Novel mRNA element, system, and use

Abstract

The present invention provides an mRNA element, a system, and a use. A UPA-containing uncapped linear mRNA of the present invention can be efficiently and stably expressed without adding a 5' cap or adding a nucleic acid modification in an in vitro transcription synthesis process, and can be used as an infectious disease vaccine or tumor vaccine expression system.

Description

Novel mRNA elements, systems, and applications thereof

[0001] This application claims priority to Chinese patent application 202411371662X with a filing date of 2024 / 9 / 29, and Chinese patent application 202510228599.2 with a filing date of 2025 / 2 / 27. This application incorporates the entirety of the aforementioned Chinese patent applications. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and specifically relates to a novel IRES element and a novel cap-independent linear mRNA expression system. BACKGROUND

[0003] In recent years, mRNA has been widely used in the field of virus prevention and personalized tumor treatment as a direct template for protein synthesis. As a new generation of vaccine technology, mRNA vaccines have the advantages of short development cycle, strong immunogenicity, programmable design, and strong scalability in production. During the COVID-19 pandemic, mRNA vaccines have been successfully applied, demonstrating excellent safety and significant immune protection effects 1 . In addition, mRNA vaccines also show great potential in the prevention of other viruses such as influenza virus, Zika virus, and AIDS, and several candidate vaccines have entered clinical trials 2 . In addition to viral vaccines, mRNA has also shown important breakthroughs in the field of tumor treatment, especially personalized tumor vaccines 3 . Personalized mRNA tumor vaccines are based on the neoantigens of a patient's tumor and are designed to encode specific mRNA, which can induce a specific T cell immune response when delivered to the body, achieving precise targeted killing of cancer cells. Compared with traditional tumor vaccines, personalized mRNA tumor vaccines have higher specificity and flexibility, and can simultaneously encode and express multiple tumor neoantigens, thereby more comprehensively activating a polyclonal T cell immune response against tumors. This multi-antigen expression characteristic not only enhances the breadth and strength of the immune response, but also provides a more effective solution to tumor heterogeneity. Multiple studies have shown that personalized mRNA tumor vaccines have entered clinical trials and have shown reliable safety and positive therapeutic effects in melanoma, non-small cell lung cancer, and pancreatic cancer 4,5 . In summary, mRNA technology not only has broad application prospects in virus prevention and control, but also has great potential in the field of personalized tumor treatment.

[0004] Currently, mRNA technology mainly includes conventional linear mRNA, self-replicating mRNA (saRNA) and circular RNA (circRNA). Conventional linear mRNA is the most mature form, which relies on the 5' cap structure-mediated translation mechanism, has the advantages of efficient expression and relatively simple preparation, and has been widely used in vaccines and protein replacement therapy. However, its disadvantages include easy degradation, low stability, the need for cap structure, and often the need for nucleic acid modification. Self-replicating mRNA can significantly reduce the required dose and improve the persistence of protein expression by self-amplification in cells through RNA-dependent RNA polymerase (RdRp). However, due to its large molecule, delivery and synthesis are more complex, and it may trigger an overactive immune response. Circular RNA is more stable than linear mRNA due to its 5' and 3' covalent closure, which can reduce nuclease degradation and is suitable for long-term protein expression, gene therapy and new vaccine development. However, its translation relies on IRES or m6A-mediated mechanisms, which may affect translation efficiency, and the production process is not yet mature, still facing low cyclization efficiency, insufficient yield, and difficult purification. SUMMARY

[0005] To solve the above technical problems, the present application provides a novel structure of cap-free and non-dependent linear mRNA (referred to as cap-free linear mRNA), which does not require the addition of a 5' cap or nucleic acid modification during in vitro transcription synthesis.

[0006] The first aspect of the present application provides a cap-free linear mRNA, which comprises the following elements in order from 5' to 3': one or more tandem 5' protection sequences (UPA), IRES, 5' UTR, protein coding region of interest, 3' UTR and optionally polyA tail, wherein the 5' protection sequence comprises an anti-exoribonuclease RNA (xrRNA) and an RNA-binding protein binding motif.

[0007] In some embodiments, the number of repetitions of the 5' protection sequence in the mRNA is 1-5, preferably 2.

[0008] In some embodiments, the xrRNA is derived from the 3' UTR of a flavivirus.

[0009] In some embodiments, the flavivirus is selected from the group consisting of cell-fusing agent virus (CFAV), dengue virus (DENV), Usutu virus (USUV), Yellow fever virus (YFV) and Zika virus (ZIKV).

[0010] In some embodiments, the 3' UTR of the flavivirus comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 12-16.

[0011] In some embodiments, the xrRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NOs: 17-33.

[0012] In some embodiments, the RNA binding protein binding motif is selected from a poly-nucleotide polyA or other RNA binding protein binding motif; preferably, the RNA binding protein binding motif has a length of 36-60 bp; preferably, the poly-nucleotide motif has a length of 48 bp.

[0013] In some embodiments, the xrRNA is linked to the polyA to form an xrRNA-PolyA sequence.

[0014] In some embodiments, the xrRNA is UX1 derived from Ussuri virus 3' UTR, the UX1 comprises a nucleotide sequence as set forth in SEQ ID NO: 1; the poly-nucleotide motif is polyA; and the 5' protection sequence xrRNA-PolyA is defined as UPA sequence.

[0015] In some embodiments, the UPA comprises a nucleotide sequence as set forth in SEQ ID NO: 8.

[0016] In some embodiments, the UPA is repeated in the mRNA for 1-5 times, preferably 2 times.

[0017] In some embodiments, the IRES comprises a nucleotide sequence as set forth in SEQ ID NO: 3 or 4.

[0018] In some embodiments, the 5' UTR comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 5, 36, 37, and / or the 3' UTR comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 6, 7, 38, 39.

[0019] In some embodiments, the protein of interest comprises a tumor antigen, a bacterial antigen, or a viral antigen.

[0020] In some embodiments, the tumor antigen is a tumor neoantigen, a tumor associated antigen, or a tumor specific antigen.

[0021] In some embodiments, the tumor is cervical cancer or melanoma.

[0022] In some embodiments, the mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 51, 52, 54, 55.

