Optimized 5' utrs and uses thereof

Abstract

The present application discloses a modified 5' UTR and mRNA comprising the modified 5' UTR and uses thereof.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drugs, specifically relating to a nucleic acid construct containing an optimized 5'UTR and its application in the prevention or treatment of diseases. Background Technology

[0002] mRNA therapy is a rapidly developing field and has been used in the expression of therapeutic proteins and vaccines. mRNA, as a novel nucleic acid drug, has unique advantages in vaccines and treatments. The rapid response, safety, and efficacy of mRNA vaccines have been validated in the response to the SARS-CoV-2 global pandemic. Despite recent clinical successes, problems such as the thermal instability of mRNA and low protein expression levels remain. This application aims to improve the expression level of the protein encoded by mRNA by screening and optimizing the 5'UTR of mRNA, thereby enhancing the immune response of mRNA vaccines and the therapeutic effect of mRNA drugs. It can be widely applied to mRNA vaccines and has excellent prospects for clinical drug application. Summary of the Invention

[0003] In one aspect, the present invention provides an optimized 5'UTR, characterized in that three random bases NNN are inserted after the transcription start site AGG of the 5'UTR, wherein in the three random bases NNN, the first N is selected from any one of A, T, C and G; the second N is selected from any one of A, T, C and G; and the third N is selected from any one of A, T, C and G.

[0004] In some embodiments, the three random bases NNN are selected from any of the following 64 combinations: AAA, TAA, CAA, GAA, AAT, TAT, CAT, GAT, AAC, TAC, CAC, GAC, AAG, TAG, CAG, GAG, ATA, TTA, CTA, GTA, ATT, TTT, CTT, GTT, ATC, TTC, CTC, ATG, TTG, CTG, GTG, ACA, TCA, CCA, GCA, ACT, TCT, CCT, GCT, ACC, TCC, CCC, GCC, ACG, TCG, CCG, GCG, AGA, TGA, CGA, GGA, AGT, TGT, CGT, GGT, AGC, TGC, CGC, GGC, AGG, TGG, CGG, GGG.

[0005] In some embodiments, the three random bases NNN are selected from any of the following 50 combinations: CAG, TGG, ACC, AGG, TCG, TAA, ATA, CAT, ATC, CTA, TGT, TGC, CGT, TGA, TTC, TTA, TAT, TAG, TAC, CAA, TTG, GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0006] In some embodiments, the three random bases NNN are selected from any of the following 49 combinations: TGG, ACC, AGG, TCG, TAA, ATA, CAT, ATC, CTA, TGT, TGC, CGT, TGA, TTC, TTA, TAT, TAG, TAC, CAA, TTG, GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0007] In some embodiments, the three random bases NNN are selected from any of the following 29 combinations: GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0008] In some embodiments, the optimized 5'UTR of the present invention may be derived from the β-globin gene (HBB), ribosomal protein genes (such as RpL38, RpS25), actin genes (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etching virus (TEV), a disordered short 5'-UTR (scrUTR), or complement factor 3 (C3). In a preferred embodiment, the 5'UTR of the present invention is selected from the human β-globin gene (hHBB) 5'UTR, RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR, or SARS-CoV2 5'UTR. In some embodiments, the nucleotide sequence of the 5'UTR is selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0009] In one aspect, the present invention provides a nucleic acid construct or mRNA molecule comprising an optimized 5'UTR as described herein. In one specific embodiment, the nucleic acid construct or mRNA molecule of the present invention further comprises one or more of the following elements: (a) a 5' cap; (b) an open reading frame (ORF); (c) a 3' untranslated region (3'UTR); and (d) a poly-A tail. In some embodiments, the 3'UTR comprises a 3'UTR derived from an albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, collagen α gene (e.g., collagen α1(I) gene), cytochrome b α subunit gene, poliovirus gene, Sindbis virus gene, brome mosaic virus gene, Zika virus gene, or dengue virus gene. In some embodiments, the 3'UTR comprises a 3'UTR derived from hHBB. In some embodiments, the nucleotide sequence of the hHBB 3'UTR is as shown in SEQ ID NO:2. In some embodiments, the optimized 5'UTR and the 3'UTR are derived from the same gene. In some embodiments, the optimized 5'UTR and the 3'UTR are derived from the human β-globin gene (hHBB), whose sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0010] In a preferred embodiment, the ORF encodes a fluorescent protein, a luciferase protein drug (e.g., an antibody), or an antigen.

[0011] In some embodiments, the antigen is derived from an infectious disease, an autoimmune disease, and / or an allergic disease; or, the antigen is an antigen, epitope, protein, or peptide derived from a pathogen or tumor.

[0012] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is selected from: liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer, bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof.

[0013] In some embodiments, the tumor-derived antigen is selected from NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or combinations thereof.

[0014] In some embodiments, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof. In some embodiments, the viral antigen is selected from antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or Epstein-Barr virus.

[0015] In some embodiments, the coronavirus is SARS-CoV-2, and the coronavirus antigen is the complete spike protein or its RBD domain. In some embodiments, the spike protein is selected from the spike protein of any of the following viral strains: SARS-CoV-2, SARS-CoV-2Alpha, SARS-CoV-2Beta, SARS-CoV-2Gamma, SARS-CoV-2Kappa, SARS-CoV-2Delta, or SARS-CoV-2Omicron.

[0016] In some embodiments, the influenza virus is an influenza A virus or an influenza B virus, and the antigen is a hemagglutinin protein (HA), neuraminidase (NA), and / or nucleoprotein (NP). In some embodiments, the influenza virus antigen is selected from the hemagglutinin protein, neuraminidase, and / or nucleoprotein of any strain of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria, or influenza B virus Yamagata. In some embodiments, the respiratory syncytial virus antigen is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the respiratory syncytial virus antigen is selected from the F protein, G protein, nucleocapsid protein, or matrix protein of a subtype A strain or a subtype B strain. In some embodiments, the respiratory syncytial virus antigen comprises a pre-fusion conformation F protein (pre-F), a post-fusion conformation F protein (post-F), or a mixture of both. In some embodiments, the F protein is the pre-fusion conformation F protein (pre-F).

[0017] In some embodiments, the antigen of the human metapneumovirus (hMPV) is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the antigen is the F protein of human metapneumovirus (hMPV).

[0018] In some embodiments, the antigen of the chicken infectious bronchitis virus is a Spike protein, an M protein, a nucleocapsid protein, or an envelope protein. In some embodiments, the antigen is the S1 region or the RBD domain of the Spike protein.

[0019] In some embodiments, the Nipah virus antigen is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the antigen is an F protein or a G protein.

[0020] In some embodiments, the antigens of the monkeypox virus are selected from inner membrane proteins M1R, H3L, E8L, and A29L, outer envelope proteins A35R and B6R, and accessory protein L5L. In some embodiments, the antigens of the monkeypox virus are A29L, M1R, B6R, and / or A35R.

[0021] In some embodiments, the fungal antigen is selected from antigens of the genera *Candida*, *Aspergillus*, *Mucor*, *Rhizopus*, *Epidermophyton*, *Malassezia*, *Preumocystis*, *Penicillium*, *Alternaria*, *Cladosporium*, *Botrytis*, *Aureobasidium*, *Fusarium*, or *Trichoderma*.

[0022] In some embodiments, the bacterial antigen is selected from antigens of actinomycetes, Bacillus, Bacteroides, Enterococcus, Listeria, Mycobacterium, Streptococcus pneumoniae, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, or Streptococcus.

[0023] In some embodiments, the antibody is an antibody capable of specifically binding to any one or more of the above-mentioned antigens.

[0024] In some implementations, the antibody is an autoantibody.

[0025] In some embodiments, the antibody is an antibody that specifically binds to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and / or LAG3.

[0026] In a preferred embodiment, the nucleotide sequence of the ORF of the present invention is selected from the sequences shown in any one of SEQ ID NO: 7-31. In some embodiments, the ORF comprises an amino acid sequence as shown in any one of SEQ ID NO: 32-56.

[0027] In some embodiments, the 5'UTR described above is the 5'UTR of the human β-globin gene (hHBB). In some embodiments, the optimized 5'UTR described above increases the expression level of the fluorescent protein Luciferase (Fluc) by at least 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or higher.

[0028] In another aspect, the present invention provides the use of the nucleic acid constructs or mRNA molecules described herein in any of the following: (1) preparation of vaccines; (2) encoding viral antigens in or in vitro in a subject; (3) preparation of drugs encoding viral antigens in or in vitro in a subject; (4) for the preparation of drugs (e.g., antibodies).

[0029] In another aspect, the present invention provides a vaccine comprising a nucleic acid construct or mRNA molecule as described herein. In some embodiments, the nucleic acid construct or mRNA molecule is encapsulated in lipid nanoparticles (LNPs).

[0030] In another aspect, the present invention provides a vector comprising a nucleic acid construct or mRNA molecule as described in the present invention.

[0031] In another aspect, the present invention provides a host cell comprising a vector as described herein.

[0032] In another aspect, the present invention provides a method for preparing mRNA molecules as described herein, comprising: synthesizing a gene encoding an ORF protein and a template DNA sequence containing mRNA transcription-related elements; preparing a template by primer PCR amplification; and obtaining mRNA by transcription using a co-transcriptional capping method.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising: a nucleic acid construct or mRNA molecule as described herein, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0034] In another aspect, the present invention provides a product or kit comprising: a nucleic acid construct or mRNA molecule as described herein, a vaccine, and / or a pharmaceutical composition.

[0035] In another aspect, the present invention provides a method for treating and / or preventing disease and / or symptoms in a subject caused by infection with a pathogen (e.g., a virus), comprising administering to a subject in need an effective amount of a nucleic acid construct or mRNA molecule, vaccine, pharmaceutical composition, and / or product or kit as described herein.

[0036] In another aspect, the present invention provides the use of nucleic acid constructs or mRNA molecules, vaccines, pharmaceutical compositions, and / or products or kits as described herein in the preparation of medicaments for treating and / or preventing diseases and / or symptoms in subjects caused by infection with pathogens (e.g., viruses).

[0037] In some embodiments, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof. In some embodiments, the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, and more preferably SARS-CoV-2, influenza A virus, or influenza B virus.

[0038] In some embodiments, the pathogen is associated with a protein drug (e.g., an antibody) or antigen encoded by an ORF contained in the nucleic acid construct or mRNA molecule.

[0039] In some exemplary embodiments, the pathogen is an influenza virus, the antigen is an antigen derived from the influenza virus, and the protein drug is an antibody that specifically binds to the influenza virus antigen. In some exemplary embodiments, the pathogen is a respiratory syncytial virus (RSV), the antigen is an antigen derived from the RSV, and the protein drug is an antibody that specifically binds to the RSV antigen.

