Optimized 3'utr and uses thereof

Abstract

The present application belongs to the field of nucleic acid drugs, and particularly relates to an optimized 3'UTR and a nucleic acid construct or mRNA molecule comprising the same. The present application also relates to the use of the optimized 3'UTR, the nucleic acid construct or the mRNA molecule in the prevention or treatment of diseases.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drugs, specifically relating to an optimized 3'UTR and a nucleic acid construct or mRNA molecule containing it. This application also relates to the use of the optimized 3'UTR, nucleic acid construct, or mRNA molecule in the prevention or treatment of diseases. Background Technology

[0002] mRNA, as a novel nucleic acid drug, has unique advantages in the fields of vaccines and treatments. The rapid response capability, safety, and efficacy of mRNA vaccines have been validated in the response to the global COVID-19 pandemic caused by SARS-CoV-2. However, mRNA technology is still in its early stages, and issues such as thermal instability and low protein expression levels remain to be addressed.

[0003] mRNA has a modular structure, including a 5' cap, a 5' untranslated region (UTR), a target gene coding region, a 3' untranslated region, and a polyA tail. The 3' UTR is crucial for maintaining mRNA stability and correct spatiotemporal translation; it contains sequence motifs that bind to different effectors (such as RNA-binding proteins, miRNAs, or other ncRNAs). Therefore, optimizing the 3' UTR to increase the expression level of the protein encoded by the mRNA is essential for enhancing the immune response of mRNA vaccines and the therapeutic efficacy of mRNA drugs.

[0004] The 3'UTR sequences used in current mRNA drugs are mainly derived from natural genes, such as the human β-globin gene (hHBB). The advantages of natural UTR sequences based on high-throughput screening over hHBB 3'UTR sequences are not significant (Orlandini et al., Mol Ther, 2019; Seo et al., Cell, 2023).

[0005] Therefore, it is necessary to optimize the 3'UTR sequence based on structural features to improve the expression level of the protein encoded by the mRNA. Summary of the Invention

[0006] The optimized 3'UTR provided in this application can increase the expression level of proteins encoded by mRNA (e.g., antigens, protein drugs (e.g., antibodies)). Therefore, mRNA vaccines containing the optimized 3'UTR provided in this application exhibit good immune response levels (e.g., the ability to induce high titers of neutralizing antibodies, the ability to induce specific T-cell immune responses), especially at low doses (e.g., 0.1 μg) while still maintaining good immune response levels. Thus, the inventors have completed this application.

[0007] Optimized 3'UTR

[0008] Therefore, in a first aspect, this application provides an optimized 3'UTR comprising the 3'UTR sequence of a natural human β-globin gene (hHBB) and / or the 3'UTR sequence of a natural SARS-CoV-2, and an xrRNA sequence or a portion thereof from the 3'UTR sequence of one or more other viruses; or, This application provides an optimized 3'UTR, which inserts and / or deletes at least a portion of the sequence in the stem-loop structure and / or pseudoknot structure of the 3'UTR sequence of the natural human β-globin gene (hHBB) and / or the 3'UTR sequence of the natural SARS-CoV-2.

[0009] In some embodiments, the optimized 3'UTR, compared to its derived natural 3'UTR sequence (e.g., the 3'UTR sequence of the natural human β-globin gene (hHBB) and / or the 3'UTR sequence of the natural SARS-CoV-2), improves the stability and / or translation efficiency of nucleic acid constructs (e.g., mRNA) containing the 3'UTR.

[0010] In some embodiments, the optimized 3'UTR, compared to its derived natural 3'UTR sequence (e.g., the 3'UTR sequence of the natural human β-globin gene (hHBB) and / or the 3'UTR sequence of the natural SARS-CoV-2), results in an increased expression level of the protein encoded by the nucleic acid construct (e.g., mRNA) containing the 3'UTR.

[0011] In some embodiments, the optimized 3'UTR, compared to its derived native 3'UTR sequence (e.g., the 3'UTR sequence of the native human β-globin gene (hHBB) and / or the 3'UTR sequence of the native SARS-CoV-2), has enhanced immunogenicity of the protein encoded by the nucleic acid construct (e.g., mRNA) containing the 3'UTR.

[0012] In some implementations, the additional virus is a virus other than SARS-CoV-2.

[0013] Existing technologies have disclosed various xrRNA sequences, among which the Flaviviridae family is the most thoroughly studied and classic class of viruses. The Flaviviridae family includes: the genus Flavivirus (e.g., Zika virus, dengue virus (types 1-4), West Nile virus, yellow fever virus, Japanese encephalitis virus); the genus Plaguevirus (e.g., bovine viral diarrhea virus); and the genus Hepatitis C virus (hepatitis C virus), etc.

[0014] Therefore, in some embodiments, the additional virus is selected from Zika virus (ZIKA), Tamana bat virus (TABV), dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, hepatitis C virus, or any combination thereof.

[0015] In some embodiments, the xrRNA sequence in the 3'UTR sequence of Zika virus (ZIKA) is shown in SEQ ID NO:42. In some embodiments, the xrRNA sequence in the 3'UTR sequence of Tamana bat virus (TABV) is shown in SEQ ID NO:41.

[0016] In some embodiments, the xrRNA sequence in the 3'UTR sequence of the one or more additional viruses is linked to the 5' end and / or 3' end of the 3'UTR sequence of the native hHBB or SARS-CoV-2.

[0017] In some embodiments, the xrRNA sequence in the 3'UTR sequence of the one or more additional viruses may or may not contain a spacer nucleotide sequence (e.g., G) between it and the 3'UTR sequence of the native hHBB or SARS-CoV-2.

[0018] In some implementations, the optimized 3'UTR contains xrRNA sequences from one, two, or more additional viral 3'UTR sequences.

[0019] In some implementations, when the optimized 3'UTR contains xrRNA sequences from two or more other viral 3'UTR sequences, these xrRNA sequences are distinct from each other.

[0020] In some implementations, when the optimized 3'UTR contains xrRNA sequences from two or more additional viral 3'UTR sequences, the xrRNA sequences from the additional viral 3'UTR sequences may or may not contain spacer nucleotide sequences (e.g., G).

[0021] Optimized 3'UTR-29

[0022] In some implementations, the optimized 3'UTR comprises, from the 5' end to the 3' end, the 3'UTR sequence of the native hHBB and the xrRNA sequence in the 3'UTR sequence of TABV.

[0023] In some embodiments, the 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1. In some embodiments, the xrRNA sequence in the 3'UTR sequence of the TABV is shown in SEQ ID NO: 41. In some embodiments, the sequence of the optimized 3'UTR is shown in SEQ ID NO: 36.

[0024] Optimized 3'UTR-30

[0025] In some implementations, the optimized 3'UTR comprises, from the 5' end to the 3' end, the xrRNA sequence in the 3'UTR sequence of ZIKA and the 3'UTR sequence of native hHBB.

[0026] In some embodiments, the 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1. In some embodiments, the xrRNA sequence in the 3'UTR sequence of the ZIKA is shown in SEQ ID NO: 42. In some embodiments, the sequence of the optimized 3'UTR is shown in SEQ ID NO: 37.

[0027] Optimized 3'UTR-31

[0028] In some embodiments, the optimized 3'UTR comprises, from the 5' end to the 3' end, the xrRNA sequence in the 3'UTR sequence of ZIKA, the xrRNA sequence in the 3'UTR sequence of ZIKA, and the 3'UTR sequence of native hHBB.

[0029] In some embodiments, the 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1. In some embodiments, the xrRNA sequence in the 3'UTR sequence of the ZIKA is shown in SEQ ID NO: 42. In some embodiments, the sequence of the optimized 3'UTR is shown in SEQ ID NO: 38.

[0030] Optimized 3'UTR-32

[0031] In some implementations, the optimized 3'UTR comprises, from the 5' end to the 3' end, the 3'UTR sequence of the native hHBB and the xrRNA sequence in the 3'UTR sequence of ZIKA.