[0023] In some embodiments, the infectious disease is caused by one or more viruses selected from the group consisting of HPV, HIV, EBV, and HBV.

[0024] The second aspect of the present application provides a vector comprising the capless linear mRNA of the first aspect of the present application.

[0025] The third aspect of the present application provides a cell comprising the capless linear mRNA of the first aspect of the present application or the vector of the second aspect of the present application.

[0026] The fourth aspect of the present application provides a nano-lipid particle comprising the capless linear mRNA of the first aspect of the present application.

[0027] The fifth aspect of the present application provides a pharmaceutical composition comprising the capless linear mRNA of the first aspect of the present application, and / or the vector of the second aspect of the present application, and / or the cell of the third aspect of the present application, and / or the nano-lipid particle of the fourth aspect of the present application.

[0028] In some embodiments, the pharmaceutical composition is a vaccine or other therapeutic protein.

[0029] In some embodiments, the other therapeutic protein comprises an antibody, a cytokine, and / or an enzyme.

[0030] The sixth aspect of the present application provides use of the capless linear mRNA of the first aspect of the present application, and / or the vector of the second aspect of the present application, and / or the cell of the third aspect of the present application, and / or the nano-lipid particle of the fourth aspect of the present application, and / or the pharmaceutical composition of the fifth aspect of the present application in the preparation of a tumor or infectious disease drug.

[0031] In some embodiments, the tumor is cervical cancer or melanoma.

[0032] Preferably, the virus is selected from the group consisting of HPV, HIV, EBV, or HBV; preferably, the protein of interest is HPV E6E7 fusion protein; preferably, the mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 51-56

[0033] The seventh aspect of the present application provides an internal ribosome entry site (IRES), the IRES comprising a nucleotide sequence as set forth in SEQ ID NO: 57 or a variant sequence thereof, the variant sequence retaining a start translation function equivalent to or better than that of SEQ ID NO: 57.

[0034] In some embodiments, the variant sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 57.

[0035] In some embodiments, the variant comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 58-68.

[0036] The eighth aspect of the present application provides use of the IRES of the seventh aspect of the present application in the preparation of a circular RNA or a linear RNA.

[0037] In some embodiments, the linear RNA is a linear mRNA.

[0038] In some embodiments, the linear mRNA is a capless linear mRNA.

[0039] In some embodiments, the capless linear mRNA is as described in the first aspect of the present application.

[0040] The advantages of the present application over the prior art are that:

[0041] 1. The EV-A IRES screened by the present application not only has higher translation efficiency than the reported CVB3 and 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 to the wild-type EV-A IRES, and the sequence length is reduced by 17%-21% compared to the wild-type (750 nt for wild-type, 618 nt for EV-A-S1 / S3, and 592 nt for EV-A-S2). The shorter IRES sequence means that the capacity of the RNA platform (circular or linear) to load sequences is improved. The EV-A IRES and its mutants can be used for circular RNA or linear drug platforms.

[0042] 2. The UPA and EV-A-S1 / EV-A-S2 sequences designed by the present application are used to replace the 5' cap of traditional mRNA to form UPA capless linear mRNA. The UPA capless linear mRNA does not require a 5' cap structure and nucleotide modification, which reduces production costs and simplifies production processes, and is particularly suitable for large-scale vaccine production and personalized tumor treatment.

[0043] 3. Improved mRNA stability and expression persistence: By introducing the UX1 sequence and polyA (UPA sequence) derived from the 3'UTR of flaviviruses, the degradation of mRNA by exonuclease XRN-1 is effectively hindered, and the stability of mRNA is improved. The design of two UPA in series (2UPA) further enhances the persistence of in vivo expression, and its stability is even better than that of traditional capped mRNA (such as luciferase activity in mice for up to 120h).

[0044] 4. Compact structure and high safety: The UX1 sequence is only 88nt, containing only two small stem-loop structures, avoiding potential safety hazards that long sequences (such as sfRNA) may bring. Experiments have shown that the UPA uncapped linear mRNA vaccine does not cause significant toxicological reactions or organ damage in mice, and has good safety.

[0045] 5. Efficient translation and immune effect: EV-A-S1, EV-A-S2 IRES can efficiently initiate translation of uncapped mRNA, combined with UPA and optimized UTR sequences (such as β-globin UTR), to achieve efficient and stable expression of proteins. In the tumor treatment model, the UPA uncapped linear mRNA vaccine induced high levels of tumor-specific T cells and significantly inhibited tumor growth.

[0046] 6. The UPA-E6E7 and 2UPA-E6E7 uncapped linear mRNA vaccines designed by the present application exhibit good safety and tumor treatment effect in mouse models, and have great potential for clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0047] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the following drawings:

[0048] Figure 1 shows the schematic diagram of the UPA uncapped linear mRNA design. The UPA and 2UPA uncapped linear mRNA sequences designed by the present application are arranged in the order of UPA (UX1+polyA) or 2UPA, EV-A-S1 IRES, 5'UTR, target gene, 3'UTR, and polyA tail from 5' to 3' end.

[0049] Figure 2 shows the screening of 5' terminal protection structure of uncapped linear mRNA. (A) Schematic diagram of xrRNA and Dumbbell structure in the 3'UTR of CFAV, DENV, USUV, YFV, ZIKV virus genome RNA and naming. (B) Comparison of luciferase activity in cell supernatant after the structure in Figure (A) is constructed to the 5' end of uncapped linear GLUC mRNA and transfected into HEK293T cells for 48h.

[0050] Figure 3 Non-coding region sequence screening. (A) Schematic diagram of non-coding region screening. (B) Sequence screening between UX1 and EV-A-S1. (C) 3' UTR screening. (D) 5' UTR screening.

[0051] Figure 4 UX1 modification and nucleic acid modification. (A) UX1 secondary structure and pseudoknot (PK) schematic diagram analyzed by SHAPE-Map. (B) UX1 mutant screening. (C) m6A, mlY, N4-Ac-C three modified nucleotides were used for mRNA synthesis at the ratio of 5%, 20%, 100%, and the GLUC activity of these mRNAs was compared.

