Circular RNA vaccine platform for efficient protein expression in immune cells and application thereof

Abstract

The application provides a novel IRES sequence and variants thereof, untranslated region sequences suitable for circular RNA. A circular RNA drug comprising the above IRES sequence, untranslated region sequence, and protein coding sequence is provided. The circular RNA drug platform of the application has high protein translation efficiency (particularly in immune cells), a compact sequence length, compatibility with a variety of protein sequences, and drugs based on the circular RNA platform exhibit good therapeutic effects in animal tumor models.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a novel IRES sequence, a circular RNA drug platform comprising the novel IRES sequence and applications thereof. BACKGROUND

[0002] Current mRNA drugs mainly adopt the form of linear mRNA with 5' end cap structure and polyA tail. In the past decades, the adoption of co-transcriptional or enzymatic capping, 1-methyl-pseudouridine (m1Ψ) modification, 5' and 3' untranslated region optimization (UTR), LNP delivery and other technologies has improved the translation efficiency and stability of mRNA drugs, and thus greatly improved the drugability of mRNA drugs. The widespread use of mRNA drugs has shown the great potential of mRNA drugs. At the same time, emerging RNA drug platforms, self-replicating RNA and circular RNA, have also attracted more and more attention.

[0003] Circular RNA is a single-stranded closed loop RNA, and its biggest feature is that it has no end, so it is more stable than linear RNA. In recent years, there have been several preclinical studies using circular RNA for the prevention of new coronal virus and the treatment of tumors, and the results of these studies show that circular RNA has excellent efficacy. Unlike linear mRNA which relies on 5' cap to initiate translation, circular RNA mainly relies on internal ribosome entry site (IRES) to initiate translation. The IRES of circular RNA determines the expression level of circular RNA drug, so the screening and optimization of it is a crucial step in the development of circular mRNA drug. In 2018, Wesselhoeft et al. compared the translation efficiency of several viral IRES and inferred endogenous IRES in circular RNA, and screened the IRES from CVB3 virus which had the best performance in circular RNA. In 2021, Jiali Yang et al. screened the IRES EV29 which had higher efficiency than CVB3. A larger scale screening and modification study was published by Chen et al. in 2023, they used a modular construction platform to compare the efficiency of dozens of IRES, and screened the more efficient HRV-B3, which improved the expression level of circular RNA by several times.

[0004] Although previous studies have screened a large number of critical translation elements of circular RNAs, these screenings have not considered the cell and tissue preference of IRES and have not been performed for specific cells and tissues. For example, the development of circular RNAs as vaccines requires efficient expression in immune cells, particularly professional antigen-presenting cells such as dendritic cells (DCs), to achieve optimal immune activation. In addition, the translation initiation of viral IRES is achieved by recruiting different translation initiation factors and IRES trans-acting factors (ITAFs) through multiple domains therein. Therefore, the modification of the structure of IRES can further enhance the translation efficiency of IRES. However, previous studies have not analyzed the secondary structure of IRES and have not systematically optimized IRES in combination with the secondary structure. In summary, screening IRES with more efficient translation in specific cells or tissues and targeted optimization in combination with RNA secondary structure analysis have the potential to greatly improve the expression effect of circular RNAs, and thus achieve reduced required dosing or improved efficacy at the same dose. SUMMARY

[0005] To solve the above technical problems, the present application designs a circular RNA drug platform with higher expression level in immune cells than existing circular RNA designs by screening and mutation of the translation element IRES, which can efficiently express protein drugs or vaccines in immune cells, thereby achieving stronger immune and therapeutic effects. Specifically,

[0006] The first aspect of the present application provides an internal ribosome entry site IRES, which comprises a nucleotide sequence shown in SEQ ID NO: 1 or a variant sequence thereof, the variant sequence retaining equivalent or superior initiation translation function to SEQ ID NO: 1.

[0007] In some embodiments, the IRES has tissue or cell preference; preferably, the cell is an immune cell.

[0008] In some embodiments, the variant sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1.

[0009] In some embodiments, the variant comprises a nucleotide sequence shown in any one of SEQ ID NOs: 2-4.

