Annular RNA (Ribonucleic Acid) vaccine platform for high-efficiency protein expression of immune cells and application of annular RNA vaccine platform

Abstract

The invention provides a novel IRES sequence suitable for circular RNA and a variant and an untranslated region sequence of the IRES sequence. The invention provides a circular RNA drug containing the IRES sequence, the untranslated region sequence and the protein coding sequence. The circular RNA drug platform has efficient protein translation ability (especially in immune cells), simplified sequence length and compatibility to various protein sequences, and the drug based on the circular RNA platform shows a good treatment effect in an animal tumor model.

Description

Technical Field

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

[0002] Current mRNA drugs mainly adopt the most classic form of linear mRNA with a 5' end cap structure and a polyadenylic acid tail (polyA). In the past few 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 have improved the translation efficiency and stability of mRNA drugs, thereby greatly improving the drugability of mRNA drugs. The widespread use of mRNA drugs has made people see the huge potential of mRNA drugs. At the same time, emerging RNA drug platforms, self-replicating RNA and circular RNA, are also attracting more and more attention.

[0003] Circular RNA is a single-stranded closed circular RNA. Its biggest feature is that it has no ends, so it is more stable than linear RNA. In recent years, many preclinical studies have used circular RNA for the prevention of new coronavirus and the treatment of tumors. The results of these studies show that circular RNA has excellent therapeutic effects. Unlike linear mRNA, which relies on the 5' cap to initiate translation, circular RNA mainly relies on the internal ribosome entry site (IRES) to initiate translation. The IRES of circular RNA determines the expression level of circular RNA drugs, so its screening and optimization is a crucial step in the development of circular mRNA drugs. In 2018, Wesselhoeft et al. compared the translation efficiency of several viral IRES and inferred endogenous cellular IRES on circular RNA, and screened out that the IRES from the CVB3 virus had the best performance on circular RNA. In 2021, Jiali Yang et al. screened IRES EV29, which is more efficient 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 out the more efficient HRV-B3, which increased the expression level of circular RNA several times.

[0004] Although previous studies have screened a large number of key translation elements of circular RNA, these screens did not consider the cell and tissue preferences of IRES, and did not screen for specific cells and tissues. For example, the development of circular RNA as a vaccine requires effective expression in immune cells, especially professional antigen-presenting cells (such as dendritic cells (DCs)), to achieve the best immune activation effect. 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 in it. Therefore, modifying the structure of IRES may further enhance the translation efficiency of IRES. However, previous studies did not analyze the secondary structure of IRES, nor did they systematically optimize and modify IRES in combination with the secondary structure. In short, screening IRES that is more efficiently translated in specific cells or tissues, and combining RNA secondary structure analysis for targeted optimization, has the potential to greatly improve the expression effect of circular RNA, thereby reducing the required dosage or improving the efficacy at the same dose. Summary of the invention

[0005] To solve the above technical problems, the present invention designs a circular RNA drug platform with a higher expression level in immune cells than existing circular RNA designs through the screening and mutation modification 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] In a first aspect, the present invention provides an internal ribosome entry site (IRES), wherein the IRES comprises a nucleotide sequence as shown in SEQ ID NO: 1 or a variant sequence thereof, wherein the variant sequence retains a translation initiation function equivalent to or better than that of SEQ ID NO: 1.

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

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

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

[0010] The second aspect of the present invention provides use of the IRES described in the first aspect of the present invention in preparing circular RNA.

[0011] The third aspect of the present invention provides use of the combination of the IRES and polyA50 described in the first aspect of the present invention in preparing circular RNA; wherein the polyA50 is as shown in SEQ ID NO:16.

[0012] The fourth aspect of the present invention provides a circular RNA, which comprises the IRES described in the first aspect of the present invention, a target protein coding sequence and a non-translated region sequence.

[0013] In certain embodiments, the circular RNA further comprises a circularization remnant sequence.

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

[0015] In certain embodiments, the target protein is selected from tumor antigens, infectious disease antigens, chimeric antigen receptors (CARs), TCR complexes, antibodies, cytokines, cas proteins, and other therapeutic proteins.

[0016] In certain embodiments, 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 shown in any one of SEQ ID NOs: 27, 29, and 31.

[0017] In certain embodiments, the infectious disease antigens include novel coronavirus, influenza virus, RSV, HMPV, rabies virus, and HIV virus antigens.