[0032] In some embodiments, the 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1. In some embodiments, the xrRNA sequence in the 3'UTR sequence of the ZIKA is shown in SEQ ID NO: 42. In some embodiments, the sequence of the optimized 3'UTR is shown in SEQ ID NO: 39.

[0033] Optimized 3'UTR-33

[0034] In some embodiments, the optimized 3'UTR comprises, from the 5' end to the 3' end, the xrRNA1 and xrRNA2 sequences of the ZIKA 3'UTR sequence and the 3'UTR sequence of the native hHBB.

[0035] In some embodiments, the 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1. In some embodiments, the xrRNA1 and xrRNA2 sequences in the 3'UTR sequence of the ZIKA are shown in SEQ ID NO: 45. In some embodiments, the sequence of the optimized 3'UTR is shown in SEQ ID NO: 40.

[0036] In some implementations, the optimized 3'UTR inserts and / or deletes at least a portion of the sequence in the stem-loop structure and / or pseudoknot structure of the 3'UTR sequence of natural hHBB and / or the 3'UTR sequence of natural SARS-CoV-2 to disrupt or modify the stem-loop structure and / or pseudoknot structure.

[0037] In some embodiments, the optimized 3'UTR inserts and / or deletes at least a portion of the sequence in the stem-loop structure and / or pseudo-knot structure of the 3'UTR sequence of native hHBB and the 3'UTR sequence of native SARS-CoV-2.

[0038] In some implementations, the optimized 3'UTR inserts and / or deletes at least a portion of the sequence in the stem-loop structure and / or pseudo-knot structure of the natural SARS-CoV2 3'UTR sequence.

[0039] In some implementations, the optimized 3'UTR inserts and / or deletes at least a portion of the sequence in the stem-loop structure of the natural hHBB 3'UTR sequence.

[0040] Optimized 3'UTR-22

[0041] In some embodiments, the sequence shown in SEQ ID NO:43 is inserted into the stem-loop structure of the 3'UTR sequence of the natural human β-globin gene (hHBB). In some embodiments, a portion of the sequence is also deleted from the stem-loop structure of the 3'UTR sequence of the natural human β-globin gene (hHBB).

[0042] In some implementations, the optimized 3'UTR sequence is as shown in SEQ ID NO: 29.

[0043] Optimized 3'UTR-23

[0044] In some embodiments, the sequence shown in SEQ ID NO: 44 is inserted into the stem-loop structure of the 3'UTR sequence of the natural human β-globin gene (hHBB). In some embodiments, a portion of the sequence is also deleted from the stem-loop structure of the 3'UTR sequence of the natural human β-globin gene (hHBB).

[0045] In some implementations, the optimized 3'UTR sequence is as shown in SEQ ID NO: 30.

[0046] As used herein, when referring to "natural hHBB 3'UTR sequence," the sequence shown in SEQ ID NO: 1 is used for description. However, those skilled in the art will understand that there are multiple versions of natural hHBB 3'UTR sequence that have substantially the same biological function, but may still have minor sequence differences from one another. Therefore, in this application, natural hHBB 3'UTR sequence is not limited to the sequence shown in SEQ ID NO: 1, but is intended to cover all known sequences. Therefore, in this application, the term "natural hHBB 3'UTR sequence" should include various naturally occurring, biologically functional hHBB 3'UTR sequences, including, for example, the hHBB 3'UTR sequence shown in SEQ ID NO: 1 and its naturally occurring variants or truncated versions.

[0047] In some embodiments, the 3'UTR sequence of the natural hHBB is as shown in SEQ ID NO: 1.

[0048] As used herein, when referring to “natural SARS-CoV-2 3'UTR sequence,” the sequence shown in SEQ ID NO: 2 is used for description. However, those skilled in the art will understand that there are multiple versions of the natural SARS-CoV-2 3'UTR sequence that have substantially the same biological function, but may still have minor sequence differences from one another. Therefore, in this application, the natural SARS-CoV-2 3'UTR sequence is not limited to the sequence shown in SEQ ID NO: 2, but is intended to cover all known sequences. Therefore, in this application, the term “natural SARS-CoV-2 3'UTR sequence” should include various naturally occurring, biologically functional SARS-CoV-2 3'UTR sequences, including, for example, the hHBB 3'UTR sequence shown in SEQ ID NO: 2 and its naturally occurring variants or truncated versions.

[0049] In some implementations, the 3'UTR sequence of the natural SARS-CoV-2 is shown in SEQ ID NO: 2.

[0050] In some embodiments, the optimized 3'UTR sequence is as shown in any one of SEQ ID NO: 8-40.

[0051] Nucleic acid constructs or mRNA molecules

[0052] In a second aspect, this application provides a nucleic acid construct comprising the optimized 3'UTR described in the first aspect. In some embodiments, it further comprises one or more of the following elements: (a) a 5' cap; (b) a 5' untranslated region (5'UTR); (c) an open reading frame (ORF); and (d) a poly-A tail.

[0053] In a second aspect, this application provides an mRNA molecule comprising the optimized 3'UTR described in the first aspect. In some embodiments, it further comprises one or more of the following elements: (a) a 5' cap; (b) a 5' untranslated region (5'UTR); (c) an open reading frame (ORF); and (d) a poly-A tail.

[0054] The nucleic acid constructs or mRNAs described in this application are not limited to a specific production method. For example, they can be produced via cell-free in vitro transcription. There are generally two ways to add a 5' cap: one is to obtain mRNA through in vitro transcription and then further process it with a capping enzyme; the other is to directly obtain capped mRNA by adding a trinucleotide capping analog to the in vitro transcription system. The latter is called co-transcriptional capping, and its advantages are fewer production steps and a single product, making it the mainstream trend in mRNA drug production. However, using a capping analog requires fixing the 5'UTR start sequence to AGG, which may alter the 5'UTR structure and affect translation efficiency. In particular, this invention optimizes the 5'UTR sequence used in the co-transcriptional capping method, improving the expression level of the protein encoded by the mRNA.

[0055] Currently, the 5'UTR sequences used in mRNA drugs typically come from two sources: one is the 5'UTR of a natural gene and its derived sequences (such as Pfizer-BioNTech's COVID-19 vaccine BNT162b2), and the other is obtained by screening from artificially designed and synthesized sequence libraries (represented by Moderna's COVID-19 vaccine mRNA-1273). The source of the 5'UTR in this invention is not limited. Generally, it can be derived from the 5'UTR of any natural gene and its derived sequences, and it can originate from animals, plants, fungi, bacteria, or viruses. Generally, it can originate from any species, such as humans, baboons, monkeys, mice, chickens, zebrafish, Arabidopsis thaliana, yeast, Escherichia coli, influenza virus, dengue virus, coronavirus, etc. Generally, it can 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 5'UTR sequence generated by an algorithm (PJ Sample et al., NatBiotechnol 2019 DOI: 10.1038 / s41587-019-0164-5; S. Castillo-Hair et al., Nature Communications 2024 DOI: 10.1038 / s41467-024-49508-2).

[0056] In some embodiments, the 5'UTR is selected from the β-globin gene (HBB), ribosomal protein genes (such as RpL38, RpS25), actin genes (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etching virus (TEV), a disordered short 5'-UTR (scrUTR), or the 5'UTR of complement factor 3 (C3).

[0057] In some embodiments, the 5'UTR and 3'UTR in the nucleic acid construct / RNA molecule of this disclosure are of the same or different origins, for example, derived from the same or different genes. In some embodiments, the 5'UTR and 3'UTR in the nucleic acid construct / RNA molecule of this disclosure are derived from the same or different species. In some embodiments, the 5'UTR and the optimized 3'UTR are derived from the same gene.

[0058] In some embodiments, the nucleic acid construct includes an optimized 5'UTR as described in the text and a 3'UTR as described above, wherein the optimized 5'UTR and the 3'UTR are derived from the same gene, for example, from the human β-globin gene (hHBB). In some embodiments, the optimized 5'UTR is derived from the 5'UTR of natural hHBB, the sequence of which is shown in SEQ ID NO: 3.