[0010] The second aspect of the present application provides use of the IRES of the first aspect of the present application in the preparation of a circular RNA.

[0011] The third aspect of the present application provides a combination of the IRES and polyA50 according to the first aspect of the present application for use in the preparation of a circular RNA; wherein the polyA50 is as shown in SEQ ID NO: 16.

[0012] The fourth aspect of the present application provides a circular RNA comprising the IRES according to the first aspect of the present application, a protein coding sequence of interest and an untranslated region sequence.

[0013] In some embodiments, the circular RNA further comprises a circularization residual sequence.

[0014] In some embodiments, the circular RNA does not comprise a circularization residual sequence.

[0015] In some embodiments, wherein the protein of interest is selected from the group consisting of tumor antigens, infectious disease antigens, chimeric antigen receptors (CARs), TCR complexes, antibodies, cytokines, cas proteins and other therapeutic proteins.

[0016] In some embodiments, wherein the tumor antigen comprises a cervical cancer antigen, a melanoma antigen, a nasopharyngeal carcinoma antigen and / or a lung cancer antigen; preferably, the circular RNA has a nucleotide sequence as shown in any one of SEQ ID NOs: 27, 29, 31.

[0017] In some embodiments, the infectious disease antigen comprises a novel coronavirus, an influenza virus, an RSV, an HMPV, a rabies virus, an HIV virus antigen.

[0018] In some embodiments, wherein the untranslated region sequence is polyA50, the sequence of which is as shown in SEQ ID NO: 16.

[0019] The fifth aspect of the present application provides a pharmaceutical composition comprising the circular RNA according to the fourth aspect of the present application and a delivery vehicle; preferably, the delivery vehicle comprises a nano-lipid particle (LNP), a virus-like particle (VLP) and / or a polymeric nanoparticle.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1) The EV-A IRES screened in this application not only has higher translation efficiency than the reported CVB3, HRV-B3 IRES in human embryonic kidney cell line HEK293T, but also has the highest translation efficiency in mouse dendritic cell line DC2.4 and human monocyte cell line THP-1. The translation efficiency of the modified EV-A IRES mutant is further improved by 50% compared with the wild-type EV-A IRES, and the sequence length is reduced by 17%-21% compared with the wild-type (750nt for wild-type, 618nt for EV-A-S1 / S3, 592nt for EV-A-S2). The shorter IRES sequence means that the ability of the circular RNA platform to load sequences is improved. EV-A IRES and its mutants can be used for circular RNA drug platforms.

[0022] 2) The polyA50 screened as the untranslated region sequence of the circular RNA drug platform can achieve the highest translation efficiency.

[0023] 3) The various tumor antigen drugs encoded by the circular RNA drug platform induced high levels of specific immune responses in mice, and showed better tumor prevention and treatment effects than traditional linear mRNA. BRIEF DESCRIPTION OF DRAWINGS

[0024] Other features, objects, and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments, when read in conjunction with the accompanying drawings:

[0025] Figure 1 A schematic diagram showing the design of the circular RNA drug platform. The circular RNA drug platform mainly consists of IRES sequence (using EV-A IRES or its mutants EV-A-S1, EV-A-S2, EV-A-S3), untranslated region sequence, and protein coding sequence of the desired drug, and their relative positions are shown in the figure. The yellow part in the figure is the unnecessary circularization residual sequence, which does not exist in some RNA circularization technology design.

[0026] Figure 2 High-efficiency translation IRES screening. (A) High-efficiency translation IRES screening in HEK239T and DC2.4 cells using dual fluorescence reporter plasmid technology. (B), (C) Screening of high-efficiency IRES in HEK239T and DC2.4 cells using synthetic circular FLUC RNA. The left panel shows a schematic diagram of the RNA circularization method. (D), (E) Comparison of IRES translation efficiency in THP-1, HEK239T, and DC2.4 using synthetic circular EGFP RNA.

