Optimized 5 'UTR and application thereof

Abstract

The invention discloses a modified 5 'UTR (Untranslated Region), mRNA (messenger Ribonucleic Acid) containing the modified 5' UTR and application of the modified 5 'UTR.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nucleic acid drugs, and particularly relates to a nucleic acid construct comprising an optimized 5'UTR and its use for preventing or treating diseases. BACKGROUND

[0002] mRNA therapy is a rapidly developing field and has been used in the field of expressing therapeutic proteins and vaccines. mRNA as a new type of nucleic acid drug has unique advantages in the field of vaccines and treatment, and the rapid response capability, safety and effectiveness of mRNA vaccines have been verified in response to the global pandemic of new crown caused by SARS-CoV-2. Despite recent clinical successes, there are still problems of mRNA heat instability and low protein expression. The present application improves the expression level of the protein encoded by mRNA by screening and optimizing the 5'UTR of mRNA, enhances the immune response of mRNA vaccine and the therapeutic effect of mRNA drug, and can be universally applied to mRNA vaccine, which has excellent clinical drug application prospect. SUMMARY

[0003] In one aspect, the present application provides an optimized 5'UTR, characterized in that 3 random bases NNN are inserted after the transcription initiation site AGG of the 5'UTR, wherein 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 certain embodiments, the 3 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, GTC, 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 certain embodiments, the 3 random bases NNN are selected from any one 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 certain embodiments, the 3 random bases NNN are selected from any one 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 certain embodiments, the 3 random bases NNN are selected from any one 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 certain embodiments, the optimized 5' UTR of the present application can be derived from a beta globin gene (HBB), a ribosomal protein gene (e.g., RpL38, RpS25), an actin gene (e.g., hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etch virus (TEV), scrambled short 5'-UTR (scrUTR), or complement factor 3 (C3). In a preferred embodiment, the 5' UTR of the present application is selected from a human beta globin gene (hHBB) 5' UTR, RpL38 5' UTR, mActb 5' UTR, hActb 5' UTR, or SARS-CoV2 5' UTR. In certain 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 application provides a nucleic acid construct or mRNA molecule comprising an optimized 5' UTR as described in the present application. In a specific embodiment, the nucleic acid construct or mRNA molecule of the present application 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); (d) a poly-adenosine (poly-A) tail. In certain embodiments, the 3' UTR comprises a 3' UTR derived from an albumin gene, an alpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, a collagen alpha gene (e.g., a collagen alpha 1 (I) gene), a cytochrome b alpha subunit gene, a poliovirus gene, a Sindbis virus gene, a brome mosaic virus gene, a Zika virus gene, or a dengue virus gene. In certain embodiments, the 3' UTR comprises a 3' UTR derived from hHBB. In certain embodiments, the nucleotide sequence of the 3' UTR of hHBB is set forth in SEQ ID NO: 2. In certain embodiments, the optimized 5' UTR and the 3' UTR are derived from the same gene. In certain embodiments, the optimized 5' UTR and the 3' UTR are derived from a human beta globin gene (hHBB), the sequences of which are set forth 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, a drug (e.g., an antibody), or an antigen.

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

[0012] In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer and bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof.

[0013] In certain embodiments, the tumor-derived antigen is selected from the group consisting of NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or a combination thereof.

[0014] In certain embodiments, the pathogen is selected from the group consisting of a virus, a bacterium, a fungus, a mycoplasma, a chlamydia, or any combination thereof. In certain embodiments, the viral antigen is selected from the group consisting of an antigen of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, avian infectious bronchitis virus, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or Epstein-Barr virus.

[0015] In certain embodiments, the coronavirus is SARS-COV-2, and the coronavirus antigen is a full-length spike protein or its RBD domain. In certain embodiments, the spike protein is selected from the group consisting of spike protein of any viral strain of 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.

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

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

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

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

[0020] In certain embodiments, the antigen of monkeypox virus is selected from the group consisting of internal membrane proteins M1R, H3L, E8L, and A29L, and external envelope proteins A35R and B6R, accessory protein L5L. In certain embodiments, the antigen of monkeypox virus is A29L, M1R, B6R, and / or A35R.

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

[0022] In certain embodiments, the bacterial antigen is selected from an antigen of Actinomyces, Bacillus, Bacteroides, Enterococcus, Listeria, Mycobacterium, Pneumococcus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, or Streptococcus.

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

[0024] In certain embodiments, the antibody is an autoantibody.

[0025] In certain 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 according to the present application is selected from the sequence set forth in any one of SEQ ID NOs: 7-31. In certain embodiments, the ORF comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 32-56.

[0027] In certain embodiments, the 5' UTR as described above is a human beta globin gene (hHBB) 5' UTR. In certain embodiments, the optimized 5' UTR as described above increases the expression 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 more.

[0028] In another aspect, the present application provides use of the nucleic acid construct or mRNA molecule according to the present application in any one of: (1) preparing a vaccine; (2) encoding a viral antigen in or outside a subject; (3) preparing a medicament for encoding a viral antigen in or outside a subject; (4) for preparing a medicament (e.g., an antibody).

[0029] In yet another aspect, the present application provides a vaccine comprising the nucleic acid construct or mRNA molecule according to the present application. In certain embodiments, the nucleic acid construct or mRNA molecule is encapsulated in a lipid nanoparticle (LNP).

[0030] In another aspect, the present application provides a vector comprising the nucleic acid construct or mRNA molecule according to the present application.

[0031] In another aspect, the present application provides a host cell comprising the vector according to the present application.

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

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

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

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

[0036] In another aspect, the present application provides a use of a nucleic acid construct or mRNA molecule, a vaccine, a pharmaceutical composition, and / or a product or kit as described herein in the manufacture of a medicament for treating and / or preventing a disease and / or a symptom caused by a pathogen (e.g., a virus) infection in a subject.

[0037] In certain embodiments, the pathogen is selected from a virus, a bacterium, a fungus, a mycoplasma, a chlamydia, or any combination thereof. In certain embodiments, the virus is an influenza virus, a respiratory syncytial virus, a coronavirus, a human metapneumovirus, a chicken infectious bronchitis virus, a Nipah virus, a monkeypox virus, more preferably the SARS-CoV-2, an influenza A virus, or an influenza B virus.

[0038] In certain embodiments, the pathogen is related to a protein drug (e.g., an antibody) or an antigen encoded by an ORF comprised in the nucleic acid construct or mRNA molecule.

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

[0040] In certain embodiments, the disease and / or symptom comprises a simple 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, severe acute respiratory syndrome (SARS).

[0041] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

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

[0043] In another aspect, the present application provides use of a nucleic acid construct or mRNA molecule, a vaccine, a pharmaceutical composition, and / or a product or kit as described herein in the manufacture of a medicament for inducing a neutralizing antibody response and / or a T cell immune response in a subject.

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

[0045] In certain embodiments, the virus is an influenza virus, a respiratory syncytial virus, a coronavirus, a human metapneumovirus, a chicken infectious bronchitis virus, a Nipah virus, a monkeypox virus.

[0046] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

[0047] In another aspect, the optimized 5’ UTR as described before is used for the manufacture of a nucleic acid construct or mRNA molecule.

[0048] In another aspect, the optimized 5’ UTR as described before is used for increasing the expression level of a protein encoded by a nucleic acid construct or mRNA molecule comprising the same.

[0049] In another aspect, the optimized 5’ UTR as described before is used for increasing the immunogenicity of a vaccine, a pharmaceutical composition, a product or a kit comprising the nucleic acid construct or mRNA molecule.

[0050] Beneficial effects

[0051] The optimized 5’ UTR provided in the present application can increase the expression level of a protein (e.g., an antigen, a protein drug (e.g., an antibody)) encoded by an mRNA. Therefore, an mRNA vaccine comprising the optimized 5’ UTR provided in the present application has a better level of immune response (e.g., can induce a high titer of neutralizing antibodies, can induce a specific T cell immune response), thereby being able to better prevent and / or treat a disease and / or a symptom caused by a pathogen (e.g., a virus) infection. Therefore, the optimized 5’ UTR provided in the present application and the mRNA molecule comprising the same have a wide application prospect in the field of vaccines, gene therapy, antibody therapy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Structure diagram of DNA template and mRNA product of co-transcription capping method for producing mRNA, and 5'UTR optimization method used in the present application.

