Nucleic acid constructs and uses thereof

Abstract

The present disclosure relates to a nucleic acid construct and its use. Specifically, the present disclosure relates to a nucleic acid construct comprising an engineered UTR and its use for preventing or treating a disease (e.g., preventing a viral infection), wherein the UTR can significantly improve the expression efficiency of a target gene in the nucleic acid construct.

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on April 27, 2022, bearing application number CN202210456261.9 and entitled "Nucleic Acid Construct and Use Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure is in the field of nucleic acid drugs, and specifically relates to nucleic acid constructs containing engineered UTRs and their use to prevent or treat diseases (e.g., viral infections). [Background technology]

[0003] Vaccines are an important area of ​​pharmaceutical research, and existing vaccines include inactivated vaccines, adenovirus vector vaccines, attenuated influenza virus vector vaccines, recombinant protein vaccines, and nucleic acid vaccines (including RNA vaccines and DNA vaccines). mRNA is the active component of RNA vaccines, which mainly includes a cap structure (Cap), a 5' untranslated region (UTR), an open reading frame (ORF) encoding an antigen protein, a 3' UTR, and a poly(A) tail structure. Among them, the 5' UTR is the key to recruit ribosomes to mRNA and initiate codon selection, and plays an important role in regulating translation efficiency and modeling the cellular proteome (Ivanov, et al. Science. 2016, 352 (6292): 1413-1416.). Eukaryotic 3'UTRs have multiple regulatory motifs and can be recognized by microRNAs (miRNAs) and rbps to control mRNA stability, positioning and translation (Mazumder et al., 2003; Mayr, 2017). The polyA tail (at the 3' end of the mRNA) is another factor that determines mRNA stability and protein levels. Alternative 3'UTRs not only affect mRNA stability and translation, but also control mRNA positioning (Tushev et al., 2018). The design and optimization of mRNA, especially the selection and use of its UTR, are important parts of the entire preparation process of mRNA vaccine products. Therefore, the development of highly efficient UTRs remains an urgent need in the mRNA drug field.

[0004] The present disclosure provides an mRNA vaccine with a new UTR structure, which has the advantages of stable and highly efficient expression of target genes, and can be universally used in mRNA vaccines (e.g., influenza or COVID-19 vaccines) to regulate the expression of target genes, and has the prospect of being used as an excellent clinical drug. Summary of the Invention

[0005] The present disclosure provides a nucleic acid element and a nucleic acid construct capable of regulating expression of a target gene, wherein the nucleic acid construct comprises at least one nucleic acid element capable of regulating expression of a target gene.

[0006] Nucleic Acid Elements In some embodiments, the nucleic acid element is a 5' untranslated region element (5'UTR).

[0007] In some embodiments, the 5'UTR is selected from the 5'UTR or a derived sequence thereof from any one of the genes, such as Rho GTPase activating protein (ARHGAP), heat shock 27 kDa protein 1 (HSPB1), hemoglobin subunit beta (HBB), and CC motif chemokine ligand 13 (CCL13), or is the 5'UTR or a derived sequence thereof from the above genes.

[0008] In some embodiments, the 5'UTR in the nucleic acid construct is a 5'UTR sequence from an ARHGAP gene or a derived sequence thereof, or is a 5'UTR sequence of the ARHGAP gene or a derived sequence thereof. In some embodiments, the ARHGAP comprises ARHGAP1, ARHGAP2, ARHGAP3, ARHGAP4, ARHGAP5, ARHGAP6, ARHGAP7(DLC1), ARHGAP8, ARHGAP9, ARHGAP10, ARHGAP12, ARHGAP13(SRGAP1), ARHGAP14(SRGAP2), ARHGAP15, ARHGAP17(RICH1), ARHGAP18, ARHGAP19, ARHGAP20, ARHGAP21, ARHGAP22, ARHGAP23, ARHGAP24, ARHGAP25, ARHGAP26.

[0009] In some embodiments, the 5'UTR is a 5'UTR sequence from, or a derivative of, the ARHGAP15 gene, or a derivative thereof. In some embodiments, the ARHGAP15 gene is from any species, such as, for example, human ARHGAP15, baboon ARHGAP15, monkey ARHGAP15, mouse ARHGAP15, etc.

[0010] In some embodiments, the 5'UTR is a 5'UTR sequence from the human ARHGAP15 gene or a derived sequence thereof, or is a 5'UTR sequence of the gene or a derived sequence thereof. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is a nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0011] In some embodiments, the 5'UTR comprises at least one point mutation that can be used to inhibit initiation of translation from an ATG within the UTR. In some embodiments, the point mutation is at any one or more of A, T, or G of the ATG sequence in the 5'UTR. In some embodiments, the point mutation is at the A site of the ATG sequence in the 5'UTR. In some embodiments, the mutation is A to G, C, or T. In some embodiments, the mutation is A to GTG, CTG, or TTG. Furthermore, the mutation can be used to inhibit initiation of translation from an ATG within the UTR.

[0012] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises at least one point mutation that can be used to inhibit initiation of translation from the ATG within the UTR. In some embodiments, the point mutation is selected from any one or more of A, T, or G of the ATG sequence in the 5'UTR. In some embodiments, the point mutation is generated at the A site of the ATG sequence in the 5'UTR. In some embodiments, the mutation is A to G, C, or T.

[0013] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence set forth in SEQ ID NO:44, or the 5'UTR comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:44, wherein N1 is selected from A, G, C, or T.

[0014] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to GTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:2 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0015] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to CTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO: 42, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0016] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to TTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0017] In some embodiments, the 5'UTR is a 5'UTR truncation. In some embodiments, the 5'UTR truncation still retains a similar activity function as the native 5'UTR, for example, still retains the function of regulating the target gene expression protein encoded by the ORF. In some embodiments, the 5'UTR truncation has an enhanced activity function, for example, an enhanced function of regulating the target gene expression protein encoded by the ORF, compared to the native 5'UTR.

[0018] In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and / or deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and retaining a nucleotide sequence at the 3' end. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction and retaining a nucleotide sequence at the 5' end. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction. In some embodiments, the 5'UTR truncate further comprises a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation does not include a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation retains a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of the length of the native 5'UTR sequence.

[0019] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises or is an ARHGAP15 5'UTR truncation, in some embodiments, the truncation scheme comprises deleting a contiguous nucleotide sequence at the 5' end of the ARHGAP15 5'UTR in the 5' to 3' direction and / or deleting a contiguous nucleotide sequence at the 3' end in the 3' to 5' direction.