[0027] Figure 3Figure 1 shows the EVA IRES secondary structure analysis and sequence optimization. (A) The shape reactivity and predicted secondary structure of EV-A IRES based on reactivity data, the red dashed box indicates the truncation position of the truncated mutant, and the blue dashed box indicates the mutation position of the multi-point mutant. (B), (C), (D) Translation efficiency comparison of EV-A mutants and wild type. (E) The shape reactivity and predicted secondary structure of EV-A-S1 based on reactivity data.

[0028] Figure 4 Figure 2 shows the untranslated region sequence screening. (A) Untranslated region sequence position schematic diagram and translation efficiency comparison of Spacer sequence. (B) Translation efficiency comparison of 3UTR sequence. Wherein, control refers to the control without inserting sequence at 3'UTR and Spacer position.

[0029] Figure 5 Figure 3 shows the tumor prevention effect of the circular RNA vaccine. (A) Circular OVA vaccine design and mouse treatment time points. (B) The proportion of T cells positive for MHC-I tetramer of OVA antigen peptide (SIINFEKL). (C) The proportion of initial T cells (T), central memory T cells (Central memory T), effector / effector memory T cells (Effector / Effector memory T) in peripheral blood mononuclear cells (PBMC) of mice. (D) Tumor volume of mice. (E) Tumor volume of mice in each group. (F) Survival curve of mice. Wherein, Control group refers to the injection of liposomes without mRNA.

[0030] Figure 6 Figure 4 shows the tumor treatment effect of the circular RNA B16F10 neoantigen vaccine. (A) Circular B16 vaccine design and antigen sequence list. (B) Mouse treatment time points. (C) The proportion of IFNγ+ T cells in spleen lymphocytes stimulated by circular B16 mRNA. (D) Tumor volume of mice. (E) Tumor volume of mice in each group. Wherein, Control group refers to the injection of liposomes without mRNA.

[0031] Figure 7 Figure 5 shows the tumor treatment effect of the circular RNA HPV E6E7 antigen vaccine. (A) Circular E6E7 vaccine design and mouse treatment time points. (B) The proportion of IFNγ+ T cells in spleen lymphocytes stimulated by HPV E6, E7 antigen peptide. (C) Tumor volume of mice. (D) Survival curve of mice. (E) Tumor volume of mice in each group. Wherein, Control group refers to the injection of liposomes without mRNA. DETAILED DESCRIPTION

[0032] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort belong to the scope of protection of the present application.

[0033] Unless otherwise defined, the technical terms or scientific terms used herein should be understood as the usual meanings understood by those of ordinary skill in the art to which the present application belongs.

[0034] Example 1 Screening of efficient IRES

[0035] Cyclic RNAs cannot undergo 5' cap-dependent translation initiation, but use IRES or m6A modification to initiate translation. In order to improve the expression level of cyclic RNA, especially in immune cells, to be suitable for tumor vaccine, the present application first screened IRES with high translation level in immune cells.

[0036] Previous studies have shown that viral IRES has the highest translation efficiency on cyclic mRNA, so most of the candidate IRESs of the present application are derived from viruses. A total of 29 IRESs were screened, including 22 viral type IRESs, 4 viral type II or type III IRESs and 3 IRESs derived from mammalian cells (A). First, the non-cap-dependent translation efficiency of these IRESs in HEK293T and mouse dendritic cell line DC2.4 was compared using dual luciferase reporter plasmids (B). Figure 2 A). First, the non-cap-dependent translation efficiency of these IRESs in HEK293T and mouse dendritic cell line DC2.4 was compared using dual luciferase reporter plasmids (B). Figure 2A) The method was as follows: The IRES sequence was chemically synthesized by Genewiz and cloned into the dual-luciferase reporter plasmid pRF, located between the RLUC and FLUC coding sequences. 100 ng of dual-luciferase reporter plasmids containing different IRES were transfected into 10,000 HEK293T or DC2.4 cells in 100 μL wells of a 96-well plate using Hieff Trans liposome transfection reagent (Yisheng Biotechnology, 40802ES01). After 24 hours, the cells were lysed, and fluorescence values ​​were detected using a dual-luciferase reporter gene assay kit (Yisheng Biotechnology, catalog number 11402ES60) according to the manufacturer's instructions. The FLUC / RLUC ratio was considered as IRES activity. Consistent with previous studies, type I IRES generally exhibited higher translation efficiency compared to other types and mammalian IRES. As the inventors anticipated, the translation efficiency of IRES showed intercellular variability. For example, the translation efficiency of human rhinovirus HRV-C20 in HEK293T is about twice that of CVB3, but only 20% of that in DC2.4. The results of this round of screening show that the IRES of human enterovirus EV-A has the highest translation efficiency in both cell lines.