[0053] Figure 2 Optimized 5'UTR mRNA synthesized by in vitro transcription and detection of expression of eGFP and FLuc reporter gene in cells.

[0054] Figure 3 5'UTR optimization method can improve the expression level of GFP expressed by various natural 5'UTRs.

[0055] Figure 4 5'UTR optimization method can improve the expression level of respiratory syncytial virus pre-fusion conformation F protein expressed by mRNA.

[0056] Figure 5 5'UTR optimization method can improve the expression level of influenza virus H1N1, H5N1 HA antigen expressed by mRNA.

[0057] Figure 6 mRNA vaccine design, synthesis, LNP encapsulation, cell expression of seasonal influenza virus H1N1, H3N2, B-Victoria strain HA antigen, and IgG antibody titer detection, virus neutralization, virus protection experiment results after immunizing BALB / c mice.

[0058] Figure 7 Cell expression of influenza virus H5N1 mRNA-HA vaccine, IgG antibody titer, virus neutralization, virus protection experiment results after immunizing BALB / c mice, SPF chickens.

[0059] Figure 8 Cell expression of mRNA-HA neck antigen of influenza virus H1N1, IgG antibody titer, virus protection experiment results after immunizing BALB / c mice.

[0060] Figure 9 Cell expression of influenza virus H1N1 mRNA-NA vaccine, IgG antibody titer, virus protection experiment results after immunizing BALB / c mice.

[0061] Figure 10 Cell expression of influenza virus PR8 (H1N1) mRNA-NP vaccine, IgG antibody titer after immunizing BALB / c mice by different routes.

[0062] Figure 11Design, synthesis, cell expression of bivalent mRNA vaccine of SARS-CoV-2 Spike protein RBD region, and detection of IgG antibody titer, pseudovirus neutralization experiment, and cell immunity detection after immunization of BALB / c mice.

[0063] Figure 12 Synthesis, cell expression of mRNA vaccine of pre-fusion conformation F protein of respiratory syncytial virus, and detection of IgG antibody titer, virus neutralization experiment, and cell immunity detection after immunization of BALB / c mice.

[0064] Figure 13 Detection of intracellular expression of human metapneumovirus mRNA-F vaccine by immunofluorescence.

[0065] Figure 14 Synthesis and intracellular expression of chicken infectious bronchitis virus Spike protein mRNA-S1 and mRNA-RBD vaccines.

[0066] Figure 15 Synthesis and intracellular expression detection of Nipah virus F protein, G protein mRNA vaccine.

[0067] Figure 16 Synthesis and intracellular expression detection of monkeypox virus L5L protein mRNA vaccine.

[0068] Figure 17 Synthesis, intracellular expression detection, and virus neutralization experiment of mRNA expressing heavy chain and light chain of anti-RSV monoclonal antibody D25.

[0069] Sequence information

[0070] The description of the sequences involved in the present application is provided in the following table.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] DETAILED DESCRIPTION

[0086] TERMINOLOGY

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

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

[0089] As used herein, “messenger RNA” (“mRNA”) refers to a nucleic acid comprising an open reading frame encoding a protein. mRNA has a modular structure, including a 5’ cap (5’ Cap), a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding a protein, a 3’ untranslated region (3’ UTR), and a polyadenylate (polyA) tail.

[0090] As used herein, an "open reading frame encoding a protein" refers to a nucleic acid sequence comprising a coding sequence that, when the open reading frame is translated, results in the production of a protein. An open reading frame typically begins with a start codon, e.g., AUG in an RNA sequence (ATG in a DNA sequence), and ends with a stop codon, e.g., UAG, UAA, or UGA in an RNA sequence (TAG, TAA, or TGA in a DNA sequence).

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

[0092] As used herein "Lipid Nanoparticles (LNPs)" refers to nanoparticles composed of lipid molecules. Typically having a particle size range of 20-200 nanometers, containing ingredients such as cationic lipids, phospholipid lipids, PEGylated lipids, etc. Lipid nanoparticles are used to encapsulate and deliver biological macromolecules (such as mRNA, DNA, etc.) and can effectively protect these macromolecules from degradation by enzymes in the body, facilitating their entry into target cells. The lipid nanoparticles comprise a cationic lipid, a neutral phospholipid, a steroidal lipid, and a polyethylene glycol-lipid. In some embodiments, the cationic lipid is DLin-MC3-DMA, SM102; the polyethylene glycol-lipid is DMG-PEG2000; the neutral phospholipid is DSPC; and the steroidal lipid is cholesterol.

[0093] 5' Cap

[0094] A 5' cap is an entity, typically a modified nucleotide entity, which is typically added at the 5' end of a mature mRNA. A 5' cap can typically be formed from a modified nucleotide, in particular from a derivative of a guanine nucleotide. Preferably, the 5' cap is attached at the 5' end via a 5'-5'- triphosphate linkage. The 5' cap can be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of an RNA.

[0095] mRNA drugs are produced in vitro by cell-free transcription, and the 5' cap is usually added in two ways: one is to further process the mRNA obtained by in vitro transcription with capping enzyme, and the other is to add a trinucleotide cap analog directly to the in vitro transcription system to obtain capped mRNA. The latter is called co-transcriptional capping, which has the advantages of fewer production steps and single product, and is the mainstream trend of mRNA drug production. However, the use of cap analogs requires the 5'UTR start sequence to be fixed as AGG, which may change the structure of the 5'UTR and affect the translation efficiency. In particular, the present application optimizes the 5'UTR sequence used in the co-transcriptional capping method, thereby improving the expression level of the protein encoded by the mRNA.

[0096] Optimized 5'UTR

[0097] As used herein, 5' untranslated region (5'UTR) means a specific portion of a messenger RNA (mRNA) that is located 5' of the open reading frame of the mRNA. Typically, the 5'UTR starts at the transcription start site and ends one nucleotide before the start codon of the open reading frame.

[0098] In the context of the present application, the 5'UTR corresponds to the sequence of the mature mRNA located between the 5' cap and the start codon. Preferably, the 5'UTR corresponds to the sequence extending from the nucleotide located 3' of the 5'-cap (preferably from the nucleotide located immediately 3' of the 5' cap) to the nucleotide located 5' of the start codon of the protein coding region (preferably to the nucleotide located immediately 5' of the start codon of the protein coding region). The nucleotide located immediately 3' of the 5' cap of the mature mRNA typically corresponds to the 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] The 5'UTR sequences currently used in mRNA drugs are usually derived from two sources: one is the 5'UTR of natural genes and their derived sequences (such as the Pfizer-BioNTech COVID-19 vaccine BNT162b2), and the other is obtained by screening from a library of artificially designed and synthesized sequences (represented by the Moderna COVID-19 vaccine mRNA-1273). Artificial de novo synthesis is a huge workload, and the 5'UTR sequences screened have no significant advantage over natural sequences (Ye et al, 2023 PNAS DOI: 10.1073 / pnas.2311752120; Castillo-Hair et al, 2024 Nature Communications DOI: 10.1038 / s41467-024-49508-2), so the 5'UTR sequences in current mRNA drugs are usually derived from natural highly expressed genes.

[0100] In the context of the present application, the term "5' UTR of a gene", such as "5' UTR of the human beta globin gene (hHBB)", is a sequence corresponding to the 5' UTR of a mature mRNA derived from the gene, i.e. a mRNA obtained by transcription of the gene and maturation of the immature mRNA. Similarly, the term "3' UTR of a gene", such as "3' UTR of the human beta globin gene (hHBB)", is a sequence corresponding to the 3' UTR of a mature mRNA derived from the gene, i.e. a mRNA obtained by transcription of the gene and maturation of the immature mRNA.

