA / RNA Mini Kit (QIAGEN) with incolumn DNase 1 digestion to obtain DNA-free RNA. The integrity of extracted RNA was analyzed using the Agilent RNA 6000 Pico Kit (Agilent). An aliquot of 1 pg total RNA (RIN > 8) was used to prepare mRNA-seq libraries following the instruction of Illumina stranded mRNA-seq preparation Guide (Illumina). In brief, mRNA was captured by oligo (dT) magnetic beads from total RNA, cation fragmented, and reverse transcribed into cDNA. cDNA was end repaired, ligated with RNA index anchor and PCR amplified to produce indexed libraries with IDT for illumined UD indexes. Following quality control, the resulting barcoded libraries were pooled in equal molarities and were paired-end (2 x 150 bp) sequenced on the NextSeq 2000 instrument (Illumina). The quality of RNA-seq reads was assessed with FastQC vO.11.9. Reads from individual samples were aligned using STAR aligner version 2.7.6a to mouse reference genome GRCm39. Raw gene counts of mapped reads were aggregated using featureCounts. The differential gene expression analysis was performed with Bioconductor package DESeq2 vl.30.0 using the normalized and filtered counts per gene from the RNA-seq data. Differentially expressed genes with adjusted P< 0.05 were subject to Gene Ontology and KEGGPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 pathway enrichment analyses using the GSEA. The visualization of upregulated genes and associated Gene Ontology' functions when comparing the three biological conditions was achieved using the Triwise R package. The Triwise R package permitted two-dimensional visualization of differential gene expression patterns when comparing three biological conditions. It filters and calculates the average expression in the three biological conditions for each gene and then transforms this gene expression matrix to barycentric coordinates, reducing the three-dimensional matrix by one and retaining only the expression changes between samples. Limma package is then used to determine differentially expressed genes. These barycentric coordinates can then be plotted in a two-dimensional dot plot where the direction of a gene (a single dot) indicates in which condition(s) the gene is upregulated, while the distance from the origin represents the strength of upregulation.

[0159] circRNA-seq and circRNA variant calling for circRNA authenticity analysis. circRNA-seq was conducted for circRNA-SIINFEKL that was extracted from transfected cells using AllPrep DNA / RNA / miRNA Universal Kit (QIAGEN), with untransfected circRNA-SIINFEKL as a control. circRNA libraries were constructed using 2pg total RNA (RIN > 7), and ribosomal RNA was depleted using Ribo-Zero as well as an enzymatic depletion method (Illumina) and was purified with AMPure XP Beads (Beckman Coulter). Linear RNA was then digested using RNase R (Biosearch Technologies). The resulting circRNA was purified with AMPure XP Beads. circRNA was then cation fragmented at 95 °C for 2 minutes. Next, reverse transcription and library construction were performed using Illumina. Stranded Total RNA Prep, followed by ligation with Ribo-Zero Plus Sample Preparation Guide (Illumina). Final libraries were size analysis by Agilent HS DNA chips (Agilent) and quantified by Qubit dsDNA HS Assay (Invitrogen). Following quality control, the resulting barcoded libraries were pooled in equal molarities and were paired-end (2 x 150 bp) sequenced on a NextSeq 2000 instrument (Illumina). The quality of raw FASTQ reads was assessed using FastQC vO.11.9. For read mapping, a reference fasta composed of two copies of circRNA-SIINFEKL sequence less one nucleotide was used. Reads were mapped either in a paired-end mode (PE) or in a single-end (SE) mode following read merging step. Here the reads were merged with either bbmerge function from BBTools package or merged reads (FLASh). The mapping step was performed with bowtie2 (bt2) with default settings, as well as with NextGenMap setting a constrain of minimum 90% identity' (-i 0.9) over 75% of read length (-R 0.75) for a read to be considered as mapped. Finally, the variant calling step was done using the LoFreq tool. In brief, the mapping errors were corrected using ‘lofreq viterbi’, followed by indel quality' insertion with ‘lofreq indelqual’, followed by base and indel alignment qualities insertions with lofreq alnquaf. The final calling step was performed using lofreq calf with options set to ‘no-default-filter- bonf 1-sig 1-call-indels1. To calculate the nucleotide authenticity rate of circRNA, reference fasta was divided into left and right flanks. Allele frequencies of left and rightflank variants detected with LoFreq were summed and divided by the length of the flank. The resulting value was then used to calculate the nucleotide rates of circRNA.PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601

[0160] Animal studies. All animal work was conducted in compliance to the Guide for the Care and Use of Animals under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University and University of Michigan. Vaccines were injected into mice subcutaneously either into mouse footpad for vaccine delivery study or into the mouse tail base for immune analysis or therapy studies. Both subcutaneous and intramuscular vaccine administrations were shown to elicit a comparable magnitude and quality of antigen-specific cellular or humoral immune responses46.

[0161] In vivo tissue and cell distribution of circRNA in mice. To study in vivo tissue distribution of circRNA, circRNA was labelled with IR800 via a 30-mer cDNA. IR800-circRNA loaded in LNPs (0.5 nmol circRNA) were subcutaneously administered in the foot pad of albino C57BL / 6 mice (6-8 weeks). A series of days later, mice were imaged for IR800 fluorescence using an IVIS Lumina system (PerkinElmer). In another cohort, 24 hours after administration, major organs were isolated and imaged on an IVIS Lumina system. The images were processed using Living Image analysis software (PerkinElmer).

[0162] To study intranodal cell distribution of circRNA by single cell flow cytometric analysis, C57BL / 6 mice were subcutaneously administered with nanoparticles of 0.5 nmol Cy5 labelled circRNA at tail base. Twenty -four hours later, draining inguinal lymph nodes were collected, and lymph node single cells were prepared by passing minced lymph nodes through 70 pm cell strainers. Cells were then washed with and resuspended in cell staining buffer for staining using the following antibodies (BioLegend): Brilliant Violet 421 anti-mouse CD45, Alexa Fluor 594 anti-mouse CDllc, FITC antimouse CD lib, APC / Cy7 anti-mouse CD8a, PerCP / Cy5.5 anti-mouse CD4, Brilliant Violet 605 antimouse CD103, PE / Cy7 anti-mouse CD205, APC / Cy7 anti-mouse F4 / 80, PE / Cy5 anti-mouse CDllb and APC anti-mouse NK1.1. Zombie Aqua Fixable Viability Kit (BioLegend) was used to stain dead cells. Cells were then washed and analyzed by flow cytometry.|0163] Serum cytokine and chemokine measurement by Luminex. C57BL / 6 mice were immunized, and blood was collected under isoflurane anesthesia 12 or 24 hours later. Blood was centrifuged for 5 minutes at 13,000 rpm, and sera were collected and stored at -80 °C. Pre-selected panels of cytokines and chemokines in the above samples were measured by Luminex (University of Virginia Flow Cytometry Core).