[0040] In some implementations, the disease and / or symptoms include uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, and severe acute respiratory syndrome (SARS).

[0041] In some implementations, the subject is a mammal, such as a mouse or a human.

[0042] In another aspect, the present invention provides a method for inducing a subject to produce a neutralizing antibody response and / or a T-cell immune response, comprising administering to a subject in need an effective amount of a nucleic acid construct or mRNA molecule, vaccine, pharmaceutical composition, and / or product or kit as described herein.

[0043] In another aspect, the present invention provides the use of nucleic acid constructs or mRNA molecules, vaccines, pharmaceutical compositions, and / or products or kits as described herein in the preparation of medicaments for inducing neutralizing antibody responses and / or T-cell immune responses in subjects.

[0044] In some embodiments, the neutralizing antibody response is a neutralizing antibody response against a viral antigen, and the T-cell immune response includes CD4+ and / or CD8+ T-cell immune responses.

[0045] In some implementations, the virus is an influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, or monkeypox virus.

[0046] In some implementations, the subject is a mammal, such as a mouse or a human.

[0047] In another aspect, the optimized 5'UTR, as described above, is used to prepare nucleic acid constructs or mRNA molecules.

[0048] In another aspect, the optimized 5'UTR, as described above, is used to enhance the expression level of proteins encoded by nucleic acid constructs or mRNA molecules containing it.

[0049] In another aspect, the optimized 5'UTR, as described above, is used to improve the immunogenicity of vaccines, pharmaceutical compositions, products, or kits containing the nucleic acid constructs or mRNA molecules.

[0050] Beneficial effects

[0051] The optimized 5'UTR provided in this application can increase the expression level of proteins encoded by mRNA (e.g., antigens, protein drugs (e.g., antibodies)). Therefore, mRNA vaccines containing the optimized 5'UTR provided in this application exhibit better immune response levels (e.g., the ability to induce high titers of neutralizing antibodies, the ability to induce specific T-cell immune responses), thereby providing better prevention and / or treatment of diseases and / or symptoms caused by pathogen (e.g., virus) infection. Therefore, the optimized 5'UTR provided in this application and the mRNA molecules containing it have broad application prospects in the fields of vaccines, gene therapy, and antibody therapy. Attached Figure Description

[0052] Figure 1 The diagram shows the structure of the DNA template and mRNA product for mRNA production via co-transcription capping, as well as the 5'UTR optimization method used in this invention.

[0053] Figure 2 Optimized 5'UTR mRNA synthesized from in vitro transcription was demonstrated, and the expression of eGFP and FLuc reporter genes in cells was detected.

[0054] Figure 3 The study demonstrated that the 5'UTR optimization method can improve the level of GFP expressed in various natural 5'UTRs.

[0055] Figure 4 The study demonstrated that the 5'UTR optimization method can increase the level of pre-fusion conformation F protein of respiratory syncytial virus expressed by mRNA.

[0056] Figure 5 The study showed that the 5'UTR optimization method can increase the level of HA antigen of influenza virus H1N1 and H5N1 expressed in mRNA.

[0057] Figure 6 The study showcases the design, synthesis, LNP encapsulation, and cell expression of mRNA vaccines expressing the HA antigens of seasonal influenza viruses H1N1, H3N2, and B-Victoria strains, as well as the results of IgG antibody titer detection, virus neutralization, and virus protection experiments after immunizing BALB / c mice.

[0058] Figure 7 The results show the cell expression of influenza virus H5N1 mRNA-HA vaccine and the IgG antibody titer, virus neutralization, and virus protection after immunization of BALB / c mice and SPF chickens.

[0059] Figure 8 The results show the cell expression of the mRNA-HA neck antigen of influenza virus H1N1, the IgG antibody titer after immunization of BALB / c mice, and the results of the virus protection experiment.

[0060] Figure 9 The results show the cell expression of influenza virus H1N1 mRNA-NA vaccine, IgG antibody titer after immunization of BALB / c mice, and the results of the virus protection experiment.

[0061] Figure 10 The cell expression of influenza virus PR8(H1N1) mRNA-NP vaccine and the IgG antibody titers after immunization of BALB / c mice via different routes are shown.

[0062] Figure 11This study demonstrates the design, synthesis, and cell expression of a bivalent SARS-CoV-2 Spike protein RBD region mRNA vaccine, as well as the results of IgG antibody titer detection, pseudovirus neutralization experiment, and cell-mediated immunity assay after immunization of BALB / c mice.

[0063] Figure 12 This study demonstrates the synthesis and cellular expression of an mRNA vaccine containing the pre-fusion conformation F protein of respiratory syncytial virus (RSV), as well as the detection of IgG antibody titers, virus neutralization assays, and cellular immunity assays after immunization of BALB / c mice.

[0064] Figure 13 The results show the intracellular expression of human metapneumovirus mRNA-F vaccine as detected by immunofluorescence.

[0065] Figure 14 The synthesis and intracellular expression of the Spike protein mRNA-S1 and mRNA-RBD vaccines against chicken infectious bronchitis virus were demonstrated.

[0066] Figure 15 The synthesis and intracellular expression of Nipah virus F protein and G protein mRNA vaccines were demonstrated.

[0067] Figure 16 The synthesis and intracellular expression of the monkeypox virus L5L protein mRNA vaccine were demonstrated.

[0068] Figure 17 The study demonstrated the mRNA synthesis, intracellular expression detection, and virus neutralization assay of the D25 heavy and light chains expressing the anti-RSV monoclonal antibody.

[0069] Sequence information

[0070] The descriptions of the sequences involved in this application are provided in the table below.

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[0085] Detailed Implementation

[0086] Terminology Definition

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains.

[0088] As used herein, “nucleic acid” or “polynucleotide” refers to an organic molecule containing two or more covalently bonded nucleotides. As used herein, “nucleotide” refers to an organic molecule containing: 1) a nucleoside, which contains a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically linked by a phosphodiester bond, in which the 3' carbon of the sugar of the first nucleotide is linked to the 5' carbon of the sugar of the second nucleic acid via a bridging phosphate group. Typically, the bridging phosphate ester contains two non-bridging oxygen atoms that are bonded only to the phosphorus atom of the phosphate ester, and two bridging oxygen atoms, each linking the phosphorus atom to the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the nucleotide sequence in a nucleic acid, if the 3' carbon of the first nucleotide is linked to the 5' carbon of the second nucleotide, the first nucleotide is said to be 5' (upstream) of the second nucleotide. Similarly, if the 5' carbon of the second nucleotide is linked to the 3' carbon of the first nucleotide, the second nucleotide is said to be 3' (downstream) of the first nucleotide. Nucleic acid sequences are usually read in 5'->3' order, starting with a 5' nucleotide and ending with a 3' nucleotide.

[0089] As used herein, “messenger RNA” (“mRNA”) refers to a nucleic acid containing an open reading frame that encodes a protein. mRNA has a modular structure, including a 5' cap, a 5' untranslated region (5'UTR), an open reading frame (ORF) that encodes the protein, a 3' untranslated region (3'UTR), and a polyadenylated nucleotide (polyA) tail.

[0090] As used in this article, an "open reading frame encoding a protein" refers to a nucleic acid sequence containing a coding sequence that, when translated, leads to the production of a protein. Open reading frames typically begin with a start codon, such as AUG in an RNA sequence (ATG in a DNA sequence), and end with a stop codon, such as UAG, UAA, or UGA in an RNA sequence (TAG, TAA, or TGA in a DNA sequence).

[0091] DNA or RNA sequences encode genes using codons. A codon is a set of three nucleotides within a nucleic acid (such as DNA or RNA) sequence. An anticodon is a set of three nucleotides within a nucleic acid, such as transfer RNA (tRNA), that is complementary to the codon, allowing the codon of the first nucleic acid to associate with the anticodon of the second nucleic acid via hydrogen bonds between the codon and anticodon bases. For example, the codon 5'-AUG-3' on mRNA has a corresponding anticodon 3'-UAC-5' on tRNA. During translation, tRNA with an anticodon complementary to the codon to be translated associates with the codon on mRNA, typically to deliver the amino acid corresponding to the codon to be translated, or to promote translation termination and the release of the translated polypeptide from the ribosome.

[0092] As used herein, "lipid nanoparticles (LNPs)" refers to nanoscale particles composed of lipid molecules. They typically have a particle size range of 20-200 nanometers and contain components such as cationic lipids, phospholipids, and PEGylated lipids. Lipid nanoparticles are used to encapsulate and deliver biomolecules (such as mRNA and DNA) and can effectively protect these macromolecules from degradation by enzymes in vivo, promoting their entry into target cells. The lipid nanoparticles contain cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol-lipids. In some embodiments, the cationic lipids are DLin-MC3-DMA or SM102; the polyethylene glycol-lipids are DMG-PEG2000; the neutral phospholipids are DSPC; and the steroidal lipids are cholesterol.

[0093] 5' Cap

[0094] A 5' cap is an entity, typically a modified nucleotide entity, that is usually capped at the 5' end of mature mRNA. The 5' cap can typically be formed from a modified nucleotide, particularly a derivative of guanine nucleotides. Preferably, the 5' cap is attached to the 5' end via a 5′-5′-triphosphate bond. The 5' cap can be methylated, for example, m7GpppN, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of RNA.

[0095] mRNA drugs are produced in a cell-free environment via in vitro transcription. The 5' cap is typically added in two ways: one is by further processing the mRNA obtained through in vitro transcription with a capping enzyme; the other is by directly obtaining capped mRNA by adding a trinucleotide capping analog to the in vitro transcription system. The latter, known as co-transcriptional capping, has the advantages of fewer production steps and a single product, making it the mainstream trend in mRNA drug production. However, using a capping analog requires fixing the 5'UTR start sequence to AGG, which may alter the 5'UTR structure and affect translation efficiency. Specifically, this invention optimizes the 5'UTR sequence used in the co-transcriptional capping method, improving the expression level of the protein encoded by the mRNA.

[0096] Optimized 5'UTR

[0097] As used herein, the 5' untranslated region (5'UTR) refers to a specific portion of messenger RNA (mRNA) located at the 5' end of the open reading frame (ORF) of the mRNA. Typically, the 5'UTR begins at the transcription start site and terminates one nucleotide before the start codon of the ORF.