[0020] In some embodiments, the truncation method includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 3' end. In some embodiments, the truncation method for the 5'UTR truncation includes deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 5' end. In some embodiments, the truncation method for the 5'UTR truncation is deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction.

[0021] In some embodiments, the truncations retain a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% in length compared to the native ARHGAP15 5'UTR (SEQ ID NO:1).

[0022] In some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises 5 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises 7 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 7 ... The 5'UTR truncation comprises a sequence of 7 to 59 consecutive nucleotides in the sequence shown in SEQ ID NO: 2 or 44, and for example, the 5'UTR truncation comprises a sequence of 5, 7, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62 consecutive nucleotides in the sequence shown in SEQ ID NO: 2 or 44.

[0023] In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation (i.e., the starting nucleotide of the 5'UTR truncation) is a nucleotide at any one of positions 1 to 57, by natural counting, in the sequence shown in SEQ ID NO: 2 or 44. In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation is a nucleotide at any one of positions 1 to 13 or 17 to 29, by natural counting, in the sequence shown in SEQ ID NO: 2 or 44. For example, the nucleotide at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57.

[0024] Exemplarily, the 5'-terminal nucleotide of the 5'UTR truncation and the length of the contiguous nucleotide sequence in the sequence shown in SEQ ID NO: 2 contained in the 5'UTR truncation are as shown in the table below.

[0025] [Table 1-1] [Table 1-2]

[0026] In some embodiments, the length of the 5'UTR truncation is 59bp, 55bp, 51bp, 47bp, 43bp, 39bp, 35bp, 31bp, 27bp, 23bp, 19bp, 15bp, 11bp, 7bp.

[0027] In some embodiments, the ARHGAP15 5'UTR truncation further comprises a point mutation of any of the above embodiments. In some embodiments, the ARHGAP15 5'UTR truncation further comprises a nucleotide sequence in which ATG is mutated to GTG. In some embodiments, the 5'UTR derived from the ARHGAP15 gene comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 28 to 30, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the nucleotide sequence set forth above.

[0028] In some embodiments, the ARHGAP15 5'UTR truncation does not include any of the point mutations of the above embodiments. In some embodiments, the 5'UTR of the ARHGAP15 gene includes a nucleotide sequence containing CTATAAT as set forth in SEQ ID NOs: 31 to 40, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the above sequences, or is the above nucleotide sequence.

[0029] In some embodiments, the 5'UTR truncation comprises at least the nucleotide sequence set forth in CTATAAT or SEQ ID NO:193.

[0030] In some embodiments, the ARHGAP15 5'UTR truncation comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 41, 76, 137 to 196, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence.

[0031] In some embodiments, the 5'UTR derived from the HSPB1 gene comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 5, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 3 to 5, or is the above-mentioned nucleotide sequence.

[0032] In some embodiments, the 5'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-2, 3-5, 28-43, 76, 137-196, or a sequence called CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the above sequences, or is the above nucleotide sequence.

[0033] nucleic acid construct The present disclosure relates to (a) an open reading frame (ORF); (b) a 5' untranslated region element (5'UTR).

[0034] In some embodiments, the ORF is a polynucleotide sequence that encodes a target gene protein.

[0035] In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous.

[0036] In some embodiments, the 5'UTR in the nucleic acid construct is located upstream of an open reading frame. In some embodiments, the 5'UTR in the nucleic acid construct is located at the 5' end of an open reading frame.

[0037] In some embodiments, the 5'UTR in the nucleic acid construct is selected from the 5'UTR or a derived sequence thereof derived from any one of the genes, such as Rho GTPase activating protein (ARHGAP), heat shock 27 kDa protein 1 (HSPB1), hemoglobin subunit beta (HBB), and CC motif chemokine ligand 13 (CCL13), or is the 5'UTR or a derived sequence thereof of the above genes.

[0038] In some embodiments, the 5'UTR in the nucleic acid construct is a 5'UTR sequence from an ARHGAP gene or a derived sequence thereof, or is a 5'UTR sequence of the ARHGAP gene or a derived sequence thereof. In some embodiments, the ARHGAP comprises ARHGAP1, ARHGAP2, ARHGAP3, ARHGAP4, ARHGAP5, ARHGAP6, ARHGAP7(DLC1), ARHGAP8, ARHGAP9, ARHGAP10, ARHGAP12, ARHGAP13(SRGAP1), ARHGAP14(SRGAP2), ARHGAP15, ARHGAP17(RICH1), ARHGAP18, ARHGAP19, ARHGAP20, ARHGAP21, ARHGAP22, ARHGAP23, ARHGAP24, ARHGAP25, ARHGAP26.

[0039] In some embodiments, the 5'UTR in the nucleic acid construct is a 5'UTR sequence from the ARHGAP15 gene or a derived sequence thereof, or is a 5'UTR sequence from the ARHGAP15 gene or a derived sequence thereof. In some embodiments, the ARHGAP15 gene is from any species, such as human ARHGAP15, baboon ARHGAP15, monkey ARHGAP15, mouse ARHGAP15, etc.

[0040] In some embodiments, the 5'UTR in the nucleic acid construct is a 5'UTR sequence derived from the human ARHGAP15 gene or a derived sequence thereof, or is the 5'UTR sequence of the human ARHGAP15 gene or a derived sequence thereof. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0041] In some embodiments, the 5'UTR in the nucleic acid construct comprises at least one point mutation that can be used to inhibit initiation of translation from an ATG within the UTR. In some embodiments, the point mutation is selected from any one or more of A, T, or G of the ATG sequence in the 5'UTR. In some embodiments, the point mutation is generated at the A site of the ATG sequence in the 5'UTR. In some embodiments, the mutation is A to G, C, or T. In some embodiments, the mutation is A to GTG, CTG, or TTG. Furthermore, the mutation can be used to inhibit initiation of translation from an ATG within the UTR.

[0042] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises at least one point mutation that can be used to inhibit initiation of translation from the ATG within the UTR. In some embodiments, the point mutation is selected from any one or more of A, T, or G of the ATG sequence in the 5'UTR. In some embodiments, the point mutation is generated at the A site of the ATG sequence in the 5'UTR. In some embodiments, the mutation is A to G, C, or T.

[0043] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence set forth in SEQ ID NO:44, or the 5'UTR comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:44, wherein N1 is selected from A, G, C, or T.