[0164] Tetramer staining on T cells. Mouse CD8+and CD4 T cells were stained for antigen-specific tetramers using PE-conjugated tetramers (NIH Tetramer Core Facility). In brief, peripheral blood was collected from the vaccinated mice and blood cells were enriched by centrifugation. Red blood cells were lysed using ACK lysis buffer for 10 min at room temperature. Blood clots were removed using a filter. Cells were washed twice in PBS and then stained using a Zombie Aqua Fixable Viability Kit (BioLegend) for 20 minutes at room temperature. Staining was quenched and cells were washed with FCS buffer (PBS buffer with 0.1% FBS). Cells were then blocked with anti-CD16 / CD32 (BioLegend)PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 for 10 minutes, followed by adding staining cocktail (PerCP / Cy5.5 anti-mouse CD3, APC / Cy7 antimouse CD8a, Brilliant Violet 421 anti -mouse PD-1 and tetramer-PE) and staining at room temperature for 30 min. Cells were then washed and 100 pl Cytofix was added into each well to resuspend cells before fixation at 4 °C for 20 minutes. Cells were then washed with Perm / Wash buffer and resuspended for flow cytometric analysis.

[0165] Intracellular cytokine staining in T cells. Peripheral blood was collected from immunized mice. Red blood cells were removed using ACK lysis buffer, and the obtained lymphocytes were transferred into U-bottom 96-well plates in 200 pl T cell culture media (RPMI-1640, 10% FBS, 100 U ml-1 penicillin / streptomycin, 50 pM (3 -mercaptoethanol, 1 x MEM non-essential amino acid solution and 1 mM sodium pyruvate). Lymphocytes were pulsed with antigen peptides (40 pg ml-1) for 4 hours, followed by addition of GolgiPlug Protein Transport Inhibitor containing brefeldin A (Thermo Fisher Scientific). The sequences of antigen peptides (CSBio) are shown in Table 3. Cells were then placed in a culture incubator for 6 hours before incubation with anti-CD16 / CD32 for 10 minutes at room temperature. Cells were stained with APC / Cy7 anti-mouse CD8a, PerCP / Cy5.5 anti-mouse CD4 and Zombie Aqua Fixable Viability Kit for 20 minutes at room temperature. Cells were washed and subsequently fixed using Cytofix (BD Biosciences), washed, and permeabilized in 200 pl Cytoperm solution (BD Biosciences). Cells were then washed using Perm / Wash buffer (BD Biosciences), and permeabilized cells were then stained using PE anti-mouse IFNy (BioLegend) and FITC anti-mouse TNF (BioLegend). Stained cells were washed for flow cytometric analysis.

[0166] Table 3. Peptides

[0167] Immune memory. Peripheral blood was collected from mice to analyze memory T cells. Immune memory was analyzed by flow cytometric analysis of peripheral lymph node homing receptor, CD62L, and adhesion molecule, CD44. In brief, red blood cells were lysed using ACK lysis buffer, and blood cells were then collected by centrifugation and washing with FCS buffer. Cells were then blocked with anti-CD16 / CD32 in FCS buffer for 10 minutes, followed by adding CD8-APC-Cy7, CD44-PE- Cy5, CD62L-FITC and dead cell-staining DAPI. Cells were stained at room temperature for 30 minutes and were then washed. Cytofix (100 pl) was added into each well to resuspend cells and cells were then incubated at 4 °C for 20 minutes. Cells were then washed with Perm / Wash buffer and resuspended forPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 flow cytometric analysis. Central memory CD8+T cells were analyzed as CD44hlCD62LhlCD8+T cells; effector memory CD8+T cells had variable to low levels of CD62L and high CD44 levels; and naive CD8+T cells had high levels of CD62L and low to intermediate levels of CD44. To verify immune memory, immunized mice were challenged by s.c. inoculation of tumor cells (3 X 105) on the right shoulder. Tumor size was monitored every' 3 days thereafter. Tumor volume was calculated using the formula volume = (length x width2) / 2 and analyzed using GraphPad Prism 7.

[0168] Tumor immunotherapy. About 3 x 105EG7.OVA, MC38, TC-1, B16F10 and SM-1 Braf7'"" cells, respectively, were subcutaneously inoculated on the shoulder of female C57BL / 6 mice (6-8 weeks; The Jackson Laboratory; n= 6-8). Tumor growth was monitored by caliper measurement. Mice were euthanized when the maximal tumor dimension reached 2 cm, or the tumor volume exceeded 2,000 mm3or developed ulceration. Mice were treated 6 days after tumor inoculation when tumors reached approximately 50 mm3. Typical doses: 5 pg RNA vaccine, 5pg CpG+10 pg antigen peptides or proteins, and 200 pg anti-PD-1 or anti-CTLA-4 (Bio X Cell, NH). Vaccine doses used in SM-1 Braf1'50® melanoma therapy: 30 pg circRNA, 15 pg circRNA CpG and 30 pg antigenic peptides. Vaccines were subcutaneously injected at mouse tail base to allow lymphatic draining every 6 days three times, and anti-PD-1 or anti-CTLA-4 was injected intraperitoneally every 3 days five times. For lymphocyte depletion, anti-CD4, anti-CD8 and anti-NKl.l (200 pg) were intraperitoneally injected every 3 days five times. Tumor size and mouse weight were monitored every 3 days. Tumor volumes were calculated and analyzed. Results were analyzed using GraphPad Prism 7.