[0101] In some aspects, the present disclosure provides optimized 5' UTRs. In one particular embodiment, a new 5' UTR is created by adding 3 random bases after the transcription start site AGG of the 5' UTR used in the co-transcriptional capping method. The present inventors surprisingly found that the newly constructed 5' UTR is able to increase the expression level of the protein encoded by the mRNA, making the optimized mRNA molecule have a broad application prospect in the fields of vaccines, gene therapy, antibody therapy, etc.

[0102] In one particular embodiment, the present disclosure provides an optimized 5' UTR which has 3 random bases NNN inserted after the transcription start site AGG of the 5' UTR. In one embodiment, the 3 random bases NNN are any 3 out of the 4 bases A, T, C and G arranged in combination (in addition to AGG). For example, in the 3 random bases NNN, the first N can be selected from any one of A, T, C and G; the second N can be selected from any one of A, T, C and G; and the third N can be selected from any one of A, T, C and G. Thus, 64 possible combinations of the 3 random bases NNN are generated.

[0103] In certain embodiments, the 3 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, GTC, 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 certain embodiments, the 3 random bases NNN are selected from any one 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 certain embodiments, the 3 random bases NNN are selected from any one 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 certain embodiments, the 3 random bases NNN are selected from any one 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 the present application is not limited. Generally, it can be derived from the 5'UTR of any natural gene and its derivative sequences, which can be derived from animals, plants, fungi, bacteria or viruses. Generally, it can be derived from any species, such as human, baboon, monkey, mouse, chicken, zebrafish, Arabidopsis thaliana, yeast, Escherichia coli, influenza virus, dengue virus, coronavirus, etc. Generally, it can be derived 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 algorithm generated 5'UTR sequence (P.J. Sample 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 and the full text thereof are hereby incorporated by reference. For example, the optimized 5'UTR of the present application can be derived from the beta globin gene (HBB), ribosomal protein genes (such as RpL38, RpS25), actin genes (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etch virus (TEV), scrambled short 5'-UTR (scrUTR) or complement factor 3 (C3). In certain embodiments, the sequences of hHBB 5'UTR, RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR and SARS-CoV2 5'UTR are set forth 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%, 100% identity thereto. In certain embodiments, these sequences have the same or similar activity compared to the sequences they are derived from.

[0108] In some embodiments, the optimized 5'UTR of the present application is a 5'UTR truncation, i.e., one or more nucleotides are deleted while 3 random bases NNN are inserted after the 5'UTR transcription start site AGG. In some embodiments, the 5'UTR truncation still maintains similar active functions as the untruncated optimized 5'UTR. In some embodiments, the optimized 5'UTR of the present application further comprises one or more point mutations, i.e., one or more nucleotide changes (e.g., addition, deletion, and / or substitution) exist while 3 random bases NNN are inserted after the 5'UTR transcription start site AGG. In preferred embodiments, such nucleotide deletion or change is able to further improve the expression level of the encoded protein.

[0109] The source of the 3'UTR of the present application 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 human, baboon, monkey, mouse, chicken, zebrafish, Arabidopsis thaliana, yeast, Escherichia coli, influenza virus, dengue virus, coronavirus, etc. (J. J. Seo, Cell 2023 DOI: 10.1016 / j.cell.2023.06.007; A. K. Morrow, bioRxiv 2024 DOI: 10.1101 / 2024.10.07.616676), or mutants of these 3'UTRs (T. Fu, et al. Nat Commun 2024, DOI: 10.1038 / s41467-024-46795-7). All 3'UTR sequences listed in the above references and the full texts thereof are hereby incorporated by reference.

[0110] For example, the 3'UTR element can be derived from the following genes: albumin gene, a-globin gene, b-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, collagen a gene (e.g., collagen a1 (I) gene), cytochrome b a subunit gene, poliovirus gene, Sindbis virus gene, brome mosaic virus gene, Zika virus gene, and dengue virus gene.

[0111] In embodiments of the application, the optimized 5' UTR and the 3' UTR are derived from the same gene, for example, from the human beta globin gene (hHBB), the sequences of which are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. In certain embodiments, these sequences have the same or similar activity as compared to the sequences from which they are derived (e.g., SEQ ID NO: 1 and SEQ ID NO: 2).

[0112] Nucleic acid constructs and RNA molecules

[0113] In some aspects, the present disclosure provides nucleic acid constructs comprising an optimized 5' UTR as described herein and an open reading (ORF) encoding a gene of interest. In preferred embodiments, the nucleic acid construct further comprises a 5' cap (5' Cap), a 3' untranslated region (3' UTR), and a polyadenylate (polyA) tail.

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

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

[0116] In some embodiments, the open reading frame of the present disclosure 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 the present disclosure comprises 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 the present disclosure are of the same or different origin, e.g., derived from the same or different genes. In some embodiments, the 5' UTR and 3' UTR in the nucleic acid construct / RNA molecule of the present disclosure are derived from the same species or different species.

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

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

[0119] In some embodiments, the nucleic acid construct comprises an optimized 5' UTR as described herein and a 3' UTR as described above, the optimized 5' UTR and the 3' UTR are derived from the same gene, e.g., from the human beta globin gene (hHBB), the sequences of which are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. In certain embodiments, these sequences have the same or similar activity as compared to the sequences from which they are derived (e.g., SEQ ID NO: 1 and SEQ ID NO: 2).

[0120] In some embodiments, the nucleic acid construct / RNA molecule of the present disclosure further comprises: (d) a poly-adenosine (poly-A) tail.

[0121] In some embodiments, the poly-A tail in the nucleic acid construct is downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is 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, 500 nucleotides in length.

[0122] In some embodiments, the poly-A tail includes, but is not limited to, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40 late polyA.

[0123] In the present disclosure, in any of the nucleic acid constructs described above, the ORF comprises a nucleotide sequence encoding at least one polypeptide or protein. In some embodiments, the nucleotide sequence can 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 set forth 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 set forth in SEQ ID NO: 30 or SEQ ID NO: 31.

[0126] In some embodiments, the polypeptide or protein encoded by the ORF is a viral antigen. Illustratively, the viral antigen includes, but is not limited to, an antigen 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 Epstein-Barr virus, etc.

[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 the group consisting of a spike protein (S protein or Spike protein), an envelope protein (E protein), a membrane protein (M protein), and a nucleocapsid protein (N protein). In some embodiments, the structural protein is a spike protein. In some embodiments, the spike protein is a SARS-COV-2 spike protein. In some embodiments, the SARS-COV-2 spike protein is selected from the group consisting of a spike protein of any of SARS-COV-2 (e.g., wild-type SARS-COV-2), 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), SARS-COV-2 Omicron (B.A.4), and the like.

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

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

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

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

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

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

[0134] In some embodiments, the nucleotide sequence of the ORF encoding a polypeptide or protein is selected from the sequence set forth in any one of SEQ ID NOs: 7-31, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. In certain embodiments, these sequences have the same or similar activity as compared to the sequence from which they are derived (e.g., SEQ ID NOs: 7-31).

[0135] In some embodiments, the ORF encoding a polypeptide or protein comprises a codon-optimized nucleotide sequence comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a nucleotide sequence encoding a protein set forth in SEQ ID NOs: 32-56. In certain embodiments, these sequences have the same or similar activity as compared to the sequence from which they are derived (e.g., SEQ ID NOs: 32-56).

[0136] In preferred embodiments, the ORF encoding a polypeptide or protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 32-56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. In certain embodiments, these sequences have the same or similar activity as compared to the sequence from which they are derived (e.g., SEQ ID NOs: 32-56).

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

[0138] In certain embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding an additional protein or polypeptide. In certain embodiments, the additional protein or polypeptide is selected from the group consisting of: a signal peptide, a multimerization domain (e.g., a dimerization domain, a trimerization domain), a tag, or any combination thereof.

[0139] Polynucleotide

[0140] The present disclosure also provides an isolated polynucleotide encoding one or more polypeptides or proteins or viral antigens as described above.