[0018] In certain embodiments, the untranslated region sequence is polyA50, and the sequence is shown in SEQ ID NO:16.

[0019] The fifth aspect of the present invention provides a pharmaceutical composition, which includes the circular RNA described in the fourth aspect of the present invention and a delivery vector; preferably, the delivery vector includes nanolipid particles (LNP), virus-like particles (VLP) and / or polymer nanoparticles.

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

[0021] 1) The EV-A IRES screened by the present invention not only has a higher translation efficiency than the reported CVB3 and HRV-B3 IRES in the human embryonic kidney cell line HEK293T, but also has the highest translation efficiency in the mouse dendritic cell line DC2.4 and the human monocytic cell line THP-1. The translation efficiency of the modified EV-A IRES mutant is further increased by 50% relative to the wild-type EV-A IRES, and the sequence length is reduced by 17%-21% relative to the wild type (wild type 750nt, EV-A-S1 / S3 618nt, EV-A-S2592nt). 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 in the circular RNA drug platform.

[0022] 2) PolyA50 was screened as the untranslated region sequence of the circular RNA drug platform, which 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 THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1 The schematic diagram of circular RNA drug platform design is shown. The circular RNA drug platform mainly consists of three parts: IRES sequence (using EV-A IRES or its mutants EV-A-S1, EV-A-S2, EV-A-S3), untranslated region sequence, and target drug protein coding sequence. Their relative positions are shown in the figure. The yellow part in the figure is a non-essential cyclization residual sequence, which does not exist in some RNA cyclization technology designs.

[0026] Figure 2 Shows the screening of efficient translation IRES. (A) The dual fluorescence reporter plasmid technology was used to screen efficient translation IRES in HEK239T and DC2.4 cells. (B) and (C) Synthetic circular FLUC RNA was used to screen efficient IRES in HEK239T and DC2.4 cells. The left figure is a schematic diagram of the RNA circularization method. (D) and (E) Synthetic circular EGFP RNA was used to compare the IRES translation efficiency in THP-1, HEK239T and DC2.4.

[0027] Figure 3Shows EVA IRES secondary structure analysis and sequence optimization. (A) Chemical modification reactivity (Shape reactivity) of EV-A IRES and predicted secondary structure based on reactivity data. The red dotted box in the figure indicates the truncation position of the truncation mutant, and the blue dotted box indicates the mutation position of the multi-point mutant. (B), (C), (D) Comparison of translation efficiency between EV-A mutants and wild type. (E) Chemical modification reactivity (Shape reactivity) of EV-A-S1 and predicted secondary structure based on reactivity data.

[0028] Figure 4 Shows the screening of untranslated region sequences. (A) Schematic diagram of the location of untranslated region sequences and comparison of translation efficiency of spacer sequences. (B) Comparison of translation efficiency of 3'UTR sequences. Among them, control refers to the control without inserted sequences at the 3'UTR and spacer positions.

[0029] Figure 5 Showing the tumor prevention effect of circular RNA vaccine. (A) Circular OVA vaccine design and mouse treatment time points. (B) The proportion of T cells positive for OVA antigen peptide (SIINFEKL) MHC-I tetramer. (C) Naive T cells in mouse peripheral blood mononuclear cells (PBMC) ( (D) Tumor volume of mice. (E) Tumor volume of mice in each group. (F) Survival curve of mice. The control group refers to the group injected with liposomes without mRNA.

[0030] Figure 6 The tumor therapeutic effect of the circular RNA B16F10 neoantigen vaccine is shown. (A) Circular B16 vaccine design and antigen sequence list. (B) Mouse treatment time points. (C) Proportion of IFNγ+ T cells in spleen lymphocytes after circular B16 mRNA stimulation (D) Mouse tumor volume. (E) Tumor volume of mice in each group. Among them, the Control group refers to the group injected with liposomes without mRNA.