[0059] In some embodiments, the 3' UTR in the nucleic acid construct / RNA molecule of this disclosure is located downstream of the open reading frame. In some embodiments, the 3' UTR in the nucleic acid construct is located at the 3' end of the open reading frame. In some embodiments, the nucleic acid construct / RNA molecule of this disclosure further includes a 5' untranslated region element (5' UTR) as described above. In some embodiments, the 5' UTR in the nucleic acid construct is located upstream of the open reading frame. In some embodiments, the 5' UTR in the nucleic acid construct is located at the 5' end of the open reading frame.

[0060] In some embodiments, the nucleic acid construct / RNA molecule of this disclosure further includes: (d) a poly-A tail.

[0061] In some embodiments, the poly-A tail in the nucleic acid construct is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the nucleic acid construct. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, or 500 nucleotides long.

[0062] In some embodiments, the poly-A tail includes, but is not limited to, HGH polyA, SV40polyA, BGHpolyA, rbGlob polyA, or SV40late polyA.

[0063] In some embodiments, in any of the above-described nucleic acid constructs, the ORF comprises a nucleotide sequence encoding at least one polypeptide or protein. In some embodiments, the nucleotide sequence may be a codon-optimized nucleotide sequence. In some embodiments, the ORF is a polynucleotide sequence encoding a target gene protein.

[0064] In some embodiments, the ORF encodes a fluorescent protein, luciferase, protein drug (e.g., antibody), or antigen.

[0065] In some implementations, the polypeptide or protein encoded by the ORF is a viral antigen. Exemplarily, viral antigens include, but are not limited to, antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or EB virus.

[0066] In some embodiments, the viral antigen is a coronavirus antigen. In some embodiments, the coronavirus is a human-infecting coronavirus, such as SARS-CoV-2 (COVID-19), SARS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, or MERS-CoV. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus antigen is a structural protein. In some embodiments, the structural protein is selected from spike protein (S protein or Spike protein), envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein). In some embodiments, the structural protein is a spike protein. In some embodiments, the spike protein is the SARS-CoV-2 spike protein. In some implementations, the SARS-COV-2 spike protein is selected from the spike protein of any of the following viral strains: SARS-COV-2 (e.g., wild-type SARS-COV-2), SARS-COV-2Alpha (B.1.1.7), SARS-COV-2Beta (B.1.351), SARS-COV-2Gamma (P.1), SARS-COV-2Kappa (B.1.617.1), SARS-COV-2Delta (B.1.617.2), SARS-COV-2Omicron (B.1.1.529), and SARS-COV-2Omicron (BA4).

[0067] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A or influenza B viruses; exemplary examples include influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H5N8, influenza A virus H2N2, influenza A virus H7N9, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., influenza B virus / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of the influenza virus, such as hemagglutinin (HA), neuraminidase (NA), ion channel protein M2, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is the HA protein of an influenza virus, such as the HA protein of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific embodiments, the influenza virus antigen is the NA protein of an influenza virus, such as the NA protein of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific implementations, the influenza virus antigen is the NP protein of an influenza virus, such as the NP protein of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), and influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).

[0068] In a preferred embodiment, the antigen of the respiratory syncytial virus is the pre-fusion conformation F protein (pre-F).

[0069] In a preferred embodiment, the antigen of the human metapneumovirus (hMPV) is the F protein.

[0070] In a preferred embodiment, the antigen of the chicken infectious bronchitis virus is the S1 region or the RBD domain of the Spike protein.

[0071] In a preferred embodiment, the antigens of the Nipah virus are the F protein and the G protein.

[0072] In a preferred embodiment, the antigen of the monkeypox virus is the L5L protein.

[0073] In some embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase.

[0074] In some specific embodiments, the nucleotide sequence of the ORF encoding the polypeptide or protein is selected from the sequence shown in SEQ ID NO:4 or SEQ ID NO:7, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. In some embodiments, these sequences have the same or similar activity compared to the sequences from which they are derived.

[0075] In some embodiments, the polypeptide or protein encoded by the ORF is a protein drug or antigen.

[0076] In some specific embodiments, the nucleotide sequence of the ORF encoding the polypeptide or protein is selected from the sequence shown in SEQ ID NO:5 or SEQ ID NO:6, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. In some embodiments, these sequences have the same or similar activity compared to the sequences from which they are derived.

[0077] In some embodiments, the ORF is a polynucleotide sequence encoding the target gene protein. In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous.

[0078] In some embodiments, the antigen is derived from an infectious disease, an autoimmune disease, and / or an allergic disease. Alternatively, the antigen may be an antigen, epitope, protein, or peptide derived from a pathogen or tumor.

[0079] In some implementations, the tumor is a solid tumor.

[0080] In some embodiments, the tumor is selected from: liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer and bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof.

[0081] In some embodiments, the tumor-derived antigen is selected from NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or combinations thereof.

[0082] In some embodiments, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof.

[0083] In some embodiments, the antigen of the virus is selected from the antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus or EB virus.

[0084] In some embodiments, the coronavirus is SARS-CoV-2, and the coronavirus antigen is the complete spike protein or its RBD domain. In still some embodiments, the spike protein is selected from the spike protein of any of the following viral strains: SARS-CoV-2, SARS-CoV-2 Alpha, SARS-CoV-2 Beta, SARS-CoV-2 Gamma, SARS-CoV-2 Kappa, SARS-CoV-2 Delta, or SARS-CoV-2 Omicron.

[0085] In some embodiments, the influenza virus is an influenza A virus or an influenza B virus, and the antigen is a hemagglutinin protein (HA), neuraminidase (NA), and / or nucleoprotein (NP). In still other embodiments, the influenza virus antigen is selected from the hemagglutinin protein, neuraminidase, and / or nucleoprotein of any of the following strains: influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria, and influenza B virus Yamagata.

[0086] In some embodiments, the respiratory syncytial virus (RSV) antigen is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the RSV antigen is selected from the F protein, G protein, nucleocapsid protein, or matrix protein of subtype A strains or subtype B strains. In some embodiments, the RSV antigen comprises a pre-fusion conformation F protein (pre-F), a post-fusion conformation F protein (post-F), or a mixture of both. In some embodiments, the RSV antigen is the pre-fusion conformation F protein (pre-F).

[0087] In some embodiments, the antigen of the human metapneumovirus (hMPV) is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the antigen is the F protein of human metapneumovirus (hMPV).

[0088] In some embodiments, the antigen of the infectious bronchitis virus (IBV) is a Spike protein, an M protein, a nucleocapsid protein, or an envelope protein. In some embodiments, the antigen of the IBV is the S1 region or the RBD domain of the Spike protein.

[0089] In some embodiments, the antigen of the Nipah virus is an F protein, a G protein, a nucleocapsid protein, or a matrix protein. In some embodiments, the antigen of the Nipah virus is either an F protein or a G protein.

[0090] In some embodiments, the antigens of the monkeypox virus are selected from inner membrane proteins M1R, H3L, E8L, and A29L, outer envelope proteins A35R and B6R, and accessory protein L5L. In some embodiments, the antigens of the monkeypox virus are A29L, M1R, B6R, and / or A35R.

[0091] In some embodiments, the fungal antigen is selected from antigens of the genera *Candida*, *Aspergillus*, *Mucor*, *Rhizopus*, *Epidermophyton*, *Malassezia*, *Preumocystis*, *Penicillium*, *Alternaria*, *Cladosporium*, *Botrytis*, *Aureobasidium*, *Fusarium*, or *Trichoderma*.

[0092] In some embodiments, the bacterial antigen is selected from antigens of actinomycetes, Bacillus, Bacteroides, Enterococcus, Listeria, Mycobacterium, Streptococcus pneumoniae, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, or Streptococcus.

[0093] In some embodiments, the antibody is an antibody capable of specifically binding to any one or more of the antigens.