[0169] Statistical analysis. Data represent mean ± s.e.m., unless denoted otherwise. Statistical analysis was performed in GraphPad Prism Software version 5.0. P values were calculated by one-way ANOVA with Bonferroni post-test, unless denoted otherwise.Example 2:Optimization and characterization of highly stable small circRNA vaccines

[0170] Small circRNAs consist of minimal elements: codon-optimized peptide-antigen-encoding RNA and a short internal ribosome entry site (IRES) and a Kozak consensus sequence that recruit ribosomes for peptide translation (Fig. 1). Using MHC-I -restricted ovalbumins?as a model antigen, circRNA were prepared as follows: (1) circularizing 51-phosphate-RNA using T4 RNA ligases and complementary 30-mer DNA splints; (2) removing linear / lariat RNA and DNA using DNase I and exonuclease T or RNase R22; (3) purification to remove immunostimulatory small DNA / RNA fragments, enzymes and nucleotides (Fig. 14); and (4) verification by Sanger sequencing (Fig. 2A) and gel electrophoresis with an estimated circularization rate of 32% (Fig. 14). A short IRES that efficiently recruits ribosomes and initiates translation is critical for the optimal immunomodulatory efficacy of small circRNA vaccines23. As shown by H-2Kb / SIINFEKL staining on vaccine-treated DCs, circRNA-SIINFEKL with 71-mer crucifer-infecting tobacco mosaic virus (crTMV) IRES permitted efficient antigen presentation, relative to circRNA-SIINFEKL with short IRES(es) long interspersedPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 nuclear elements 1 (LINE1) and RNA binding motif protein 3 (RBM3), the corresponding linear RNA (liRNA, linearized circRNA with the nicking site between IRES and coding sequence, without 5' cap or 3' poly (A) or untranslated regions (UTRs) (Fig. 2A), and CpG oligonucleotide-adjuvanted OVA protein24'26(Fig. 2B, Fig. 2C, Fig. 15A, and Table 1). Consistently, crTMV permitted circRNA- SIINFEKL to efficiently prime SIINFEKL-specific B3Z CD8+T cell hybridoma (Fig. 2B, Fig. 2C and Fig. 2D). Deletion of IRES or Kozak sequence reduced antigen presentation and T cell priming (Fig. 15B). Therefore, crTMV IRES was selected for further studies. Interestingly, relative to state-of-the-art 5-methoxyuridine-modified OVA-encoding mRNA (5moU-mRNA-OVA), circRNA-SIINFEKL enhanced and prolonged antigen presentation and T cell activation (Fig. 2B, Fig. 2D and Fig. 15B). A model Flag peptide -encoding circRNA (circRNA-Flag) efficiently expressed Flag products in live mouse DC2.4 cells, as shown by anti-Flag immunostaining (Fig. 2E).

[0171] mRNA can be degraded by ubiquitous biological and environmental RNases, especially exonucleases that are primarily responsible for mRNA degradation1, which limits mRNA shelf-life, biological half-life, antigen translation efficiency and duration, and consequently immunomodulatory efficacy. The absence of termini in circRNA prevents exonuclease degradation17. To study the biostability of circRNA in live cells, a model circRNA in which the coding RNA was substituted with a Anorogenic RNA aptamer, Broccoli21was synthesized. Upon transfection, the resulting ciwBroccoli showed biostability in live DCs and the Broccoli Auorescence sustained for at least 7 days despite signal dilution during cell division, in contrast to almost complete Auorescence decay of linearized circ Broccoli within 12 hours (Fig. 2F, Fig. 2G, Fig. 2H and Fig. 16A). Exogenous RNA, including circRNA, can be subject to mutation owing to RNA editing by endogenous editors, such as cytidine deaminases AID and APOBEC28, and adenosine deaminase ADAR29. Yet, such circRNA mutation may affect the ability of IRES and Kozak sequence to initiate peptide translation and alter peptide sequences. To study this, mouse bone marrow-derived dendritic cells (BMDCs) were transfected with circRNA- SIINFEKL. Twenty-four hours later, total circRNA was isolated from cells, and circRNA-seq analysis of circRNA-SIINFEKL showed no detectable circRNA mutations (Fig. 21), indicating the high sequence authenticity of this small circRNA in live cells. Modification-free small circRNA showed superior thermostability than not only the corresponding small liRNA but also 5moU-mRNA-OVA. Specifically, as estimated with a one-phase decay model, upon storage in phosphate -buffered saline (PBS) at -20 °C, 4 °C and 23 °C, the half-lives of circRNA-SIINFEKL are 401, 78 and 16 days, respectively, in contrast to 143, 44 and 6 days, respectively, for 5moUmRNA-OVA, and 2.6, <0.5 and <0.5 days, respectively, for liRNA-SIINFEKL (Fig. 2J and Fig. 16B). Consistently, when transfected into DCs, small circRNA-Flag fragments, showed durable Flag expression for at least 7 days when the experiment ended, in contrast to less than 3 days of protein expression by 5moU-mRNA-fLuc (Fig. 2K). It was found that, circBroccoli-loaded LNPs (Fig. 2L) retained strong Auorescence intensity upon transfection in DCs after storage in solutions at 4 °C or -20 °C (supplemented with sucrose) for 70 days, verifying great thermostability of circRNA LNPs (Fig. 2M). circRNA is eventually degraded byPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 hydrolysis30, endonuclease degradation31and exonucleases upon circRNA linearization. Large circRNA has been often used for drug and vaccine development so far11. It was hypothesized that because small circRNA typically has fewer sites of hydrolysis and endonuclease cleavage than large circRNA, small circRNA is overall more stable than large circRNA. To test this, 1,760-mer large circRNA consisting of a long IRES (CVB3) and RNA encoding SARS-CoV-2 spike protein receptor-binding domain (RBD) were synthesized (Fig. 17). Upon incubation in diluted fetal bovine serum (FBS) followed by gel electrophoresis, 111-mer small circRNA-SIINFEKL showed higher stability than large circRNA-RBD (Fig. 2N and Fig. 20). Overall, small circRNA showed biostability and thermostability, which are important for their long shelf-life, sustained antigen translation, and robust and durable immunomodulation.Example 3:LNPs efficiently delivered small circRNA to lymph nodes and APCs to elicit T cell responses