[0031] Figure 7 The tumor treatment effect of circular RNA HPV E6E7 antigen vaccine is shown. (A) Circular E6E7 vaccine design and mouse treatment time points. (B) The proportion of IFNγ+ T cells in spleen lymphocytes after stimulation with HPV E6 and E7 antigen peptides. (C) Mouse tumor volume. (D) Mouse survival curve. (E) Tumor volume of mice in each group. Among them, the Control group refers to the group injected with liposomes without mRNA. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0034] Example 1 Screening of Highly Efficient Translation IRES

[0035] Circular RNA cannot perform 5' cap-dependent translation initiation, but uses IRES or m6A modification to initiate translation. In order to increase the expression level of circular RNA, especially in immune cells, so as to be suitable for tumor vaccines, the present invention first screened IRES with high translation level in immune cells.

[0036] Previous studies have shown that viral IRES has the highest translation efficiency on circular mRNA, so most of the candidate IRES of the present invention are derived from viruses. A total of 29 IRES were screened, including 22 viral type I IRES, 4 viral type II or III IRES and 3 mammalian cell-derived IRES ( Figure 2 A). First, we compared the cap-independent translation efficiency of these IRESs in HEK293T and mouse dendritic cell line DC2.4 using dual-luciferase reporter plasmids ( Figure 2A), the method is: the IRES sequence was chemically synthesized by Jinweizhi and cloned into the dual luciferase reporter plasmid pRF, located between the RLUC and FLUC coding sequences. 100ng of dual fluorescence reporter plasmids containing different IRES were transfected into 10,000 HEK293T or DC2.4 cells in 100μL per well of a 96-well plate using Hieff Trans liposome transfection reagent (Yisheng Biological, 40802ES01). After 24 hours, the cells were lysed and the fluorescence value was detected using a dual luciferase reporter gene detection kit (Yisheng Biological, Cat. No. 11402ES60) according to the manufacturer's instructions. The ratio of FLUC / RLUC values ​​is considered to be IRES activity. Consistent with previous studies, type I IRES generally has stronger translation efficiency than other types and mammalian IRES. As expected by the inventors, the translation efficiency of IRES varies from cell to cell. For example, the translation efficiency of human rhinovirus HRV-C20 is about twice that of CVB3 in HEK293T, but only 20% of the latter in DC2.4. The results of this round of screening showed that the IRES of human enterovirus EV-A had the highest translation efficiency in both cell lines.

[0037] To further verify the performance of IRES with high translation efficiency on circular RNA, the seven IRES with the highest expression levels were constructed into circular RNA, and the circular firefly luciferase FLUC mRNA ( Figure 2 B). The expression of these IRES in circular RNA is different from that in the dual-luciferase reporter system. This may be due to the fact that the reporter plasmid may undergo unexpected shearing and fail to produce complete RNA, or the spatial structure of the IRES in circular RNA is different from that in linear RNA. However, whether on the dual-luciferase plasmid or on the circular RNA, the IRES from Enterovirus A (EV-A) of the Enterovirus genus has the highest relative translation efficiency ( Figure 2 B). The IRES of human rhinovirus HRVB3 and coxsackievirus CVB3 reported in previous studies also have very good translation efficiency, but in HEK293T and DC2.4 ( Figure 2C) are slightly inferior to the IRES of EV-A. In order to explore the adaptability of EV-A IRES to different coding sequences, the mRNA of the circular enhanced fluorescent protein EGFP was constructed, and the EGFP expression level was compared 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 that is suitable for different coding sequences and has high translation efficiency in conventional cell lines, mouse and human immune cell lines, and is an excellent circular RNA translation element.

[0038] Example 2 EVA IRES secondary structure analysis and sequence optimization

[0039] Previous studies have shown that the five domains of the viral type 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, and domains II and IV are responsible for recruiting ITAFs such as hnRNP A1 and PCBP1 / 2 to promote the stability of the IRES structure and the recruitment of initiation factors. Ribosome scanning occurs in domain VI, and there are relatively few reports on the function of domain III. The inventor believes that mutations and simplifications of various domains of EV-AIRES may stabilize the structure of IRES and thus improve the ability of EV-A IRES to initiate translation, or transform it into a shorter IRES. Therefore, the present invention first uses SHAPE-MaP technology to analyze the structure of EV-A IRES, and after determining the internal structure distribution of EV-A, a series of mutations and simplifications are designed in a targeted manner, and their translation efficiencies are compared.