[0141] In some embodiments, the present disclosure provides the use of any one of the nucleic acid constructs or RNA molecules or polynucleotides described herein in any one of: (1) the manufacture of a vaccine; (2) the encoding of a viral antigen in or in vitro; (3) the manufacture of a medicament for the encoding of a viral antigen in or in vitro; (4) a medicament.

[0142] In some embodiments, the nucleic acid construct or RNA molecule or polynucleotide in the present disclosure encodes a protein of interest, wherein the open reading frame (ORF) has enhanced expression of the protein of interest.

[0143] Vector

[0144] The present disclosure also provides a vector comprising a nucleic acid construct or RNA molecule described herein, wherein the nucleic acid construct / RNA molecule can be present in and / or can be part of the vector, such as a plasmid, a cosmid, a YAC, or a viral vector. The vector can be an expression vector, i.e., a vector that provides for expression of the polypeptide encoded by the nucleic acid construct or RNA. The expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, terminators, etc.). Selection of such elements and their sequences for expression in a particular host is a matter of choice, as is well within the level of ordinary skill in the art. Control elements and other elements useful or necessary for expression of the polypeptide-encoding nucleic acids of the present disclosure are, for example, promoters, terminators, selectable markers, leader sequences, reporter genes, etc.

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

[0146] In some embodiments, the vector of the present disclosure further comprises a promoter, e.g., the promoter is at the 5' end of the 5' UTR of the nucleic acid construct, e.g., the promoter is a T7 promoter, a T7lac promoter, a Tac promoter, a Lac promoter, a Trp promoter.

[0147] Host cells

[0148] The present disclosure also provides a host cell comprising a nucleic acid construct, RNA molecule, or polynucleotide described herein. In some embodiments, the cell is capable of expressing one or more polypeptides encoded by a nucleic acid construct or RNA of the present 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 strains, Proteus strains, and Pseudomonas strains, and Gram-positive bacterial strains such as Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.

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

[0151] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0152] However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins can also be used in the present disclosure.

[0153] Production or preparation methods

[0154] The present disclosure provides a method of making a nucleic acid construct or RNA of the present disclosure, as well as a method of making a polypeptide encoded thereby.

[0155] Methods and reagents, e.g., particular suitable vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, etc., for preparing nucleic acid constructs or RNA, and polypeptides encoded thereby, are known in the art. Similarly, protein isolation and purification techniques suitable for use in manufacturing the polypeptides of the disclosure in the methods of the disclosure are known to those of skill in the art.

[0156] In some embodiments, the method of preparing a nucleic acid construct or RNA comprises culturing the aforementioned host cell and recovering the nucleic acid construct or RNA produced from the culture. Nucleic acid constructs or RNA of the disclosure, and polypeptides encoded thereby, can also be obtained by other methods of production known in the art, such as chemical synthesis, including solid or liquid phase synthesis.

[0157] In some embodiments, the method of preparing an RNA molecule comprises preparing a nucleic acid construct or vector and then using the nucleic acid construct or vector for reverse transcription to obtain the RNA molecule. In some particular embodiments, the method further comprises adding a 5’ Cap to the 5’ end of the RNA molecule.

[0158] Vaccine

[0159] Gene vaccine

[0160] As used herein, a “gene vaccine” is a vaccine for genetic inoculation, generally understood as a “third generation” vaccine. It typically consists of a genetically engineered nucleic acid molecule encapsulated in the lipid nanoparticle (LNP) that allows a peptide or protein (antigen) specific to a pathogen or tumor to be expressed in vivo. A gene vaccine is expressed after administration to a patient and taken up by cells. Expression of the administered nucleic acid leads to production of the encoded protein. When these proteins are recognized as foreign by the patient’s immune system, an immune response is elicited.

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

[0162] Monovalent vaccine

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

[0164] Multivalent / combination vaccine

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

[0166] In some embodiments, in the monovalent vaccine and the multivalent / combination vaccine described above, 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 selected from the group consisting of 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 a SARS-COV-2 spike protein. In some embodiments, the SARS-COV-2 spike protein is selected from the group consisting of spike protein of any of 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), SARS-COV-2 Omicron (BA.4), and the like.

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

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

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

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

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

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

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

[0174] The present disclosure also provides a multivalent / combination vaccine comprising RNA encoding one or more coronaviruses or antigens of one or more different organisms. That is, the vaccines of the present disclosure 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] The present disclosure also provides a pharmaceutical composition comprising the nucleic acid construct described herein, the polynucleotide described herein, the vaccine described herein, and / or the vector described herein, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant, specifically, the pharmaceutical composition is a solid preparation, an injection, a preparation for external use, a spray, a liquid preparation, or a complex preparation.

[0177] In certain embodiments, the adjuvant is selected from a metal salt, 3-D-mono-phosphoryl lipid A (MPL), saponin, an oil and water emulsion, a liposome, a nanoparticle, or any combination thereof.

[0178] In certain embodiments, the pharmaceutically acceptable carrier can be a sterile liquid such as water and oil, including those derived from petroleum, animals, plants, or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In certain preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

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

[0180] The pharmaceutical compositions of the present application can be administered by a variety of suitable means. Suitable means of administration include, but are not limited to, parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0181] Product or kit

[0182] The present disclosure provides a product or kit comprising a nucleic acid construct described herein, a polynucleotide described herein, a vaccine described herein, a vector described herein, and / or a pharmaceutical composition described herein. The kit can be used to provide related detection or diagnostic uses.

[0183] Methods and uses

[0184] The present disclosure also provides the use of a therapeutically and / or prophylactically effective amount of a nucleic acid construct described herein, a polynucleotide described herein, a vaccine described herein, a vector described herein, a pharmaceutical composition described herein, and / or a product or kit described herein in the preparation of a medicament for the treatment and / or prevention of a disease.

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

[0186] The present disclosure also provides a method, comprising administering to a subject in need thereof an effective amount of a nucleic acid construct described herein, a polynucleotide described herein, a vaccine described herein, a vector described herein, a pharmaceutical composition described herein, and / or a product or kit described herein, which is capable of inducing a neutralizing antibody response and / or a T cell immune response in the subject, e.g., a neutralizing antibody response against a viral antigen, e.g., a CD4+ and / or CD8+ T cell immune response against a viral antigen. Wherein the subject has an increase in the subject's antigen antibody titer following vaccination relative to the antigen antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against the antigen.

[0187] In some embodiments, the disease comprises a viral infectious disease or a viral infection related respiratory disease. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a human 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 influenza virus, such as an influenza A virus or an influenza B virus. Illustratively, the influenza virus is an influenza A virus H1N1, an influenza A virus H3N2, an influenza A virus H5N1, an influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), an influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the viral infection related respiratory disease (e.g., SARS-CoV-2 infection related respiratory disease) comprises simple infection such as fever, cough, and sore throat, headache, rhinitis, etc., pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxemic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.

[0188] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments, various purposes and advantages of the present application will become apparent to those skilled in the art.

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

[0190] Figure 1 The structure of the DNA template for the co-transcriptional capping method of mRNA production and the mRNA product of mRNA, as well as the 5'UTR optimization method used in this study, are shown. Table 1 lists the three base sequences added in the 5'UTR produced and tested in this study.

[0191] Table 1. Three-base (NNN) sequence and number

[0192]

[0193] Example Two: Regulating the translation efficiency of eGFP and Fluc reporter genes by 5'UTR optimization

[0194] With the combination of the existing human beta globin gene (hHBB) 5'UTR and 3'UTR (SEQ ID NO: 1, SEQ ID NO: 2), 3 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 eGFP (SEQ ID NO: 7) and Luciferase (SEQ ID NO: 8) mRNA constructs were assembled. The in vitro transcription template was amplified by PCR, and the sequence of the amplification primer was

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

[0196] R-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGG(SEQ ID NO:58).