[0098] In the context of this invention, the 5'UTR corresponds to the sequence of mature mRNA located between the 5' cap and the start codon. Preferably, the 5'UTR corresponds to the sequence extending from the 3' nucleotide at the 5' cap (preferably from the 3' nucleotide immediately following the 5' cap) to the 5' nucleotide at the start codon of the protein-coding region (preferably to the 5' nucleotide immediately following the start codon of the protein-coding region). The 3' nucleotide immediately following the 5' cap of the mature mRNA typically corresponds to a transcription start site. The term "corresponds to" means that the 5'UTR sequence can be an RNA sequence, such as in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to said RNA sequence.

[0099] Currently, the 5'UTR sequences used in mRNA drugs typically come from two sources: one is the 5'UTR of natural genes and its derived sequences (such as Pfizer-BioNTech's COVID-19 vaccine BNT162b2), and the other is obtained by screening from artificially designed and synthesized sequence libraries (represented by Moderna's COVID-19 vaccine mRNA-1273). Artificial de novo synthesis is extremely labor-intensive, and the advantages of screened 5'UTR sequences compared to natural sequences are not significant (Ye et al, 2023 PNAS DOI:10.1073 / pnas.2311752120; Castillo-Hair et al, 2024 Nature Communications DOI:10.1038 / s41467-024-49508-2). Therefore, the 5'UTR sequences in current mRNA drugs are usually derived from naturally expressed high-expression genes.

[0100] In the context of this invention, the term "5'UTR of a gene," such as "5'UTR of the human β-globin gene (hHBB)," corresponds to the sequence of the 5'UTR of the mature mRNA derived from that gene (i.e., the mRNA obtained through transcription of the gene and maturation of immature mRNA). Similarly, the term "3'UTR of a gene," such as "3'UTR of the human β-globin gene (hHBB)," corresponds to the sequence of the 3'UTR of the mature mRNA derived from that gene (i.e., the mRNA obtained through transcription of the gene and maturation of immature mRNA).

[0101] In some respects, this disclosure provides an optimized 5'UTR. In one specific embodiment, three random bases are added after the transcription start site AGG of the 5'UTR used in the co-transcription capping method to construct a new 5'UTR. The inventors have surprisingly discovered that the newly constructed 5'UTR can improve the expression level of the protein encoded by the mRNA, making the optimized mRNA molecule a promising candidate for applications in vaccines, gene therapy, antibody therapy, and other fields.

[0102] In one specific embodiment, this disclosure provides an optimized 5'UTR with a 3-base NNN inserted after the transcription start site AGG. In one embodiment, the 3-base NNN is obtained by permuting any three of the four bases A, T, C, and G (excluding AGG). For example, in the 3-base NNN, the first N can be selected from any of A, T, C, and G; the second N can be selected from any of A, T, C, and G; and the third N can be selected from any of A, T, C, and G. Thus, a total of 64 possible combinations of the 3-base NNN are generated.

[0103] In some embodiments, the three random bases NNN are selected from any of the following 64 combinations: AAA, TAA, CAA, GAA, AAT, TAT, CAT, GAT, AAC, TAC, CAC, GAC, AAG, TAG, CAG, GAG, ATA, TTA, CTA, GTA, ATT, TTT, CTT, GTT, ATC, TTC, CTC, ATG, TTG, CTG, GTG, ACA, TCA, CCA, GCA, ACT, TCT, CCT, GCT, ACC, TCC, CCC, GCC, ACG, TCG, CCG, GCG, AGA, TGA, CGA, GGA, AGT, TGT, CGT, GGT, AGC, TGC, CGC, GGC, AGG, TGG, CGG, GGG.

[0104] In some embodiments, the three random bases NNN are selected from any of the following 50 combinations: CAG, TGG, ACC, AGG, TCG, TAA, ATA, CAT, ATC, CTA, TGT, TGC, CGT, TGA, TTC, TTA, TAT, TAG, TAC, CAA, TTG, GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0105] In some embodiments, the three random bases NNN are selected from any of the following 49 combinations: TGG, ACC, AGG, TCG, TAA, ATA, CAT, ATC, CTA, TGT, TGC, CGT, TGA, TTC, TTA, TAT, TAG, TAC, CAA, TTG, GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0106] In some embodiments, the three random bases NNN are selected from any of the following 29 combinations: GGT, GAA, AGA, TCC, TTT, CTG, GAC, CTT, CCG, GAG, GTA, TCA, CCA, GTC, CGA, GGC, GCA, CGC, CCT, CCC, GGA, CGG, GAT, GTT, GGG, CTC, GCT, GCG, GCC.

[0107] The source of the optimized 5'UTR of this invention is not limited. Generally, it can be derived from the 5'UTR of any natural gene and its derived sequences, and it can be derived from animals, plants, fungi, bacteria, or viruses. Generally, it can be derived from any species, such as humans, baboons, monkeys, mice, chickens, zebrafish, Arabidopsis thaliana, yeast, Escherichia coli, influenza virus, dengue virus, coronavirus, etc. Generally, it can originate from any artificially synthesized 5'UTR sequence, which can be derived from any artificially designed 5'UTR sequence (such as US16441647), or from any 5'UTR library or 5'UTR sequence generated by an algorithm (PJSample et al., Nat Biotechnol 2019 DOI:10.1038 / s41587-019-0164-5; S. Castillo-Hair et al., Nature Communications 2024 DOI:10.1038 / s41467-024-49508-2). All 5'UTR sequences listed in the above patents and documents, as well as their full texts, are hereby incorporated into this document. For example, the optimized 5'UTR of the present invention can be derived from β-globin genes (HBB), ribosomal protein genes (such as RpL38, RpS25), actin genes (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etching virus (TEV), disordered short 5'UTRs (scrUTRs), or complement factor 3 (C3). In some embodiments, the sequences of hHBB 5'UTR, RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR, and SARS-CoV2 5'UTR are as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100% identity with them. In some implementations, these sequences have the same or similar activity as the sequences from which they are derived.

[0108] In some embodiments, the optimized 5'UTR of the present invention is a truncated 5'UTR, i.e., the insertion of three random bases NNN after the 5'UTR transcription start site AGG, while simultaneously deleting one or more nucleotides. In some embodiments, the truncated 5'UTR retains similar functional activity as the untruncated optimized 5'UTR. In some embodiments, the optimized 5'UTR of the present invention further comprises one or more point mutations, i.e., the insertion of three random bases NNN after the 5'UTR transcription start site AGG, while simultaneously altering one or more nucleotides (e.g., addition, deletion, and / or substitution). In a preferred embodiment, such nucleotide deletions or alterations can further enhance the expression level of the encoded protein.

[0109] The source of the 3'UTR of this invention is not limited. Generally, it can be derived from the 3'UTR of any natural gene, which can be derived from animals, plants, fungi, bacteria, or viruses. Generally, it can be derived from any species, such as humans, baboons, monkeys, mice, chickens, zebrafish, Arabidopsis thaliana, yeast, Escherichia coli, influenza virus, dengue virus, coronavirus, etc. (JJSeo, Cell2023 DOI:10.1016 / j.cell.2023.06.007; AKMorrow, bioRxiv 2024 DOI:10.1101 / 2024.10.07.616676), or mutants of these 3'UTRs (T.Fu, et al. Nat Commun2024, DOI:10.1038 / s41467-024-46795-7). All 3'UTR sequences listed in the above literature, along with their full texts, are cited in this paper.

[0110] For example, the 3'UTR element may be derived from the following genes: albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, collagen α gene (e.g., collagen α1(I) gene), cytochrome b α subunit gene, poliovirus gene, Sindbis virus gene, brome mosaic virus gene, Zika virus gene, and dengue virus gene.

[0111] In embodiments of the invention, the optimized 5'UTR and the 3'UTR are derived from the same gene, for example, from the human β-globin gene (hHBB), with sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with them. In some embodiments, these sequences have the same or similar activity compared to their derived sequences (e.g., SEQ ID NO:1 and SEQ ID NO:2).

[0112] Nucleic acid constructs and RNA molecules

[0113] In some aspects, this disclosure provides nucleic acid constructs comprising the optimized 5'UTR and open reading comprehension (ORF) encoding the target gene as described herein. In a preferred embodiment, the nucleic acid construct further comprises a 5' cap, a 3' untranslated region (3'UTR), and a polyadenylated (polyA) tail.

[0114] In some aspects, this disclosure provides RNA molecules. In a particular embodiment, the RNA molecule is a modified mRNA comprising a 5' cap, an optimized 5' UTR as described herein, an ORF encoding a target gene, a 3' untranslated region (3' UTR), and a polyadenylated (polyA) tail.

[0115] In some embodiments, the ORF is a polynucleotide sequence encoding a target gene protein. In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous. In some embodiments, the 5' UTR in the nucleic acid construct is located upstream of the open reading frame. In some embodiments, the 5' UTR in the nucleic acid construct is located at the 5' end of the open reading frame. In some embodiments, the nucleic acid construct / RNA molecule of this disclosure further includes a 3' untranslated region element (3' UTR) as described above. In some embodiments, the 3' UTR in the nucleic acid construct / RNA molecule of this disclosure is located downstream of the open reading frame. In some embodiments, the 3' UTR in the nucleic acid construct is located at the 3' end of the open reading frame.

[0116] In some embodiments, the open reading frame described herein is derived from a different gene than the 5' UTR and / or the 3' UTR. In some embodiments, the nucleic acid construct / RNA molecule of this disclosure contains at least one open reading frame, at least one 5' UTR, or at least one 3' UTR. In some embodiments, the 5' UTR and 3' UTR in the nucleic acid construct / RNA molecule of this disclosure are from the same or different sources, for example, from the same or different genes. In some embodiments, the 5' UTR and 3' UTR in the nucleic acid construct / RNA molecule of this disclosure are from the same or different species.

[0117] In some implementations, the nucleic acid construct includes a 5' UTR and a 3' UTR, wherein:

[0118] The 5'UTR is selected from the 5'UTR or a derivative sequence of any gene such as ARHGAP (e.g., ARHGAP15), HSPB1, HBB, CCL13, etc., and the 3'UTR is selected from the 3'UTR or a derivative sequence of any gene such as HBB, ARHGAP15, CORO1A, HPX, etc.

[0119] In some embodiments, the nucleic acid construct includes an optimized 5'UTR as described in the text and a 3'UTR as described above, wherein the optimized 5'UTR and the 3'UTR are derived from the same gene, such as the human β-globin gene (hHBB), and their sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with them. In some embodiments, these sequences have the same or similar activity compared to their derived sequences (e.g., SEQ ID NO:1 and SEQ ID NO:2).

[0120] In some embodiments, the nucleic acid construct / RNA molecule disclosed herein further includes: (d) a polyadenylate (poly-A) tail.