[0052] Figure 5 Evaluation of cap-independent linear mRNA based on UPA sequence. (A) Protein expression of cap-independent linear mRNA of UPA, 2UPA, 3UPA, and cap-independent linear mRNA with EV-A-S1 only or PolyA+EV-A-S1, and capped mRNA (CAP), capped modified mRNA (mCAP) transfected HEK293T cells for 48h. (B) Cell supernatant of mRNA transfected Hela cells in Figure (A) was collected and washed from day 1 to day 5, and the GLUC activity of the supernatant relative to day 1 was detected. (C) Total Flux of mice changed with time after injection of UPA, 2UPA, mCAP, CAP four kinds of FLUC mRNA into mice muscle. (D) Animal live fluorescence picture of data in Figure (C).

[0053] Figure 6 UPA intracellular binding protein analysis. (A) Schematic diagram of UPA sequence binding protein analysis: avidin aptamer sequence was added after UPA sequence to bind to streptavidin magnetic beads to separate the binding proteins (Pull down) in Hela cells, and mass spectrometry analysis was performed on the differential bands to find candidate proteins, and Western blot and RNA immunoprecipitation analysis (RIP) were performed. The aptamer sequence alone was used as a control group. (B) Silver staining result of Pull down separated protein gel. (C) Western blot verification result of candidate protein. (D) RIP verification result of candidate protein. The numerical value above the column chart indicates the fold enrichment of the corresponding protein group relative to the control IGG group to UPA RNA.

[0054] Figure 7 Safety evaluation of UPA cap-independent linear mRNA vaccine in mice. (A) Body weight change of mice after injection of UPA-E6E7, 2UPA-E6E7, mCAP-E6E7 three kinds of vaccines. (B) Serum ALT level of mice before and after two doses of vaccine. (C) Serum AST level of mice before and after two doses of vaccine. (D) Histological staining of major organs of mice two weeks after the second dose of vaccine.

[0055] Figure 8 UPA capless linear mRNA tumor vaccine treatment efficacy evaluation. (A) B16F10-OVA tumor volume data. (B) Body weight change of mice in the same period. (C) Tumor volume of individual mice in four groups of mCAP-FLUC, UPA-OVA, 2UPA-OVA, mCAP-OVA administration and pictures of subcutaneous tumors peeled off after euthanasia on the 20th day after tumor inoculation, the circle indicates the tumor-free mouse. (D) Representative results of flow cytometry analysis of OVA antigen peptide (SIINFEKL) MHC tetramer positive T cells and statistics.

[0056] Figure 9 Capless linear mRNA 5' end protection structure screening. (A) TC-1 tumor volume data. (B) Body weight change of mice in the same period. (C) Tumor volume of individual mice in four groups of mCAP-FLUC, UPA-E6E7, 2UPA-E6E7, mCAP-E6E7 administration and pictures of subcutaneous tumors on the 23rd day after tumor inoculation. (D) Representative results of flow cytometry analysis of IFNy positive T cells and statistics.

[0057] Figure 10 shows efficient translation IRES screening. (A) Screening of efficient translation IRES in HEK239T and DC2.4 cells using dual fluorescence reporter plasmid technology. (B), (C) Screening of efficient IRES in HEK239T and DC2.4 cells using synthetic circular FLUC RNA, the left panel is a schematic diagram of RNA circularization method. (D), (E) Comparison of IRES translation efficiency in THP-1, HEK239T and DC2.4 using synthetic circular EGFP RNA.

[0058] Figure 11 shows EVA IRES secondary structure analysis and sequence optimization. (A) Shape reactivity of EV-A IRES and predicted secondary structure based on reactivity data, the red dashed box in the figure 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) Comparison of translation efficiency of EV-A mutants with wild type. (E) Shape reactivity of EV-A-S1 and predicted secondary structure based on reactivity data. DETAILED DESCRIPTION

[0059] TERMS DEFINITION

[0060] As used herein, “xrRNA” or “anti-exoribonuclease RNA” or “exoribonuclease-resistant RNA” is an RNA molecule that resists degradation by host exonucleases through a special three-dimensional structure, which is widely present in the genomes of Flaviviridae viruses (such as dengue virus, Zika virus, West Nile virus, etc.). The xrRNA forms a stable pseudoknot structure or stem-loop structure through base pairing, which hinders the degradation of exonucleases (such as Xrn1 of host cells) in the 5'→3' direction. Its nucleotide sequence is highly conserved in Flaviviridae, and is usually located in the 3' non-coding region (3'-UTR) of the viral genomic RNA.

[0061] As used herein, “RNA-binding protein binding motif” refers to a sequence on the RNA that binds to the RNA-binding protein motif.

[0062] Design of uncapped linear RNA

[0063] The present application provides a design method of uncapped linear mRNA and UPA uncapped linear mRNA obtained by the method. The UPA uncapped linear mRNA of the present application does not need to add 5' cap or use capping enzyme to cap during in vitro transcription synthesis, nor does it need to add nucleic acid modification, and can be synthesized using classic ribonucleotides under the catalysis of RNA polymerase, and can be expressed efficiently and stably, and can be used as a tumor vaccine expression system, and has stronger induction of immunity and tumor inhibition effect than conventional mRNA.

[0064] In some embodiments, the design and optimization process of the uncapped linear mRNA of the present application mainly includes the following steps:

[0065] 1) First, the structure UX1 in the 3'UTR of the flavivirus that protects the uncapped linear mRNA was screened and obtained, and was combined with EV-A-S1 IRES to design uncapped linear mRNA.

[0066] 2) The untranslated region sequence of the uncapped linear mRNA was further screened and optimized, and it was determined that the combination of UX1 and PolyA (UPA) at the 5' end can make the uncapped linear mRNA achieve the highest expression level.

[0067] 3) The protein expression level and stability of UPA uncapped linear mRNA, uncapped linear mRNA without UPA sequence, and CleanCap capped linear mRNA were evaluated and compared in cell lines and in mice, and it was found that the protein expression level and stability of two tandem UPA (2UPA) uncapped linear mRNA in cell lines were close to those of capped mRNA, and had better expression stability in mice.