[0044] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to GTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:2 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0045] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to CTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO: 42, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0046] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which ATG has been mutated to TTG. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0047] In some embodiments, the 5'UTR in the nucleic acid construct of the present disclosure is a 5'UTR truncation.In some embodiments, the 5'UTR truncation still retains the activity function similar to that of the natural 5'UTR, for example, still retains the function of regulating the target gene expression protein encoded by the ORF.In some embodiments, the 5'UTR truncation has an enhanced activity function, for example, an enhanced function of regulating the target gene expression protein encoded by the ORF, compared to the natural 5'UTR.

[0048] In some embodiments, the cleavage method of the 5'UTR cleavage product includes deleting a consecutive nucleotide sequence at the 5' end in the sequence direction from 5' to 3' end and / or deleting a consecutive nucleotide sequence at the 3' end in the sequence direction from 3' to 5' end. In some embodiments, the cleavage method of the 5'UTR cleavage product includes deleting a consecutive nucleotide sequence at the 5' end in the sequence direction from 5' to 3' end and retaining a nucleotide sequence at the 3' end. In some embodiments, the cleavage method of the 5'UTR cleavage product includes deleting a consecutive nucleotide sequence at the 3' end in the sequence direction from 3' to 5' end and retaining a nucleotide sequence at the 5' end. In some embodiments, the cleavage method of the 5'UTR cleavage product includes deleting a consecutive nucleotide sequence at the 5' end in the sequence direction from 5' to 3' end, and the cleavage method of the 5'UTR cleavage product includes deleting a consecutive nucleotide sequence at the 5' end in the sequence direction from 3' to 5' end.

[0049] In some embodiments, the 5'UTR truncation further comprises a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation does not comprise a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation retains a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% in length compared to the length of the sequence of the native 5'UTR.

[0050] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises or is an ARHGAP15 5'UTR truncation, in some embodiments, the truncation scheme comprises deleting a contiguous nucleotide sequence at the 5' end of the ARHGAP15 5'UTR in the 5' to 3' direction and / or deleting a contiguous nucleotide sequence at the 3' end in the 3' to 5' direction.

[0051] In some embodiments, the truncation method includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 3' end. In some embodiments, the truncation method for the 5'UTR truncation includes deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 5' end. In some embodiments, the truncation method for the 5'UTR truncation is deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction.

[0052] In some embodiments, the truncations retain a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% in length compared to the native ARHGAP15 5'UTR (SEQ ID NO:1).

[0053] In some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the nucleotide sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises 5 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises 7 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the nucleotide ... sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, in some embodiments, the 5'UTR truncation comprises at least 7 contiguous nucleotides of the sequence shown in SEQ ID NO: 2 or 44, The 5'UTR truncation comprises a sequence of 7 to 59 consecutive nucleotides in the sequence shown in SEQ ID NO:2, and for example, the 5'UTR truncation comprises a sequence of 5, 7, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62 consecutive nucleotides in the sequence shown in SEQ ID NO:2.

[0054] In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation (i.e., the starting nucleotide of the 5'UTR truncation) is a nucleotide at any one of positions 1 to 57, by natural counting, in the sequence shown in SEQ ID NO: 2 or 44. In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation is a nucleotide at any one of positions 1 to 13 or 17 to 29, by natural counting, in the sequence shown in SEQ ID NO: 2 or 44. For example, the nucleotide at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57.

[0055] Exemplarily, the 5'-terminal nucleotide of the 5'UTR truncation and the length of the contiguous nucleotide sequence in the sequence shown in SEQ ID NO: 2 contained in the 5'UTR truncation are as shown in the table below.

[0056] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0057] In some embodiments, the length of the 5'UTR truncation is 59bp, 55bp, 51bp, 47bp, 43bp, 39bp, 35bp, 31bp, 27bp, 23bp, 19bp, 15bp, 11bp, 7bp.

[0058] In some embodiments, the ARHGAP15 5'UTR truncation further comprises a point mutation of any of the above embodiments. In some embodiments, the ARHGAP15 5'UTR truncation further comprises a nucleotide sequence in which ATG is mutated to GTG. In some embodiments, the 5'UTR derived from the ARHGAP15 gene comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 28 to 30, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the nucleotide sequence set forth above.

[0059] In some embodiments, the ARHGAP15 5'UTR truncation does not include the point mutation of any of the above embodiments. In some embodiments, the 5'UTR of the ARHGAP15 gene includes a nucleotide sequence shown in SEQ ID NO: 31 to 40, CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the nucleotide sequence shown in SEQ ID NO: 31 to 40.

[0060] In some embodiments, the 5'UTR truncation comprises at least the nucleotide sequence set forth in CTATAAT or SEQ ID NO:193.

[0061] In some embodiments, the ARHGAP15 5'UTR truncation comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 41, 76, 137 to 196, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence.

[0062] In some embodiments, the 5'UTR derived from the HSPB1 gene comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 5, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 3 to 5, or is the above-mentioned nucleotide sequence.

[0063] In some embodiments, the 5'UTR in the nucleic acid construct described herein comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-2, 3-5, 28-43, 76, 137-196, or a sequence called CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the above-mentioned nucleotide sequence.

[0064] In some specific embodiments, the 5'UTR in any one of the above nucleic acid constructs comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 5, 28 to 43, 76, 137 to 196, or the sequence CTATAAT.

[0065] In some embodiments, the nucleic acid construct of the disclosure further comprises (c) a 3' untranslated region element (3'UTR).

[0066] In some embodiments, the open reading frame described in the present disclosure is derived from a gene different from the 5'UTR and / or the 3'UTR. In some embodiments, the nucleic acid construct 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 of the present disclosure are derived from the same or different, e.g., from the same or different genes. In some embodiments, the 5'UTR and 3'UTR in the nucleic acid construct of the present disclosure are derived from the same or different species.

[0067] In some embodiments, the 3'UTR in the nucleic acid construct of the present 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.

[0068] In some embodiments, the 3'UTR in the nucleic acid construct of the present disclosure is selected from the 3'UTR or a derived sequence thereof derived from any one of genes such as hemoglobin subunit beta (HBB), ARHGAP15, coronin 1A (CORO1A), and hemopexin (HPX), or is the 3'UTR or a derived sequence thereof of the above genes.

[0069] In some embodiments, the 3'UTR is a 3'UTR sequence derived from the HBB or ARHGAP15 gene or a derived sequence thereof, or is the 3'UTR sequence of the ARHGAP15 gene or a derived sequence thereof.