[0121] In some embodiments, the poly-A tail in the nucleic acid construct is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the nucleic acid construct. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, or 500 nucleotides long.

[0122] In some implementations, the poly-A tail includes, but is not limited to, HGH polyA, SV40polyA, BGHpolyA, rbGlob polyA, or SV40late polyA.

[0123] In this disclosure, in any of the above-described nucleic acid constructs, the ORF comprises a nucleotide sequence encoding at least one polypeptide or protein. In some embodiments, the nucleotide sequence may be a codon-optimized nucleotide sequence.

[0124] In some embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase. In specific embodiments, the sequence of the ORF is shown in SEQ ID NO:7 or SEQ ID NO:8.

[0125] In some embodiments, the polypeptide or protein encoded by the ORF is a protein drug (e.g., an antibody). In specific embodiments, the sequence of the ORF is shown in SEQ ID NO:30 or SEQ ID NO:31.

[0126] In some implementations, the polypeptide or protein encoded by the ORF is a viral antigen. Exemplarily, viral antigens include, but are not limited to, antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or EB virus.

[0127] In some embodiments, the viral antigen is a coronavirus antigen. In some embodiments, the coronavirus is a human-infecting coronavirus, such as SARS-CoV-2 (COVID-19), SARS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, or MERS-CoV. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus antigen is a structural protein. In some embodiments, the structural protein is selected from spike protein (S protein or Spike protein), envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein). In some embodiments, the structural protein is a spike protein. In some embodiments, the spike protein is the SARS-CoV-2 spike protein. In some implementations, the SARS-COV-2 spike protein is selected from the spike protein of any of the following viral strains: SARS-COV-2 (e.g., wild-type SARS-COV-2), SARS-COV-2Alpha (B.1.1.7), SARS-COV-2Beta (B.1.351), SARS-COV-2Gamma (P.1), SARS-COV-2Kappa (B.1.617.1), SARS-COV-2Delta (B.1.617.2), SARS-COV-2Omicron (B.1.1.529), and SARS-COV-2Omicron (BA4).

[0128] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A or influenza B viruses; exemplary examples include influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H5N8, influenza A virus H2N2, influenza A virus H7N9, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., influenza B virus / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of the influenza virus, such as hemagglutinin (HA), neuraminidase (NA), ion channel protein M2, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is the HA protein of an influenza virus, such as the HA protein of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific embodiments, the influenza virus antigen is the NA protein of an influenza virus, such as the NA protein of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific implementations, the influenza virus antigen is the NP protein of an influenza virus, such as the NP protein of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).

[0129] In a preferred embodiment, the antigen of the respiratory syncytial virus is the pre-fusion conformation F protein (pre-F).

[0130] In a preferred embodiment, the antigen of the human metapneumovirus (hMPV) is the F protein.

[0131] In a preferred embodiment, the antigen of the chicken infectious bronchitis virus is the S1 region or the RBD domain of the Spike protein.

[0132] In a preferred embodiment, the antigens of the Nipah virus are the F protein and the G protein.

[0133] In a preferred embodiment, the antigen of the monkeypox virus is the L5L protein.

[0134] In some specific embodiments, the nucleotide sequence of the ORF encoding the polypeptide or protein is selected from the sequence shown in any one of SEQ ID NO: 7-31, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. In some embodiments, these sequences have the same or similar activity compared to the sequences from which they are derived (e.g., SEQ ID NO: 7-31).

[0135] In some embodiments, the ORF encoding the polypeptide or protein comprises a codon-optimized nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence encoding the protein shown in SEQ ID NO:32-56. In some embodiments, these sequences have the same or similar activity as the sequences from which they are derived (e.g., SEQ ID NO:32-56).

[0136] In a preferred embodiment, the ORF encoding a polypeptide or protein comprises an amino acid sequence as shown in any of SEQ ID NO: 32-56 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. In some embodiments, these sequences have the same or similar activity as the sequences from which they are derived (e.g., SEQ ID NO: 32-56).

[0137] In some embodiments, this disclosure provides a nucleic acid construct comprising, from the 5' to 3' direction, an optimized 5'UTR, ORF, 3'UTR, and a poly-A tail as described herein. In some embodiments, the 5'UTR comprises or is a nucleotide sequence as shown in SEQ ID NO: 1 or any of 3-6, the ORF comprises or is a nucleotide sequence as shown in SEQ ID NO: 7-31, and the 3'UTR comprises or is a nucleotide sequence as shown in SEQ ID NO: 2.

[0138] In some embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding an additional protein or polypeptide. In some embodiments, the additional protein or polypeptide is selected from: signal peptides, multimerizing domains (e.g., dimerizing domains, trimerizing domains), tags, or any combination thereof.

[0139] Polynucleotides

[0140] This disclosure also provides an isolated polynucleotide that encodes one or more polypeptides or proteins or viral antigens as described above.

[0141] In some implementations, this disclosure provides the use of any of the nucleic acid constructs, RNA molecules, or polynucleotides described herein in any of the following: (1) preparation of a vaccine; (2) encoding a viral antigen in or in a subject; (3) preparation of a medicament encoding a viral antigen in or in a subject; (4) for the preparation of a medicament.

[0142] In some embodiments, the nucleic acid constructs, RNA molecules, or polynucleotides of this disclosure encode a target protein, wherein the open reading frame (ORF) has enhanced target protein expression.

[0143] carrier

[0144] This disclosure also provides a vector comprising the nucleic acid construct or RNA molecule described herein, wherein the nucleic acid construct / RNA molecule may be present in the vector and / or may be part of the vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be an expression vector, i.e., a vector that provides expression of the nucleic acid construct or RNA-encoded polypeptide. The expression vector typically comprises at least one nucleic acid of this disclosure, operably linked to one or more suitable expression regulatory elements (e.g., promoters, terminators, etc.). Selection of said elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the polypeptides encoded by this disclosure include, for example, promoters, terminators, selection markers, leader sequences, reporter genes, etc.

[0145] The nucleic acid constructs / RNA molecules disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the nucleotide sequence information disclosed herein, and / or can be isolated from suitable natural sources.

[0146] In some embodiments, the vector disclosed herein further includes a promoter, for example, the promoter is located at the 5' end of the 5' UTR of the nucleic acid construct, such as the T7 promoter, T7lac promoter, Tac promoter, Lac promoter, or Trp promoter.

[0147] host cells

[0148] This disclosure also provides a host cell comprising the nucleic acid constructs, RNA molecules, or polynucleotides described herein. In some embodiments, the cell is capable of expressing one or more nucleic acid constructs or RNA-encoded polypeptides of this disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0149] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0150] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0151] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0152] However, this disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0153] Production or preparation method

[0154] This disclosure provides a method for preparing the nucleic acid construct or RNA of this disclosure, and a method for preparing the polypeptide encoded therefrom.

[0155] Methods and reagents for preparing nucleic acid constructs or RNA and their encoded polypeptides, such as specific suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, protein separation and purification techniques suitable for manufacturing the polypeptides encoded by this disclosure are well known to those skilled in the art.

[0156] In some embodiments, the method for preparing the nucleic acid construct or RNA includes culturing the aforementioned host cells and recovering the resulting nucleic acid construct or RNA from the culture. The nucleic acid construct or RNA of this disclosure, and its encoded polypeptide, can also be obtained by other production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0157] In some embodiments, the method for preparing RNA molecules includes: preparing a nucleic acid construct or vector, and then using the nucleic acid construct or vector to perform reverse transcription to obtain RNA molecules. In some specific embodiments, the method further includes adding a 5' cap to the 5' end of the RNA molecule.

[0158] vaccine

[0159] Gene vaccines

[0160] As used in this article, "gene vaccines" refer to vaccines administered via gene delivery, often understood as "third-generation" vaccines. They typically consist of genetically modified nucleic acid molecules encapsulated in lipid nanoparticles (LNPs), allowing peptides or proteins (antigens) specific to pathogens or tumor antigens to be expressed in vivo. Gene vaccines are expressed after administration to a patient and uptake by cells. The expression of the administered nucleic acid leads to the production of the encoded protein. When these proteins are recognized as foreign substances by the patient's immune system, an immune response is triggered.

[0161] This disclosure provides a vaccine comprising the nucleic acid construct, the RNA, or the polynucleotide described in this invention. In some embodiments, the nucleic acid construct or RNA encodes one or more antigens of one or more viral strains. The vaccine provided in this disclosure may be a monovalent vaccine, a multivalent vaccine, or a combination vaccine.

[0162] Monovalent vaccine

[0163] This disclosure provides a monovalent vaccine comprising an antigen encoding an organism. In some embodiments, the monovalent vaccine comprises an antigen encoding a viral strain.

[0164] Multivalent / Combination Vaccines

[0165] In some embodiments, the vaccine may include nucleic acid constructs, RNA or polynucleotide molecules, or multiple nucleic acid constructs, RNA or polynucleotide molecules encoding two or more antigens of the same or different species. In some embodiments, the vaccine includes RNA or multiple RNAs encoding two or more antigens of the same or different viral strains. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more viral antigens.

[0166] In some embodiments, in the above-described monovalent and multivalent / combination vaccines, the antigen is a coronavirus antigen, such as SARS-CoV-2 (COVID-19), SARS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, or MERS-CoV. In some embodiments, the coronavirus antigen is a structural protein, such as a protein selected from spike protein (S protein or Spike protein), envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein). In some embodiments, the structural protein is a spike protein, such as the SARS-CoV-2 spike protein. In some implementations, the SARS-COV-2 spike protein is selected from the spike protein of any of the following viral strains: SARS-COV-2 (e.g., wild-type SARS-COV-2 mRNA), SARS-COV-2 Alpha (B.1.1.7), SARS-COV-2 Beta (B.1.351), SARS-COV-2 Gamma (P.1), SARS-COV-2 Kappa (B.1.617.1), SARS-COV-2 Delta (B.1.617.2), SARS-COV-2 Omicron (B.1.1.529), and SARS-COV-2 Omicron (BA.4).

[0167] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A or influenza B viruses; exemplary examples include influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H5N8, influenza A virus H2N2, influenza A virus H7N9, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of the influenza virus, such as hemagglutinin (HA), neuraminidase (NA), ion channel protein M2, matrix protein M1, nucleoprotein NP, etc. In some specific implementations, the influenza virus antigen is the HA, NA, or NP protein of the influenza virus, such as the HA, NA, or NP proteins of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).

[0168] In a preferred embodiment, the antigen of the respiratory syncytial virus is the pre-fusion conformation F protein (pre-F).

[0169] In a preferred embodiment, the antigen of the human metapneumovirus (hMPV) is the F protein.