[0197] The mRNA was synthesized in vitro by co-transcription capping method, and the IVT system was configured as follows:

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

[0199]

[0200]

[0201] After adding the template, incubate at 37°C for 2h, add 1 μL DNase I to each reaction and incubate at 37°C for 30min to digest the DNA template, and use LiCl precipitation method to precipitate the RNA at -80°C. Centrifuge to collect the precipitated mRNA, centrifuge at 14,000 rpm for 10 min at 4°C, and a white RNA precipitate appears at the bottom of the centrifuge tube; after discarding the supernatant, add 1 mL of pre-cooled 70% ethanol, vortex to wash the RNA, and repeat the washing once; dry at room temperature until the precipitate becomes translucent, add RNase-free H2O to dissolve the RNA, and use NanoDrop One to measure the mRNA concentration, and 1% TBE agarose gel electrophoresis to detect the quality of the mRNA. The results are shown in Figure 2 A, and a high-quality mRNA product was obtained.

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

[0203] After the mRNA transfected cells were cultured for 24 h, the high-content cell imaging analysis system was used to scan and quantify the GFP fluorescence intensity in the 96-well plate to analyze the effect of NNN sequence on GFP expression. The results are shown in Figure 2 C, the insertion of NNN sequence can enhance the expression of GFP, and the insertion of 57 NNN sequences significantly enhances the expression of GFP.

[0204] The same method was used to prepare luciferase-expressing mRNA Figure 2 After the mRNA transfected cells were cultured for 24 h, the cells were lysed, and luciferin substrate was added to detect the biological luminescence at 560 nm to analyze the effect of NNN sequence on luciferase expression. The results are shown in Figure 2 D, the insertion of NNN sequence can enhance the expression of luciferase, and the insertion of 50 NNN sequences significantly enhances the expression of luciferase. Among them, 1 has a 10%-20% increase, 20 has a 20%-50% increase, and 29 has a more than 50% increase.

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

[0206] The combination of RpL38, mActb, hActb, SARS-CoV2 5'UTR (SEQ ID NO: 3-6) and hHBB 3'UTR was used to assemble mRNA constructs encoding GFP, with 50-GAT / 58-GCT / 64-GGG sequences inserted between the T7 promoter and these 5'UTRs. IVT, mRNA transfection of cells and high-content cell imaging analysis system quantification of GFP expression were performed by the same method. The results are shown in Figure 3 Regardless of which 5'UTR is combined, the insertion of GAT, GCT and GGG sequences significantly enhances the expression of GFP.

[0207] Example Four: Optimized 5'UTR enhances the expression of RSV virus antigens

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

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

[0210] Synthetic DNA template containing mRNA transcription related elements was synthesized, the original sequence was the combination of human beta globin gene (hHBB) 5'UTR and 3'UTR (SEQ ID NO: 1, SEQ ID NO: 2), and the HA antigen sequence of influenza H1N1 and H5N1 (SEQ ID NO: 9, SEQ ID NO: 12) was assembled. The H1N1-HA optimized sequence contained 10 hHBB 5'UTR inserted with NNN; the H5N1-HA optimized sequence contained 11 hHBB 5'UTR inserted with NNN. Template preparation was performed by primer PCR amplification. mRNA was obtained by transcription using co-transcription method, and BHK21 cells were transfected with mRNA for expression verification by indirect fluorescent immunoassay. Cells were fixed with 4% paraformaldehyde 24h after transfection, and after washing the cells, the cells were blocked and perforated with 1% BSA containing 0.2% Triton X-100 (diluted with PBS), followed by specific antibody incubation for 1h, and after washing the cells, the secondary antibody conjugated with AF488 was incubated for 1h, and after incubation, the cells were washed and stored in PBS. The expression of HA in the cells was quantified using a high-content cell imaging analysis system, and the results are shown in FIGs. 5A and 5B. The insertion of these NNN sequences all enhanced the expression of HA. Figure 5

[0211] Example Six: Mouse immunogenicity test of seasonal influenza HA mRNA vaccine​​

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

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

[0214] The mRNA-LNP was transfected into BHK-21 cells for expression verification, and the expression of HA was detected by immunoblotting. Immunoblotting: One day before transfection, the cultured BHK-21 cells were transfected with mRNA-LNP at a concentration of 0.25×10 6 ​Inoculated with the concentration of 1 μg / mL in 12-well plates, 1 mL per well. mRNA transfection system: 1 μg mRNA, 1.6 μL Lipo8000, 50 μL opti-MEM per well. The transfected cells were incubated in a 37°C cell incubator for 24 h. RIPA mild cell lysis buffer was used to lyse the cells, 200 μL per well, on ice for 20 min. After lysis, the supernatant was collected by centrifugation at 4°C, mixed with the loading buffer and boiled in a water bath for 10 min. The obtained sample was used for SDS-PAGE electrophoresis. The electrophoresis conditions were constant voltage of 160 V until the target bands were separated; semi-dry transfer was used to transfer the protein to the PVDF membrane, and the transfer conditions were 25 W for 30 min; the transferred membrane was blocked in 5% skim milk for 1 h, incubated with the first antibody for 1 h, and then incubated with the corresponding second antibody for 1 h after TBST washing; the results were analyzed by ECL luminescence after washing the membrane. The results are shown in Figure 6 As shown in FIG. 8, the HA proteins were well expressed.

[0215] BALB / c female 6-week-old mice were randomly divided into groups of 5, and were immunized with monovalent or trivalent mRNA vaccines (Trivalent Influenza Vaccines, TRV) at doses of 0.1, 1, and 10 μg per mouse, and were immunized again 3 weeks later. At the same time, a commercially available quadrivalent inactivated influenza vaccine (QIV) was used as a positive control at a dose of 2 μg per mouse; mRNA-GFP was used as a negative control at a dose of 10 μg per mouse. Blood samples were collected at 3 weeks after the initial immunization and 3 weeks after the booster immunization, and the HA IgG antibody titers were detected by ELISA. The HA protein was used to coat the enzyme-labeled plate and incubated at 4°C overnight, the blocking solution (5% skim milk prepared with PBS) was added and incubated at 37°C for 1 h, the plate was washed with PBST, 3-fold gradient-diluted inactivated serum was added and incubated at 37°C for 1 h, the plate was washed, the anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 h, and finally the color developing substrate was added for color development, the reaction was terminated, the absorbance value was read at 450 nm using an enzyme-labeled instrument, and the antibody titer induced by the vaccine was calculated. The results are shown in Figure 6 As shown in FIG. 8, the mRNA vaccine can induce a good immune response level.

[0216] Neutralization titers of sera from mice immunized with mRNA vaccines were determined. The sera from mice three weeks after the booster immunization were inactivated at 56°C for 30 min, and then diluted by 3-fold gradient from an initial dilution of 1:50; 100 FFU of A / California / 07 / 2009 (H1N1) virus was incubated with the serum at 37°C for 1 h, the cells were washed with PBS, and 100 μL of the mixture was added to the cells for incubation at 37°C for 1 h; 2% sodium carboxymethylcellulose (CMC, 2X) was mixed with MEM medium at a volume ratio of 1:1 to prepare the overlay solution, and TPCK trypsin was added to a final concentration of 2 μg / mL; the virus supernatant was discarded from the cell plate, the cells were washed once with PBS, 100 μL of the overlay solution was added to each cell well, and the plate was incubated in a 37°C incubator for 24 h; the neutralization titers of the sera were calculated according to the immune spots, and the results are shown in Figure 6 F.

[0217] Three weeks after the booster immunization, the mice were intranasally infected with 5LD50 of A / California / 07 / 2009 (H1N1), A / Hong Kong / 1 / 68 (H3N2), and B / Guangzhou / 0215 / 2012 (Victoria) viruses, respectively, and the body weight of the mice was measured daily after the challenge, and the death of the mice was monitored, and the results are shown in Figure 6 G, which shows that the mRNA vaccine has a good protective effect on the CA07, HK68, and GZ12 viruses in mice.

[0218] Example Seven: Immunogenicity of an 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, which contained a combination of the 5' UTR and 3' UTR of the human beta globin gene (hHBB) inserted with AGA optimized sequences. The template was prepared by primer PCR amplification, and mRNA was obtained by transcription using the co-transcriptional capping method. The mRNA was transfected into BHK-21, HeLa, and 293T17 cells for expression verification, and the expression of the original H5N1 Texas HA protein (Texas-WT) and the mutant protein (Mut, Texas-343-345del) was detected by immunoblotting, and the results are shown in Figure 7 A, which shows that all the HA proteins have good expression in different cell lines.