[0037] To further verify the performance of highly efficient IRES on circular RNA, the seven IRES with the highest expression levels were constructed into circular RNA. Using the previously reported PIE circularization method (Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation ineukaryotic cells. Nat Commun. 2018; 9(1):2629. doi:10.1038 / s41467-018-05096-6), circular firefly luciferase FLUC mRNA was synthesized. Figure 2 B). The expression of these IRES in circular RNA differs from that in dual-luciferase reporter systems, possibly due to unintended splicing of the reporter plasmid, resulting in incomplete RNA production, or differences in the spatial structure of IRES in circular RNA compared to linear RNA. However, regardless of whether on dual-luciferase plasmids or circular RNA, IRES from Enterovirus A (EV-A) exhibit the highest relative translation efficiency. Figure 2 B). Previous studies have reported that IRES of human rhinovirus HRVB3 and Coxsackievirus CVB3 also have very good translation efficiency, but in HEK293T and DC2.4 ( Figure 2C) all of which are inferior to EV-A IRES. To investigate the adaptability of EV-A IRES to different coding sequences, we constructed a circular enhanced green fluorescent protein (EGFP) mRNA and compared the EGFP expression levels in HEK293T, DC2.4 and human monocytic cell line THP-1 Figure 2 D, Figure 2 E). The results showed that EV-A IRES still had the highest translation efficiency. The above results show that EV-A IRES is an IRES suitable for different coding sequences and has high translation efficiency in conventional cell lines, mice and human immune cell lines, and is an excellent circular RNA translation element.

[0038] Example 2 Secondary structure analysis and sequence optimization of EV-A IRES

[0039] Previous studies have shown that the five domains of viral type I IRES recruit different translation initiation factors and ITAFs to initiate translation. Among them, domain V directly interacts with eIF4G and eIF4A to participate in the assembly of the 48S ribosome complex, domains II and IV are responsible for recruiting ITAFs such as hnRNP A1 and PCBP1 / 2 to promote IRES structure stability and initiation factor recruitment, ribosome scanning occurs in domain VI, and there are fewer reports about the function of domain III. The inventors believe that mutations and simplification of each domain of EV-A IRES may stabilize the structure of the IRES and thus improve the ability of EV-A IRES to initiate translation, or modify it into a shorter IRES. Therefore, the inventors first analyzed the structure of EV-A IRES using SHAPE-MaP technology, determined the internal structure distribution of EV-A, and then designed a series of mutations and simplifications and compared their translation efficiencies.

[0040] According to the secondary structure model of EV-A IRES based on SHAPE-MaP reactivity, this IRES also has the typical secondary structure of a type I IRES, including the common domains I to VI Figure 3 A). However, a unique stem-loop structure is present in EV-A IRES, located after DVI, which is named DVII. According to previous reports, DII, DIV and DV are relatively conserved and have important functions; DIII is not well defined in function but is relatively conserved in sequence, and DVI is relatively variable; DI is mainly involved in viral genome replication rather than translation. Based on this, the optimization strategy of the present application includes mutating important domains and deleting non-critical sequences.