[0068] 4) Further assessment of the safety of UPA capless linear mRNA vaccine, the results show that the vaccine does not cause significant toxicological changes in mice. Finally, using UPA capless linear mRNA vaccine to treat B16F10-OVA and HPV-related TC-1 mouse tumors, it is found that UPA capless linear mRNA vaccine induces high levels of tumor-specific immunity and significantly inhibits tumor growth.

[0069] In some embodiments, the UPA capless linear mRNA of the present application is as shown in FIG. 1. The sequence of the UPA capless linear mRNA from 5' to 3' is UX1 (an xrRNA sequence of Ussuri virus USUV) (SEQ ID NO: 1), polyA (polyadenylate sequence) (SEQ ID NO: 2), EV-A-S1 or EV-A-S2 (engineered enterovirus EV-A IRES) (SEQ ID NO: 3, 4), 5'UTR (such as SEQ ID NO: 5 or others), target gene (drug protein or vaccine), 3'UTR (3' untranslated region sequence) (such as SEQ ID NO: 6, 7 or others), polyA tail (polyadenylate tail) (SEQ ID NO: 10). Among them, the role of UX1 and polyA sequence is to bind protein in cells to stabilize capless linear mRNA and hinder the degradation of XRN-1 enzyme; and the function of EV-A-S1 is to initiate the translation of capless linear mRNA to efficiently express the target gene. The UX1 sequence is only 88 nt long, containing only two small stem-loop structures.

[0070] The combination of UX1 and polyA in the present application which plays a protective role is called UPA sequence (SEQ ID NO: 8), and this cap-independent linear mRNA is called UPA capless linear mRNA. The UPA sequence at the 5' end of the UPA capless linear mRNA uses 2 repeated UPA sequences (2UPA (SEQ ID NO: 9)), after which the intracellular stability is further improved, the target gene can be expressed more stably and continuously, and better efficacy is achieved.

[0071] In order to make the objects, 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 creative labor fall within the scope of protection of the present application.

[0072] Unless otherwise defined, the technical terms or scientific terms used herein should be understood as their ordinary meanings to those of ordinary skill in the art to which the present application belongs.

[0073] Example 1 Synthesis of UPA capless linear mRNA

[0074] UPA capless linear mRNA plasmid construction: T7 promoter (SEQ ID NO: 11), UPA (SEQ ID NO: 8) or 2UPA sequence (SEQ ID NO: 9), EV-A-S1 IRES (SEQ ID NO: 3), human beta-globin 5’UTR (SEQ ID NO: 5), gene of interest, human HBA1 3’UTR (SEQ ID NO: 6), and 105nt long poly(A) sequence were inserted in pUC57 plasmid as template plasmid for UPA capless linear mRNA. DNA fragments were synthesized by Genewiz and amplified by PCR. Seamless cloning step was done using ClonExpress II One-Step Cloning Kit from Norgen Biotek.

[0075] In vitro transcription synthesis of UPA capless linear mRNA: T7 High Yield RNA Synthesis Kit from Norgen Biotek was used. ATP, UTP, CTP, GTP and 10x Transcription Buffer were mixed sequentially, linearized template plasmid DNA (500 ng per 20ul system) and T7 RNA Polymerase Mix were added, mixed after adding appropriate amount of RNase Free Water, and reacted at 37°C for 2h. RNA was purified and quality tested.

[0076] In vitro transcription synthesis of capped linear mRNA: All capped linear mRNA templates were constructed using mRNA Template Cloning Kit from Takara Bio. (Bodipy, Cat# 6143). The template plasmid in this kit contains T7 promoter, human beta-globin 5’UTR, human HBA1 3’UTR and 105nt long poly(A) sequence. T7 High Yield RNA Synthesis Kit for Co-transcription from Norgen Biotek was used. ATP, N1-Me-Pseudo UTP (modified) or UTP (unmodified), CTP, GTP and cap1 m7GAG cap and 10x Transcription Buffer were mixed sequentially, linearized template plasmid DNA (500 ng per 20ul system) and T7 RNA Polymerase Mix were added, mixed after adding appropriate amount of RNase Free Water, and reacted at 37°C for 2h. RNA was purified and quality tested.

[0077] Example 2 Screening of capless linear mRNA 5’ end protection structure

[0078] The inventors previously designed EV-A-S1 IRES through large-scale screening and engineering, which can replace the 5’ cap structure of traditional mRNA to efficiently initiate the translation of target protein. However, another function of the cap of linear mRNA is to hinder the degradation of the major RNA 5’ exonuclease XRN-1 in cells, thereby achieving stable expression of mRNA. Therefore, it is also necessary to find a sequence that can play a similar blocking role and be combined with EV-A-S1 IRES for constructing capless linear mRNA for stable expression. According to the literature, the 3’ UTR of flavivirus RNA genome contains structures that can hinder the degradation of XRN-1, including xrRNA structure and Dumbbell structure. Therefore, the inventors analyzed the secondary structures of the 3’ UTR sequences of CFAV, DENV, USUV, YFV, and ZIKV flaviviruses (SEQ ID NO: 12-16) using RNAfold, and found 18 xrRNA and Dumbbell type structures (FIG. 2A) (SEQ ID NO: 1, 17-33). These structures were constructed one by one at the 5’ end of linear Gaussian luciferase (GLUC) mRNA with EV-A-S1 IRES (polyA was used to separate EV-A-S1 IRES to avoid mutual influence between secondary structures). By comparing the luciferase activity of the cell supernatant of HEK239T cells transfected with these capless linear mRNAs for 48 h (cells were transfected using CALNP mRNA reagent from Beijing Donna Pharmaceutical, and luciferase activity was detected using the Biyun Tian Gaussian luciferase reporter gene detection kit), the inventors found that the luciferase expression level of capless linear mRNA with UX1, DX1, ZX1, DD1, and DX2 at the 5’ end was significantly higher than that of capless linear mRNA without protection structure before EV-A-S1 IRES (FIG. 2B). Among them, the addition of UX1 from USUV (Ussuutu virus) before EV-A-S1 IRES increased the luciferase expression level to about 6.5 times that of the no-protection structure group. The above results show that the addition of certain flavivirus 3’ UTR structures at the 5’ end of capless linear mRNA indeed improves the expression level of capless linear mRNA. Among them, the UX1 (SEQ ID NO: 1) structure has the greatest effect on improving the expression level of capless linear mRNA.