[0220] The prepared mRNA was mixed with the lipid mixture containing SM102 cationic lipid at a volume ratio of 3:1 by microfluidic encapsulation, mixed and concentrated to prepare mRNA-LNP vaccine. BALB / c female mice were randomly divided into groups of 8, and immunized at different doses of 1, 5 μg per mouse, and immunized again 3 weeks later. Blood was collected at 2 weeks after immunization to detect HA protein antibody titer, and the results are shown in Figure 7 B. The mRNA vaccine can induce a good immune response level after the first and second immunization, and shows a dose-dependent manner. The neutralization experiment was performed using influenza A virus A / Ostrich / Hebei / B430-1 / 240418 (H5N1) of evolutionary branch 2.3.4.4b, and the results are shown in Figure 7 C. The mRNA vaccine induced high neutralizing antibodies against H5N1. As shown in Figure 7 D, 7E, the virus protection experiment was performed using influenza A virus A / Ostrich / Hebei / B430-1 / 240418 (H5N1), and the immunization of 1 μg and 5 μg mRNA vaccine can completely protect the mice, which confirms that the optimized mRNA vaccine prepared in this example can prevent influenza virus.

[0221] The mRNA was transfected into chicken DF-1 cells for expression verification, and the expression of original H5N1 Texas HA protein (Texas-WT) and mutant protein (Mut, Texas-343-345del) was detected by immunoblotting, and the results are shown in Figure 7 F. All HA proteins have good expression. 10-day-old SPF chicks were randomly divided into groups of 10, and immunized at a dose of 30 μg per mouse. Blood was collected at 1 week after the first immunization, and equal dose booster immunization was performed, and blood was collected at 2 weeks after the booster immunization. The HA protein antibody titer was detected, and the results are shown in Figure 7 G. The H5N1 mRNA-HA vaccine can induce a good immune response level in chickens. As shown in Figure 7 H, 7I, the virus protection experiment was performed using H5 influenza virus DKLH and DKFJ of evolutionary branch 2.3.4.4b, and the mRNA vaccine can completely protect the chickens. It is confirmed that the optimized mRNA vaccine prepared in this example can prevent influenza virus.

[0222] Example Eight: Mouse immunogenicity detection of classical influenza strain HA conserved neck domain mRNA vaccine

[0223] A gene expressing the conserved neck domain of the HA protein of influenza strain A / New Caledonia / 20 / 1999 (H1N1) (SEQ ID NO: 13) and a template DNA sequence containing mRNA transcription-related elements, including a combination of the 5'UTR and 3'UTR of the human β-globin gene (hHBB) with an AGA optimized sequence inserted, was synthesized and prepared by primer PCR amplification. mRNA was obtained by transcription using the co-transcriptional capping method ( Figure 8 A) mRNA was transfected into BHK-21 cells for expression verification, and indirect immunofluorescence was used to detect the expression of the conserved neck domain (H1stem) of the HA protein. 24 hours after transfection, the cells were fixed with 2% paraformaldehyde and hybridized with the human CR6261 antibody for primary antibody hybridization. The antibody can bind to the H1stem and the primary antibody was washed off. The cells were incubated with a 488-labeled fluorescent secondary antibody for 1 hour at room temperature. The cells were then photographed using a fluorescence microscope. The results are shown in Figure 2. Figure 8 As shown in B, 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 by microfluidic encapsulation and concentrated to prepare an mRNA-LNP vaccine. BALB / c female mice were randomly divided into 5 / group and given a primary immunization at a dose of 5 μg / mouse. A secondary immunization was performed 3 weeks later. Blood samples were collected to measure HA protein antibody titers 3 weeks after the primary immunization and 3 weeks after the booster immunization. The results are as follows: Figure 8 As shown in C. mRNA vaccines can induce good immune response levels after the first and second immunizations.

[0225] On the 28th day after the second vaccination, mice were challenged with 5LD50 doses of A / Puerto Rico / 8 / 34 (H1N1, PR8) virus by intranasal injection. The body weight of mice was measured every day after the challenge, and the mortality of mice was monitored. Figure 8 As shown in D, a 5 μg dose of H1stem-mRNA vaccine has a 100% protective effect against influenza virus PR8 virus.

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

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

[0228] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipid at a volume ratio of 3:1 by microfluidic encapsulation, mixed and concentrated to prepare mRNA-LNP vaccine. BALB / c female mice were randomly divided into groups of 5, and immunized for the first time at a dose of 5 μg per mouse, and immunized for the second time 3 weeks later. Blood samples were taken at 3 weeks after the first immunization and 3 weeks after the booster immunization to detect NA protein antibody titers, and the results are shown in Figure 9 C. The mRNA vaccine can induce a good level of immune response after the first and second immunization.

[0229] On day 21 after the second immunization, the mice were infected with 5xLD50 dose of CA07 virus by intranasal challenge. The body weight of the mice was measured daily after the challenge, and the death of the mice was monitored, and the results are shown in Figure 9 D, 9E, the 5 μg dose of mRNA-NA vaccine has a 100% protective effect against CA07 virus.

[0230] Example Ten: Mouse immunogenicity detection of influenza virus NP mRNA vaccine

[0231] The gene of influenza virus PR8 virus nucleoprotein (NP) and the template DNA sequence containing mRNA transcription related elements (SEQ ID NO: 15) were synthesized, which contained the combination of 5'UTR and 3'UTR of human beta globin gene (hHBB) inserted with AGA optimized sequence. The template was prepared by primer PCR amplification, and the mRNA was obtained by co-transcriptional capping method Figure 10 A). The mRNA was transfected into BHK-21 cell line for expression verification, and the expression of NP protein was detected by immunoblotting, and the results are shown in Figure 10 B, the NP protein can be well expressed.

[0232] The prepared mRNA was mixed with a lipid mixture containing SM102 cationic lipid at a volume ratio of 3:1 by microfluidic encapsulation, mixed and concentrated to prepare mRNA-LNP vaccine. C57BL / 6 female mice were randomly divided into groups of 5, and immunized for the first time at a dose of 5 μg per mouse, and the immunization routes included: intramuscular injection (i.m.), subcutaneous injection (s.c.), intravenous injection (i.v.), intranasal (i.n.) and aerosol inhalation. Immunized for the second time 3 weeks later. Blood samples were taken at 3 weeks after the first immunization and 3 weeks after the booster immunization to detect protein antibody titers, and the results are shown in Figure 10The mRNA vaccines delivered by intramuscular, subcutaneous, intravenous routes can induce better immune response level after the first and second immunization; the nasal drops and atomization can induce significant immune response, and the other several immunization routes can induce more significant immune response.

[0233] Example XI: Mouse immunogenicity test of new crown RBD mRNA vaccine

[0234] Synthetic DNA sequence (SEQ ID NO: 16) expressing the RBD domain of the new crown Spike protein and template DNA sequence containing mRNA transcription related elements, which contains the combination of 5’UTR and 3’UTR of human beta globin gene (hHBB) inserted with AGA optimized sequence. Template preparation is carried out by primer PCR amplification, and mRNA is obtained by transcription with co-transcriptional capping method Figure 11 A, 11B), transfect BHK-21 cells with mRNA for expression verification, detect the expression of RBD dimer by immunoblotting, and the results are shown in Figure 11 C, the RBD dimer protein has good expression.

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

[0236] BALB / c female mice were randomly divided into groups of 8, and immunized for the first time at a dose of 10 μg per mouse, and immunized for the second time 3 weeks later. Blood samples were taken at 3 weeks after the first immunization and 3 weeks after the booster immunization to detect IgG antibody titers. The results are shown in Figure 11 D, the mRNA vaccine can induce better immune response level after the first and second immunization.