[0172] Nanocarriers are important for the delivery and immunomodulation of mRNA vaccines32. To select a nanocarrier for small circRNA vaccines, liposomes and three LNPs (with ionizable lipids SM- 102, Dlin-MC3-DMA and Dlin-KC2-DMA, respectively) were tested, two classes of the most successful nucleic acid nanocarriers thus far32. circRNA-SIINFEKL and control 5moU-mRNA-OVA were loaded into these nanocarriers (N / P ratio 6). SM-102 LNPs showed comparable encapsulation efficiencies for mRNA (86.3%) and circRNA (86.9%). Owing to the small size of small circRNA, 185 copies of circRNA-SIINFEKL were calculated to be loaded per LNP with 170 nm in diameters, in contrast to 14 copies of 5moU-mRNA-OVA per LNP. Upon subcutaneous injection at the tail base of C57BL / 6 mice, H-2Kb-SIINFEKL tetramer staining showed that SM-102 LNPs of 5ug circRNA- SIINFEKL (10-15% typical doses of T cell mRNA vaccines33) elicited the most frequent SIINFEKL±CD8+T cells among peripheral blood mononuclear cells (PBMCs) (Fig. 3A and Fig. 3B). Furthermore, as shown by Luminex, among all formulations, SM-102 LNPs of circRNA-SIINFEKL elicited the lowest levels of systemic chemokines associated with reactogenicity, a common side effect of mRNA vaccines (Fig. 3C and Fig. 18). This indicates the good safety utility of small circRNA vaccines. Taken together, SM-102 LNP was selected as the nanocarriers for small circRNA vaccines. In vivo imaging system (IVIS) imaging showed that, for at least 8 days after subcutaneous injection in mice, SM-102 LNPs enhanced the accumulation of IR800-labelled circRNA (IR800-circRNA) in draining lymph nodes, which harbor various lymphocytes and orchestrate immunomodulation (Fig. 3D, Fig. 3E and Fig. 3F). This provides the basis for sustained antigen expression and T cell priming over a prolonged duration. Lymph nodes harbor various APC subsets that have differential vaccine uptake and immune-stimulation efficacies. circRNA uptake by intranodal APCs by flow cytometry 24 hours after subcutaneous injection of Cy5-circRNA LNPs was studied in mice. The present disclosure found that LNPs efficiently delivered circRNA to intranodal APC subsets that are important to present peptide epitopes and elicit antigen-specific T cell responses, including CD8+T cell-priming CDl lc+CDl lb- CD8+classical DCs (eDCs), CD4+T cell-priming CDl lc+CDl lb+CD4±CD103- CD205+eDCs andPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601CDllc+CDllb-CD8- CD4- CD103+migratory eDCs (Fig. 3G). Moreover, within only 0.5 hour after transfection in cultured DCs, LNPs permitted rapid endosome escape of circRNA to the cytosol to allow peptide translation (Fig. 3H, Fig. 31 and Fig. 19). The present disclosure also found that liposomes efficiently delivered small circRNA to draining lymph nodes and intranodal APCs in mice (Fig. 9 and Fig. 10) and elicited SIINFEKL-specific CD8+T cell responses more efficiently than 5moU-mRNA- OVA and CpG-adjuvanted OVA (Fig. 11 A, Fig. 1 IB and Fig. 11C). Meanwhile, presumably owing to vaccine-induced immuno-stimulation, liposomal circRNA-SIINFEKL upregulated expression of the immune checkpoint programmed death receptor 1 (PD-1) on T cells, especially SIINFEKL-specific CD8+T cells (Fig. 1 ID, Fig. 1 IE and Fig. 1 IF), providing an opportunity for ICB combination therapy with circRNA vaccines. The present disclosure found that liposomal circRNA vaccine-immunized mice resisted challenge with EG7.OVA tumor cells, without significant weight loss, indicating the good safety utility of these vaccines (Fig. 11G and Fig. 11H). Overall, nanocarriers such as LNPs efficiently delivered small circRNA vaccines to lymph nodes and pivotal intranodal APCs to elicit potent T cell responses with great safety utility.Example 4:Small circRNA mediated immunomodulation with low PKR activation

[0173] Various types of RNAhave been found to elicit innate immunity by activating endosomal PRRs such as Toll-like receptor 3 (TLR3), TLR7, TLR8 or cytosolic PRRs (such as RIG-I and melanoma differentiation-associated protein 5 (MDA5))34. Combined with the immunostimulatory ability of nanocarriers (for example, LNPs), mRNA vaccines elicit innate immunity that provides APCs with pro- inflammatory cytokines and co-stimulation signals for antigen presentation and T cell priming. To investigate the impact of small circRNA vaccines on immunomodulation, RNA-seq genome-wide transcriptomic analysis of mouse BMDCs LNP -transfected were conducted with circRNA-SIINFEKL and 5moU-mRNA-OVA, respectively, for 24 hours, with PBS or blank LNPs as controls. About 560 genes were grouped by their involvement in inflammation, migration, antigen uptake and presentation, PRRs (such as RIG-I-like receptors (RLRs), TLRs and C-type lectin receptors (CLRs)), MHC-I and MHC-II, or miscellaneous immune-related genes. From the hierarchical clustering of these differentially expressed genes (Fig. 4A and Fig. 4B), upregulated PRRs were identified and activated immune-related pathways by LNP-transfected circRNA relative to blank LNPs. Overall, relative to blank LNPs, circRNA-SIINFEKL showed low upregulation of the above immune-related genes. For example, circRNA upregulated the transcriptions of Ddx58 (also known as Rigi) and Clec4, whereas 5moU-mRNA-OVA activated RLRs (IfM (also known as Mda5 ), Oaslb, Eff2sl and Eff2ak2 (Pk )) and Tlr3 (Fig. 4C) that are characteristic of long dsRNA / ssRNA PRR activation. KEGG pathway analysis further confirmed that RNA sensor genes such as Rigi were activated by circRNA-SIINFEKL. The differential PRR activation profiles between circRNA-SIINFEKL and 5moU-mRNA-OVA may be due to structural differences such as the absence of long dsRNA in small circRNA. Furthermore, circRNA-SIINFEKL matured DCs, as indicated by enhanced gene activities of MHC-I (H2-KE) andPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 co-stimulatoiy molecules (such as Cd40, Cd80 and Cd86) (Fig. 4C), which was supported by the elevated expression levels of co-stimulatory factors CD80 and CD86 (Fig. 20). Moreover, circRNA- SIINFEKL triggered a multifaceted shift to inflammation in DCs, with upregulation of NLR (Nlrc4) and pro-inflammatory cytokines ( Tnf and Ill2rb]). circRNA-SIINFEKL also upregulated some immunosuppressive mediators, which presumably act as endogenous negative regulators of innate immune activation. Some of these genes encode therapeutic targets, for example, programmed death ligand 1 (PD-L1; encoded by Cd274), that hold use for combination therapy with circRNA vaccines. To validate PRRs for circRNA, HEK-Lucia RIG-I, HEK-Blue hTLR3 and HEK-Blue hTLR7 reporter cells were respectively transfected with circRNA-SIINFEKL with controls, including large circRNA- OVA, 5moU-mRNA-OVA and blank transfection agent, as well as RNA immuno-stimulant polyinosinic-polycytidylic acid (poly(LC)) as a positive control. circRNA-OVA comprises IRES CVB3 and a coding sequence (CDS) for OVA, and was synthesized by IVT and permuted intron-exon I (PIE- I)-mediated RNA circularization (Fig. 21A and Fig. 21B and Fig. 41). As aresult, circRNA-SIINFEKL activated RIG-I, but not TLR3 or TLR7 (Fig. 4D). RIG-I-dependent circRNA sensing was further validated in model RM1 cells with knockout of interferon-I3 (IFN(3) promoter stimulator I (IPS- 1), an adaptor required by RIG-I-mediated type I IFN induction (Fig. 4E). Lastly, relative to large circRNA- RBD and 5moU-mRNA-OVA, small circRNA-SIINFEKL showed low PKR phosphorylation (pPKR) in cells, indicating low PKR activation (Fig. 4F, Fig. 4G, Fig. 21C and Fig. 21D). The present disclosure demonstrates that, relative to large circRNA and mRNA often with long dsRNA, the minimal long dsRNA in small circRNA minimizes pPKR inhibition of protein translation. Consistently, relative to large circRNA-RBD, small circRNA-SIINFEKL resulted in low cytotoxicity in HEK293T cells (Fig. 4H). Overall, these results reveal the immunomodulatory mechanism of small circRNA vaccines with low PKR activation and low cytotoxicity.Example 5:Benchmarking small circRNA vaccines against modified mRNA, large circRNA and unmodified mRNA vaccines