[0070] In some embodiments, the 3'UTR from the HBB gene comprises or is the nucleotide sequence set forth in SEQ ID NO:7, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0071] In some embodiments, the 3'UTR from the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:8 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0072] In some embodiments, the 3'UTR from the CORO1A gene comprises or is the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0073] In some embodiments, the 3'UTR from the HPX gene comprises or is the nucleotide sequence set forth in SEQ ID NO:10, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0074] In some embodiments, the 3'UTR in the nucleic acid construct of the present disclosure comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 7 to 10, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In some specific embodiments, the 3'UTR comprises or is a nucleotide sequence set forth in SEQ ID NO: 7 or 8.

[0075] In some embodiments, the nucleic acid construct comprises a 5' UTR and a 3' UTR, The 5'UTR is selected from the 5'UTR derived from any one of genes such as ARHGAP (e.g., ARHGAP15), HSPB1, HBB, CCL13, etc., or a derived sequence thereof, of the above-mentioned genes, and the 3'UTR is selected from the 3'UTR derived from any one of genes such as HBB, ARHGAP15, CORO1A, HPX, etc., or a derived sequence thereof, of the above-mentioned genes.

[0076] In some embodiments, the nucleic acid construct comprises a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following: the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is the 5'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 5'UTR of the ARHGAP15 gene or a derived sequence thereof, and the 3'UTR is the 3'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 3'UTR of the ARHGAP15 gene or a derived sequence thereof, the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, or a sequence derived therefrom, of the HBB gene; the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, The 5'UTR is a 5'UTR from the HSPB1 gene or a derived sequence thereof, or a 5'UTR of the HSPB1 gene or a derived sequence thereof, and the 3'UTR is a 3'UTR from the HPX gene or a derived sequence thereof, or a 3'UTR of the HPX gene or a derived sequence thereof, or the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is the 5'UTR from the CCL13 gene or a derived sequence thereof, or the 5'UTR of the CCL13 gene or a derived sequence thereof, and the 3'UTR is the 3'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 3'UTR of the ARHGAP15 gene or a derived sequence thereof; the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, The 5'UTR is a 5'UTR derived from the CCL13 gene or a derived sequence thereof, and the 3'UTR is a 3'UTR derived from the HPX gene or a derived sequence thereof, or a 3'UTR derived from the HPX gene or a derived sequence thereof.

[0077] In some embodiments, a nucleic acid construct of the disclosure comprises a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following: the 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence shown in SEQ ID NO: 7, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, the 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence shown in SEQ ID NO: 8, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, the 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence shown in SEQ ID NO: 9, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, the 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:8 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:10 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:8 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:10 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:8 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, The 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0078] the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:10 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, In some embodiments, the nucleic acid construct comprises a 5' UTR and a 3' UTR, The 5'UTR comprises a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or is the above-mentioned nucleotide sequence, and the 3'UTR is a 3'UTR derived from the HBB, ARHGAP15, CORO1A, or HPX gene or a derived sequence thereof, or is the 3'UTR of the above-mentioned gene or a derived sequence thereof.

[0079] In some specific embodiments, the nucleic acid construct comprises a 5' UTR and a 3' UTR, The 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2, 28 to 43, 76, 137 to 196, and CTATAAT, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 7 to 10, or is the above-mentioned nucleotide sequence.

[0080] In some embodiments, a nucleic acid construct of the disclosure further comprises (d) a polyadenylic acid (poly-A) tail.

[0081] 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 at the 3' end of the nucleic acid construct. In some embodiments, the length of the poly-A tail is at least about 50, 100, 150, 200, 300, 400, 500 nucleotides.

[0082] In some embodiments, the poly-A tail includes, but is not limited to, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0083] In some specific embodiments, the poly-A tail comprises or is the nucleotide sequence set forth in SEQ ID NO: 16 or 135.

[0084] In some embodiments, in the 5' to 3' direction, the nucleic acid construct of any one of the above comprises any one of i) to iv): i) 5'UTR and open reading frame (ORF); ii) the open reading frame (ORF) and the 3'UTR; iii) the 5'UTR, the open reading frame (ORF), and the 3'UTR; iv) 5'UTR, open reading frame (ORF), 3'UTR, and poly-A tail.

[0085] Among them, the ORF is derived from a gene different from the 5'UTR and / or the 3'UTR.

[0086] In some embodiments, the 5'UTR in i), iii) to iv) is selected from a 5'UTR derived from any one of genes such as ARHGAP, HSPB1, HBB, CCL13, or a derived sequence thereof (e.g., the 5'UTR of ARHGAP15 or a derived sequence thereof).

[0087] In some embodiments, the 3'UTR in ii) to iv) is selected from a 3'UTR derived from any one of genes such as HBB, ARHGAP15, CORO1A, HPX, etc., or a derived sequence thereof (e.g., the 3'UTR of HBB or ARHGAP15 or a derived sequence thereof).

[0088] In some embodiments, the poly-A tail in iv) to iv) comprises a nucleotide sequence as set forth in SEQ ID NO: 16 or 135, for example.

[0089] In some embodiments, the 5'UTR in i) comprises or is a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 5, 28 to 43, 76, 137 to 196, and CTATAAT.

[0090] In some embodiments, the 3'UTR in ii) comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 7 to 10.

[0091] In some embodiments, the 5'UTR in iii) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 5, 28 to 43, 76, 137 to 196, or CTATAAT, or is the above-mentioned nucleotide sequence, and the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 7 to 10, or is the above-mentioned nucleotide sequence.

[0092] In some embodiments, the 5'UTR in iv) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 5, 28 to 43, 76, 137 to 196, CTATAAT, or is the above-mentioned nucleotide sequence, the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 7 to 10, or is the above-mentioned nucleotide sequence, and the poly-A tail comprises a nucleotide sequence set forth in SEQ ID NO: 16 or 135, or is the above-mentioned nucleotide sequence.

[0093] In the present disclosure, in any one of the 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.

[0094] In some embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase.

[0095] In some embodiments, the polypeptide or protein encoded by the ORF is a viral antigen. Exemplary viral antigens include, but are not limited to, influenza virus, coronavirus, respiratory syncytial virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or Epstein-Barr virus.

[0096] In some embodiments, the viral antigen is a coronavirus antigen. In some embodiments, the coronavirus is a coronavirus that infects humans, 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 a spike protein (Spike protein, S protein, or Spike protein), an envelope protein (envelope protein, E protein), a membrane protein (membrane protein, M protein), and a nucleocapsid protein (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 spike protein of any of the following viral strains: 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 (BA4), etc.