[0170] In a preferred embodiment, the antigen of the chicken infectious bronchitis virus is the S1 region or the RBD domain of the Spike protein.

[0171] In a preferred embodiment, the antigen of the Nipah virus is either the F protein or the G protein.

[0172] In a preferred embodiment, the antigen of the monkeypox virus is the L5L protein.

[0173] In some embodiments, two or more different RNAs (e.g., mRNA) can be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs can be formulated separately in individual lipid nanoparticles, and the lipid nanoparticles can then be combined as a single vaccine composition (e.g., comprising multiple RNAs encoding multiple antigens) or administered individually.

[0174] This disclosure also provides multivalent / combination vaccines that contain RNA encoding one or more coronaviruses or antigens of one or more different organisms. That is, the vaccines disclosed herein can be multivalent / combination vaccines that target one or more antigens of the same strain / species, or one or more antigens of different strains / species.

[0175] Pharmaceutical Composition

[0176] This disclosure also provides a pharmaceutical composition comprising the nucleic acid constructs, polynucleotides, vaccines, and / or carriers described herein, as well as pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a combination preparation.

[0177] In some embodiments, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles, or any combination thereof.

[0178] In some embodiments, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including petroleum-derived, animal-, plant-based, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0179] In some embodiments, the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0180] The pharmaceutical compositions of the present invention can be administered by various suitable methods. Suitable methods of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0181] Product or reagent kit

[0182] This disclosure provides a product or kit comprising the nucleic acid constructs, polynucleotides, vaccines, vectors, and / or pharmaceutical compositions described herein. The kit may be used for related detection or diagnostic purposes.

[0183] Methods and uses

[0184] This disclosure also provides the use of therapeutic and / or preventive effective amounts of the nucleic acid constructs, polynucleotides, vaccines, vectors, pharmaceutical compositions, and / or products or kits described herein in the preparation of medicaments for the treatment and / or prevention of diseases.

[0185] This disclosure also provides a method for treating and / or preventing disease, comprising administering to a subject in need a therapeutically and / or preventively effective amount of the nucleic acid construct, polynucleotide, vaccine, vector, pharmaceutical composition, and / or product or kit described herein.

[0186] This disclosure also provides a method comprising administering to a subject in need an effective amount of the nucleic acid construct, polynucleotide, vaccine, vector, pharmaceutical composition, and / or product or kit described herein, which is capable of inducing a neutralizing antibody response and / or T-cell immune response in the subject, such as a neutralizing antibody response against a viral antigen, or a CD4+ and / or CD8+ T-cell immune response against a viral antigen. The subject's antigen-antibody titer increases after vaccination relative to the antigen-antibody titer of a subject vaccinated with a conventional vaccine against the antigen at an effective prophylactic dose.

[0187] In some embodiments, the disease includes viral infectious diseases or respiratory diseases associated with viral infection. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a human-infecting coronavirus, such as SARS-CoV-2 (COVID-19), SARS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, or MERS-CoV. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the virus is an influenza virus. In some embodiments, the coronavirus is a human-infecting influenza virus, such as influenza A virus or influenza B virus. Exemplarily, the influenza virus is influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some implementations, the viral infection-related respiratory diseases (e.g., SARS-CoV-2 infection-related respiratory diseases) include uncomplicated infections such as fever, cough and sore throat, headache, rhinitis, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.

[0188] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of this application. Various objects and advantages of this application will become apparent to those skilled in the art from the following detailed description of preferred embodiments.

[0189] Example 1. Characterization of mRNA 5'UTR sequence

[0190] Figure 1 This paper presents a schematic diagram of the DNA template and mRNA product used in the co-transcriptional capping method for producing mRNA, as well as the 5'UTR optimization method used in this study. Table 1 lists the three base sequences added to the 5'UTR produced and tested in this study.

[0191] Table 1. Tribase (NNN) Sequences and Numbers

[0192]

[0193] Example 2: Optimizing the translation efficiency of eGFP and Fluc reporter genes using 5'UTR

[0194] Using existing combinations of the human β-globin gene (hHBB) 5'UTR and 3'UTR (SEQ ID NO:1, SEQ ID NO:2), three random nucleotide sequences were inserted between the T7 promoter and the 5'UTR. A total of 64 base combinations were tested in vitro (Table 1), and mRNA constructs encoding eGFP (SEQ ID NO:7) and Luciferase (SEQ ID NO:8) were assembled. The in vitro transcription template was amplified by PCR, using primer sequences as follows:

[0195] F-ATTCTAATACGACTCACTATAAGG NNN ACATTTGCTTCTGA CACA(SEQ ID NO:57)

[0196] R-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGG (SEQ ID NO: 58).

[0197] mRNA was synthesized in vitro via co-transcriptional capping, and the IVT system was prepared according to the following system:

[0198] Table 2. IVT reaction system (20 μL)

[0199]

[0200]

[0201] After adding the template, the mixture was incubated at 37°C for 2 hours. For each reaction, 1 μL of DNase I was added and incubated at 37°C for 30 minutes to digest the DNA template. RNA was then precipitated using LiCl at -80°C. The precipitated mRNA was collected by centrifugation and centrifuged at 14,000 rpm for 10 minutes at 4°C until a white RNA precipitate appeared at the bottom of the centrifuge tube. The supernatant was discarded, and 1 mL of pre-chilled 70% ethanol was added. The RNA was vortexed and washed once. The precipitate was dried at room temperature until it became translucent. RNase-free H2O was added to fully dissolve the RNA, and the mRNA concentration was determined using NanoDrop One. The mRNA quality was assessed by 1% TBE agarose gel electrophoresis. Results are as follows: Figure 2 As shown in Figure A, a high-quality mRNA product was obtained.

[0202] The synthesized mRNA was transfected into the BHK-21 cell line using lipo8000, and the cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. One day before transfection, the cells were cultured at a concentration of 0.15 / 0.3 × 10⁻⁶ mRNA. 6 Cells were seeded at a concentration of 100 μL / mL in 96-well plates. The mRNA transfection system consisted of 50 ng mRNA, 0.16 μL Lipo8000, and 5 μL opti-MEM per well. Transfected cells were cultured at 37°C for 24 h for subsequent analysis.

[0203] After mRNA transfection and cell culture for 24 h, the fluorescence intensity of GFP in 96-well plates was scanned and quantified using a high-content cell imaging analysis system to analyze the effect of the NNN sequence on GFP expression. Results are as follows: Figure 2 As shown in Figure C, the insertion of NNN sequences can enhance GFP expression, and the insertion of 57 NNN sequences significantly enhances GFP expression.

[0204] The same method was used to prepare mRNA expressing luciferase. Figure 2 B) After transfecting cells with mRNA and culturing them for 24 h, the cells were lysed, and a Luciferin substrate-based detection reagent was added. Bioluminescence was detected at 560 nm to analyze the effect of the NNN sequence on luciferase expression. The results are as follows: Figure 2 As shown in Figure D, the insertion of NNN sequences can enhance luciferase expression. All 50 NNN sequences significantly enhanced luciferase expression. Among them, one sequence showed an expression increase of 10%-20%, 20 sequences showed an increase of 20%-50%, and 29 sequences showed an increase of over 50%.

[0205] Example 3: Effects of NNN combined with different 5'UTRs on GFP expression in BHK-21 mRNA

[0206] A GFP-encoding mRNA construct was assembled by inserting 50-GAT / 58-GCT / 64-GGG sequences between the T7 promoter and these 5'UTRs using a combination of RpL38, mActb, hActb, SARS-CoV2 5'UTR (SEQ ID NO:3-6), and hHBB 3'UTR. GFP expression was then quantified using IVT, mRNA transfection, and a high-content cell imaging system. Results are as follows: Figure 3 As shown, regardless of which 5'UTR is used, the insertion of GAT, GCT, and GGG sequences significantly enhanced GFP expression.

[0207] Example 4: Optimized 5'UTR enhances RSV viral antigen expression

[0208] A template DNA sequence containing mRNA transcription-related elements was artificially synthesized. The original sequence was a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) (SEQ ID NO:1, SEQ ID NO:2), which was used to assemble the RSV-preF antigen sequence (SEQ ID NO:17). The optimized sequence contained 18 inserted NNNs in the hHBB 5'UTR. Template preparation was performed by primer PCR amplification. mRNA was obtained by co-transcriptionalization, and expression was verified by transfecting BHK21 cells with indirect fluorescent immunoassay. 24 h after transfection, cells were fixed with 4% paraformaldehyde, washed, and then blocked and perforated with 1% BSA (diluted in PBS) containing 0.2% Triton X-100. Cells were then incubated with specific antibodies for 1 h, washed, and incubated with AF488-conjugated secondary antibody for 1 h. After incubation, cells were washed again and stored in PBS. RSV expression in cells was quantified using a high-content cell imaging analysis system. The results are shown below. Figure 4 As shown, the insertion of these NNN sequences all enhanced the expression of RSV.

[0209] Example 5: Optimized 5'UTR enhances the expression of influenza virus HA antigen in cells.

[0210] Template DNA sequences containing mRNA transcription-related elements were artificially synthesized. The original sequences were combinations of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) (SEQ ID NO:1, SEQ ID NO:2), which were then assembled into HA antigen sequences for influenza H1N1 and H5N1 (SEQ ID NO:9, SEQ ID NO:12). The optimized H1N1-HA sequence contained 10 NNN-inserted hHBB 5'UTRs; the optimized H5N1-HA sequence contained 11 NNN-inserted hHBB 5'UTRs. Templates were prepared by primer PCR amplification. mRNA was obtained by co-transcriptionalization, and expression was verified by transfecting BHK21 cells with indirect fluorescent immunoassay. 24 h after transfection, cells were fixed with 4% paraformaldehyde. After washing, cells were blocked and perforated with 1% BSA (diluted in PBS) containing 0.2% Triton X-100. Cells were then incubated with specific antibodies for 1 h, washed, and incubated with AF488-conjugated secondary antibody for 1 h. After incubation, cells were washed again and stored in PBS. High-content cell imaging analysis was used to quantify HA expression in cells. The results are as follows: Figure 5 As shown in A and 5B, the insertion of these NNN sequences all enhanced the expression of HA.

[0211] Example 6: Immunogenicity test of seasonal influenza HA mRNA vaccine in mice

[0212] Based on the WHO-recommended seasonal influenza vaccine strains for 2023-2024, trivalent influenza vaccines were designed using the hemagglutinin protein (HA) of strains A / Wisconsin / 67 / 2022 (H1N1), A / Darwin / 6 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage). Figure 6 A, SEQ ID NO: 9-11). A template DNA sequence containing elements related to in vitro transcription of mRNA was artificially synthesized, comprising a combination of hHBB 5'UTR and 3'UTR with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification. mRNA was obtained by transcription using a co-transcriptional capping method. Figure 6 B).