[0174] Current mRNA vaccines use long poly(A) and artificial caps, as well as modified nucleotides for modified mRNA, which together result in balanced mRNA biostability, protein production and immuno-stimulation. Moreover, synthetic large circRNAs have been engineered as a novel class of mRNA vaccines against SARS-CoV-217. The immunomodulatory efficacies of modification-free circRNA-SIINFEKL was first studies as a benchmark against three forms of modified mRNA vaccines in vitro and in mice: (1) protein-encoding 5moU-mRNA-OVA, (2) SIINFEKL-encoding 5moU-mRNA (5moUmRNA-SIINFEKL) and (3) pseudouridine ('P)-mRNA-SIINFEKL. All these mRNAs have 573' UTRs, 3' ADO and CleanCap (Tables 1 and 2). After LNP transfection into DC2.4 cells, circRNA- SIINFEKL elicited a highly rapid, abundant and durable SIINFEKL antigen presentation over 3 days (Fig. 5 A and Fig. 22). Next, C57BL / 6 mice (6-8 weeks) were immunized with these RNAs at escalating doses (1, 3, 10, 30 and 100 pg), respectively, followed by monitoring their potency and duration ofPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601SIINFEKL-specific CD8+T cell responses over 180 days, body weights and innate immune responses to evaluate T cell responses and vaccine safety (Fig. 5B). After two doses, tetramer staining (day 21) showed that while all these vaccines showed dose-dependent SIINFEKL±CD8+T cell responses, circRNA-SIINFEKL elicited the largest fractions of SIINFEKL±CD8 T cells among all PBMC CD8+T cells (Fig. 5C and Fig. 23). As low as 2 x Ipg circRNA-SIINFEKL elicited 2.6- and 7.3-fold PBMC SIINFEKL±CD8+T cell fractions relative to that by 5moU-mRNA-SIINFEKL and 'P-mRNA- SIINFEKL, respectively. Furthermore, at higher doses, circRNA-SIINFEKL caused less mouse body weight loss and more rapid weight recovery than benchmark mRNAs, indicating the superior safety of small circRNA vaccines (Fig. 5D and Fig. 24). A second booster of circRNA-SIINFEKL (day 28) substantially expanded the repertoire of SIINFEKL-specific CD8+T cells, which plateaued at -75% peripheral SIINFEKL-specific PBMC CD8+T cells (day 35) at a dose of 3 X 30 pg (Fig. 25). Over days 21-180, while all RNA vaccines showed dose-dependent SIINFEKL-specific T cell responses, the area under the curve (AUC) of SIINFEKL-specific PBMC CD8+T cell fractions elicited by circRNA- SIINFEKL was higher than that by all the benchmark mRNA vaccines (Fig. 5E). Moreover, on day 35, the total counts of SIINFEKL±CD8+T cells in peripheral blood and spleen per mouse induced by as low as 10 pg circRNA-SIINFEKL were estimated to be over 2-fold typical dose of adoptive TCR-T cells used for mouse solid tumor immunotherapy35(Fig. 5F). These findings indicates that the utility of small circRNA vaccines for in vivo generation of therapeutically significant dose of TCR-T cells for tumor immunotherapy, which bypasses any ex vivo TCR-T cell engineering and manufacturing. The T cell responses induced by circRNA-SIINFEKL waned less rapidly than that by modified mRNA, although high-dose 5moU-mRNA-OVA, which produces more immunogenic OVA than SIINFEKL, also elicited up to -70% SIINFEKL-specific PBMC CD8+T cells (day 35). Specifically, on day 180, 8- 30% SIINFEKL-specific PBMC CD8+T cells persisted with 10-100 pg circRNA-SIINFEKL, whereas such T cell fractions elicited by modified mRNA-SIINFEKL waned almost completely and that by 10- 100 pg 5moU-mRNA OVA waned to 3-20%. These findings indicates that the circRNA-SIINFEKL elicited superior T cell memory than these benchmark mRNAs. The immune memory was verified by elevated total fractions of CD62L-CD44hi effector memory T (TEM) cells and D62L+CD44hi central memory T (TCM) cells, especially SIINFEKL memory T cells (Fig. 26). Consistently, although the total counts of PBMC and splenic SIINFEKL±CD8+T cells elicited by all vaccines reduced on day 180 relative to day 35, the T cell repertoires elicited by circRNA vaccines persisted for a longer duration than those elicited by benchmark mRNA (Fig. 5F). It was indicated that additional booster circRNA immunization would maintain the high T cell repertoires, when needed. Immuno-stimulation is often accompanied by immune exhaustion, as characterized by the upregulated expression of immune checkpoints such as PD-1. Levels of PD-1 in total PBMC CD8+T cells in the immunized mice showed dose-dependent PD-1 upregulation by day 35 (Fig. 27), and PD-1 levels had largely recovered by day 49. The immune checkpoint upregulation can sensitize T cells for ICB, providing an opportunity to combine ICB with circRNA vaccines for optimal cancer therapeutic efficacy. Next, circRNA-PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601SIINFEKL were benchmarked against unmodified OVA-encoding mRNA-OVA and large circRNA- OVA vaccines for their abilities to elicit T cell responses in mice. circRNA-OVA consists of an efficient long IRES (CVB3) and encodes highly immunogenic full-protein OVA (Fig. 21A and Fig. 21B). Upon immunization in C57BL / 6 mice (6-8 weeks), circRNA-SIINFEKL elicited higher fractions of PBMC SIINFEKL±CD8+T cells than unmodified mRNA-OVA and large circRNA-OVA at equivalent doses over 120 days (Fig. 5G and Fig. 12).