[0097] In some embodiments, the SARS-COV-2 spike protein comprises or is an amino acid sequence set forth in any one of SEQ ID NOs: 12, 14, 21, 23, 25, 80, or 136, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto.

[0098] In some embodiments, the ORF comprises a codon-optimized nucleotide sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a wild-type nucleotide sequence encoding a SARS-COV-2 antigen (e.g., wild-type SARS-COV-2, SEQ ID NO:81).

[0099] In some embodiments, the ORF encoding the polypeptide or protein comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 13, 15, 20, 22, 24, 81, or 97, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0100] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A virus or influenza B virus, and illustratively, the influenza virus is influenza A virus H1N1, influenza A virus H3N2, influenza A virus H3N8, influenza A virus H2N2, influenza A virus H5N1, 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 an influenza virus, such as hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is an 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), or influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific embodiments, the influenza virus antigen is an NA protein of an influenza virus, such as the NA protein of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), or influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).

[0101] In some embodiments, the HA protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83-86, 98-101, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto, or is any of the above sequences.

[0102] In some embodiments, the ORF comprises a codon-optimized nucleotide sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a wild-type nucleotide sequence encoding an HA antigen.

[0103] In some embodiments, the ORF comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 87-90, 102-105, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above sequence.

[0104] In some embodiments, the ORF comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 93 to 96, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above sequence.

[0105] In some embodiments, the present disclosure provides a nucleic acid construct comprising, in order from 5' to 3', a 5'UTR, an ORF, a 3'UTR, and a poly-A tail. In some embodiments, the 5'UTR comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 1-5, 28-43, 76, 137-196, CTATAAT, the ORF comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 13, 15, 20, 22, 24, 81, 97, 87-90, 93-96, 102-105, the 3'UTR comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 7-10, and the poly-A tail comprises or is a nucleotide sequence set forth in SEQ ID NO: 16 or 135.

[0106] Illustratively, the nucleic acid construct comprises the nucleotide sequences shown in SEQ ID NOs:18-19.

[0107] In the present disclosure, including any embodiment thereof, the nucleic acid construct is a nucleic acid molecule such as a cDNA.

[0108] RNA molecule The present disclosure relates to (a) an open reading frame (ORF); (b) a 5' untranslated region element (5'UTR).

[0109] In some embodiments, the ORF is a polynucleotide sequence that encodes a target gene protein.

[0110] In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous.

[0111] In some embodiments, the 5'UTR in the RNA molecule is located upstream of an open reading frame. In some embodiments, the 5'UTR in the RNA molecule is located at the 5' end of an open reading frame.

[0112] In some embodiments, the 5'UTR in the RNA molecule is selected from the 5'UTR or a derived sequence thereof from any one of the genes, such as Rho GTPase activating protein (ARHGAP), heat shock 27 kDa protein 1 (HSPB1), hemoglobin subunit beta (HBB), and CC motif chemokine ligand 13 (CCL13), or is the 5'UTR or a derived sequence thereof from the above genes.

[0113] In some embodiments, the 5'UTR in the RNA molecule is a 5'UTR sequence from an ARHGAP gene or a derived sequence thereof, or is a 5'UTR of the ARHGAP gene or a derived sequence thereof. In some embodiments, the ARHGAP comprises ARHGAP1, ARHGAP2, ARHGAP3, ARHGAP4, ARHGAP5, ARHGAP6, ARHGAP7(DLC1), ARHGAP8, ARHGAP9, ARHGAP10, ARHGAP12, ARHGAP13(SRGAP1), ARHGAP14(SRGAP2), ARHGAP15, ARHGAP17(RICH1), ARHGAP18, ARHGAP19, ARHGAP20, ARHGAP21, ARHGAP22, ARHGAP23, ARHGAP24, ARHGAP25, ARHGAP26.

[0114] In some embodiments, the 5'UTR is a 5'UTR sequence or a derived sequence thereof from the ARHGAP15 gene, or a 5'UTR sequence or a derived sequence thereof from the ARHGAP15 gene, in some embodiments, the ARHGAP15 gene is from any species, such as human ARHGAP15, baboon ARHGAP15, mouse ARHGAP15, etc.

[0115] In some embodiments, the 5'UTR is a 5'UTR sequence derived from the human ARHGAP15 gene or a derived sequence thereof, or is the 5'UTR of the ARHGAP15 gene or a derived sequence thereof. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO: 45 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0116] In some embodiments, the 5'UTR in the RNA molecule of the present disclosure comprises at least one point mutation, including a point mutation in any embodiment of the nucleic acid construct. In some embodiments, the mutation is an A to G, C, or U.

[0117] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence set forth in SEQ ID NO:79, or the 5'UTR comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:79, wherein N2 is selected from A, G, C, or U.

[0118] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which AUG is mutated to GUG. In some embodiments, the 5'UTR from the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO: 46, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0119] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which AUG is mutated to CUG. In some embodiments, the 5'UTR from the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:77 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0120] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises a nucleotide sequence in which AUG is mutated to UUG. In some embodiments, the 5'UTR from the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:78 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0121] In some embodiments, the 5'UTR in the RNA molecule of the present disclosure is a 5'UTR truncation. In some embodiments, the 5'UTR truncation still retains the activity function similar to that of the natural 5'UTR, for example, still retains the function of regulating the target gene expression protein encoded by the ORF.

[0122] In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and / or deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and retaining a nucleotide sequence at the 3' end. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction and retaining a nucleotide sequence at the 5' end. In some embodiments, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction. In some embodiments, the 5'UTR truncate further comprises a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation does not include a point mutation of any of the above embodiments. In some embodiments, the 5'UTR truncation retains a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of the length of the native 5'UTR sequence.

[0123] In some embodiments, the 5'UTR sequence from the ARHGAP15 gene comprises or is an ARHGAP15 5'UTR truncation, in some embodiments, the truncation scheme comprises deleting a contiguous nucleotide sequence at the 5' end of the ARHGAP15 5'UTR in the 5' to 3' direction and / or deleting a contiguous nucleotide sequence at the 3' end in the 3' to 5' direction.