[0237] Vero cells were plated in 96-well plates at 0.15 x 10 6 Each well contains 100 μL, and the neutralization experiment was performed after 12 h. The serum was diluted 100 times, 150 μl was added to four repeated wells, 50 μl was taken to the next well for 1:3 gradient dilution, and 100 TCID50 of VSV-Spike pseudovirus was added to each well. Shake well, incubate at 37°C for 1 h, replace the original culture medium of the cells with the virus-serum mixture, and perform Luciferase bioluminescence assay after 24 h. The results show that the vaccine has neutralization effect on different strain pseudoviruses of SARS-CoV-2 Figure 11 E).

[0238] Three weeks after the second immunization, the spleen of the mice was harvested and ground for spleen lymphocyte separation. The red blood cells were removed by red blood cell lysis buffer and the spleen cells were diluted to 2.5×10 6 / mL, 100μL was added to each well and cultured in a 96-well cell culture plate. An NC control group and a novel coronavirus peptide library stimulation group were set up. After 72 hours of culture, the ELISpots (3321-4HST-2) kit was used for detection, and the spot values ​​were read 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 Test of Respiratory Syncytial Virus mRNA Vaccine in Mice A DNA sequence (SEQ ID NO: 17) expressing the pre-fusion conformation F protein (pre-F) of respiratory syncytial virus 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 AGA optimized sequence inserted. Template preparation was performed by primer PCR amplification, and mRNA was obtained by transcription using the co-transcriptional capping method ( Figure 12 A), mRNA quality was tested by capillary electrophoresis, and the results were 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 immunoblotting. The results are shown in Figure 12 As shown in 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 by microfluidic encapsulation and concentrated to prepare an mRNA-LNP vaccine. BALB / c female mice aged 5-7 weeks were randomly divided into 16 groups and given a primary immunization with the mRNA vaccine at a dose of 5 μg / mouse. A secondary immunization was performed 3 weeks after the primary immunization and 3 weeks after the booster immunization. Blood samples were collected to detect anti-RSV pre-F or post-F IgG antibody titers 3 weeks after the primary immunization and 3 weeks after the booster immunization. The results are shown in Figure 2. Figure 12 As shown in D, 12E, and 12F, mRNA vaccines can induce a good level of immune response against prefusion F protein after one and two immunizations.

[0241] The titer of neutralizing antibodies in mouse serum after immunization with mRNA vaccine was determined. Mouse serum was heat-inactivated at 56°C for 30 minutes, then diluted 1:100, and then diluted 3-fold. 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 hour. 100 μL of serum-virus mixture was transferred to a monolayer of HEp-2 cells (3x10 4In 96-well plates (100 μL / well), the cells were incubated at 37 °C for 48 h, then washed with PBS and fixed with 4% paraformaldehyde. The infection of RSV virus was measured by indirect immunostaining. The serum neutralization titers were calculated according to the intensity of immunofluorescence signal, and the results are shown in Table 1. Figure 12 As shown in FIG. G, the RSV mRNA vaccine can induce high neutralizing antibodies against RSV type A classical strain (A2) and clinical strain (ON1), and type B (BA9).

[0242] The T cell immune response of mice after mRNA vaccine immunization was detected. The mouse spleen was ground to obtain a single cell suspension and the red blood cells were removed, and the treated spleen cells were counted and diluted to 2 x 10 7 100 μL of spleen cells were added to the RSV (A2) F0 peptide pool (2 μg / mL), 37 °C for 1 h, then GolgiStop / GolgiPlug diluent was added, and the incubation was continued at 37 °C for 7 h. The cells were washed twice with PBS, stained with dead and live cell dye, then the cell surface proteins were labeled with fluorescently labeled antibodies, including anti-CD3e FITC, anti-CD4 PE-Cy7, anti-CD8a BV510, anti-CD69 Alexa 700, anti-CD44 BV421 and anti-CD62L BV650, 4 °C, dark incubation for 30 min. The 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-2 PE and anti-IL-4 BV786. The samples were analyzed by flow cytometry, and the results are shown in FIG. H, I and I. Figure 12 As shown in FIG. H, I and I, the mRNA vaccine induced excellent T cell immune response.

[0243] On day 21 after the second immunization, female BALB / c mice (8 per group) were infected with 1 x 10 6 p.f.u dose of RSV A2 virus by intranasal infection, and the protection of the vaccine to the mice was analyzed by detecting the RSV viral load in the lungs of the mice on day 4 after the infection, and the results are shown in FIG. J. Figure 12 As shown in FIG. J, the mRNA vaccine has a good protective effect on RSV A2 virus.

[0244] Example XIII: Preparation and expression of mRNA vaccine of hMPV

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

[0246] Example Fourteen: Cell expression and chicken virus protection experiment of infectious bronchitis virus (IBV) antigen

[0247] Chicken infectious bronchitis is a disease that occurs in chickens caused by infectious bronchitis virus (IBV), which is an acute and highly contagious viral respiratory disease, and is one of the important diseases in the poultry industry. The disease is mainly prevented, and the most important thing is to do a good job of vaccine immunization.

[0248] Four different mRNA vaccines of IBV strain Spike protein S1 region or RBD domain were designed, and the DNA template sequence (SEQ ID NO: 19-26) of the antigen and the template DNA containing mRNA transcription related elements were synthesized, which contained the combination of human beta globin gene (hHBB) 5' UTR and 3' UTR with AGA optimized sequence inserted. Template preparation was performed by primer PCR amplification, and mRNA was obtained by co-transcriptional capping method Figure 14 A). mRNA was transfected into chicken DF1 cells for expression verification, and the expression of antigen with FLAG tag was detected by immunoblotting, and the results are shown in Figure 14 B, which shows that IBV S1 and RBD proteins have good expression in cells.

[0249] Example Fifteen: 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 diseases and fatal encephalitis. Currently, there is no treatment or vaccine available for humans or animals. A DNA sequence expressing Nipah virus F protein (SEQ ID NO: 27), a DNA sequence of G protein (SEQ ID NO: 28), and a template DNA sequence for 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 AGA optimized sequence inserted. Template preparation was performed by primer PCR amplification. Transcription was performed using the co-transcriptional capping method to obtain mRNA ( Figure 15 A), mRNA was transfected into BHK-21 cell line for expression verification, and the expression of NiV-F and NiV-G proteins was detected by immunoblotting. The results are shown in Figure 15 As shown in B, NiV-F and NiV-G proteins can be well expressed.

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

[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 AGA optimized sequence inserted. Template preparation was performed by primer PCR amplification, and mRNA was obtained by transcription using the co-transcriptional capping method ( Figure 16 A). The mRNA was transfected into BHK-21 cell line for expression verification, and the expression of MPXV-L5L protein was detected by immunoblotting. The results are shown in Figure 2. Figure 16 As shown in B, MPXV-L5L protein can be well expressed.

[0253] Example 17: Anti-RSV nucleic acid antibody drug

[0254] DNA sequences expressing the heavy and light chains of RSV monoclonal antibody D25 (SEQ ID NOs: 30 and 31) and template DNA sequences for 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 AGA optimized sequence inserted. Template preparation was performed by primer PCR amplification, and mRNA was obtained by transcription using the co-transcriptional capping method ( Figure 17 A). The mRNA was transfected into the BHK-21 cell line for expression verification. The expression of D25 antibody protein at different time points was detected by ELISA. The neutralization activity of D25 antibody against RSV-A2 virus was verified by in vitro neutralization experiment. The results are shown in Figure 2. Figure 17 As shown in B, D25 antibody protein can be well expressed in BHK-21 cells, with sustained expression for about 3-4 days, and has good neutralizing activity ( Figure 17 C).

Claims

1. An optimized 5'UTR, characterized in that Three random bases NNN are inserted after the 5'UTR transcription start site AGG, 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; Preferably, 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, GTC, 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; Preferably, the three random bases NNN are selected from any one 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; Preferably, the three random bases NNN are selected from any one 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; Preferably, the three random bases NNN are selected from any one 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.