[0175] The next step was to evaluate vaccine -elicited innate immunity associated with reactogenicity and proinflammation, a panel of serum chemokines and pro-inflammatory cytokines were measured by Luminex 12 hours after the third dosing. Overall, relative to modified mRNAs and large circRNA- OVA, small circRNA-SIINFEKL induced lower levels of chemokines and cytokines that are often associated with immunotoxicity (Fig. 5H and Fig. 28). The data indicated good safety utility of small circRNA vaccines. Overall, these results demonstrate the superior or at least comparable T cell responses and safety of small circRNA vaccines relative to modified mRNA and large circRNA vaccines.

[0176] Immunosenescence during ageing makes older people vulnerable to immune disorders and impairs vaccines' immunomodulatory efficacy36. Thus, vaccines are highly desired to elicit potent and durable immunity in older people. In aged C57BL / 6 mice (1 year old) (Fig.5I and Fig.5 J) circRNA- SIINFEKL (5 pg. days 0 and 14) elicited 184% more frequent PBMC SIINFEKL±CD8+T cells than 5moU-mRNA-OVA and 242% more than CpG-adjuvanted OVA (day 21), with potent T cell polyfunctionality and immune memory that protected mice from EG7.OVA cell challenge (Fig. 51, Fig. 5 J. Fig. 5K and Fig. 29). Taken together, this showed the ability of small circRNAvaccines to potentiate the T cell responses with memory in immunosenescent aged mice.Example 6:Broad application of circRNA vaccines for various peptide immunogens

[0177] Modular small circRNA vaccines can be adjusted to encode versatile peptide immunogens for broad applications. Beside MHC-I-restricted immunogens, MHC-II restricted immunogens elicit CD4+helper and effector T cell responses that are important for disease prophylaxis and immunotherapy. In C57BL / 6 mice, circRNA encoding MHCIL restricted OVA323-339 (named as ISQ) (5 pg; days 0 and 14) elicited ISQ-specific polyfunctional CD4+T cells, as shown by intracellular IFNy / TNF (formerly known as TNFa) staining (day 21) (Fig. 6A and Fig. 6B). circRNA-ISQ enhanced the ability of MHC- I-restricted circRNA-SIINFEKL to elicit SIINFEKL±CD8+T cell response. (Fig. 6C). Moreover, circRNA-ISQ+circRNA-SIINFEKL outperformed 5moU-mRNAOVA, which encodes OVA protein with both SIINFEKL and ISQ epitopes, to elicit PBMC SIINFEKL±CD8+T cell response. Furthermore, small circRNA vaccines are applicable to tumor-associated antigens and tumor neoantigens for cancer immunotherapy. For example, circRNA encoding MHC-I-restricted ADPGK, an MC38 tumor cellspecific neoantigen38, elicited dose-dependent T cell responses with memory in mice (Fig. 6D, Fig. 6EPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 and Fig. 30). circRNA-ADPGK (2 x 5pg) elicited 5-fold ADPGK ±CD8+T cells, relative to liRNA- ADPGK or CpG-adjuvanted ADPGK peptide; meanwhile, circRNA-ADPGK elevated the PD-1 level on CD8+T cells, which can sensitize these cells for anti-PD-1 ICB in cancer combination immunotherapy (Fig. 6F). Multivalent vaccines can overcome the challenges of tumor antigenic heterogeneity and tumor cell immune evasion39. Bivalent circRNA encoding MHC-I-restricted melanoma-associated antigens mouse TRP2i8o-is» and human gpl0023-33, the latter priming T cells to recognize both human and mouse gplOO40, elicited bispecific CD8+T cells (Fig. 6G).