[0040] According to the EV-A IRES secondary structure model based on SHAPE-MaP reactivity, this IRES also has a typical type I IRES secondary structure, including several common domains I to VI ( Figure 3 A). However, a unique stem-loop structure also appeared in EV-A IRES, located after DVI, which was named DVII. According to previous reports, the three domains of DII, DIV and DV are relatively conservative and have important functions; although the function of DIII is unclear, the sequence is relatively conservative, and DVI is relatively variable; DI is mainly involved in the replication of the viral genome rather than translation. Based on this, the optimization strategy of the present invention includes mutating important domains and deleting non-critical sequences.

[0041] The method of optimizing the important domain of the present invention is to increase the GC pairing ratio of the longer stem to improve the stability of the secondary structure without changing the secondary structure and the sequence of the binding region of important proteins (such as PCBP1 / 2, eIF4G). According to this strategy, 6 mutants of EV-A were designed and constructed on the circular GLUC mRNA. 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 wild-type EV-A, these mutations did not significantly improve the translation efficiency. Among them, the 4m2 and 4m3 mutations of DIV and the 5m1 mutation of DV had no obvious change on the translation efficiency. The remaining mutants, 2m1 of DII, 4m1 of DIV and 5m2 of DV, all significantly 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 present invention has successively shortened several non-critical sequences of DI (90nt), Linker between DI and DII (30nt), DVI (42nt), and DVII (32nt) of EV-A ( Figure 3 A), the fluorescence signal results show that the deletion of the three structures DI, DVI, and DVII has no effect on the translation function of IRES, but the loss of Linker significantly reduces the translation efficiency ( Figure 3 C). Based on the above results, the inventors decided to combine truncated DI, DVI and DVII that do not affect the function, and the mutation 5m1 that slightly improves the translation efficiency. The combined results showed that the truncated EV-A-S1 (simultaneous truncations of DI and DVI) and the truncated EV-A-S2 (simultaneous truncations of DI, DVI and DVII) significantly improved the translation efficiency of EV A by about 50%, while the truncated EV-A-S3 (mutation 5m1 based on EV-A-S1) did not further improve the translation efficiency ( Figure 3 D). The inventors speculate that this result may be because the truncation of non-core regions will not affect the core domain, and after truncating multiple non-functional structures, the IRES structure is more simple and stable. To verify this conjecture, the inventors analyzed the IRES structure of truncated DI and DII at the same time. The SHAPE-MaP activity results showed that the signal of the truncated EV-A-S1 core region is almost completely consistent with the wild-type IRES ( Figure 3 E). 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 and DVII structure next to DII where the nucleic acid sequence is not deleted also disappear at the same time ( Figure 3E). This result supports the inventors' conjecture that deleting multiple non-critical structures does make IRES more streamlined and stable. In short, after the transformation based on SHAPE-MaP structural analysis, the modified EV-A IRES is not only more streamlined (750nt shortened to EV-A-S1 / S3 618nt / EV-A-S2 592nt), but also has significantly improved translation efficiency.

[0043] Example 3 Untranslated region sequence screening

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

[0045] The circular GLUC mRNA was also used to screen the UTR sequence. For the Spacer sequence, Apt-EIF4G, β-globin 5'UTR, and polyA50 were compared. The results showed that the GLUC signal was the highest when polyA50 was used as the 5'Spacer ( Figure 4 A). For 3'UTR sequences, the screening included HBA1 3UTR, polyAC, polyA50, and β-globin 3'UTR. In addition, considering that the 3'UTR sequence of the virus may also be involved in the translation of viral RNA, the screening also included the 3'UTR of CVB3, EVA, HRV-B3, and SINV viruses. The results showed that the best spacer sequence in the circular 3'UTR position was still polyA50 ( Figure 4 B) In conclusion, the polyA50 sequence can best promote the function of IRES regardless of whether it is the 5'Spacer or 3'UTR of circular RNA. This may be due to the simple spatial structure of the polyA sequence and its ability to recruit PABP, which promotes the interaction between IRES and 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 start sequence, polyA50 sequence as the untranslated region sequence, and places the coding sequences of ovalbumin OVA, mouse melanoma tandem antigen B16-8, and human cervical cancer virus HPV antigen E6E7 after IRES to design three circular RNA vaccines, namely circular OVA, circular B16-8, and circular E6E7 ( Figure 5 A, 6A, 7A). The three circular RNA vaccines were produced using the PIE cyclization 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 mouse tumor models.