2. The 5'UTR according to claim 1, wherein the 5'UTR is selected from the group consisting of a β-globin gene (HBB), a ribosomal protein gene (such as RpL38, RpS25), an actin gene (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etch virus (TEV), a scrambled short 5'-UTR (scrUTR), or a 5'UTR of complement factor 3 (C3); Preferably, the 5'UTR is selected from hHBB 5'UTR, RpL38 5'UTR, mActb 5'UTR, hActb 5'UTR or SARS-CoV2 5'UTR; preferably, 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.

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

4. The nucleic acid construct or mRNA molecule according to claim 3, 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); (d) a polyadenylic acid (poly-A) tail; Preferably, the 3'UTR comprises a 3'UTR derived from an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, a collagen α gene, an α-subunit of cytochrome b gene, a poliovirus gene, a Sindbis virus gene, a brome mosaic virus gene, a Zika virus gene, or a dengue virus gene; More preferably, the 3'UTR comprises a 3'UTR derived from hHBB; more preferably, the nucleotide sequence of the 3'UTR of hHBB is shown in SEQ ID NO: 2; More preferably, the optimized 5'UTR and the 3'UTR are derived from the same gene; most preferably, the optimized 5'UTR and the 3'UTR are derived from human beta globin gene (hHBB), and their sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

5. The nucleic acid construct or mRNA molecule according to claim 3 or 4, wherein The ORF encodes a fluorescent protein, luciferase, a protein drug (eg, an antibody) or an antigen.

6. The nucleic acid construct or mRNA molecule according to claim 5, wherein 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 a tumor; Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from the group consisting of liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer and bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof; Preferably, the tumor-derived antigen is selected from NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or a combination thereof; Preferably, the pathogen is selected from viruses, bacteria, fungi, mycoplasmas, chlamydia, or any combination thereof; Preferably, the viral antigen is selected from the group consisting of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus, and Epstein-Barr virus antigens; Preferably, the coronavirus is SARS-COV-2, and the coronavirus antigen is a complete spike protein or its RBD domain; more preferably, the spike protein is selected from the spike protein of any one of the virus strains of SARS-COV-2, SARS-COV-2Alpha, SARS-COV-2Beta, SARS-COV-2Gamma, SARS-COV-2Kappa, SARS-COV-2Delta or SARS-COV-2Omicron; Preferably, the influenza virus is influenza A virus or influenza B virus, and the antigen is hemagglutinin protein (HA), neuraminidase (NA) and / or nucleoprotein (NP); more preferably, the influenza virus antigen is selected from the hemagglutinin protein, neuraminidase and / or nucleoprotein of any one strain of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria, and influenza B virus Yamagata; Preferably, the antigen of the respiratory syncytial virus is F protein, G protein, nucleocapsid protein or matrix protein; preferably, the antigen of the respiratory syncytial virus is selected from the F protein, G protein, nucleocapsid protein or matrix protein of a subtype A strain or a subtype B strain; preferably, the antigen of the respiratory syncytial virus comprises F protein in a pre-fusion conformation (pre-F), F protein in a post-fusion conformation (post-F), or a mixture of the two; preferably, the antigen of the respiratory syncytial virus is F protein in a pre-fusion conformation (pre-F); Preferably, the antigen of human metapneumovirus (hMPV) is F protein, G protein, nucleocapsid protein or matrix protein; preferably, the antigen is F protein of human metapneumovirus (hMPV); Preferably, the antigen of the infectious bronchitis virus of chicken is Spike protein, M protein, nucleocapsid protein or envelope protein; preferably, the antigen of the infectious bronchitis virus of chicken is the S1 region or RBD domain of the Spike protein; Preferably, the antigen of the Nipah virus is F protein, G protein, nucleocapsid protein or matrix protein; Preferably, the antigen of the Nipah virus is F protein or G protein; Preferably, the antigen of the monkeypox virus is selected from the inner membrane proteins M1R, H3L, E8L and A29L, and the outer envelope proteins A35R and B6R, and the accessory protein L5L; preferably, the antigen of the monkeypox virus is A29L, M1R, B6R and / or A35R; Preferably, the fungal antigen is selected from antigens of Candida, Aspergillus, Mucor, Rhizopus, Epidermophyton, Malassezia, Preumocystis, Penicillium, Alternaria, Cladosporium, Botrytis, Aureobasidium, Fusarium or Trichoderma; Preferably, the bacterial antigen is selected from antigens of Actinomycetes, Bacillus, Bacteroides, Enterococcus, Listeria, Mycobacterium, Pneumococcus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus or Streptococcus.

7. The nucleic acid construct or mRNA molecule according to claim 5 or 6, wherein The antibody is an antibody capable of specifically binding to any one or more antigens; Preferably, any one or more antigens are as defined in claim 6; Preferably, the antibody is an autoantibody; Preferably, 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.

8. The nucleic acid construct or mRNA molecule according to any one of claims 3 to 7, wherein The nucleotide sequence of the ORF is selected from the sequence shown in any one of SEQ ID NOs: 7-31; Preferably, the ORF comprises an amino acid sequence as shown in any one of SEQ ID NOs: 32-56.

9. Use of the nucleic acid construct or mRNA molecule of any one of claims 3 to 8 in any of the following: (1) preparing a vaccine; (2) encoding a viral antigen in a subject in vivo or in vitro; (3) preparing a drug that encodes a viral antigen in a subject in vivo or in vitro; (4) for preparing a drug (e.g., an antibody).

10. A vaccine comprising the nucleic acid construct or mRNA molecule according to any one of claims 3 to 8; preferably, the nucleic acid construct or mRNA molecule is encapsulated in lipid nanoparticles (LNPs).

11. A vector comprising the nucleic acid construct or mRNA molecule according to any one of claims 3 to 8. A host cell comprising the vector according to claim 11 .

13. A method for preparing the mRNA molecule according to any one of claims 3 to 8, comprising: The gene encoding the protein of the ORF and the template DNA sequence containing the mRNA transcription-related elements are synthesized, the template is prepared by primer PCR amplification, and the mRNA is transcribed using the co-transcriptional capping method.

14. A pharmaceutical composition comprising: the nucleic acid construct or mRNA molecule according to any one of claims 3 to 8, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

15. A product or kit comprising the nucleic acid construct or mRNA molecule according to any one of claims 3 to 8, the vaccine according to claim 10, and / or the pharmaceutical composition according to claim 14.

16. A method for treating and / or preventing a disease and / or symptom caused by infection with a pathogen (e.g., a virus) in a subject, comprising administering to a subject in need thereof an effective amount of the nucleic acid construct or mRNA molecule of any one of claims 3 to 8, the vaccine of claim 10, the pharmaceutical composition of claim 14, and / or the product or kit of claim 15; Preferably, the pathogen is selected from viruses, bacteria, fungi, mycoplasmas, chlamydia, or any combination thereof; Preferably, the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus, Nipah virus, monkeypox virus, and more preferably, the virus is SARS-CoV-2, influenza A virus, or influenza B virus; Preferably, the disease and / or symptoms include simple 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, severe acute respiratory syndrome (SARS); Preferably, the subject is a mammal, for example, a mouse or a human.

17. A method for inducing a neutralizing antibody response and / or a T cell immune response in a subject, comprising administering to a subject in need thereof an effective amount of the nucleic acid construct or mRNA molecule of any one of claims 3 to 8, the vaccine of claim 10, the pharmaceutical composition of claim 14, and / or the product or kit of claim 15; Preferably, the neutralizing antibody response is a neutralizing antibody response against viral antigens, and the T cell immune response includes a CD4+ and / or CD8+ T cell immune response; Preferably, the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus, Nipah virus, monkeypox virus; Preferably, the subject is a mammal, for example, a mouse or a human.

18. Use of the optimized 5'UTR of claim 1 or 2 in any one or more of the following: (i) preparing a nucleic acid construct or mRNA molecule; (ii) improving the expression level of a protein encoded by a nucleic acid construct or mRNA molecule comprising the same; or (iii) improving the immunogenicity of a vaccine, pharmaceutical composition, product or kit comprising the nucleic acid construct or mRNA molecule.

Images