[0079] UX1 (SEQ ID NO: 1)

[0080] Example 3 Untranslated region sequence screening and UX1 modification and nucleic acid modification

[0081] After the previous screening, the inventors chose the most efficient UX1 as the 5' end protection sequence for the capless linear mRNA. To further optimize the expression level of the capless linear mRNA, the inventors also screened the spacer sequence between UX1 and EV-A-S1 IRES, the 5' and 3' untranslated region sequence of the mRNA using the capless linear GLUC mRNA (Figure 3A). The results showed that the addition of polyA between UX1 and EV-A-S1 significantly improved the expression level of luciferase compared to the addition of no sequence or other polymeric sequences such as PolyC (SEQ ID NO: 34) and PolyT (SEQ ID NO: 35) (Figure 3B). The inventors then compared the difference in effect when several 5'UTR (SEQ ID NO: 5, 36, 37) and 3'UTR (SEQ ID NO: 6, 7, 38, 39) sequences were used in the capless linear mRNA and found that the capless linear mRNA with both 3'UTR and 5'UTR adopting the UTR of beta-globin had the highest translation efficiency (Figures 3C, 3D).

[0082] Previous studies have shown that the inhibition of XRN-1 by xrRNA is regulated by the pseudoknot structure in the structure, so modifying the pseudoknot structure of UX1 may further improve its effect in capless linear mRNA. To verify this assumption, the inventors first analyzed the secondary structure of UX1 using SHAPE-Map technology and analyzed the pseudoknot structure of UX1 using IPknot software and found that UX1 has two pseudoknot structures, PK1 and PK2 (Figure 4A). The inventors designed UX1 mutants with longer pseudoknot sequences (Long-PK1 (SEQ ID NO: 40), Long-PK2 (SEQ ID NO: 41)) or increased GC ratio of PK2 (mPK2 (SEQ ID NO: 42)) and found that the three mutants did not enhance but rather reduced the expression of capless linear GLUC mRNA in the capless linear GLUC mRNA (Figure 4B). This indicates that simply modifying the length or GC ratio of the pseudoknot cannot enhance the effect of UX1, but rather may change the stability of the overall structure of UX1.

[0083] Nucleic acid modifications such as pseudouridine can regulate the stability and translation efficiency of mRNA. Therefore, we tried to introduce nucleic acid modifications into cap-free linear mRNA and evaluated the protein expression level. During the in vitro transcription of cap-free linear GLUC mRNA, different proportions of m6A, m1y, and N4-Ac-C modified nucleotides were incorporated, respectively. After transfecting HEK293T cells for 48 h, the GLUC luciferase activity was detected. The results showed that with the increase of the proportion of the three nucleic acid modifications, the expression of cap-free linear GLUC mRNA decreased (Figure 4C). This may be due to the fact that the functions of UX1 and EV-A-S1 IRES in cap-free linear mRNA both depend on the formation of special secondary structures, and the introduction of non-classical nucleotides changes the secondary structure of these sequences, destroying their functions.

[0084] Example 4 Expression evaluation of cap-free linear mRNA based on UPA sequence

[0085] Based on the previous experimental results, it was determined that the capless linear mRNA with U X1 at the 5' end and a Poly A sequence (hereinafter referred to as UPA sequence) has the highest protein expression level. Next, a capless linear mRNA with one (UPA) (SEQ ID NO: 43), two (2UPA) (SEQ ID NO: 44) and three (3UPA) (SEQ ID NO: 45) tandem UPA sequences at the 5' end was designed. These UPA capless linear mRNAs were compared with linear mRNAs capped by CleanCap technology (CAP without nucleic acid modification and m1Ψ modified mCAP) (SEQ ID NO: 48) and capless linear mRNAs without UPA (pA-EV-A-S1 (SEQ ID NO: 46), EV-A-S1 (SEQ ID NO: 47)). It was found that the introduction of UPA indeed significantly improved the expression of capless linear mRNA, but the expression level was still slightly lower than that of linear mRNA capped by CleanCap technology (Figure 5A). By comparing multiple tandem UPA capless linear mRNAs, the inventors found that the capless linear mRNA with two tandem UPAs had the highest expression level (Figure 5A). Further investigation of the stability of capless linear mRNA based on UPA sequence for long-term protein expression in Hela cell line showed that the expression stability of 2UPA and 3UPA capless linear mRNA in cells was comparable, and was basically consistent with that of linear mRNA capped by CleanCap technology. The stability of single UPA capless linear mRNA was slightly lower than that of capped mRNA, while the expression stability of capless linear mRNA with only Poly A and EV-A-S1 or only EV-A-S1 was significantly lower than that of other mRNAs (Figure 5B). In order to further evaluate the ability of UPA capless linear mRNA drug to express drug proteins in mammals, BALB / c mice were injected with SM102 LNP-encapsulated UPA, 2UPA capless FLUC (firefly luciferase) mRNA and CAP, mCAP FLUC mRNA in the muscle. Unlike the results in Hela cells, the luciferase activity in mice was continuously detected for 120 h, and it was found that the luciferase level of 2UPA capless FLUC was continuously higher than that of mCAP FLUC and CAP FLUC, which were modified and unmodified, respectively, from 48 h to 120 h (Figures 5C, 5D).

[0086] In summary, the tandem UPA sequence significantly enhances the protein expression stability of capless linear mRNA, and the capless linear mRNA with two tandem UPA sequences has a stronger ability to continuously express proteins in mammals than the m1Ψ modified CleanCap capped linear mRNA.