[0213] SM102, DSPC, cholesterol, and DMG-PEG2000 were mixed in ethanol at a molar ratio of 50:10:38.5:1.5. Then, using a microfluidic mixer, the lipids were mixed with mRNA (dissolved in 50 mM sodium acetate, pH 4.0) at a volume ratio of 1:3. The total flow rate for both mixtures was set to 12 mL / min, with a flow rate ratio of 1:3. The mRNA-LNP was dialyzed with PBS for 4 h to remove acetate and ethanol, and then dialyzed for 2 h with the PBS replaced. The mRNA-LNP was dialyzed to neutral pH, and its particle size and distribution were analyzed using a Malvern nanoparticle size analyzer. The encapsulation efficiency of the mRNA was determined using the Ribogreen method (Thermo Fisher assay). The results are as follows: Figure 6 As shown in C, the encapsulated vaccine has an average particle size of 60–80 nm, uniform particle size distribution (PDI<0.1), and an encapsulation rate of greater than 90%, indicating good quality.

[0214] mRNA-LNP was transfected into BHK-21 cells for expression validation, and HA expression was detected by Western blotting. Western blotting: One day before transfection, cultured BHK-21 cells were injected with 0.25 × 10⁻⁶ cells. 6Cells were seeded at a concentration of 1 / mL into 12-well plates, 1 mL per well. The mRNA transfection system consisted of 1 μg mRNA, 1.6 μL Lipo8000, and 50 μL opti-MEM per well. Transfected cells were cultured at 37°C for 24 h. Cells were lysed using RIPA mild cell lysis buffer, 200 μL per well on ice for 20 min. After lysis, the supernatant was collected by centrifugation at 4°C, mixed with loading buffer, and boiled in a water bath for 10 min. The resulting samples were used for SDS-PAGE electrophoresis. Electrophoresis conditions were constant voltage 160V until the target bands separated. A semi-dry transfer method was used to transfer proteins to PVDF membranes at 25W for 30 min. The transferred membranes were blocked in 5% skim milk for 1 h, incubated with primary antibody for 1 h, washed with TBST, and then incubated with the corresponding secondary antibody for 1 h. After washing, the results were analyzed by ECL chemiluminescence. Results are as follows: Figure 6 As shown in Figure D, HA proteins are all well expressed.

[0215] Six-week-old female BALB / c mice were randomly assigned to groups of five and immunized initially with monovalent or trivalent mRNA vaccines (TRV) at doses of 0.1, 1, or 10 μg / mouse, followed by a second immunization three weeks later. A commercially available quadrivalent inactivated influenza vaccine (QIV) was used as a positive control at a dose of 2 μg / mouse; mRNA-GFP was used as a negative control at a dose of 10 μg / mouse. Blood samples were collected three weeks after the initial immunization and three weeks after the booster immunization, and HAIgG antibody titers were measured by ELISA. The ELISA plate was coated with HA protein and incubated overnight at 4°C. Blocking buffer (5% skim milk powder prepared with PBS) was added and incubated at 37°C for 1 hour. After washing with PBST, serially diluted inactivated serum (3-fold serial dilution) was added and incubated at 37°C for 1 hour. After washing again, anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 hour. Finally, the chromogenic substrate was added for color development, and the reaction was terminated. The absorbance was read at 450 nm using an ELISA reader, and the vaccine-induced antibody titer was calculated. Results are as follows: Figure 6 As shown in E, mRNA vaccines can induce a good level of immune response.

[0216] The neutralizing titer of serum from mice immunized with mRNA vaccine was determined. Serum from mice three weeks after booster immunization was inactivated at 56°C for 30 min, initially diluted 1:50, and then serially diluted 3-fold. 100 FFU of A / California / 07 / 2009 (H1N1) virus was incubated with serum at 37°C for 1 h. Cells were washed with PBS, and 100 μL of the mixture was added to the cells and incubated at 37°C for 1 h. A covering medium was prepared by mixing 2% sodium carboxymethyl cellulose (CMC, 2X) with MEM medium at a 1:1 volume ratio, and TPCK trypsin was added to a final concentration of 2 μg / mL. The viral supernatant in the cell plate was discarded, the cells were washed once with PBS, and 100 μL of the covering medium was added to each well. The cells were incubated at 37°C for 24 h. The serum neutralizing titer was calculated based on the immunospots. The results are shown below. Figure 6 As shown in F.

[0217] Three weeks after booster immunization, mice were challenged via nasal drops with 5 LD50 doses of A / California / 07 / 2009 (H1N1), A / Hong Kong / 1 / 68 (H3N2), and B / Guangzhou / 0215 / 2012 (Victoria) viruses, respectively. Mouse body weight was measured daily after challenge, and mortality was monitored. Results are as follows: Figure 6 As shown in G, the mRNA vaccine has a good protective effect against CA07, HK68, and GZ12 viruses in mice.

[0218] Example 7: Immunogenicity test of influenza H5N1 HA mRNA vaccine in mice

[0219] A template DNA sequence containing the HA gene (SEQ ID NO:12) of highly pathogenic avian influenza A / Texas / 37 / 2024 (H5N1) and mRNA transcription-related elements was synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. The mRNA was transfected into BHK-21, HeLa, and 293T17 cells for expression verification. The expression of the original H5N1 TexasHA protein (Texas-WT) and the mutant protein (Mut, Texas-343-345del) was detected by Western blotting. The results are as follows: Figure 7 As shown in Figure A, all HA proteins were well expressed in different cell lines.

[0220] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipids at a volume ratio of 3:1 via microfluidic encapsulation and concentration to prepare the mRNA-LNP vaccine. BALB / c female mice were randomly divided into groups of 8 and primary immunized with doses of 1 or 5 μg / mouse, respectively. A secondary immunization was administered 3 weeks later. Blood samples were collected 2 weeks post-immunization to detect HA protein antibody titers. The results are as follows: Figure 7 As shown in B, the mRNA vaccine induced a good level of immune response after both primary and secondary immunizations, demonstrating a dose-dependent effect. Neutralization experiments were conducted using influenza virus A / Ostrich / Hebei / B430-1 / 240418(H5N1) from phylogenetic clade 2.3.4.4b, and the results are shown in Figure B. Figure 7 As shown in Figure C, the mRNA vaccine induced a high level of neutralizing antibodies against H5N1. Figure 7 As shown in D and 7E, the virus protection experiment was conducted using influenza virus A / Ostrich / Hebei / B430-1 / 240418(H5N1). Immunization with 1 μg and 5 μg of mRNA vaccine both completely protected mice, confirming that the optimized mRNA vaccine prepared in this embodiment can prevent influenza virus.

[0221] mRNA was transfected into chicken DF-1 cells for expression validation. The expression of the original H5N1 Texas HA protein (Texas-WT) and the mutant protein (Mut, Texas-343-345del) was detected by Western blotting. The results are as follows: Figure 7 As shown in F, all HA proteins were well expressed. Ten-day-old SPF chicks were randomly divided into groups of 10 and immunized with a dose of 30 μg / chick. Blood samples were collected one week after the initial immunization, followed by a booster immunization with the same dose. Blood samples were collected two weeks after the booster immunization to detect HA protein antibody titers. The results are shown in F. Figure 7 As shown in G, the H5N1 mRNA-HA vaccine can induce a good level of immune response in chickens. Figure 7 As shown in H and 7I, virus protection experiments were conducted using H5 influenza viruses DKLH and DKFJ from evolutionary branch 2.3.4.4b. The mRNA vaccines provided complete protection to chickens. This confirms that the optimized mRNA vaccine prepared in this embodiment can prevent influenza virus infection.

[0222] Example 8: Immunogenicity assay of mouse vaccine based on conserved HA neck domain mRNA of classic influenza strain.

[0223] A gene (SEQ ID NO:13) expressing the conserved neck domain of the HA protein from influenza strain A / New Caledonia / 20 / 1999 (H1N1) and a template DNA sequence containing mRNA transcription-related elements were synthesized. This template included a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA optimized sequence. Template preparation was performed by primer PCR amplification. MRNA was obtained by co-transcriptional capping. Figure 8 A) mRNA was transfected into BHK-21 cells for expression verification. Indirect immunofluorescence was used to detect the expression of the conserved neck domain (H1stem) of the HA protein. Twenty-four hours after transfection, cells were fixed with 2% paraformaldehyde and hybridized with human CR6261 antibody for 1 hour at room temperature. This antibody binds to H1stem. After washing off the primary antibody, cells were incubated with 488-labeled fluorescent secondary antibody for 1 hour at room temperature. Images were then taken using a fluorescence microscope. The results are shown below. Figure 8 As shown in Figure B, the H1stem protein is well expressed.

[0224] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipids at a volume ratio of 3:1 via microfluidic encapsulation and concentration to prepare the mRNA-LNP vaccine. BALB / c female mice were randomly divided into groups of 5 and immunized initially at a dose of 5 μg / mouse. A second immunization was administered 3 weeks later. Blood samples were collected at 3 weeks after the initial immunization and 3 weeks after the booster immunization to detect HA protein antibody titers. The results are as follows: Figure 8 As shown in C. mRNA vaccines can elicit a good level of immune response after primary and secondary immunizations.

[0225] On day 28 after the second immunization, mice were challenged with 5LD50 doses of A / Puerto Rico / 8 / 34 (H1N1,PR8) virus via intranasal instillation. Mouse body weight was measured daily after challenge, and mortality was monitored. Results are as follows: Figure 8 As shown in D, a 5 μg dose of the H1stem-mRNA vaccine provides 100% protection against the influenza PR8 virus.

[0226] Example 9: Immunogenicity assay of influenza NA protein mRNA vaccine in mice

[0227] A template DNA sequence containing the gene (SEQ ID NO: 14) of neuraminidase (NA) from influenza virus A / California / 07 / 2009 (H1N1, CA07) and mRNA transcription-related elements was synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 9A). mRNA was transfected into the BHK-21 cell line for expression validation. NA protein expression was detected by Western blotting, and the results are as follows: Figure 9 As shown in B, NA protein can be well expressed.

[0228] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipids at a volume ratio of 3:1 via microfluidic encapsulation and concentration to prepare the mRNA-LNP vaccine. BALB / c female mice were randomly divided into groups of 5 and immunized initially at a dose of 5 μg / mouse. A second immunization was administered 3 weeks later. Blood samples were collected at 3 weeks after the initial immunization and 3 weeks after the booster immunization to detect NA protein antibody titers. The results are as follows: Figure 9 As shown in C. mRNA vaccines can elicit a good level of immune response after primary and secondary immunizations.