[0094] In some implementations, any one or more antigens are as defined above.

[0095] In some implementations, the antibody is an autoantibody.

[0096] In some embodiments, the antibody is an antibody that specifically binds to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and / or LAG3.

[0097] In some embodiments, the nucleic acid construct comprises, from 5' to 3', a 5' UTR, an ORF, an optimized 3' UTR as described herein, and a poly-A tail, in sequence. In some embodiments, the 5' UTR comprises or is a nucleotide sequence as shown in SEQ ID NO: 3, the ORF comprises or is a nucleotide sequence as shown in any of SEQ ID NO: 4-7, and the 3' UTR comprises or is a nucleotide sequence as shown in any of SEQ ID NO: 8-40.

[0098] In some embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding an additional protein or polypeptide. In some embodiments, the additional protein or polypeptide is selected from: signal peptides, multimerizing domains (e.g., dimerizing domains, trimerizing domains), tags, or any combination thereof.

[0099] carrier

[0100] In a second aspect, this application also provides a vector comprising the nucleic acid construct or mRNA molecule described in the first aspect.

[0101] In some embodiments, the vector may be, for example, a plasmid, a sticky-terminal plasmid, a YAC, or a viral vector. The vector may be an expression vector, i.e., a vector that provides a nucleic acid construct or an RNA-encoding polypeptide expression vector. The expression vector typically contains at least one nucleic acid of this disclosure, operably linked to one or more suitable expression regulatory elements (e.g., promoters, terminators, etc.). Selecting these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art.

[0102] The vector of this application may contain a variety of elements, including but not limited to one or more of the following: origin of replication; optional marker gene; one or more expression control elements, such as transcription control elements (e.g., promoter, enhancer, terminator) and / or one or more translation signals; and signal sequences or leader sequences for targeting the secretion pathway in selected host cells.

[0103] host cells

[0104] In a third aspect, this application also provides a host cell comprising the vector described in the second aspect.

[0105] In some embodiments, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells.

[0106] In some embodiments, the eukaryotic cells are mammalian cells, such as mouse cells or human cells.

[0107] Host cells include, for example, insect cells, mammalian cells, avian cells, bacterial cells, and yeast cells. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, and Schneider S2 cells. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Avian cells include, for example, chicken embryonic stem cells, chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).

[0108] Preparation method

[0109] In a fourth aspect, this application provides a method for preparing the mRNA molecule as described above, comprising: synthesizing a gene encoding an ORF protein and a template DNA sequence containing mRNA transcription-related elements; preparing the template by primer PCR amplification; and obtaining mRNA by transcription using a co-transcription capping method.

[0110] In a fourth aspect, this application provides a method for preparing the nucleic acid constructs as described above, comprising: culturing the aforementioned host cells and recovering the resulting nucleic acid constructs from the culture.

[0111] The nucleic acid constructs or mRNAs disclosed herein can also be obtained by other production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis. In some embodiments, the method for preparing mRNA molecules includes: preparing the nucleic acid construct or vector as described above, and then performing reverse transcription using the nucleic acid construct or vector to obtain mRNA molecules. In some specific embodiments, the method further includes adding a 5' cap to the 5' end of the RNA molecule.

[0112] vaccine

[0113] In a fifth aspect, this application provides a vaccine comprising a nucleic acid construct or mRNA molecule as described above.

[0114] In some embodiments, the nucleic acid construct or mRNA molecule is encapsulated in lipid nanoparticles (LNPs). In some embodiments, the vaccine is expressed after administration to a patient and uptake by cells. Expression of the administered nucleic acid leads to the production of the encoded protein. When these proteins are recognized as foreign by the patient's immune system, an immune response is triggered.

[0115] In some implementations, the vaccine also contains adjuvants and / or buffer solutions.

[0116] In some embodiments, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles, or any combination thereof.

[0117] In some embodiments, the vaccine is administered via a method selected from intravenous injection, transmucosal delivery, sublingual, nasal, intrathecal, bronchial, rectal, percutaneous, or inhalation.

[0118] In some embodiments, the vaccine is applied to the mucosal surfaces of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa). In some embodiments, the vaccine induces an immune response in the mucosa (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa).

[0119] In some implementations, the vaccine is delivered via nasal or inhalation.

[0120] In some embodiments, the vaccine of the present invention is administered via mucosal delivery. Specific methods of administration can be found, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995, regarding mucosal delivery techniques, including intranasal mucosal delivery.

[0121] In some embodiments, the vaccine of the present invention is administered via the dermis or transdermal route. For specific administration methods, see, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995.

[0122] In some embodiments, the vaccine is a monovalent vaccine that contains an antigen encoding an organism. In some embodiments, the monovalent vaccine contains an antigen encoding a viral strain.

[0123] In some embodiments, the vaccine is a multivalent / combination vaccine. In some embodiments, the vaccine may comprise nucleic acid constructs, RNA or polynucleotide molecules, or multiple nucleic acid constructs, RNA or polynucleotide molecules encoding two or more antigens of the same or different species. In some embodiments, the vaccine comprises RNA or multiple RNAs encoding two or more antigens of the same or different viral strains. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more viral antigens.

[0124] Pharmaceutical Composition

[0125] In a sixth aspect, this application provides a pharmaceutical composition comprising: a nucleic acid construct or mRNA molecule as described above, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0126] In some preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from pH adjusters (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic, or nonionic surfactants such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0127] In some preferred embodiments, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including petroleum-derived, animal-, plant-derived, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0128] In some preferred embodiments, the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0129] In some preferred embodiments, the pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder (e.g., lyophilized powder), sustained-release formulation, etc.

[0130] The pharmaceutical compositions of the present invention can be administered by various suitable methods. Suitable methods of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In some preferred embodiments, the pharmaceutical compositions are formulated into pharmaceutical preparations suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans according to conventional procedures.

[0131] Typically, pharmaceutical compositions intended for injection (e.g., intravenous administration, such as by bolus or continuous infusion) are sterile and isotonic. If desired, such pharmaceutical compositions may also contain solubilizers and local anesthetics such as ergotamine to reduce pain at the injection site. Furthermore, pharmaceutical compositions intended for injection may contain preservatives. In some preferred embodiments, pharmaceutical compositions intended for injection may also be available in unit dose form (e.g., stored in ampoules or in multi-dose containers).

[0132] In some embodiments, the pharmaceutical compositions of the present invention may contain additional active ingredients, such as additional vaccines, antiviral agents, and / or monoclonal antibodies.

[0133] Reagent test kit

[0134] In a seventh aspect, this application provides a kit comprising the nucleic acid construct or mRNA molecule as described above, the vaccine as described above, and / or the pharmaceutical composition as described above.

[0135] Uses and methods

[0136] On the other hand, this application provides the use of the nucleic acid constructs or mRNA molecules as described above, the vaccines as described above, the pharmaceutical compositions as described above, or the kits as described above, in the preparation of a medicament for treating and / or preventing a subject from having a disease and / or symptoms caused by infection with a pathogen (e.g., a virus).

[0137] On the other hand, this application provides a method for treating and / or preventing a disease, comprising administering to a subject in need a therapeutic and / or preventive amount of the aforementioned nucleic acid construct or mRNA molecule, the aforementioned vaccine, the aforementioned pharmaceutical composition, or the aforementioned kit.

[0138] In some embodiments, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof.

[0139] In some embodiments, the virus is an influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, monkeypox virus, and more preferably SARS-CoV-2, influenza A virus, or influenza B virus.

[0140] In some implementations, the disease and / or symptoms include uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, and severe acute respiratory syndrome (SARS).

[0141] In some implementations, the subject is a mammal, such as a mouse or a human.

[0142] On the other hand, this application provides the use of the nucleic acid constructs or mRNA molecules as described above, the vaccines as described above, the pharmaceutical compositions as described above, or the kits as described above, in the preparation of a medicament for inducing a subject to produce a neutralizing antibody response and / or a T-cell immune response.