[0178] T cell vaccines hold great utility to prevent and treat pathogenic viral infections and associated cancers, such as SARS-CoV-2 and human papillomavirus (HPV) infections, and HPV-associated cancers41. Relative to long B cell epitopes that are often susceptible to mutations, T cell peptide epitopes are short (8-20 amino acids) and highly conserved owing to less susceptibility' to mutations. This makes T cell epitopes attractive to elicit broadly responsive T cell responses against rapidly mutating pathogens, such as SARS-CoV-2. As a step to this end, circRNA encoding MHC-I-restricted HPV16 E749 5741outperformed liRNA-E?49 57 and CpG-adjuvanted E749 57 peptides to elicit potent and long- lasting E?49 57-specific CD8+T cell response in mice (Fig. 6H, Fig. 61 and Fig. 31). Likewise, circRNA encoding MHC-I-restricted SARS-CoV-2 spike protein RBD440 459 elicited potent and long-lasting RBD440 459±CD8+T cells, as shown by tetramer staining over 70 days and intracellular cytokine staining (Fig. 61 and g. 6K). Taken together, these results demonstrate the broad applicability of small circRNA vaccines to elicit T-cell responses.Example 7:Low-dose circRNA vaccines reduced tumor immunosuppression for potent immunotherapy|0179] Tumor microenvironment (TME) is primarily where tumor cells suppress anti-tumor immunity2. In MC38 tumors, MC38-specific neoantigen vaccine circRNA-ADPGK, when combined with anti-PD-1, reduced immunosuppressive myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3+CD25+regulatory T (Treg) cells, increased the tumor infiltration of total CD8+T cells and ADPGICPCD8+T cells, and the ratio of enhanced CD8+T cells to Tregcells in the tumor, which predicts tumor therapeutic efficacy (Fig. 7A, Fig. 7B, Fig. 7C and Fig. 7D). RNA-seq of tumor tissue and transcriptome analysis showed that circRNA-ADPGK and circRNA-ADPGK+anti-PD-1 triggered a multifaceted shift of the tumor to an inflamed and tumoricidal microenvironment, with upregulated gene transcripts related to pro-inflammatory cytokines, leukocyte-recruiting chemokines, factors associated with DC maturation and T cell priming, and natural killer (NK) cell and macrophage activation (Fig. 7E and Fig. 7F and Fig. 7G). These changes of the cellular and molecular tumor immune microenvironment are indicated to be important attributes to the tumor therapeutic efficacy. As a result, circRNA-ADPGK neoantigen vaccine (3 x 5 pg) inhibited the growth of ADPGK-positive MC38 tumor (Fig. 7H). Moreover, circRNA-ADPGK potentiated the therapeutic efficacy of anti-PD-1 , which blocks the upregulated PD-1 on energic T cells (Fig. 7H). Depletion of CD8+T cells, but not CD4+T cells orPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601NK cells, abrogated the therapeutic efficacy of circRNA, verifying the central role of CD8+T cells in MHC-I restricted circRNA-ADPGK (Fig. 71).|0180] Anticancer T cell responses and TME immunity permit effective cancer immunotherapy. Given their ability to elicit potent and durable T cell responses and reduce TME immunosuppression, small circRNA vaccines were further assessed for tumor immunotherapy in multiple mouse tumor models. Low-dose circRNA-SIINFEKL (3 x 5 pg) regressed EG7.OVA tumors (~50 mm3) and prolonged mouse survival, which outperformed benchmark 5moU-mRNA-OVA and CpG-adjuvanted OVA (Fig. 12A, Fig. 12B, Fig. 12C and Fig. 12D). Depletion of CD8+T cells, but not CD4+T cells or NK cells, abrogated the therapeutic efficacy, verifying the central role of CD8+T cells in MHC-I-restricted circRNA-SIINFEKL. circRNA did not cause changes in mouse body weights, indicating its good safety utility (Fig. 12D). Moreover, small circRNA-SIINFEKL outcompeted full-protein OVA-encoding circRNA OVA with a highly efficient IRES (CVB3) to inhibit EG7.OVA tumor growth in C57BL / 6 mice (Fig. 12E). High-risk HPV, including HPV 16, causes -5% of all cancer cases. However, cunent US Food and Drug Administration (FDA)-approved HPV vaccines are unable to treat HPV associated cancer. Moreover, current immunotherapy such as ICB and adoptive cell transfer are limited by ICB resistance and poor T cell memory. Therapeutic vaccines are promising to elicit T cell response for combination immunotherapy of HPV-associated cancer. MHCI- restricted HPV16 protein E7 is an attractive oncoviral antigen because it is consistently and selectively expressed in HPV-associated cancer but not healthy cells, bypasses host immune tolerance, and is required to initiate and maintain HPV-associated cancer, which prevents cancer immune escape. Yet, current E7-based experimental therapeutic vaccines have limited efficacy against HPV-associated cancer in the clinic so far owing to low immunogenicity. Given the robust T cell response elicited by MHC-I-restricted circRNA-E7 (Fig. 6H and Fig. 61), circRNA-E7 were tested for the immunotherapy of E7-positive TC-1 tumor. Low-dose circRNA-E7 (3 x 5pg) inhibited TC-1 tumor progression via CD8+T cells (Fig. 8B and Fig. 12F). Because circRNA-E7 upregulated PD-1 on CD8+T cells, combining circRNA-E7 with anti-PD-1 further enhanced the tumor therapeutic efficacy (Fig. 8B and Fig. 8C). It was hypothesized that increasing the circRNA dose would further improve the tumor therapeutic efficacy.

[0181] Tumor antigenic heterogeneity and the resulting tumor cell immune escape represent a major obstacle against subunit vaccine-based tumor immunotherapy. Multivalent vaccines can at least partially overcome tumor antigenic heterogeneity and prevent tumor immune escape, thereby promoting tumor therapeutic efficacy. To test this using small circRNA vaccines, tetravalent small circRNA were engineered, termed as circRNA-T2 / g / Tl / Tl, which encoded MHC-I-restricted mouse TRP2iso-i9o, human gpl0023-33 and mouse TRP1455.463, as well as MHC-II-restricted mouse TRPlio6-i3o39. In poorly immunogenic B16F 10 melanoma, MHC-I / II-restricted circRNA-T2 / g / Tl / Tl (3 x 10 pg) inhibited tumor progression, in which both CD8+and CD4+T cells (Fig. 8D and Fig. 8E). Combining circRNA with both anti-PD-1 and anti-CTLA-4 further potentiated the therapeutic efficacy and prolonged mouse survival (Fig. 8D, Fig. 8E, Fig. 8F and Fig. 8G), with 2 out of 7 tumors showing complete regressionPCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601 without recurrence in 3 months (Fig. 8F). Around 50% of human melanoma contain BRAFV600Emutations, which cause constant oncogenic activation of BRAF, a serine / threonine protein kinase, and promotes melanoma tumorigenesis by, for example, evasion of antitumor immune responses, senescence and apoptosis. Syngeneic BrafV600ESMI mouse melanoma is highly aggressive and poor immunogenic with highly immunosuppressive TME, making it often resistant to immunotherapy. Thus, SMI melanoma closely resembles human BRATV600Emelanoma to predict its clinical therapeutic efficacy. Indeed, in syngeneic C57BL / 6 mice, BrafV600ESMI mouse melanoma did not respond to even dual ICB (anti-PD-1+anti-CTLA-4). By contrast, tetravalent circRNAT2 / g / Tl / Tl vaccine (3 x 30 pg) inhibited the progression of BrafV600ESMI melanoma, and combining circRNA with anti-PD-1+anti- CTLA-4 completely regressed 4 out of 8 tumors with 50% survival rate in 2 months (Fig. 8H and Fig. 81). Taken together, this demonstrates the use of small circRNA vaccines for tumor combination immunotherapy.Example 8Pulmonary delivery of small circRNA-M2e vaccines for influenza prophylaxis

[0182] Pulmonary vaccines are highly desired, especially for respiratory diseases such as flu and COVID-19. Pulmonary delivery is minimally invasive and can deliver vaccines to lung-residing immune cell repertoires, providing the opportunity to elicit both systemic immunity and mucosal immunity, the latter particularly beneficial for the prevention of respiratory infections and the treatment of respiratory immune disorders. Upon intratracheal administration in mice, LNPs enhanced circRNA- SIINFEKL accumulation in the lung and lung-residing APCs, including CDllc+CDllb“CD8+eDCs, CD 1 1 e CD 1 1 b CD4 CD 103 CD2O5 eDCs, and CD1 lb F4 / 80 macrophages (Fig. 39A, Fig. 39B, Fig. 39C and Fig. 39D). As a result, pulmonary circRNA-SIINFEKL elicited robust systemic SIINFEKL+CD8+T cell response that outperformed 5moU-mRNA-OVA and CpG-adjuvanted OVA (Fig. 39E).