[0048] 4.1 Template plasmid construction

[0049] The T7 promoter, the Anabaena 2.0PIE element 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), IRES and protein coding sequence were inserted into the pUC57 plasmid and used as a template plasmid for circular mRNA. All linear mRNA templates were constructed using the mRNA template cloning kit of Takara Bio. (Bao Riyi, Cat. No. 6143). The template plasmid in the kit contains the T7 promoter, human β-globin 5'UTR, human HBA1 3'UTR and a 105nt long poly(A) sequence. The DNA fragment was synthesized by Jin Weizhi and amplified by PCR. The seamless cloning step was completed using the ClonExpressII one-step cloning kit of Novozyme.

[0050] 4.2 Linear and circular RNA synthesis and packaging

[0051] In the present invention, the preparation of linear mRNA uses NEB's HindIII endonuclease enzyme linearized plasmid as a template, and uses Yisheng's T7 high-efficiency RNA synthesis kit (Yisheng Biological, Catalog No. 10673ES50) for in vitro transcription. The mRNA is capped with Cap1 during co-transcription, and transcribes mRNA with a 105nt long polyA tail. After incubation at 37°C for 2 hours, the reaction mixture is treated with DNase I at 37°C for 20 minutes, and then purified using Thermo Scientific's GeneJET RNA purification kit and analyzed by 2% agarose gel.

[0052] For circular mRNA, PCR amplified templates were used for in vitro transcription using Yisheng's T7 High-Efficiency RNA Synthesis Kit. The reaction mixture was incubated at 37°C overnight (about 16 hours) to achieve maximum spontaneous circularization and then treated with DNase I at 37°C for 20 minutes. 1×T4 RNA ligase reaction buffer and 2mM (final concentration) GTP were then added and incubated at 55°C for 15 minutes for further circularization. After RNA was purified by the GeneJET RNA Purification Kit, it was heated at 65°C for 3 minutes. 20U RNase R (Biyuntian) and 1×RNase R reaction buffer were added to every 20μg of purified RNA and incubated at 37°C for 2 hours. Finally, the circular RNA was purified by the GeneJET RNA Purification Kit and analyzed by 4% polyacrylamide gel or 2% agarose gel.

[0053] Purify and analyze qualified circular and linear RNA using Polyplus's in vivo-jetRNA delivery reagent, and package them into RNA liposome complexes (LPX) according to the manufacturer's instructions for animal administration. In the clean bench, dilute the RNA with mRNA buffer to 50μg / ml, then add 2μl / μg of in vivo-jetRNA reagent, mix gently, and let stand at room temperature for 15 minutes before using it for animal administration.

[0054] 4.3 Tumor prevention effect of circular RNA vaccine

[0055] In order to verify the preventive effect of the circular RNA platform designed in the present invention as a tumor vaccine on tumors, OVA was selected as a model antigen and a B16F10 stable cell line expressing OVA was constructed. From 27 to 10 days before subcutaneous inoculation of B16F10 OVA tumor cells, mice were vaccinated with three injections of the vaccine by intravenous injection, and blood was collected on the third day before tumor inoculation to detect T cells in PBMC ( Figure 5A). After three injections of the vaccine, flow cytometry analysis showed that the proportion of T cells positive for the OVA antigen peptide (SIINFEKL) MHC tetramer in the three vaccine groups was significantly higher than that in the untreated group, indicating that all three RNA vaccines successfully induced OVA-specific T cells ( Figure 5 B). CircOVA and pseudouracil-modified linear m1ΨLinearOVA induced a higher proportion of OVA-specific T cells than unmodified linear LinearOVA. Further analysis of all T cell subtypes in PBMCs revealed that the CircOVA and LinearOVA groups The proportion of T cells was significantly lower than that of the untreated group, while there was no significant difference between the m1ΨLinearOVA group and 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 significantly higher proportions. The proportions of effector T cells in the three groups were significantly higher than those in the untreated group ( Figure 5 C). These results may indicate that CircOVA and LinearOVA induced a more significant overall immune response due to recognition by pattern recognition receptors of immune cells, while m1ΨLinearOVA did not significantly change the overall T cell subset ratio due to its lower immunostimulatory properties. Ten days after the third vaccine was administered, mice were subcutaneously inoculated with B16F10 OVA tumor cells, and tumor formation was subsequently monitored until 60 days after tumor inoculation. Mice in all three vaccine groups did not develop tumors during the 60 days of monitoring (CircOVA: 4 / 5, m1ΨLinearOVA: 3 / 5, LinearOVA: 2 / 5). In particular, 80% of the mice in the CircOVA group did not develop tumors, while all mice in the untreated group developed tumors in about two weeks ( Figure 5 E). On the 24th day after tumor inoculation, the tumor size of the three groups of mice given the vaccine was significantly smaller than that of the untreated group ( Figure 5 D), the tumor size of mice in the untreated group reached the humanitarian endpoint. The above results show that the circular RNA vaccine designed by the present invention shows excellent immune activation ability, T cell induction ability similar to modified linear mRNA and better tumor prevention effect.