[0143] On the other hand, this application provides methods for inducing a subject to produce a neutralizing antibody response and / or a T-cell immune response, including administering to a subject in need an effective amount of the nucleic acid construct or mRNA molecule as described above, the vaccine as described above, the pharmaceutical composition as described above, or the kit as described above.

[0144] In some embodiments, the neutralizing antibody response is a neutralizing antibody response against a viral antigen, and the T-cell immune response includes CD4+ and / or CD8+ T-cell immune responses.

[0145] In some implementations, the virus is an influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, or monkeypox virus.

[0146] In some implementations, the subject is a mammal, such as a mouse or a human.

[0147] On the other hand, this application provides the use of the optimized 5'UTR as described above in any one or more of the following: (i) preparing nucleic acid constructs or mRNA molecules; (ii) increasing the expression level of proteins encoded by nucleic acid constructs or mRNA molecules containing thereof; or (iii) improving the immunogenicity of vaccines, pharmaceutical compositions, or kits containing said nucleic acid constructs or mRNA molecules.

[0148] Terminology Definition

[0149] As used herein, the term "untranslated region" or "UTR" refers to the mRNA portions upstream of the start codon and downstream of the stop codon that are not translated and are therefore referred to as the 5' untranslated region (5''UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed using coding regions and are therefore exons when present in mature mRNA.

[0150] As used herein, the term “3' untranslated region (3'-UTR)” refers to the portion of messenger RNA (mRNA) immediately following a translation stop codon. mRNA molecules are transcribed from a DNA sequence and then translated into a protein. Several regions of the mRNA molecule are not translated into protein, including the 5' cap, 5' UTR, 3' UTR, and polyadenylated tail.

[0151] In some implementations, the 3'UTR may contain regulatory regions within the 3'-untranslated region that are known to affect polyadenylation and stability of mRNA. Many 3'-UTRs also contain AU-rich elements (AREs). Furthermore, the 3'-UTR contains the sequence AAUAAA, which directs the addition of hundreds of adenine residues, known as poly(A) tails, to the ends of the mRNA transcript.

[0152] As used herein, the term "5' untranslated region (5'UTR)" (also known as the leader sequence or leader RNA) is the region of mRNA located directly upstream of the start codon. The 5'UTR begins at the transcription start site and terminates one nucleotide (nt) before the start codon (usually AUG) of the coding region. In prokaryotes, the 5'UTR is typically 3–10 nucleotides long, while in eukaryotes it is often much longer, generally ranging from 100 to several thousand nucleotides, although shorter UTRs are sometimes observed in eukaryotes.

[0153] As used herein, the term "xrRNA" refers to a class of RNA functional elements with a specific nucleotide sequence located in the 3' untranslated region (3'UTR) of viral genomic RNA. The characteristic feature is that this sequence can fold into a unique and stable three-dimensional topological structure through intramolecular base pairing and interactions. This structure effectively resists degradation by exonucleases acting in the 5'→3' direction of the host cell, thereby producing and stably truncating subgenomic noncoding RNA products in its 3' direction.

[0154] As used herein, the term "stem-loop" or hairpin structure is a basic and ubiquitous secondary structural unit in RNA molecules. It consists of a double-stranded "stem" region of at least 4-6 consecutive nucleotides formed by complementary base pairing (typically Watson-Crick pairing), and a single-stranded "loop" region of 3 or more unpaired nucleotides. The stability of the stem region is primarily determined by the number and type of base pairs. In the art, based on existing technology, those skilled in the art can use conventional computer software (e.g., RNAfold, mfold, etc., using energy minimization or co-sequence evolution analysis algorithms; see Zuker M. "Mfold web server for nucleic acidfolding and hybridization prediction." Nucleic Acids Res. 2003) to perform free energy calculations and structural predictions on a given RNA sequence (e.g., the human β-globin gene or the 3'UTR sequence of SARS-CoV-2), thereby definitively identifying all potential stem-loop structures and their specific nucleotide composition and location.

[0155] As used herein, the term "pseudoknot structure" refers to a complex secondary structure in RNA molecules with specific topological features. Its core characteristic is that a single-stranded loop region of nucleotides pairs complementaryly with another single-stranded sequence in the same RNA molecule (usually located upstream or downstream of the loop region), forming a second stem region that "knots" together. This interrupts the previously continuous base pairing (in the first stem region), and the two stem regions are spatially stacked. According to existing technology, those skilled in the art can use specially designed bioinformatics tools (e.g., pknots and DotKnot using dynamic programming algorithms, or relevant modules in the Vienna RNA package; see Reuter JS & Mathews DH, "RNAstructure: software for RNA secondary structure prediction and analysis." BMC Bioinformatics. 2010) to analyze target sequences. These algorithms can effectively identify pseudoknot topologies defined by such nested base pairings, thereby determining the presence of pseudoknot structures in the 3'TR sequence of the human β-globin gene or SARS-CoV-2 and resolving their precise sequence boundaries.

[0156] As used herein, the term “nucleic acid” or “polynucleotide” refers to an organic molecule containing two or more covalently bonded nucleotides. As used herein, “nucleotide” refers to an organic molecule containing: 1) a nucleoside, which contains a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically linked by a phosphodiester bond, where the 3' carbon of the sugar of the first nucleotide is linked to the 5' carbon of the sugar of the second nucleic acid via a bridging phosphate group. Typically, the bridging phosphate ester contains two non-bridging oxygen atoms that are bonded only to the phosphorus atom of the phosphate ester, and two bridging oxygen atoms, each linking the phosphorus atom to the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the nucleotide sequence in a nucleic acid, if the 3' carbon of the first nucleotide is linked to the 5' carbon of the second nucleotide, the first nucleotide is said to be 5' (upstream) of the second nucleotide. Similarly, if the 5' carbon of the second nucleotide is linked to the 3' carbon of the first nucleotide, the second nucleotide is said to be 3' (downstream) of the first nucleotide. Nucleic acid sequences are usually read in 5'->3' order, starting with a 5' nucleotide and ending with a 3' nucleotide.

[0157] As used herein, the term “messenger RNA” (“mRNA”) refers to a nucleic acid containing an open reading frame that encodes a protein. mRNA has a modular structure that includes a 5' cap, a 5' untranslated region (5'UTR), an open reading frame (ORF) that encodes the protein, a 3' untranslated region (3'UTR), and a polyadenylated nucleotide (polyA) tail.

[0158] As used herein, the term "protein-coding open reading frame" refers to a nucleic acid sequence containing a coding sequence that, when translated, results in the production of a protein. Open reading frames typically begin with a start codon, such as AUG in RNA sequences (ATG in DNA sequences), and end with a stop codon, such as UAG, UAA, or UGA in RNA sequences (TAG, TAA, or TGA in DNA sequences).

[0159] DNA or RNA sequences encode genes using codons. A codon is a set of three nucleotides within a nucleic acid (such as DNA or RNA) sequence. An anticodon is a set of three nucleotides within a nucleic acid, such as transfer RNA (tRNA), that is complementary to the codon, allowing the codon of the first nucleic acid to associate with the anticodon of the second nucleic acid via hydrogen bonds between the codon and anticodon bases. For example, the codon 5'-AUG-3' on mRNA has a corresponding anticodon 3'-UAC-5' on tRNA. During translation, tRNA with an anticodon complementary to the codon to be translated associates with the codon on mRNA, typically to deliver the amino acid corresponding to the codon to be translated, or to promote translation termination and the release of the translated polypeptide from the ribosome.

[0160] As used herein, the term "lipid nanoparticles (LNPs)" refers to nanoscale particles composed of lipid molecules. They typically have a particle size range of 20-200 nanometers and contain components such as cationic lipids, phospholipids, and PEGylated lipids. Lipid nanoparticles are used to encapsulate and deliver biomolecules (such as mRNA and DNA) and effectively protect these macromolecules from degradation by enzymes in vivo, promoting their entry into target cells. The lipid nanoparticles comprise cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol-lipids. In some embodiments, the cationic lipids are DLin-MC3-DMA or SM102; the polyethylene glycol-lipids are DMG-PEG2000; the neutral phospholipids are DSPC; and the steroidal lipids are cholesterol.