[0183] The next steps of the experiment was to evaluate flu circRNA vaccine encoding influenza matrix protein-2 extracellular domain (M2e), an evolutionarily conserved yet poorly immunogenic antigen with the potential to develop universal flu vaccines47. circRNA-M2e were designed with crTMV IRES (Fig. 40A). circRNA-M2e was successfully synthesized via ligation of oligo RNA precursors, as verified by agarose gel electrophoresis (Fig. 40B). circRNA-M2e was then loaded into SM-102 LNPs with a encapsulation efficiency of 91.92 ± 2.34 %, and hydrodynamic diameters of approximately 100 nm, as verified by dynamic light scattering (DLS) and cryo-electron microscopy (crvo-EM) (Fig. 40C and Fig. 40D).

[0184] Due to the ability of circRNA to potentiate adaptive immune responses, MHC-II-restricted circRNA-M2e (3x5 pg; day 0, 14, 28) elicited 2.2- and 6.0- fold systemic M2e+CD4+T cells relative to liRNA-M2e and CpG-adjuvanted M2e (day35) in C57BL / 6 mice, as revealed by tetramer staining (Fig. 39F and Fig. 39G). Intracellular cytokine staining further verified the potent functionality of circRNA-PCT / US25 / 50083 08 October 2025 (08.10.2025)Docket No. 45500.601M2e-elicited M2e+CD4+T cells (day 35) (Fig. 39H). Further, in immunosenescent aged mice (18-20 months), circRNA-M2e elicited potent PBMC M2e+CD4+T cell response, with intriguingly 2-fold frequent PBMC M2e+CD4+T cells in male mice than that in female mice (Fig. 391). The underlying mechanism for the sex differences in circRNA-M2e-elicited T cell responses remains to be fully understood. To assess the immune-tolerability of pulmonary circRNA vaccine in C57BL / 6 mice, immune-tolerabilit -associated chemokines from serum and bronchoalveolar lavage (BAL) were measured by Luminex 12 hours and 24 hours after the third dose (Fig. 39J and Fig. 39K). circRNA induced the lowest systemic and local chemokine production. Further, serum levels of proinflammatorv cytokines TNF-a, IFN-y, IL-6, IL-12p40, and IL-12p70 were increased 12 h after circRNA administration, suggesting increased homing and activation of immune cells. Moreover, in BAL, these proinflammatory factors did not increase significantly, indicating that the pulmonary circRNA vaccine did not cause local inflammatory responses, which can avoid immune-related adverse effects associated with overly strong respiratory inflammation. As a result, pulmonary circRNA significantly protected mice from a lethal challenge of mouse-adapted H1N1 influenza A / Puerto Rico / 8 / 34 (PR8) virus [4 x LD50 (median lethal dose)] 4 weeks after the last immunization, with ~70% mouse survival and less body weight loss than controls (Fig. 39L and Fig. 39M). These results demonstrate the use of pulmonary circRNA vaccines, including circRNA-M2e as a universal flu vaccine.REFERENCES

[0185] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. 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Claims

Docket No. 45500.601ClaimsWhat is claimed:

1. A composition for inducing an immune response comprising a small circular ribonucleic acid (circRNA) molecule encoding at least one antigen or an immunogenic peptide fragment thereof, wherein tire composition is self-adjuvanted and elicits a T-cell immune response.

2. The composition of claim 1, wherein the antigen is selected from a protein, polypeptide, peptide, or fragment thereof derived from a pathogen, tumor-associated antigen, or autoantigen.

3. The composition of claim 1 or 2, wherein the immunogenic peptide fragment comprises a T-cell epitope or a multivalent peptide sequence derived from an antigen of a pathogen, tumor-associated antigen, or autoantigen.

4. The composition of claim 3, wherein the T-cell epitope is optionally an MHC class I- restricted peptide epitope or a MHC class II- restricted peptide epitope.

5. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or salt thereof.

6. A method of modulating an immune response in a subject, comprising administering to the subject a composition comprising a small circular ribonucleic acid (circRNA) molecule encoding an antigen or immunogenic peptide fragment thereof, wherein the circRNA is translated in a host cell to produce multivalent peptide antigens and activate antigen-specific T cells.

7. The method of claim 6, wherein administration of the composition enhances antigen processing and presentation by antigen-presenting cells.

8. The method of claim 6, wherein the composition enhances proteolytic degradation of the antigen.

9. The method of claim 6, wherein the composition enhances adaptive immune responses.

10. The method of claim 6, wherein the composition improves immunomodulatory efficacy relative to an unmodified antigen or a linear mRNA vaccine.

11. The method of claim 6, wherein the administration of the composition induces a stronger and longer- lasting T-cell immune response relative to a linear RNA or peptide vaccine encoding the same antigen.

12. The method of claim 6. wherein the composition is a nucleic acid vaccine, optionally delivered as a protein-encoding RNA, or a derivative thereof.Docket No. 45500.60113. The method of claim 6, wherein the composition is administered as a cancer immunotherapy, a prophylactic vaccine, or a therapeutic vaccine.

14. The method of claim 6, wherein the composition is used for prevention or treatment of a viral infection, microbial infection, cancer, autoimmune disorder, or derivative thereof.

15. The method of claim 13, wherein the prophylactic vaccine is for the prevention of a microbial infection, a viral infection, cancer, an autoimmune disease or any derivative thereof.

16. The method of claim 13, wherein the therapeutic vaccine is for the treatment of a microbial infection, a viral infection, cancer, an autoimmune disease, or any derivative thereof.

17. Use of a composition comprising any one of claims 1-5.

18. Use of a composition comprising any one of claims 1-5, for the prevention of a disease in a subject.

19. Use of a composition comprising any one of claims 1-5, for the treatment of a disease in a subject.

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