[0229] On day 21 after the second immunization, mice were challenged with 5xLD50 doses of CA07 virus via intranasal instillation. Mouse weight was measured daily after challenge, and mortality was monitored. Results are as follows: Figure 9 As shown in D and 9E, a 5 μg dose of mRNA-NA vaccine provides 100% protection against the CA07 virus.

[0230] Example 10: Immunogenicity Detection of Influenza Virus NP mRNA Vaccine in Mice

[0231] The gene for synthesizing the PR8 viral nucleoprotein (NP) of influenza virus and a template DNA sequence containing mRNA transcription-related elements (SEQ ID NO: 15) including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 10 A). mRNA was transfected into the BHK-21 cell line for expression validation. NP protein expression was detected by Western blotting, and the results are as follows: Figure 10 As shown in B, NP protein can be well expressed.

[0232] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipids at a volume ratio of 3:1 via microfluidic encapsulation and concentration to prepare the mRNA-LNP vaccine. C57BL / 6 female mice were randomly divided into groups of 5 mice each and given a primary immunization at a dose of 5 μg / mouse via intramuscular injection (IM), subcutaneous injection (SC), intravenous injection (IV), nasal instillation (IN), and nebulized inhalation. A secondary immunization was given 3 weeks later. Blood samples were collected at 3 weeks after the primary immunization and 3 weeks after the booster immunization to detect protein antibody titers. The results are as follows: Figure 10As shown in C. Intramuscular, subcutaneous, and intravenous delivery of mRNA vaccines can elicit good immune responses after the first and second immunizations; nasal drops and nebulization can induce significant immune responses, while other immunization routes can induce even more pronounced immune responses.

[0233] Example 11: Immunogenicity Detection of COVID-19 RBD mRNA Vaccine in Mice

[0234] A DNA sequence expressing the RBD domain of the SARS-CoV-2 Spike protein (SEQ ID NO: 16) and a template DNA sequence containing mRNA transcription-related elements were synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 11 A, 11B), mRNA was transfected into BHK-21 cells for expression verification. RBD dimer expression was detected by Western blotting, and the results are as follows: Figure 11 As shown in Figure C, the RBD dimer protein is well expressed.

[0235] The prepared mRNA was mixed with a lipid mixture containing MC3 cationic lipids at a volume ratio of 3:1 in a 1.5 mL centrifuge tube and dialyzed in PBS to prepare the mRNA-LNP vaccine.

[0236] Female BALB / c mice were randomly divided into groups of 8 and 10 μg / mouse for primary immunization. A second immunization was administered 3 weeks later. Blood samples were collected at 3 weeks post-primary immunization and 3 weeks post-boost immunization to measure IgG antibody titers. Results are as follows: Figure 11 As shown in D, mRNA vaccines can elicit a good level of immune response after the first and second immunizations.

[0237] Vero cells were fed at a concentration of 0.15 × 10⁻⁶. 6 Cell culture medium was seeded at 100 μL / mL in 96-well plates, 12 h in each well. After 12 h, a neutralization experiment was performed. The serum was diluted 100-fold, and 150 μL was added to four replicate wells. 50 μL of the diluted serum was then added to subsequent wells for a 1:3 serial dilution. 100 TCID50 of VSV-Spike pseudovirus was added to each well. The mixture was shaken and incubated at 37°C for 1 h. The original cell culture medium was then replaced with the virus-serum mixture. After 24 h, Luciferase bioluminescence assay was performed, and the pseudovirus neutralizing titer (pVNT50) was calculated. The results showed that the vaccine had a neutralizing effect against different strains of SARS-CoV-2 pseudovirus. Figure 11 E).

[0238] Three weeks after the second immunization, the spleens of mice were harvested, ground, and splenic lymphocytes were separated. Red blood cells were removed using erythrocyte lysis buffer, and the spleen cells were diluted to 2.5 × 10⁻⁶. 6 100 μL of the vaccine was added to each well of a 96-well cell culture plate at a concentration of 100 cells / mL. The plates were incubated with both an NC control group and a COVID-19 peptide library stimulation group. After 72 hours of culture, the cells were analyzed using an ELISpots (3321-4HST-2) kit. Spot values ​​were recorded and statistically analyzed. The results showed that the vaccine effectively induced a specific T-cell immune response. Figure 11 F, 11G).

[0239] Example 12: Immunogenicity Detection of Respiratory Syncytial Virus mRNA Vaccine in Mice A DNA sequence expressing the pre-fusion conformation F protein (pre-F) of respiratory syncytial virus (RSV) (SEQ ID NO: 17) and a template DNA sequence containing mRNA transcription-related elements were synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 12 A) The quality of mRNA was detected by capillary electrophoresis, and the results are as follows: Figure 12 As shown in B, the obtained mRNA product has high integrity. The mRNA was transfected into BHK-21 cells for expression verification, and the expression of RSV pre-F protein was detected by Western blotting. The results are as follows. Figure 12 As shown in Figure C, the pre-F protein is well expressed.

[0240] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipids at a volume ratio of 3:1 via microfluidic encapsulation and concentration to prepare the mRNA-LNP vaccine. BALB / c female 5-7 week old mice were randomly divided into groups of 16 each and received a primary immunization with the mRNA vaccine at a dose of 5 μg / mouse. A secondary immunization was administered 3 weeks later. Blood samples were collected 3 weeks after the primary immunization and 3 weeks after the booster immunization to detect anti-RSV pre-F or post-F IgG antibody titers. Results are as follows: Figure 12 As shown in D, 12E, and 12F, the mRNA vaccine can elicit a good level of immune response against the pre-fusion F protein after the first and second immunizations.

[0241] The titers of neutralizing antibodies in mouse serum following mRNA vaccine immunization were determined. Mouse serum was inactivated by heating at 56°C for 30 min, then diluted 1:100, followed by 3-fold serial dilutions. 100 p. fu of RSV A2, ON1, or BA9 virus was incubated with an equal volume of mouse serum dilution at 37°C for 1 h. 100 μL of the serum-virus mixture was transferred to HEp-2 cells containing a monolayer (3 x 10⁻⁶ cells). 4Cells were incubated in 96-well plates (cells / well) at 37°C for 48 h, followed by washing with PBS and fixing with 4% paraformaldehyde. RSV infection was measured by indirect immunostaining. Serum neutralization titers were calculated based on immunofluorescence signal intensity. Results are shown below. Figure 12 As shown in G, the RSV mRNA vaccine can induce high and neutralizing antibodies against the classical RSV type A strain (A2) and the clinical strain (ON1), as well as type B (BA9).

[0242] The T-cell immune response in mice immunized with the mRNA vaccine was detected. Mouse spleens were harvested, ground to obtain a single-cell suspension, and erythrocytes were removed. The processed spleen cells were counted and diluted to 2 x 10⁻⁶ cells. 7 Cells / mL. 100 μL of spleen cells were added to the RSV(A2)F0 peptide library (2 μg / mL) and stimulated at 37°C for 1 h. Then, GolgiStop / GolgiPlug dilution was added, and incubation continued at 37°C for 7 h. Cells were washed twice with PBS, stained with a live / dead cell dye, and then labeled with fluorescently labeled antibodies for cell surface proteins, including anti-CD3e FITC, anti-CD4 PE-Cy7, anti-CD8a BV510, anti-CD69 Alexa 700, anti-CD44 BV421, and anti-CD62L BV650. Incubation was carried out at 4°C in the dark for 30 min. Cells were permeabilized and fixed with Cytofix / Cytoperm solution and stained with fluorescently labeled anti-cytokine antibodies, including anti-IFN-γ BV711, anti-TNF BB700, anti-IL-2PE, and anti-IL-4 BV786. The samples were analyzed using flow cytometry, and the results are as follows: Figure 12 As shown in H and 12I, the mRNA vaccine induced an excellent T-cell immune response.

[0243] On day 21 after the second immunization, female BALB / c mice (n=8 per group) were challenged with 1x10g of the virus via intranasal drip. 6 The protective effect of the vaccine on mice was analyzed by detecting RSV A2 viral load in the lungs of mice on day 4 after challenge with PFU-dose RSV A2 virus. The results are as follows: Figure 12 As shown in Figure J, the mRNA vaccine has a good protective effect against RSV A2 virus.

[0244] Example 13: Preparation and Expression of hMPV mRNA Vaccine

[0245] A DNA sequence expressing human metapneumovirus (hMPV) F protein (SEQ ID NO: 18) and a template DNA sequence containing mRNA transcription-related elements were synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 13 A). mRNA was transfected into BHK-21 cells for expression verification. The expression of F protein was detected by indirect immunofluorescence staining, and the results are as follows: Figure 13 As shown in Figure B, protein F is well expressed in cells.

[0246] Example 14: Cellular Expression of Infectious Bronchitis Virus (IBV) Antigen and Experiment on Chicken Virus Protection

[0247] Infectious bronchitis in chickens is a disease caused by the infectious bronchitis virus (IBV). It is an acute, highly contagious viral respiratory disease and one of the most important infectious diseases in the poultry industry. Prevention is key, and the most important aspect is effective vaccination.

[0248] Four different IBV strains were designed to develop mRNA vaccines targeting the S1 region or RBD domain of the Spike protein. The antigen DNA template sequence (SEQ ID NO: 19-26) and template DNA containing mRNA transcription-related elements were synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 14 A). mRNA was transfected into chicken DF1 cells for expression verification. The expression of the FLAG-tagged antigen was detected by Western blotting, and the results are as follows: Figure 14 As shown in Figure B, this indicates that IBV S1 and RBD proteins are well expressed in cells.

[0249] Example 15: Preparation and Cell Expression Detection of Nipah Virus F Protein and G Protein mRNA Vaccine

[0250] Nipah virus is a zoonotic virus that can cause acute respiratory illness and fatal encephalitis. Currently, there are no treatments or vaccines available for humans or animals. This study synthesized DNA sequences expressing the Nipah virus F protein (SEQ ID NO:27), the G protein (SEQ ID NO:28), and template DNA sequences for mRNA transcription-related elements, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification. MRNA was obtained by co-transcriptional capping. Figure 15 A) mRNA was transfected into the BHK-21 cell line for expression verification. The expression of NiV-F and NiV-G proteins was detected by Western blotting, and the results are as follows: Figure 15 As shown in Figure B, NiV-F and NiV-G proteins can be well expressed.