[0087] Example 5 Analysis of UPA sequence binding proteins in cells

[0088] As a piece of artificially engineered viral sequence, UPA sequence might interact with some proteins in human cells and affect the function and safety of UPA cap-independent linear mRNA. Therefore, UPA+avidin aptamer RNA sequence (SEQ ID NO: 49) and avidin aptamer control (SEQ ID NO: 50) were designed to isolate binding proteins in Hela cell lysate and analyze (RIP) and verify the interaction of UPA sequence with them by mass spectrometry, Western blot, RNA immunoprecipitation (Figure 6A, 6B). After mass spectrometry analysis of YBX1, PABPC1, ILF2, ILF3, IGF2BP1, EIF2AK2, DDX3X, HNRNPR, the inventors verified them by Western blot, and the results showed that UPA sequence indeed enriched YBX1, PABPC1, ILF2, ILF3, IGF2BP1, EIF2AK2, HNRNPR, but not DDX3X. Further RIP experiment was used to verify the binding of RNA and these proteins in living cells, and it was found that 7 proteins enriched in UPA RNA except HNRNPR. HNRNPR is mainly distributed in the nucleus, not in the cytoplasm, which may explain why it does not enrich UPA RNA transfected into the cytoplasm. These data show that UPA sequence interacts with YBX1, PABPC1, ILF2, ILF3, IGF2BP1, EIF2AK2 in living cells. Among these proteins, ILF2, ILF2, EIF2AK2 are all immune regulation related proteins, and their binding to UPA may affect the immune induction ability of UPA cap-independent linear mRNA. PABPC1 is a polyA binding protein that can stabilize RNA and promote translation, so it can bind to the polyA of UPA sequence to enhance the stability and translation level of UPA cap-independent linear mRNA. YBX1 and IGF2BP1 are common RNA binding proteins that can stabilize RNA, and their interaction with UPA may further stabilize UPA cap-independent linear mRNA. The above results show that UPA sequence can not only stabilize RNA by blocking XRN-1, but also stabilize RNA and regulate RNA recognition and immune induction by binding to RNA binding proteins.

[0089] Example 6 Safety evaluation of UPA cap-independent linear mRNA vaccine in mice

[0090] The above data show that the design of 2UPA plus EV-A-S1 makes the uncapped linear mRNA have a similar translation level to the CleenCap-capped mRNA with a capping efficiency of about 95%, and even better in vivo sustained expression ability. The uncapped linear mRNA omits the capping step of linear mRNA, not only simplifying the production, but more importantly breaking the technical barrier of mRNA capping and cap analogs. However, considering that the triphosphate end of the uncapped linear mRNA and the viral-derived sequence both have certain immunostimulatory properties, evaluating the safety of the uncapped linear mRNA is also a crucial step in the development process. Therefore, the uncapped linear mRNA HPV E6E7 fusion protein vaccine with clinical application potential was designed, and the safety of the vaccine was evaluated in mice in vivo. The inventors injected 5ug RNA / dose / mouse of UPA-E6E7 (SEQ ID NO: 51), 2UPA-E6E7 (SEQ ID NO: 52) uncapped linear mRNA LNP vaccine (SM102) and capped modified mCAP-E6E7 (SEQ ID NO: 53) mRNA LNP vaccine (SM102) into the muscle of three groups of C57BL / 6 mice, respectively, and injected 5ug RNA / dose / mouse of the same booster needle vaccine one week later, during which the body weight and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the mice were monitored. The results showed that the body weight of the three groups of mice administered within one week after the first injection and booster vaccination did not significantly differ from the body weight change of the untreated group (Untreated) (Figure 7A). At the same time, the serum ALT and AST levels of the mice administered on the 7th day after the first injection and booster vaccination also did not significantly change relative to before administration or the untreated group (Figures 7B, 7C). Finally, the mice were euthanized two weeks after the booster vaccination, and the main internal organs were collected for histological staining. The histological results showed that the hearts, livers, spleens, lungs, and kidneys of the mice in the administration group did not have obvious drug-related pathological features (Figure 7D). The above results show that the uncapped linear mRNA vaccine of UPA and 2UPA is safe in mice at 5ug RNA / dose / mouse administered for two injections.

[0091] Example 7 Evaluation of the therapeutic effect of UPA uncapped linear mRNA tumor vaccine

[0092] C57BL / 6 mice were subcutaneously injected with B16F10-OVA tumor cells, and on the third and tenth day after cell injection, 3 ug RNA / dose / mouse of UPA, 2UPA cap0linear mRNA vaccine encoding OVA antigen (UPA-OVA (SEQ ID NO: 54), 2UPA-OVA (SEQ ID NO: 55)) and modified capped mCAP mRNA vaccine (mCAP-OVA (SEQ ID NO: 56)) were administered by intramuscular injection, and mCAP FLUC (mCAP-FLUC) as a control. The mice tumor volume was continuously measured from the 3rd to 20th day after cell inoculation, and it was found that the tumor growth of mice administered with three OVA vaccines was significantly slower than that of control group mice, among which the tumor growth of 2UPA-OVA group was the slowest (Figures 8A, 8C). Meanwhile, the measured mouse body weight results showed that no abnormal changes in mouse body weight were observed after administration of UPA, 2UPA cap0linear mRNA vaccine and modified capped mRNA vaccine, again demonstrating the safety of UPA cap0linear mRNA vaccine (Figure 8B). In order to compare the intensity of tumor antigen T cell immunity induced by the vaccine, the peripheral blood of the mice was collected one week after the second immunization to detect the proportion of T cells targeting OVA antigen in PBMC. The results of flow cytometry analysis showed that compared with the control group, three OVA vaccines significantly induced high levels of OVA antigen specific T cells (T cells positive for OVA antigen peptide (SIINFEKL, SEQ ID NO: 57) MHC tetramer: average proportion of UPA-OVA 15.8%; 2UPA-OVA 22.9%; mCAP-OVA 19.1%; mCAP-FLUC 0.7%) (Figure 8D). Consistent with the tumor growth inhibition effect, the average tumor OVA antigen specific T cell proportion of the 2UPA-OVA group was the highest. These results showed that the UPA-based cap0linear mRNA tumor vaccine had good specific immune induction effect and tumor treatment effect.