[0161] Beneficial effects

[0162] The optimized 3'UTR provided in this application can increase the expression level of proteins encoded by mRNA (e.g., antigens, protein drugs (e.g., antibodies)). Therefore, mRNA vaccines containing the optimized 3'UTR provided in this application exhibit good immune response levels (e.g., capable of inducing high titers of neutralizing antibodies, capable of inducing specific T-cell immune responses), especially at low doses (e.g., 0.1 μg). Thus, this vaccine can effectively prevent and / or treat diseases and / or symptoms caused by pathogen (e.g., virus) infection.

[0163] Therefore, the optimized 5'UTR and the mRNA molecule containing it provided in this application have broad application prospects in the fields of vaccines, gene therapy, and antibody therapy. Attached Figure Description

[0164] Figure 1 The novel 3'UTR was shown to promote the expression of the eGFP reporter gene in BHK-21 cells.

[0165] Figure 2 The novel 3'UTR was shown to increase the level of HA antigen of influenza viruses H1N1 and H5N1 expressed on mRNA.

[0166] Figure 3 The novel 3'UTR was shown to enhance the expression level of the reporter gene Luciferase in mice during both the acute and late expression phases following immunization.

[0167] Figure 4 The results show the IgG antibody titers detected in BALB / c mice immunized with the influenza virus PR8(H1N1) mRNA-HA vaccine.

[0168] Figure 5 The results show the IgG antibody titers detected in BALB / c mice immunized with the Texas (H5N1) mRNA-HA vaccine.

[0169] Sequence information

[0170] The descriptions of the sequences involved in this application are provided in the table below.

[0171] Detailed Implementation

[0172] The invention will now be described in the following non-limiting embodiments.

[0173] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0174] Example 1. Characterization of mRNA 3'UTR sequence

[0175] This embodiment is based on the 3'UTR sequence (SEQ ID NO: 1) of the natural human β-globin gene (hHBB) and the 3'UTR sequence (SEQ ID NO: 2) of the natural SARS-CoV-2, and innovatively designs to introduce the advantageous features of the viral 3'UTR to improve the stability and translation efficiency of mRNA.

[0176] A total of 33 optimized 3'UTR sequences were obtained, which were named Optimized 3'UTR-1 to Optimized 3'UTR-33, as shown in SEQ ID NO: 8 to SEQ ID NO: 40. These optimized 3'UTR sequences incorporate two types of modification approaches.

[0177] 1. Tandem with other xrRNA sequences

[0178] Specifically, the optimized 3'UTR-29 to optimized 3'UTR-33 are based on the natural hHBB 3'UTR sequence (SEQ ID NO: 1). This sequence is then tandemly linked with the xrRNA sequence from the 3'UTR sequence of another virus. The optimized 3'UTR-29 (SEQ ID NO: 36) is formed by linking a Tamanu bat virus (TABV) xrRNA sequence (SEQ ID NO: 41) to the 3' end of the natural hHBB 3'UTR sequence (SEQ ID NO: 1).

[0179] The optimized 3'UTR-30 (SEQ ID NO: 37) is a Zika virus (ZIKA) xrRNA sequence (SEQ ID NO: 42) linked to the 5' end of the natural hHBB 3'UTR sequence (SEQ ID NO: 1).

[0180] The optimized 3'UTR-31 (SEQ ID NO: 38) is formed by linking two Zika virus (ZIKA) xrRNA sequences (SEQ ID NO: 42) to the 5' end of the natural hHBB 3'UTR sequence (SEQ ID NO: 1).

[0181] The optimized 3'UTR-32 (SEQ ID NO: 39) is a Zika virus (ZIKA) xrRNA sequence (SEQ ID NO: 42) linked to the 3' end of the natural hHBB 3'UTR sequence (SEQ ID NO: 1).

[0182] The optimized 3'UTR-33 (SEQ ID NO: 40) is formed by linking the _xrRNA1 and xrRNA2 sequences (SEQ ID NO: 45) of Zika virus (ZIKA) to the 5' end of the 3'UTR sequence (SEQ ID NO: 1) of natural hHBB.

[0183] II. Sequence for Modifying Stem-Loop and False-Nodal Structures

[0184] Specifically, the optimized 3'UTR-1 to optimized 3'UTR-28 are based on the natural SARS-CoV2 3'UTR sequence (SEQ ID NO: 2) and / or the natural hHBB 3'UTR sequence (SEQ ID NO: 1), with modifications made to the stem-loop structure and / or pseudo-knot structure. The optimized 3'UTR-1, optimized 3'UTR-8 to optimized 3'UTR-17 (SEQ ID NO: 8, SEQ ID NO: 15 to SEQ ID NO: 24) are obtained by deleting a portion of the stem-loop structure and / or pseudo-knot structure of the 3'UTR sequence (SEQ ID NO: 2) of the natural SARS-CoV2 and inserting a portion of the sequence.

[0185] The optimized 3'UTR-2 to optimized 3'UTR-7 (SEQ ID NO: 9 to SEQ ID NO: 14) are obtained by deleting part of the stem-loop structure and / or pseudo-knot structure of the 3'UTR sequence of natural SARS-CoV2 (SEQ ID NO: 2) and the 3'UTR sequence of hHBB (SEQ ID NO: 1) and inserting part of the sequence.

[0186] The optimized 3'UTR-18 to optimized 3'UTR-21 (SEQ ID NO: 25 to SEQ ID NO: 28) are derived by deleting a portion of the stem-loop structure sequence of the natural hHBB 3'UTR sequence (SEQ ID NO: 1).

[0187] The optimized 3'UTR-22 (SEQ ID NO: 29) is formed by deleting a portion of the stem-loop structure sequence of the natural hHBB 3'UTR sequence (SEQ ID NO: 1) and inserting a sequence (SEQ ID NO: 43).

[0188] The optimized 3'UTR-23 (SEQ ID NO: 30) is formed by deleting a portion of the stem-loop structure sequence of the natural hHBB 3'UTR sequence (SEQ ID NO: 1) and inserting a sequence (SEQ ID NO: 44).

[0189] The optimized 3'UTR-24 to optimized 3'UTR-28 (SEQ ID NO: 31 to SEQ ID NO: 35) are obtained by deleting a portion of the stem-loop structure sequence of the natural hHBB 3'UTR sequence (SEQ ID NO: 1) and inserting a portion of the sequence.

[0190] Example 2: Novel 3'UTR enhances translation efficiency of eGFP reporter genes

[0191] Using the existing human β-globin gene (hHBB) 5'UTR (SEQ ID NO: 3) and the aforementioned 33 optimized 3'UTR sequences or the natural hHBB 3'UTR sequences, a total of 34 combinations were combined and tested in vitro to assemble an eGFP mRNA construct (SEQ ID NO: 4). The in vitro transcription template was amplified by PCR.

[0192] mRNA was synthesized in vitro via co-transcriptional capping, and the IVT system was prepared according to the following system: Table 2 IVT reaction system (20 μL)

[0193] After adding the template, the mixture was incubated at 37 °C for 2 h. For each reaction, 1 μL of DNase I was added and incubated at 37 °C for 30 min to digest the DNA template. RNA was then precipitated using LiCl at -80 °C. The precipitated mRNA was collected by centrifugation and centrifuged at 14,000 rpm for 10 min at 4 °C until a white RNA precipitate appeared at the bottom of the centrifuge tube. The supernatant was discarded, and 1 mL of pre-chilled 70% ethanol was added. The RNA was vortexed and washed once. The precipitate was dried at room temperature until it became translucent. RNase-free H2O was added to fully dissolve the RNA, and the mRNA concentration was determined using NanoDrop One. The mRNA quality was assessed by 1% TBE agarose gel electrophoresis. The results showed that high-quality mRNA products were obtained.