[0124] In some embodiments, the truncation method includes deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 3' end. In some embodiments, the truncation method for the 5'UTR truncation includes deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction of the ARHGAP15 5'UTR and retaining a nucleotide sequence at the 5' end. In some embodiments, the truncation method for the 5'UTR truncation is deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction of the ARHGAP15 5'UTR and deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction.

[0125] In some embodiments, the truncations retain a sequence that is at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% in length compared to the native ARHGAP15 5'UTR (SEQ ID NO:45).

[0126] In some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the nucleotide sequence shown in SEQ ID NO: 46 or 79, in some embodiments, the 5'UTR truncation comprises 5 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 46 or 79, in some embodiments, the 5'UTR truncation comprises 7 to 62 contiguous nucleotides of the sequence shown in SEQ ID NO: 46 or 79, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the nucleotide ... sequence shown in SEQ ID NO: 46 or 79, in some embodiments, the 5'UTR truncation comprises at least 5 contiguous nucleotides of the sequence shown in SEQ ID NO: 46 or 79, 79, and for example, the 5'UTR truncation contains a sequence of 5, 7, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62 consecutive nucleotides in the sequence shown in SEQ ID NO:2.

[0127] In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation (i.e., the starting nucleotide of the 5'UTR truncation) is a nucleotide at any one of positions 1 to 57, by natural counting, in the sequence shown in SEQ ID NO: 46 or 79. In some embodiments, the 5'-terminal nucleotide of the 5'UTR truncation is a nucleotide at any one of positions 1 to 13 or 17 to 29, by natural counting, in the sequence shown in SEQ ID NO: 46 or 79. For example, the nucleotide at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57.

[0128] In some embodiments, the length of the 5'UTR truncation is 59bp, 55bp, 51bp, 47bp, 43bp, 39bp, 35bp, 31bp, 27bp, 23bp, 19bp, 15bp, 11bp, 7bp.

[0129] In some embodiments, the ARHGAP15 5'UTR truncation further comprises a point mutation of any of the above embodiments. In some embodiments, the ARHGAP15 5'UTR truncation further comprises a nucleotide sequence in which AUG is mutated to GUG. In some embodiments, the 5'UTR derived from the ARHGAP15 gene comprises a nucleotide sequence shown in SEQ ID NO: 63 to 65, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above nucleotide sequence.

[0130] In some embodiments, the ARHGAP15 5'UTR truncation does not include the point mutation of any of the above embodiments. In some embodiments, the 5'UTR derived from the ARHGAP15 gene includes the nucleotide sequence set forth in SEQ ID NOs: 66 to 75, CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the nucleotide sequence set forth above.

[0131] In some embodiments, the 5'UTR derived from the ARHGAP15 gene comprises a sequence corresponding to the sequence set forth in SEQ ID NO: 41, 76, 137-196 (with U replacing T in the sequence), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the above-mentioned nucleotide sequence.

[0132] In some embodiments, the 5'UTR from the HSPB1 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:47 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0133] In some embodiments, the 5'UTR from the HBB gene comprises or is the nucleotide sequence set forth in SEQ ID NO:48 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0134] In some embodiments, the 5'UTR from the CCL13 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:49, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0135] In some embodiments, the 5'UTR in the RNA molecule described herein comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 45-49, 63-75, 77-78, CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. Alternatively, the 5'UTR in the RNA molecule comprises a sequence corresponding to a sequence set forth in SEQ ID NOs: 41, 76, 137-196 (U replacing T in the sequence), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0136] In some specific embodiments, the 5'UTR in any one of the above RNA molecules comprises or is any one of the nucleotide sequences set forth in SEQ ID NOs: 45-49, 63-75, 77-78, and CUAUAAU, or the 5'UTR in the RNA molecule comprises a sequence corresponding to the sequence set forth in SEQ ID NOs: 41, 76, and 137-196 (U replacing T in the sequence).

[0137] In some embodiments, the RNA molecules of the disclosure further comprise (c) a 3' untranslated region element (3'UTR).

[0138] In some embodiments, the open reading frame described in the present disclosure is derived from a different gene than the 5'UTR and / or the 3'UTR. In some embodiments, the 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 RNA molecule of the present disclosure are derived from the same or different, e.g., from the same or different genes. In some embodiments, the 5'UTR and 3'UTR in the RNA molecule of the present disclosure are derived from the same or different species.

[0139] In some embodiments, the 3'UTR in the RNA molecule of the present disclosure is located downstream of an open reading frame. In some embodiments, the 3'UTR in the RNA molecule is located at the 3' end of an open reading frame. In some embodiments, the 3'UTR in the RNA molecule of the present disclosure is selected from the 3'UTR or a derived sequence thereof from any one of the genes such as hemoglobin subunit beta (HBB), ARHGAP15, coronin 1A (CORO1A), and hemopexin (HPX), or is the 3'UTR or a derived sequence thereof of the above genes.

[0140] In some embodiments, the 3'UTR is a 3'UTR sequence derived from the HBB or ARHGAP15 gene or a derived sequence thereof, or is the 3'UTR sequence of the ARHGAP15 gene or a derived sequence thereof.

[0141] In some embodiments, the 3'UTR from the HBB gene comprises or is the nucleotide sequence set forth in SEQ ID NO:50 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0142] In some embodiments, the 3'UTR from the ARHGAP15 gene comprises or is the nucleotide sequence set forth in SEQ ID NO:51, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0143] In some embodiments, the 3'UTR from the CORO1A gene comprises or is the nucleotide sequence set forth in SEQ ID NO:52 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0144] In some embodiments, the 3'UTR from the HPX gene comprises or is the nucleotide sequence set forth in SEQ ID NO:53, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0145] In some embodiments, the 3'UTR in an RNA molecule of the present disclosure comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 50-53, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0146] In some specific embodiments, the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO: 50 or 51.

[0147] In some embodiments, the RNA molecule comprises a 5' UTR and a 3' UTR, The 5'UTR is selected from the 5'UTR derived from any one of genes such as ARHGAP (e.g., ARHGAP15), HSPB1, HBB, CCL13, etc., or a derived sequence thereof, of the above-mentioned genes, and the 3'UTR is selected from the 3'UTR derived from any one of genes such as HBB, ARHGAP15, CORO1A, HPX, etc., or a derived sequence thereof, of the above-mentioned genes.