[0251] Example 16: Preparation and Cell Expression Detection of Monkeypox Virus (MPXV) mRNA Vaccine

[0252] A template DNA sequence containing the monkeypox virus L5L gene (SEQ ID NO:29) and mRNA transcription-related elements was synthesized, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA-optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 16 A). mRNA was transfected into the BHK-21 cell line for expression validation. MPXV-L5L protein expression was detected by Western blotting, and the results are as follows: Figure 16 As shown in Figure B, the MPXV-L5L protein can be well expressed.

[0253] Example 17: Anti-RSV Nucleic Acid Antibody Drug

[0254] DNA sequences (SEQ ID NO: 30, 31) expressing the heavy and light chains of the RSV monoclonal antibody D25 and template DNA sequences for mRNA transcription-related elements were synthesized, respectively, containing a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an inserted AGA optimized sequence. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping. Figure 17 A). mRNA was transfected into the BHK-21 cell line for expression validation. The expression of D25 antibody protein at different time points was detected by ELISA. The neutralizing activity of the D25 antibody against RSV-A2 virus was verified by in vitro neutralization experiments. Results are as follows: Figure 17 As shown in Figure B, the D25 antibody protein was well expressed in BHK-21 cells, persisting for approximately 3-4 days, and exhibited good neutralizing activity. Figure 17 C).

Claims

1. An optimized 5' UTR, characterized in that, Three random bases NNN were inserted after the transcription start site AGG in the 5'UTR; The 5'UTR is selected from hHBB 5'UTR, RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR, or SARS-CoV2 5'UTR; When the 5'UTR is hHBB 5'UTR, the three random bases NNN are selected from any one of the following 64 combinations: AAA, TAA, CAA, GAA, AAT, TAT, CAT, GAT, AAC, TAC, CAC, GAC, AAG, TAG, CAG, GAG, ATA, TTA, CTA, GTA, ATT, TTT, CTT, GTT, ATC, TTC, CTC, ATG, TTG, CTG, GTG, ACA, TCA, CCA, GCA, ACT, TCT, CCT, GCT, ACC, TCC, CCC, GCC, ACG, TCG, CCG, GCG, AGA, TGA, CGA, GGA, AGT, TGT, CGT, GGT, AGC, TGC, CGC, GGC, AGG, TGG, CGG, GGG; When the 5'UTR is RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR, or SARS-CoV2 5'UTR, the three random bases NNN are selected from any one of the following three combinations: GAT, GCT, GGG; wherein, The nucleotide sequence of the 5'UTR is selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

2. A nucleic acid construct or mRNA molecule comprising the 5'UTR of claim 1.

3. The nucleic acid construct or mRNA molecule according to claim 2, further comprising one or more of the following elements: (a) a 5' cap; (b) an open reading frame (ORF); (c) a 3' untranslated region (3'UTR); and (d) a poly-A tail.

4. The nucleic acid construct or mRNA molecule according to claim 3, wherein the 3'UTR comprises a 3'UTR derived from an albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, collagen α gene, cytochrome b α subunit gene, poliovirus gene, Sindbis virus gene, brome mosaic virus gene, Zika virus gene, or dengue virus gene.

5. The nucleic acid construct or mRNA molecule according to claim 3, wherein the 3'UTR comprises a 3'UTR derived from hHBB.

6. The nucleic acid construct or mRNA molecule according to claim 3, wherein the nucleotide sequence of the 3'UTR of the hHBB is shown in SEQ ID NO:

2.

7. The nucleic acid construct or mRNA molecule according to claim 3, wherein the optimized 5'UTR and the 3'UTR are derived from the human β-globin gene (hHBB), and their sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

8. The nucleic acid construct or mRNA molecule according to claim 3, wherein, The ORF encodes a fluorescent protein, luciferase, antibody, or antigen.

9. The nucleic acid construct or mRNA molecule according to claim 8, wherein, The antigen is an antigen derived from a pathogen or tumor.

10. The nucleic acid construct or mRNA molecule according to claim 9, wherein the tumor is a solid tumor.

11. The nucleic acid construct or mRNA molecule according to claim 9, wherein the tumor is selected from: liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer, bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof.

12. The nucleic acid construct or mRNA molecule according to claim 9, wherein the tumor-derived antigen is selected from NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or combinations thereof.

13. The nucleic acid construct or mRNA molecule according to claim 9, wherein the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof.

14. The nucleic acid construct or mRNA molecule according to claim 13, wherein the antigen of the virus is selected from the antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, chicken infectious bronchitis virus, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus or EB virus.

15. The nucleic acid construct or mRNA molecule according to claim 14, wherein it has any one of the following characteristics: (1) The coronavirus is SARS-CoV-2, and the coronavirus antigen is the complete spike protein or its RBD domain; (2) The influenza virus is influenza A virus or influenza B virus, and the antigen is hemagglutinin protein (HA), ceramidinase (NA) and / or nucleoprotein (NP); (3) The antigen of the respiratory syncytial virus is the F protein, G protein, nucleocapsid protein or matrix protein; (4) The antigen of the human metapneumovirus (hMPV) is the F protein, G protein, nucleocapsid protein or matrix protein; (5) The antigens of the chicken infectious bronchitis virus are Spike protein, M protein, nucleocapsid protein or envelope protein; (6) The antigen of the Nipah virus is the F protein, G protein, nucleocapsid protein or matrix protein; (7) The antigens of the monkeypox virus are selected from the inner membrane proteins M1R, H3L, E8L and A29L, the outer membrane proteins A35R and B6R, and the accessory protein L5L.

16. The nucleic acid construct or mRNA molecule according to claim 15, wherein the spike protein is selected from the spike protein of any of the following viral strains: SARS-COV-2, SARS-COV-2 Alpha, SARS-COV-2 Beta, SARS-COV-2 Gamma, SARS-COV-2 Kappa, SARS-COV-2 Delta, or SARS-COV-2 Omicron.

17. The nucleic acid construct or mRNA molecule according to claim 15, wherein the influenza virus antigen is selected from the hemagglutinin protein, neuraminidase and / or nucleoprotein of any of the following strains: influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria, and influenza B virus Yamagata.

18. The nucleic acid construct or mRNA molecule according to claim 15, wherein the antigen of the respiratory syncytial virus is selected from the F protein, G protein, nucleocapsid protein or matrix protein of subtype A strain or subtype B strain.

19. The nucleic acid construct or mRNA molecule according to claim 15, wherein the antigen of the chicken infectious bronchitis virus is the S1 region or RBD domain of the Spike protein.

20. The nucleic acid construct or mRNA molecule according to claim 8, wherein the antigen is selected from antigens of the genera *Candida*, *Aspergillus*, *Mucor*, *Rhizopus*, *Epidermophyton*, *Malassezia*, *Preumocystis*, *Penicillium*, *Alternaria*, *Cladosporium*, *Botrytis*, *Aureobasidium*, *Fusarium*, or *Trichoderma*.

21. The nucleic acid construct or mRNA molecule according to claim 13, wherein the bacteria are selected from actinomycetes, Bacillus, Bacteroides, Enterococcus, Listeria, Mycobacterium, Streptococcus pneumoniae, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus or Streptococcus.

22. The nucleic acid construct or mRNA molecule according to claim 8, wherein the antibody is an antibody that specifically binds to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and / or LAG3.

23. The nucleic acid construct or mRNA molecule according to claim 3, wherein, The nucleotide sequence of the ORF is selected from any one of the sequences shown in SEQ ID NO:7-31.

24. The nucleic acid construct or mRNA molecule according to claim 3, wherein the ORF comprises an amino acid sequence as shown in any one of SEQ ID NO: 32-56.

25. Use of the nucleic acid construct or mRNA molecule according to any one of claims 2-24 in any of the following: (1) preparation of a vaccine; (2) encoding a viral antigen in vitro in a subject; (3) preparation of a drug that encodes a viral antigen in vivo or in vitro in a subject; (4) preparation of an antibody drug.

26. A vaccine comprising a nucleic acid construct or mRNA molecule as described in any one of claims 2-24.

27. The vaccine of claim 26, wherein the nucleic acid construct or mRNA molecule is encapsulated in lipid nanoparticles (LNP).

28. A vector comprising the nucleic acid construct or mRNA molecule as described in any one of claims 2-24.

29. A host cell comprising the vector of claim 28.

30. A method for preparing the mRNA molecule according to any one of claims 2-24, comprising: The gene encoding the ORF protein and the template DNA sequence containing mRNA transcription-related elements were prepared by primer PCR amplification, and mRNA was obtained by co-transcription capping.

31. A pharmaceutical composition comprising: a nucleic acid construct or mRNA molecule as described in any one of claims 2-24, and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition of claim 31, comprising: a pharmaceutically acceptable carrier, diluent, or adjuvant.

33. A product comprising a nucleic acid construct or mRNA molecule as described in any one of claims 2-24, a vaccine as described in claim 26 or 27, and / or a pharmaceutical composition as described in claim 31 or 32.

34. Use in the preparation of a medicine by any one of claims 2-24, the vaccine of claim 26 or 27, the pharmaceutical composition of claim 31 or 32, and / or the product of claim 33, wherein, The drug is used to treat and / or prevent diseases and / or symptoms in a subject caused by pathogen infection.

35. The use according to claim 34, wherein the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof.

36. The use as described in claim 35, wherein the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, chicken infectious bronchitis virus, Nipah virus, or monkeypox virus.

37. The use as described in claim 36, wherein the virus is SARS-CoV-2, influenza A virus, or influenza B virus.

38. The use according to claim 34, wherein the disease and / or symptoms include uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, and septic shock.

39. The use as described in claim 34, wherein the subject is a mammal.

40. Use in the preparation of a medicine by the nucleic acid construct or mRNA molecule according to any one of claims 2-24, the vaccine according to claim 26 or 27, the pharmaceutical composition according to claim 31 or 32, and / or the product according to claim 33, wherein, The drug is used to induce neutralizing antibody responses and / or T-cell immune responses in subjects.

41. The use according to claim 40, wherein the neutralizing antibody reaction is a neutralizing antibody reaction against a viral antigen, and the T cell immune response includes a CD4+ and / or CD8+ T cell immune response.

42. The use as described in claim 41, wherein the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, chicken infectious bronchitis virus, Nipah virus, or monkeypox virus.

43. The use as described in claim 40, wherein the subject is a mammal.

44. Use of the optimized 5'UTR of claim 1 in any one or more of the following: (i) preparing nucleic acid constructs or mRNA molecules; (ii) increasing the expression level of a protein encoded by a nucleic acid construct or mRNA molecule containing thereof; or (iii) increasing the immunogenicity of a product containing said nucleic acid construct or mRNA molecule.

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