[0194] The synthesized mRNA was transfected into the BHK-21 cell line using lipo8000, and the cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. One day before transfection, the cells were sputtered at 0.15 × 10⁶ cells / day. 6 Cells were seeded at a concentration of 100 μL / mL in 96-well plates. The mRNA transfection system consisted of 30 ng mRNA, 0.16 μL Lipo8000, and 5 μL opti-MEM per well. Transfected cells were cultured at 37 °C for 24 h for subsequent analysis.

[0195] After mRNA transfection and cell culture for 24-72 h, the fluorescence intensity of GFP in 96-well plates was scanned and quantified using a high-content cell imaging analysis system to analyze the effect of the optimized 3'UTR sequence on GFP expression. Results are as follows: Figure 1 As shown, all 33 optimized 3'UTR sequences enhanced GFP expression.

[0196] Example 3: Novel 3'UTR enhances the expression of HA antigen in A / Puerto Rico / 8 / 1934 (H1N1, PR8) and A / Texas / 37 / 2024 (H5N1, Texas) viruses.

[0197] Representative sequences were selected to validate enhanced expression of HA antigens from H1N1 and H5N1 influenza viruses. Template DNA sequences containing mRNA transcription-related elements were artificially synthesized, including the PR8-HA (SEQ ID NO:5) or Texas-HA antigen (SEQ ID NO:6) sequences. Template preparation was performed using primer PCR amplification.

[0198] mRNA was obtained by co-transcription and transfected into the BHK-21 cell line. Expression was verified by indirect fluorescent immunoassay. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde, washed, and then blocked and perforated with 1% BSA (diluted in PBS) containing 0.2% Triton X-100. Cells were then incubated with a specific antibody for 1 hour, washed, and incubated with AF488-conjugated secondary antibody for 1 hour. After incubation, cells were washed again and stored in PBS. High-content cell imaging analysis was used to quantify HA expression in the cells.

[0199] The results are as follows Figure 2 As shown, compared with the natural 3'UTR, the optimized 3'UTR-22, 3'UTR-23, 3'UTR-29, 3'UTR-30 and 3'UTR-32 all enhanced the expression of HA in both viruses.

[0200] Example 4: Novel 3'UTR enhances the expression of Luciferase reporter gene in mice

[0201] Representative sequences were selected to validate the expression of the Luciferase reporter gene in mice. A template DNA sequence containing mRNA transcription-related elements, including the Luciferase sequence (SEQ ID NO:7), was synthesized. Template preparation was performed using primer PCR amplification. mRNA encoding luciferase was obtained by co-transcriptionalization. The mRNA was encapsulated in LNP and injected into the right thigh muscle of BALB / c mice (5 μg / mouse). Luciferase expression was then monitored by in vivo imaging. Before imaging, each mouse was injected with 100 µL of a 30 mg / mL D-Luciferin substrate, followed by isoflurane anesthesia. Imaging was performed 20 minutes after substrate injection to ensure optimal substrate-luciferase reaction.

[0202] The results are as follows Figure 3 As shown, compared with the natural 3'UTR, both the optimized 3'UTR-22 and 3'UTR-23 significantly enhanced the expression of Luciferase in mice.

[0203] Example 5: Immunogenicity assay of influenza A / Puerto Rico / 8 / 1934 (H1N1, PR8) HA mRNA vaccine in mice

[0204] The mRNA from Example 3 (containing PR8-HA (SEQ ID NO:5)) was used in the preparation of the mRNA-LNP vaccine. SM102 (purchased from Zhejiang Shenzhou Pharmaceutical Co., Ltd.), DSPC (purchased from Nippon Seika Co., Ltd.), cholesterol (purchased from Nippon Seika Co., Ltd.), and DMG-PEG2000 (purchased from Beijing Jiankai Technology Co., Ltd.) were mixed in ethanol at a molar ratio of 50:10:38.5:1.5. Then, using a microfluidic mixer, the lipids and mRNA (dissolved in 50 mM sodium acetate, pH 4.0) were mixed at a volume ratio of 1:3, with a total flow rate of 12 mL / min and a flow rate ratio of 1:3. The mixture was dialyzed with PBS for 4 h to remove acetic acid and ethanol from the mRNA-LNP, and then dialyzed for 2 h to replace the PBS. The mRNA-LNP vaccine, dialyzed until neutral, was used for immunization.

[0205] Six-week-old female BALB / c mice were randomly divided into groups of five and immunized with mRNA vaccine at doses of 0.1, 1, and 5 μg / mouse. A second immunization was administered three weeks later. Blood samples were collected three weeks after the initial immunization and three weeks after the booster immunization for HA IgG antibody titer detection via ELISA. HA protein was used to coat ELISA plates and incubated overnight at 4°C. Blocking buffer (5% skim milk powder prepared with PBS) was added and incubated at 37°C for 1 h. After washing with PBST, serially diluted inactivated serum (3-fold) was added and incubated at 37°C for 1 h. After washing again, anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 h. Finally, chromogenic substrate was added for color development, and the reaction was terminated. The absorbance was read at 450 nm using an ELISA reader, and the vaccine-induced antibody titer was calculated.

[0206] The results are as follows Figure 4 As shown, the optimized mRNA vaccine based on 3'UTR-22 and 3'UTR-23 can induce a good level of immune response, especially at a low dose of 0.1 μg, it still produces specific binding antibodies.

[0207] Example 6: Immunogenicity assay of influenza A / Texas / 37 / 2024 (H5N1, Texas) HA mRNA vaccine in mice

[0208] The mRNA from Example 3 (containing Texas-HA (SEQ ID NO: 6)) was used in the preparation of the mRNA-LNP vaccine. The vaccine preparation method was the same as in Example 5. Six-week-old female BALB / c mice were randomly divided into groups of five and immunized with the mRNA vaccine at doses of 0.1, 1, and 5 μg / mouse. A second immunization was performed three weeks later. Blood samples were collected at three weeks after the initial immunization and three weeks after the booster immunization to detect HA IgG antibody titers by ELISA.

[0209] The results are as follows Figure 5 As shown, the optimized 3'UTR-29-based mRNA vaccine can induce a good level of immune response, especially at a low dose of 0.1 μg, it still produces specific binding antibodies.

[0210] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An optimized 3'UTR, (i) comprising the 3'UTR sequence of a natural human β-globin gene (hHBB) and / or the 3'UTR sequence of a natural SARS-CoV-2, and an xrRNA sequence or a portion thereof in the 3'UTR sequence of one or more other viruses; or, (ii) Insertion and / or deletion of at least a portion of the sequence in the stem-loop structure and / or pseudoknot structure of the 3'UTR sequence of the natural human β-globin gene (hHBB) and / or the 3'UTR sequence of the natural SARS-CoV-2.

2. The optimized 3'UTR as described in claim 1, wherein, The xrRNA sequence in the 3'UTR sequence of one or more other viruses is linked to the 5' end and / or 3' end of the 3'UTR sequence of the natural hHBB or SARS-CoV-2. Preferably, the optimized 3'UTR contains xrRNA sequences from one, two, or more additional viral 3'UTR sequences; Preferably, the additional virus is selected from Zika virus (ZIKA), Tamana bat virus (TABV), dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, hepatitis C virus, or any combination thereof; Preferably, the xrRNA sequence in the 3'UTR sequence of ZIKA is as shown in SEQ ID NO: 42; Preferably, the xrRNA sequence in the 3'UTR sequence of TABV is as shown in SEQ ID NO:

41.