[0148] In some embodiments, the RNA molecule comprises a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following: the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is the 5'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 5'UTR of the ARHGAP15 gene or a derived sequence thereof, and the 3'UTR is the 3'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 3'UTR of the ARHGAP15 gene or a derived sequence thereof, the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, or a sequence derived therefrom, of the HBB gene; the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HBB gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HPX gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the ARHGAP15 gene or a sequence derived therefrom, the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the HSPB1 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, The 5'UTR is a 5'UTR from the HSPB1 gene or a derived sequence thereof, or a 5'UTR of the HSPB1 gene or a derived sequence thereof, and the 3'UTR is a 3'UTR from the HPX gene or a derived sequence thereof, or a 3'UTR of the HPX gene or a derived sequence thereof, or the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the HBB gene or a sequence derived therefrom, the 5'UTR is the 5'UTR from the CCL13 gene or a derived sequence thereof, or the 5'UTR of the CCL13 gene or a derived sequence thereof, and the 3'UTR is the 3'UTR from the ARHGAP15 gene or a derived sequence thereof, or the 3'UTR of the ARHGAP15 gene or a derived sequence thereof; the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, and the 5'UTR is a 5'UTR derived from the CCL13 gene or a sequence derived therefrom, and the 3'UTR is a 3'UTR derived from the CORO1A gene or a sequence derived therefrom, The 5'UTR is a 5'UTR derived from the CCL13 gene or a derived sequence thereof, and the 3'UTR is a 3'UTR derived from the HPX gene or a derived sequence thereof, or a 3'UTR derived from the HPX gene or a derived sequence thereof.

[0149] In some embodiments, an RNA molecule of the present disclosure comprises a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following: The 5'UTR comprises any one of the nucleotide sequences shown in SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, and CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence shown in SEQ ID NOs: 41, 76, and 137 to 196 (a sequence corresponding to U 50 (replacing T in SEQ ID NO: 44) or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises the nucleotide sequence set forth in SEQ ID NO: 50 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the nucleotide sequence described above; The 5'UTR comprises any one of the nucleotide sequences shown in SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, and CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence shown in SEQ ID NOs: 41, 76, and 137 to 196 (a sequence corresponding to U 51 (replacing T in SEQ ID NO: 51) or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises the nucleotide sequence set forth in SEQ ID NO: 51 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the nucleotide sequence described above; The 5'UTR comprises any one of the nucleotide sequences shown in SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, and CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence shown in SEQ ID NOs: 41, 76, and 137 to 196 (a sequence corresponding to U 52 (replacing T in SEQ ID NO: 53) or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises the nucleotide sequence set forth in SEQ ID NO: 52 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the nucleotide sequence described above; The 5'UTR comprises any one of the nucleotide sequences shown in SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, and CUAUAAU, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence shown in SEQ ID NOs: 41, 76, and 137 to 196 (a sequence corresponding to U 53 (replacing T in SEQ ID NO: 53) or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises the nucleotide sequence set forth in SEQ ID NO: 53 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or is the nucleotide sequence described above; the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:47 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:50 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:47 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:51 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:47 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:52 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:47 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:53 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:48 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:50 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:48 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:51 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:48 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:52 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:48 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:53 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:49 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:50 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:49 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:51 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, the 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:49 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:52 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, The 5'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:49 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the 3'UTR comprises or is the nucleotide sequence set forth in SEQ ID NO:53 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0150] In some embodiments, the RNA molecule comprises a 5' UTR and a 3' UTR, The 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, and CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence shown in SEQ ID NOs: 41, 76, and 137 to 196 (T in the sequence is replaced with U), and the 3'UTR is a 3'UTR derived from any one of the genes HBB, ARHGAP15, CORO1A, and HPX, or a derived sequence thereof, or is the 3'UTR of the above-mentioned genes, or a derived sequence thereof.

[0151] In some specific embodiments, the RNA molecule comprises a 5'UTR and a 3'UTR, The 5'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 45 to 46, 63 to 75, 77 to 78, CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence set forth in SEQ ID NOs: 41, 76, 137 to 196 (T in the sequence is replaced with U), and the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 50 to 53, or is the above-mentioned nucleotide sequence.

[0152] In some embodiments, the RNA molecule of the present disclosure further comprises (d) a polyadenylic acid (poly-A) tail.

[0153] In some embodiments, the poly-A tail in the RNA molecule is located downstream of the 3'UTR. In some embodiments, the poly-A tail in the RNA molecule is located at the 3' end of the 3'UTR. In some embodiments, the poly-A tail is at the 3' end of the RNA molecule. In some embodiments, the length of the poly-A tail is at least about 50, 100, 150, 200, 300, 400, 500 nucleotides.

[0154] In some embodiments, the poly-A tail includes, but is not limited to, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0155] In some specific embodiments, the poly-A tail comprises or is the nucleotide sequence set forth in SEQ ID NO: 56 or 135.

[0156] In some embodiments, the RNA molecule of the present disclosure further comprises (e) a 5' cap structure (5'Cap).

[0157] In some embodiments, the 5' cap structure in the RNA molecule is located upstream of the 5' UTR. In some embodiments, the 5' cap structure in the RNA molecule is located at the 5' end of the 5' UTR. In some embodiments, the 5' cap structure is a cap structure known to those of skill in the art, such as Cap0 (methylation of the first nucleobase, e.g., m7GpppN), Cap1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap2 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap3 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap4 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap5 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap6 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap7 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap8 (additional methylation of the nucleotide adjacent to m7GpppN, e.g., m7G(5')ppp(5')(2' m7GpppN (additional methylation of the ribose of the 4th nucleotide downstream of m7GpppN), Cap4 (additional methylation of the ribose of the 4th nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.

[0158] In some embodiments, chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) is used to form the 5'-cap structure (eg, Cap0 or Cap1).

[0159] In some embodiments, the 5'-cap structure (e.g., Cap0 or Cap1) is formed by enzymatic capping using a capping enzyme (e.g., cowpox virus capping enzyme and / or a cap-dependent 2'-O methyltransferase). In some embodiments, an immobilized capping enzyme is used to add the 5' cap structure (Cap0 or Cap1). The capping methods and procedures in WO2016 / 193226 are incorporated herein in their entirety.

[0160] In some embodiments, the 5' cap structure includes, but is not limited to, ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0161] In some specific embodiments, the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG. Other 5' cap structures or cap structure analogs may also be used.

[0162] In some embodiments, in the 5' to 3' direction, the RNA molecule of any one of the above comprises any one of i) to v): i) 5'UTR and open reading frame (ORF); ii) the open reading frame (ORF) and the 3'UTR; iii) the 5'UTR, the open reading frame (ORF), and the 3'UTR; iv) the 5'UTR, the open reading frame (ORF), the 3'UTR, and the poly-A tail; v) 5' cap structure (5'Cap), 5'UTR, open reading frame (ORF), 3'UTR, and poly-A tail.