[0041] The method for optimizing important domains of the application is to improve the GC pairing ratio of the longer stem to improve the stability of the secondary structure without changing the secondary structure and without changing the sequence of the important protein (such as PCBP1 / 2, eIF4G) binding region. According to this strategy, six mutants of EV-A were designed and constructed into a circular GLUC mRNA, and the GLUC signal level was detected by transfecting HEK293T cells to compare the translation efficiency of the mutant IRES Figure 3 B). However, compared with the wild type EV-A, these mutations did not significantly improve the translation efficiency, among which the 4m2, 4m3 mutations of DIV and the 5m1 mutation of DV had no obvious change on the translation efficiency, and the remaining mutants, 2m1 of DII, 4m1 of DIV and 5m2 of DV, greatly reduced the translation efficiency of EV A. This result shows that in addition to the secondary structure, the base composition of these regions is also crucial to the translation efficiency of IRES.

[0042] On the other hand, the application further truncates the DI (90 nt) of EV-A, the Linker (30 nt) between DI and DII, DVI (42 nt), and DVII (32 nt) in turn Figure 3 A), the fluorescence signal results show that deleting DI, DVI, and DVII has no effect on the translation function of IRES, but the deletion of Linker significantly reduces the translation efficiency Figure 3 C). Based on the above results, the inventors decided to combine the truncated DI, DVI and DVII which do not affect the function, and the 5m1 mutation which slightly improves the translation efficiency. The combination results show that the truncated EV-A-S1 (DI and DVI are truncated at the same time) and the truncated EV-A-S2 (DI, DVI and DVII are truncated at the same time) both significantly improve the translation efficiency of EV A by about 50%, while the truncated EV-A-S3 (5m1 mutation based on EV-A-S1) does not further improve the translation efficiency Figure 3 D). The inventors guess that this result may be that the truncation of non-core regions does not affect the core domain, and after truncating multiple non-functional structures, the IRES structure is more single and stable. In order to verify this guess, the inventors further analyzed the IRES structure of DI and DII truncated at the same time, and the SHAPE-MaP activity results show that the signal of the core region of the truncated EV-A-S1 is almost completely consistent with that of the wild type IRES Figure 3 E). By comparing the predicted secondary structures of EV-A-S1 and wild type EV-A, it can be seen that the core structure of EV-A-S1 is perfectly preserved, the deleted structure disappears, and the small stem loop structure next to DII and the DVII structure which are not deleted also disappear Figure 3E). This result supports the inventors' hypothesis that the deletion of multiple non-essential structures indeed makes the IRES more compact and stable. In summary, after the SHAPE-MaP structure-based modification, the modified EV-A IRES is not only more compact (750 nt shortened to EV-A-S1 / S3 618 nt / EV-A-S2 592 nt), but also has significantly improved translation efficiency.

[0043] Example 3 Untranslated region sequence screening

[0044] Although the translation of the circular RNA is mediated by the IRES, the translation efficiency of the circular RNA and the expression amount of the target protein are also significantly affected by other sequences in the design of the circular RNA. For example, the residual sequence in the PIE method circularization process can inhibit the translation of the circular RNA, and the addition of a spacer sequence at both ends of the residual sequence can reduce this inhibition. Similar to the 5' and 3' untranslated regions (UTRs) of the classic linear mRNA, the spacer sequence at both ends of the residual sequence can have different effects on the translation and stability of the circular RNA due to the different relative positions of the IRES and the CDS. For convenience of distinction, the 5' spacer sequence of the IRES and the 3' spacer sequence of the CDS are referred to as the 5' Spacer and the 3' UTR of the circular RNA, respectively, and are screened separately. Figure 4 A).

[0045] The screening of the UTR sequence was also performed using the circular GLUC mRNA. For the Spacer sequence, Apt-EIF4G, β-globin 5' UTR, polyA50 were compared. The results showed that polyA50 as the 5' Spacer had the highest GLUC signal ( Figure 4 A). For the 3' UTR sequence, the screening included HBA1 3' UTR, polyAC, polyA50, β-globin 3' UTR. In addition, considering that the 3' UTR sequence of the virus can also be involved in the translation of the viral RNA, the screening also included the 3' UTR of the CVB3, EVA, HRV-B3, SINV virus. The results showed that the best spacer sequence for the circular 3' UTR position was still polyA50 ( Figure 4 B). In summary, the polyA50 sequence can best promote the function of the IRES whether it is used as the 5' Spacer or the 3' UTR of the circular RNA, which can be due to the simple spatial structure of the polyA sequence and the ability to recruit PABP, promoting the interaction of the IRES with the translation initiation factor eIF4G.