3. The optimized 3'UTR as described in claim 1 or 2, wherein, The optimized 3'UTR includes, from the 5' end to the 3' end, the following: (1) The 3'UTR sequence of natural hHBB and the xrRNA sequence in the 3'UTR sequence of TABV; for example, the optimized 3'UTR sequence is shown in SEQ ID NO: 36; (2) The xrRNA sequence in the 3'UTR sequence of ZIKA and the 3'UTR sequence of native hHBB; for example, the optimized 3'UTR sequence is shown in SEQ ID NO: 37; (3) The xrRNA sequence in the 3'UTR sequence of ZIKA, the xrRNA sequence in the 3'UTR sequence of ZIKA, and the 3'UTR sequence of native hHBB; for example, the optimized 3'UTR sequence is shown in SEQ ID NO: 38; or, (4) The 3'UTR sequence of natural hHBB and the xrRNA sequence in the 3'UTR sequence of ZIKA; for example, the sequence of the optimized 3'UTR is shown in SEQ ID NO:

39.

4. The optimized 3'UTR as described in claim 1, wherein, Insert the sequence shown in SEQ ID NO:43 or SEQ ID NO:44 into the stem-loop structure of the 3'UTR sequence of the natural human β-globin gene (hHBB); for example, the optimized 3'UTR sequence is shown in SEQ ID NO:29 or SEQ ID NO:

30.

5. The optimized 3'UTR according to any one of claims 1-4, wherein, The 3'UTR sequence of the natural hHBB is shown in SEQ ID NO: 1; Preferably, the 3'UTR sequence of natural SARS-CoV-2 is as shown in SEQ ID NO: 2; Preferably, the optimized 3'UTR sequence is as shown in any one of SEQ ID NO: 8-40.

6. A nucleic acid construct or mRNA molecule comprising the optimized 3'UTR as described in any one of claims 1-5.

7. The nucleic acid construct or mRNA molecule of claim 6, further comprising one or more of the following elements: (a) a 5' cap; (b) a 5' untranslated region (5'UTR); (c) an open reading frame (ORF); and (d) a poly-A tail. Preferably, the 5'UTR is selected from β-globin gene (HBB), ribosomal protein gene (such as RpL38, RpS25), actin gene (such as hActB, mActB), SARS-CoV2, tobacco mosaic virus (TMV), tobacco etching virus (TEV), disordered short 5'-UTR (scrUTR), or 5'UTR of complement factor 3 (C3); Preferably, the 5'UTR and the optimized 3'UTR originate from the same gene; Preferably, the optimized 5'UTR is derived from the 5'UTR of natural hHBB, and its sequence is shown in SEQ ID NO:

3.

8. The nucleic acid construct or mRNA molecule according to claim 6 or 7, wherein, The ORF encodes a fluorescent protein, luciferase, protein drug (e.g., antibody), or antigen.

9. The nucleic acid construct or mRNA molecule according to claim 8, wherein, The antigen is derived from infectious diseases, autoimmune diseases, and / or allergic diseases; or, the antigen is an antigen, epitope, protein, or peptide derived from pathogens or tumors. Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from: liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer, bile duct cancer, bladder cancer, pancreatic cancer, or any combination thereof; Preferably, the tumor-derived antigen is selected from NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or combinations thereof; Preferably, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof; Preferably, the antigen of the virus is selected from the antigens of influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, chicken infectious bronchitis virus, Nipah virus, monkeypox virus, human immunodeficiency virus, herpes simplex virus, rabies virus or EB virus. Preferably, the coronavirus is SARS-COV-2, and the coronavirus antigen is the complete spike protein or its RBD domain; more preferably, the spike protein is selected from the spike protein of any of the following viral strains: SARS-COV-2, SARS-COV-2 Alpha, SARS-COV-2 Beta, SARS-COV-2 Gamma, SARS-COV-2 Kappa, SARS-COV-2 Delta, or SARS-COV-2 Omicron. Preferably, the influenza virus is an influenza A virus or an 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 strain of influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza B virus Victoria, or influenza B virus Yamagata. Preferably, the respiratory syncytial virus antigen is an F protein, a G protein, a nucleocapsid protein, or a matrix protein; preferably, the respiratory syncytial virus antigen is selected from the F protein, G protein, nucleocapsid protein, or matrix protein of subtype A strains or subtype B strains; preferably, the respiratory syncytial virus antigen comprises a pre-fusion conformation F protein (pre-F), a post-fusion conformation F protein (post-F), or a mixture of both; preferably, the respiratory syncytial virus antigen is the pre-fusion conformation F protein (pre-F). Preferably, the antigen of the human metapneumovirus (hMPV) is an F protein, a G protein, a nucleocapsid protein, or a matrix protein; preferably, the antigen is the F protein of human metapneumovirus (hMPV). Preferably, the antigen of the chicken infectious bronchitis virus is a Spike protein, an M protein, a nucleocapsid protein, or an envelope protein; preferably, the antigen of the chicken infectious bronchitis virus is the S1 region or the RBD domain of the Spike protein. Preferably, the antigen of the Nipah virus is an F protein, a G protein, a nucleocapsid protein, or a matrix protein; preferably, the antigen of the Nipah virus is an F protein or a G protein. Preferably, the antigen of the monkeypox virus is selected from inner membrane proteins M1R, H3L, E8L and A29L, outer envelope proteins A35R and B6R, and 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 the genera *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, Streptococcus pneumoniae, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus or Streptococcus.

10. The nucleic acid construct or mRNA molecule according to claim 8 or 9, wherein, The antibody is an antibody capable of specifically binding to any one or more of the antigens; Preferably, any one or more antigens are as defined in claim 9; 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.

11. A vector comprising the nucleic acid construct or mRNA molecule as described in any one of claims 6-10.

12. A host cell comprising the vector of claim 11.

13. A method for preparing the mRNA molecule according to any one of claims 6-10, comprising: The gene encoding the ORF protein and the template DNA sequence containing mRNA transcription-related elements were prepared by primer PCR amplification, and mRNA was obtained by co-transcription capping.

14. A vaccine comprising a nucleic acid construct or mRNA molecule as described in any one of claims 6-10; Preferably, the nucleic acid construct or mRNA molecule is encapsulated in lipid nanoparticles (LNP).

15. A pharmaceutical composition comprising: a nucleic acid construct or mRNA molecule as described in any one of claims 6-10, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

16. A kit comprising a nucleic acid construct or mRNA molecule according to any one of claims 6-10, a vaccine according to claim 14, and / or a pharmaceutical composition according to claim 15.

17. Use in the preparation of a medicament of any one of claims 6-10, the vaccine of claim 14, the pharmaceutical composition of claim 15, or the kit of claim 16, for the treatment and / or prevention of a subject from disease and / or symptoms caused by infection with a pathogen (e.g., a virus); Preferably, the pathogen is selected from viruses, bacteria, fungi, mycoplasma, chlamydia, or any combination thereof; Preferably, the virus is an influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, or monkeypox virus; more preferably, the virus is SARS-CoV-2, influenza A virus, or influenza B virus. Preferably, the diseases and / or symptoms include uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, and severe acute respiratory syndrome (SARS). Preferably, the subject is a mammal, such as a mouse or a human.

18. Use of the nucleic acid construct or mRNA molecule of any one of claims 6-10, the vaccine of claim 14, the pharmaceutical composition of claim 15, or the kit of claim 16 in the preparation of a medicament, wherein the medicament is used to induce a subject to produce a neutralizing antibody response and / or a T-cell immune response; Preferably, the neutralizing antibody reaction is a neutralizing antibody reaction against viral antigens, and the T cell immune response includes CD4+ and / or CD8+ T cell immune responses; Preferably, the virus is influenza virus, respiratory syncytial virus, coronavirus, human metapneumovirus, infectious bronchitis virus of chickens, Nipah virus, or monkeypox virus. Preferably, the subject is a mammal, such as a mouse or a human.

19. Use of the optimized 5'UTR according to any one of claims 1-5 in any one or more of the following: (i) preparing nucleic acid constructs or mRNA molecules; (ii) increasing the expression level of proteins encoded by nucleic acid constructs or mRNA molecules containing therein; or (iii) improving the immunogenicity of vaccines, pharmaceutical compositions, or kits containing said nucleic acid constructs or mRNA molecules.

Images