[0056] 4.4 Tumor therapeutic effects of circular RNA tumor neoantigens and tumor-associated antigen vaccines

[0057] OVA is a highly immunogenic antigen, so the B16F10-OVA model may not truly reflect the tumor treatment scenario. Tumor mutation new antigens and virus-derived tumor-associated antigens are common targets in tumor vaccine design, so a tandem antigen vaccine encoding 8 reported B16F10 tumor mutation antigens was designed ( Figure 6 A) and HPV E6E7 fusion protein vaccine ( Figure 7 A), used to treat B16F10 and TC-1 tumor models, respectively.

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

[0059] TC-1 is a cell line that stably expresses HPV oncogenes E6 and E7 and is a commonly used HPV-related tumor model. In order to test the therapeutic effect of HPV tumor vaccine on established tumors, mice were subcutaneously inoculated with TC-1 cells for 14 days and then intravenously injected with mRNA vaccine encoding HPV E6 / E7 fusion protein. Blood was collected 7 days after the second injection of vaccine ( Figure 7 A). Flow cytometry analysis of PBMCs showed that the CircE6E7 vaccine induced the highest proportion of antigen-specific IFNγ-secreting T cells in mouse PBMCs, and the m1ΨLinear E6E7 vaccine also successfully induced a moderate level of antigen-specific IFNγ-secreting T cells. 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 ( Figure 7 B). Consistent with the induction of T cells, both CircHPV and m1ΨLinearHPV vaccines successfully cleared tumors, and tumor growth was also inhibited in the LinearHPV group ( Figure 7 C, D, E). These results indicate that the circular mRNA encoding mutant antigens and HPV antigen vaccines of the present invention induce stronger tumor antigen-specific T cell immunity in mice compared to conventional linear mRNA, and exhibit excellent tumor treatment effects.

[0060] The sequences involved in the above embodiments of the present invention are shown in Table 1 below.

[0061] Table 1 Nucleotide and amino acid sequence listing

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0073] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Internal ribosome entry site IRES, characterized in that The IRES comprises the nucleotide sequence shown in SEQ ID NO: 1 or a variant sequence thereof, wherein the variant sequence retains a translation initiation function that is equivalent to or better than that of SEQ ID NO:

1.

2. The IRES of claim 1, wherein the variant sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:

1.

3. The IRES according to claim 1 or 2, wherein the variant comprises the nucleotide sequence shown in any one of SEQ ID NOs: 2-4.

4. Use of the IRES according to any one of claims 1 to 3 in preparing circular RNA.

5. Use of the combination of the IRES according to any one of claims 1 to 3 and polyA50 in preparing circular RNA; wherein the polyA50 is as shown in SEQ ID NO:

16.

6. Circular RNA, characterized in that The method comprises the IRES of any one of claims 1 to 3, a target protein coding sequence and a non-translated region sequence; optionally, further comprising a cyclization residue sequence.

7. The circular RNA according to claim 5, wherein the target protein is selected from tumor antigens, infectious disease antigens, chimeric antigen receptors (CARs), TCR complexes, antibodies, cytokines, cas proteins and other therapeutic proteins.

8. The circular RNA according to claim 7, 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 shown in any one of SEQ ID NOs: 27, 29, and 31.

9. The circular RNA according to any one of claims 5 to 8, wherein the untranslated region sequence is polyA50, the sequence of which is shown in SEQ ID NO:

16.

10. A pharmaceutical composition, characterized in that It comprises the circular RNA and a delivery vector as described in any one of claims 5 to 7; preferably, the delivery vector comprises nanolipid particles (LNP), virus-like particles (VLP) and / or polymer nanoparticles.

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