[0046] Example 4 Design and application of circular RNA drug platform

[0047] The circular RNA drug platform uses EV-A-S1 IRES as the translation initiation sequence and the polyA50 sequence as the untranslated region sequence. It then places the coding sequences for ovalbumin (OVA), mouse melanoma tandem antigen B16-8, and human cervical cancer virus (HPV) antigen E6E7 after the IRES to design three circular RNA vaccines: circular OVA, circular B16-8, and circular E6E7. Figure 5 A, 6A, 7A). These three circular RNA vaccines were produced using the PIE circularization method reported in the literature (Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018; 9(1):2629. doi:10.1038 / s41467-018-05096-6) and applied in a mouse tumor model.

[0048] 4.1 Template plasmid construction

[0049] The pUC57 plasmid was inserted with the T7 promoter, the Anabaena 2.0 PIE element, IRES, and protein-coding sequence reported by Wesselhoeft et al. (Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation ineukaryotic cells. Nat Commun. 2018; 9(1):2629. doi:10.1038 / s41467-018-05096-6) and used as a template plasmid for circular mRNA. All linear mRNA templates were constructed using the Takara Bio mRNA Template Cloning Kit (Baori Medical, catalog number 6143). The template plasmid in this kit contained the T7 promoter, human β-globin 5'UTR, human HBA1 3'UTR, and a 105 nt long poly(A) sequence. DNA fragments were synthesized by Genewiz and amplified by PCR. Seamless cloning was performed using the Novizan ClonExpress II One-Step Cloning Kit.

[0050] 4.2 Linear and Circular RNA Synthesis and Packaging

[0051] In this invention, linear mRNA was prepared using NEB's HindIII endonuclease to linearize plasmid as template, and in vitro transcription was performed using Norgen's T7 High Yield RNA Synthesis Kit (Norgen Biotek, Cat# 10673ES50). Cap1 cap was added to mRNA during co-transcription, and mRNA with 105 nt long polyA tail was transcribed. After incubation of reaction mixture at 37℃ for 2 hours, DNase I was used to treat at 37℃ for 20 minutes, and then purified using Thermo Scientific's GeneJET RNA Purification Kit, and analyzed by 2% agarose gel.

[0052] For circular mRNA, in vitro transcription was performed using PCR amplified template, and Norgen's T7 High Yield RNA Synthesis Kit was used. Reaction mixture was incubated at 37℃ overnight (about 16 hours) to achieve maximum spontaneous circularization, and then treated with DNase I at 37℃ for 20 minutes. Then 1x T4 RNA Ligase Reaction Buffer and 2 mM (final concentration) GTP were added, and incubated at 55℃ for 15 minutes for further circularization. After RNA was purified by GeneJET RNA Purification Kit, it was heated at 65℃ for 3 minutes. 20 U RNase R (Bioteke) and 1x RNase R Reaction Buffer were added per 20 μg purified RNA, and incubated at 37℃ for 2 hours. Finally, circular RNA was purified by GeneJET RNA Purification Kit, and analyzed by 4% polyacrylamide gel or 2% agarose gel.

[0053] Purified and qualified circular and linear RNA were packaged into RNA lipoplex (LPX) for animal administration using Polyplus' in vivo-jetRNA Delivery Reagent, following the manufacturer's instruction. In a clean bench, RNA was diluted to 50 μg / ml with mRNA buffer, and then 2 μl / μg of in vivo-jetRNA reagent was added. After gentle mixing, the mixture was incubated at room temperature for 15 minutes before use for animal administration.