[0093] Example 8 UPA cap0linear mRNA HPV E6E7 vaccine treatment effect evaluation for HPV related tumor

[0094] Human papillomavirus (HPV) is closely related to the occurrence of various cancers such as cervical cancer, anal cancer, and oropharyngeal cancer, and the tumor cells of these cancers usually express HPV E6, E7 and other proteins. Therefore, the HPV E6E7 UPA cap0linear mRNA vaccine designed by the inventors has potential clinical value for treating HPV related tumors. In order to explore the tumor treatment effect of the vaccine, C57BL / 6 mice were subcutaneously injected with TC-1 cells expressing HPV E6 and E7 proteins, and when the tumor grew to the ninth day, the average tumor volume was about 50 mm 3The mice were injected with UPA-E6E7, 2UPA-E6E7, mCAP-E6E7 (3ug / dose / mouse) respectively. The results showed that the tumor volume of the mice immunized with E6E7 vaccines continuously and significantly reduced after two immunizations, while the tumor volume of the control group mice continuously increased (Fig. 9A, 9C). Meanwhile, the steady increase of the mice weight in the same period also indicated that the UPA capless linear mRNA HPV E6E7 vaccine had good safety (Fig. 9B). One week after the second immunization, the mice PBMC were isolated and stimulated with E6 and E7 derived antigen peptides (Jinsai Biotech) to analyze the proportion of E6, E7 antigen specific T cells. The results showed that the three E6E7 vaccines successfully induced a high proportion of antigen specific T cells (average proportion of IFNγ positive T cells: UPA-OVA 9.6%; 2UPA-OVA 15.5%; mCAP-OVA 13.6%; mCAP-FLUC 0.1%) (Fig. 9D). Among them, the 2UPA-E6E7 vaccine induced the highest proportion of antigen specific T cells, which was significantly higher than the UPA-E6E7 vaccine group, indicating that two tandem UPA significantly improved the immune effect of the UPA capless linear mRNA vaccine. The above results showed that the capless linear mRNA HPV E6E7 vaccine based on UPA sequence could efficiently induce antigen specific T cell immunity and treat HPV related tumors, which had great clinical application potential.

[0095] The IRES used in the above embodiments 1-8 of the present application was obtained by screening by the methods of embodiments 9-10 below.

[0096] Screening of efficient translation IRES

[0097] The candidate IRESes of the present application are mostly derived from viruses. A total of 29 IRESes were screened, including 22 viral type I IRESes, 4 viral type II or III IRESes, and 3 mammalian cell-derived IRESes (FIG. 2A). First, the cap-independent translation efficiency of these IRESes in HEK293T and mouse dendritic cell line DC2.4 was compared using dual-luciferase reporter plasmids (FIG. 10A) by synthesizing IRES sequences from Genewiz and cloning them into the dual-luciferase reporter plasmid pRF between the RLUC and FLUC coding sequences. 100 ng of dual-luciferase reporter plasmids containing different IRESes were transfected into 10,000 HEK293T or DC2.4 cells in a 96-well plate using Hieff Trans Lipid Transfection Reagent (Yoxin Biotech, 40802ES01) at 100 μL per well. After 24 hours, the cells were lysed and the luciferase values were detected using the Dual-Luciferase Reporter Assay Kit (Yoxin Biotech, Cat# 11402ES60) according to the manufacturer's instructions. The ratio of FLUC / RLUC values was taken as the IRES activity. Consistent with previous studies, type I IRESes generally have stronger translation efficiency than other types and mammalian IRESes. As expected by the inventors, the translation efficiency of IRESes has cell-to-cell differences. 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 show that the IRES of human enterovirus EV-A (SEQ ID NO: 57) has the highest translation efficiency in both cell lines.

[0098] To further validate the performance of IRES with high translation efficiency on circular RNA, the 7 IRESs with the highest expression levels were constructed into circular RNA, and circular Firefly luciferase FLUC mRNA was synthesized using the previously reported PIE circularization method (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) (FIG. 10B). The expression of these IRESs in circular RNA was different from that in the dual-luciferase reporter system, which might be due to the fact that the reporter plasmid might be cut non-expectedly and did not produce intact RNA, or the spatial structure of IRES in circular RNA was different from that in linear RNA. However, regardless of whether it was on the dual-luciferase plasmid or on the circular RNA, the IRES from Enterovirus A (EV-A) of the genus Enterovirus of the family Picornaviridae had the highest relative translation efficiency (FIG. 10B). The IRESs of human rhinovirus HRVB3 and coxsackievirus CVB3, which were reported in previous studies, also had very good translation efficiency, but were slightly inferior to the IRES of EV-A in HEK293T and DC2.4 (FIG. 10C). To explore the adaptability of the EV-A IRES to different coding sequences, circular enhanced green fluorescent protein (EGFP) mRNA was constructed, and the EGFP expression levels were compared in HEK293T, DC2.4, and human monocytic cell line THP-1 (FIG. 10D, FIG. 10E). The results showed that the EV-A IRES still had the highest translation efficiency. The above results indicate that the 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.

[0099] Example 10 Secondary structure analysis and sequence optimization of EV-A IRES

[0100] Previous studies have shown that the 5 domains of 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 48S ribosome complex, domains II and IV are responsible for recruiting hnRNP A1 and PCBP1 / 2 and other ITAFs 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 IRES and thus improve the ability of EV-A IRES to initiate translation, or modify it into a shorter IRES. Therefore, the present application first uses SHAPE-MaP technology to analyze the structure of EV-A IRES, determines the internal structure distribution of EV-A, and then designs a series of mutations and simplifications and compares their translation efficiency.

[0101] According to the secondary structure model of EV-A IRES based on SHAPE-MaP reactivity, this IRES also has the typical secondary structure of type I IRES, which includes the common domains I to VI (Figure 11A). However, a unique stem-loop structure appears in EV-A IRES, which is located after DVI and is named DVII. According to previous reports, DII, DIV and DV are relatively conservative and have important functions; DIII is not clear in function but the sequence is relatively conservative, 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.

[0102] The method for optimizing important domains of the present application is to improve the GC pairing ratio of longer stems without changing the secondary structure and without modifying the binding region sequence of important proteins (such as PCBP1 / 2, eIF4G) to improve the stability of the secondary structure. According to this strategy, 6 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 11B). However, compared with wild-type EV-A, none of these mutations significantly improved the translation efficiency. Among them, the 4m2 and 4m3 mutations of DIV and the 5m1 mutation of DV had no obvious effect on the translation efficiency, and 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.