[0054] 4.3 Tumor prevention effect of circular RNA vaccine

[0055] To verify the tumor prevention effect of the circular RNA platform designed in this invention as a tumor vaccine, OVA was chosen as a model antigen, and B16F10 stable cell line expressing OVA was constructed. Three days before subcutaneous inoculation of B16F10 OVA tumor cells, mice were inoculated with three injections of vaccine by intravenous injection, and blood was taken on the third day before tumor inoculation to detect T cells in PBMCs. Figure 5A) After the third vaccination, flow cytometry analysis showed that the proportion of T cells positive for three groups of OVA antigen peptides (SIINFEKL) MHC tetramer given by the vaccine was significantly higher than that of the untreated group, indicating that the three groups of RNA vaccines all successfully induced OVA-specific T cells Figure 5 B) Among them, CircOVA and uracil-modified linear m1ΨLinearOVA induced a higher proportion of OVA-specific T cells than unmodified linear LinearOVA. Further analysis of the subtypes of all T cells in PBMCs found that the proportion of T cells in the CircOVA and LinearOVA groups was significantly lower than that in the untreated group, while the m1ΨLinearOVA group had no significant difference from the untreated group Figure 5 C) Analysis of the proportion of Central memory T cells also showed that only the CircOVA and Linear OVA groups had a significantly higher proportion. While the proportion of effector T cells in the three vaccine groups was significantly higher than that in the untreated group Figure 5 C) These results may show that CircOVA and LinearOVA induce a more significant overall immune response due to recognition by pattern recognition receptors of immune cells, while m1ΨLinearOVA does not significantly change the proportion of T cell subgroups due to its lower immunogenicity. Ten days after the third vaccination, mice were subcutaneously inoculated with B16F10 OVA tumor cells, and tumor formation was subsequently monitored for 60 days after tumor inoculation. All mice in the three vaccine groups did not form tumors within 60 days of monitoring (CircOVA: 4 / 5, m1ΨLinearOVA: 3 / 5, LinearOVA: 2 / 5), especially in the CircOVA group, 80% of the mice did not form tumors, while all mice in the untreated group formed tumors in about two weeks Figure 5 E) On the 24th day of tumor inoculation, the tumor size of mice in the three vaccine groups was significantly smaller than that in the untreated group Figure 5 D) The tumor size of mice in the untreated group reached the humane endpoint. The above results show that the circular RNA vaccine designed by the present application shows excellent immune activation ability, similar T cell induction ability to modified linear mRNA, and better tumor prevention effect.

[0056] 4.4 Tumor treatment effect of circular RNA tumor neoantigen and tumor-associated antigen vaccine

[0057] OVA is a very immunogenic antigen, so the B16F10-OVA model may not truly reflect the tumor treatment scenario. Tumor mutation neoantigens and virus-derived tumor-associated antigens are common targets in tumor vaccine design, so a tandem antigen vaccine encoding eight B16F10 tumor mutation antigens reported in the literature was also designed​Figure 6 A) and HPV E6E7 fusion protein vaccine (B) respectively for treating B16F10 and TC-1 tumor models. Figure 7 A), respectively for treating B16F10 and TC-1 tumor models.

[0058] Three B16-8 tandem antigen vaccines were used to immunize mice on day 3 after subcutaneous inoculation of B16F10 cells, and a booster was given on day 10, and the tumor size was continuously detected until day 21 (A) and (B). On day 21, the mice were euthanized and the spleens were collected, and the proportion of IFNγ+CD8 T cells in the spleen lymphocytes after stimulation with mutant antigen mRNA was detected to detect the proportion of vaccine-induced mutant antigen-specific T cells. The results showed that the three vaccine groups all induced significant IFNγ+T cells, and the average proportion of IFNγ+T cells in the CircB16-8 group was the highest (C). The results of tumor monitoring also showed that CircB16-8 and m1ΨLinearB16-8 both significantly inhibited tumor growth (D, E). Figure 6 B) and (C). The results of tumor monitoring also showed that CircB16-8 and m1ΨLinearB16-8 both significantly inhibited tumor growth (D, E). Figure 6 C) and (D, E). Figure 6