[0103] On the other hand, the present application further truncates the DI (90 nt), the linker between DI and DII (30 nt), DVI (42 nt), and DVII (32 nt) of EV-A, which are longer non-essential sequences (Fig. 11A). The results of the fluorescent signal show that the deletion of DI, DVI, and DVII has no effect on the translation function of IRES, while the deletion of the linker significantly reduces the translation efficiency (Fig. 11C). Based on the above results, the inventors decided to combine the truncated DI, DVI, and DVII that do not affect the function, and the mutation 5m1 that slightly improves the translation efficiency. The results of the combination show that the truncated EV-A-S1 (simultaneously truncating DI and DVI) and the truncated EV-A-S2 (simultaneously truncating DI and DVI and DVII) significantly improve the translation efficiency of EV A by about 50%, while the truncated EV-A-S3 (mutating 5m1 based on EV-A-S1) does not further improve the translation efficiency (Fig. 11D). The inventors speculate that this result may be that the truncation of non-core regions does not affect the core domain, and after the truncation of multiple non-functional structures, the IRES structure is more single and stable. To verify this guess, the inventors further analyzed the IRES structure of the truncated DI and DII, 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 (Fig. 11E). 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 that are not deleted also disappear (Fig. 11E). This result supports the inventors' guess that deleting multiple non-essential structures indeed makes the IRES more compact and stable. In summary, after the modification based on the SHAPE-MaP structure analysis, the modified EV-A IRES not only becomes more compact (750 nt shortened to EV-A-S1 / S3618 nt / EV-A-S2592 nt), but also significantly improves the translation efficiency.

[0104] The sequence information involved in the above embodiments 9-10 is shown in Table 1 below.

[0105] Table 1

[0106] References:

[0107] 1. Baden LR, El Sahly HM, Essink B, et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2021;384(5):403-416. doi: 10.1056 / NEJMoa2035389

[0108] 2. Pardi N, Krammer F. mRNA vaccines for infectious diseases-advances, challenges and opportunities. Nat Rev Drug Discov. 2024;23(11):838-861. doi: 10.1038 / s41573-024-01042-y

[0109] 3. Liu C, Shi Q, Huang X, Koo S, Kong N, Tao W. mRNA-based cancer therapeutics. Nat Rev Cancer. 2023;23(8):526-543. doi: 10.1038 / s41568-023-00586-2

[0110] 4. Weber JS, Carlino MS, Khattak A, et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. Lancet. 2024;403(10427):632-644. doi: 10.1016 / S0140-6736(23)02268-7

[0111] 5. Rojas LA, Sethna Z, Soares KC, et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature. 2023;618(7963):144-150. doi: 10.1038 / s41586-023-06063-y

Claims

1. A capless linear mRNA, characterized in that, comprises, in the 5' to 3' order: 1-5 tandem 5' protection sequences, an IRES, a 5' UTR, a protein-coding region of interest, a 3' UTR, and optionally a polyA tail; wherein the 5' protection sequence comprises an anti-exoribonuclease RNA (xrRNA) and / or an RNA-binding protein binding motif.

2. The mRNA according to claim 1, characterized in that, the xrRNA is derived from a Flavivirus 3' UTR; preferably, the Flavivirus is selected from the group consisting of Cell-fusing agent virus (CFAV), Dengue virus (DENV), Usutu virus (USUV), Yellow fever virus (YFV), or Zika virus (ZIKV); preferably, the 3' UTR of the Flavivirus comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 12-16; preferably, the xrRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 1, 17-33.

3. The mRNA of claim 1, wherein the RNA-binding protein binding motif of the 5' protection sequence is selected from the group consisting of a polynucleotide motif polyA or other RNA-binding protein binding motif; the RNA-binding protein binding motif has a length of 36-60 bp; preferably, the polynucleotide motif comprises a nucleotide sequence as set forth in SEQ ID NO: 2; preferably, the polynucleotide motif is a polyA with a length of 48 bp.

4. The mRNA of any one of claims 1-3, wherein the xrRNA is UX1 derived from a Usutu virus 3' UTR, the UX1 comprises a nucleotide sequence as set forth in SEQ ID NO: 1; the RNA-binding protein binding motif is a polyA; the 5' protection sequence is a UPA sequence; preferably, the UPA comprises a nucleotide sequence as set forth in SEQ ID NO:

8.

5. The mRNA of claim 4, wherein the number of repeats of the UPA in the mRNA is 1-5, preferably 2.

6. The mRNA of any one of claims 1-5, wherein the IRES comprises a nucleotide sequence as set forth in SEQ ID NO: 3 or 4, the 5' UTR comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 5, 36, 37, and / or the 3' UTR comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 6, 7, 38, 39.

7. The mRNA of any one of claims 1-6, wherein the protein of interest comprises a tumor antigen, a bacterial antigen or a viral antigen; preferably, the tumor antigen is a tumor associated antigen or a tumor specific antigen; preferably, the tumor is cervical cancer or melanoma; preferably, the virus is selected from the group consisting of HPV, HIV, EBV or HBV; preferably, the protein of interest is a HPV E6E7 fusion protein; preferably, the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 51, 52, 54, 55.

8. A composition characterized in that, The mRNA of any one of claims 1-7, in a form selected from the group consisting of at least one of a vector, a cell, a nano-lipid particle.

9. A pharmaceutical composition, characterized by, The composition of claim 8, in a dosage form selected from the group consisting of a vaccine or a therapeutic protein preparation, preferably, the therapeutic protein comprises an antibody, a cytokine or an enzyme.

10. Use of the mRNA of any one of claims 1-7, the composition of claim 8 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating a tumor or an infectious disease; preferably, the tumor comprises cervical cancer and melanoma; preferably, the infectious disease is caused by one or more viruses selected from the group consisting of HPV, HIV, EBV and HBV.

11. An internal ribosome entry site (IRES) characterized in that, The IRES comprises the nucleotide sequence of SEQ ID NO: 57 or a variant sequence thereof, which retains a comparable or superior initiation of translation function as SEQ ID NO:

57.

12. The IRES of claim 11, wherein the variant sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:

57.

13. The IRES of claim 11 or 12, wherein the variant comprises the nucleotide sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 58-68.

14. Use of the IRES of any one of claims 11-13 in the manufacture of a circular RNA or a linear RNA; preferably, the linear RNA is a capless linear mRNA.

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