[0059] TC-1 is a cell line stably expressing HPV oncogenes E6 and E7, and is a commonly used HPV-related tumor model. To test the therapeutic effect of the HPV tumor vaccine on the already formed tumor, mRNA vaccines encoding HPV E6 / E7 fusion proteins were intravenously injected into mice 14 days after subcutaneous inoculation of TC-1 cells, and blood was collected 7 days after the second vaccine (A). Flow cytometry analysis of PBMCs showed that the CircE6E7 vaccine induced the highest proportion of antigen-specific IFNγ-secreting T cells in the mouse PBMCs, and the m1ΨLinear E6E7 vaccine also successfully induced moderate levels of antigen-specific IFNγ-secreting T cells, while the proportion of specific T cells in the Linear E6E7 group was higher than that in the control group, but the difference was not significant (B). Consistent with the induction of T cells, both the CircHPV and m1ΨLinearHPV vaccines successfully eliminated the tumor, and the tumor growth in the LinearHPV group was also inhibited (C, D, E). These results show that the circular mRNA encoding mutant antigens and HPV antigen vaccines of the present application induce stronger tumor antigen-specific T cell immunity in mice compared to conventional linear mRNA, and exhibit excellent tumor treatment effect. Figure 7 B) and (C). The results of tumor monitoring also showed that CircB16-8 and m1ΨLinearB16-8 both significantly inhibited tumor growth (D, E). Figure 7 B) and (C). The results of tumor monitoring also showed that CircB16-8 and m1ΨLinearB16-8 both significantly inhibited tumor growth (D, E). Figure 7 C, D, E) These results show that the circular mRNA encoding mutant antigens and HPV antigen vaccines of the present application induce stronger tumor antigen-specific T cell immunity in mice compared to conventional linear mRNA, and exhibit excellent tumor treatment effect.

[0060] The sequences involved in the above examples of the present application are shown in Table 1.

[0061] Table 1 Nucleotide and amino acid sequence list

[0062] ​

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] The foregoing presents and describes the basic principles and main features of the present application and the advantages thereof. It is obvious to a person skilled in the art that the present application is not limited to the details of the above-described exemplary embodiments, but that it can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, the scope of the present application being defined by the appended claims rather than the above description, and it is therefore intended that all changes and modifications that fall within the meaning and range of equivalency of the claims be embraced therein. Any reference signs in the claims should not be construed as limiting the claims to the figures in which the reference signs are used.

[0073] Furthermore, it should be understood that although the present specification has been described in accordance with embodiments, not every embodiment can include every feature described. The description is intended to cover all possible combinations of the features described. The specification has been presented for purposes of illustration to describe embodiments. It is not intended to be exhaustive or to limit the application to the precise forms disclosed. Many variations and modifications can be possible in light of the above teachings.

Claims

1. An internal ribosome entry site (IRES) characterized in that, The IRES is a nucleotide sequence as shown in any one of SEQ ID NO: 2-4.

2. Use of the IRES of claim 1 in preparing a circular RNA.

3. Use of the combination of the IRES of claim 1 and polyA50 in preparing a circular RNA; wherein the polyA50 is as shown in SEQ ID NO:

16.

4. A circular RNA, characterized in that, comprising the IRES of claim 1, a protein coding sequence of interest, and an untranslated region sequence.

5. The circular RNA of claim 4, further comprising a circularization residual sequence.

6. The circular RNA of claim 4, wherein the protein of interest is selected from the group consisting of a tumor antigen, an infectious disease antigen, a chimeric antigen receptor (CAR), a TCR complex, an antibody, a cytokine, a cas protein, and other therapeutic proteins.

7. The circular RNA of claim 6, wherein the tumor antigen comprises a cervical cancer antigen, a melanoma antigen, a nasopharyngeal carcinoma antigen, and / or a lung cancer antigen.

8. The circular RNA of claim 7, wherein the circular RNA has a nucleotide sequence as shown in any one of SEQ ID NO: 27, 29, 31.

9. The circular RNA of any one of claims 4-8, wherein the untranslated region sequence is polyA50, and the sequence is as shown in SEQ ID NO:

16.

10. A pharmaceutical composition, characterized by, comprising the circular RNA of any one of claims 4-9 and a delivery vehicle.

11. The composition of claim 10, wherein the delivery vehicle is a nano-lipid particle (LNP), a virus-like particle (VLP), or a polymeric nanoparticle.

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