[0163] Among them, the ORF is derived from a gene different from the 5'UTR and / or the 3'UTR.

[0164] In some embodiments, the 5'UTR in i), iii) to v) is selected from a 5'UTR derived from any one of genes such as ARHGAP, HSPB1, HBB, CCL13, or a derived sequence thereof (e.g., the 5'UTR of ARHGAP15 or a derived sequence thereof).

[0165] In some embodiments, the 3'UTR in ii) to v) is selected from a 3'UTR derived from any one of genes such as HBB, ARHGAP15, CORO1A, HPX, etc., or a derived sequence thereof (e.g., the 3'UTR of HBB or ARHGAP15, or a derived sequence thereof).

[0166] In some embodiments, the poly-A tail in iv)-v) comprises, for example, the nucleotide sequence set forth in SEQ ID NO:56.

[0167] In some embodiments, the 5' cap structure in v) includes, but is not limited to, Cap0, Cap1 (e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap2, Cap3, Cap4, ARCA.

[0168] In some embodiments, the 5'UTR in i) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 45 to 49, 63 to 75, 77 to 78, CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence set forth in SEQ ID NOs: 41, 76, 137 to 196 (U replaces T in the sequence).

[0169] In some embodiments, the 3'UTR in ii) comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 50 to 53.

[0170] In some embodiments, the 5'UTR in iii) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 45 to 49, 63 to 75, 77 to 78, CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequence set forth in SEQ ID NOs: 41, 76, 137 to 196 (in which T is replaced by U), and the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 50 to 53, or is the above-mentioned nucleotide sequence.

[0171] In some embodiments, the 5'UTR in iv) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 45-49, 63-75, 77-78, CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequences set forth in SEQ ID NOs: 41, 76, 137-196 (with U replacing T in the sequence), the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 50-53, or is the above-mentioned nucleotide sequence, and the poly-A tail comprises a nucleotide sequence set forth in SEQ ID NO: 56, or is the above-mentioned nucleotide sequence.

[0172] In some embodiments, the 5'UTR in v) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 45-49, 63-75, 77-78, CUAUAAU, or is the above-mentioned nucleotide sequence, or the 5'UTR comprises a sequence corresponding to the sequences set forth in SEQ ID NOs: 41, 76, 137-196 (U replaces T in the sequence), the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 50-53, or is the above-mentioned nucleotide sequence, the poly-A tail comprises a nucleotide sequence set forth in SEQ ID NO: 56, or is the above-mentioned nucleotide sequence, and the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG.

[0173] In the present disclosure, in any one of the RNA molecules, 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.

[0174] In some embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase.

[0175] In some embodiments, the polypeptide or protein encoded by the ORF is a viral antigen. Exemplary viral antigens include, but are not limited to, influenza virus, coronavirus, respiratory syncytial virus, human immunodeficiency virus, herpes simplex virus, rabies virus, or Epstein-Barr virus.

[0176] In some embodiments, the viral antigen is a coronavirus antigen. In some embodiments, the coronavirus is a coronavirus that infects humans, 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 a spike protein (Spike protein, S protein, or Spike protein), an envelope protein (envelope protein, E protein), a membrane protein (membrane protein, M protein), and a nucleocapsid protein (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 spike protein of any of the following viral strains: 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 (BA.4), etc. In some embodiments, the SARS-COV-2 spike protein comprises or is an amino acid sequence set forth in any one of SEQ ID NOs: 12, 14, 21, 23, 25, 80, or 136, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto.

[0177] In some embodiments, the ORF comprises a codon-optimized nucleotide sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a wild-type nucleotide sequence encoding a SARS-COV-2 antigen (e.g., wild-type SARS-COV-2, SEQ ID NO:82).

[0178] In some embodiments, the ORF encoding the above-mentioned polypeptide or protein comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 54 to 55, 59 to 61, 82, and 130, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned nucleotide sequence.

[0179] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A virus or influenza B virus, and illustratively, the influenza virus is influenza A virus H1N1, influenza A virus H3N2, influenza A virus H3N8, influenza A virus H2N2, influenza A virus H5N1, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of an influenza virus, such as hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is an 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), or influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific embodiments, the influenza virus antigen is an NA protein of an influenza virus, such as the NA protein of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), or influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).In some embodiments, the HA protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83-86, 98-101, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto, or is any of the above sequences.

[0180] In some embodiments, the ORF comprises a codon-optimized nucleotide sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a wild-type nucleotide sequence encoding an HA antigen.

[0181] In some embodiments, the ORF encoding the above-mentioned polypeptide or protein comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 126-129, 131-134, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above-mentioned sequence.

[0182] In some embodiments, the present disclosure provides an RNA molecule comprising, in order from 5' to 3', a 5'UTR, an ORF, a 3'UTR, and a poly-A tail. In some embodiments, the 5'UTR comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 45-49, 45-49, 63-75, 77-78, CUAUAAU, or the 5'UTR comprises a sequence corresponding to a sequence set forth in SEQ ID NOs: 41, 76, 137-196 (U replaces T in the sequence), the ORF comprises or is a nucleotide sequence set forth in SEQ ID NOs: 54-55, 59-61, 82, 126-134, the 3'UTR comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 50-53, and the poly-A tail comprises or is a nucleotide sequence set forth in SEQ ID NO: 56 or 135. In some specific embodiments, the RNA molecule comprises the nucleotide sequences set forth in SEQ ID NOs: 57-58, 106-125.

[0183] In the present disclosure, including any embodiment thereof, the RNA molecule may be mRNA.

[0184] The present disclosure is the first to use, screen, and design 5'UTR sequences derived from human Rho GTPase activating protein (ARHGAP) for recombinant expression of target genes (e.g., SARS-CoV-2 viral proteins), and successfully verify that 5'UTR derived from ARHGAP15 and its modified 5'UTR can initiate efficient expression of different target genes. Rho GTPase activating protein 15 (ARHGAP15) belongs to the ARHGAP family and is a Rac1-specific GTPase activating protein (GAP) and a major negative regulator of Rho family GTPase activity. The present disclosure provides more 5'UTR and 3'UTR selections and combinations with excellent expression efficiency, and may provide an mRNA vaccine that can efficiently prevent novel coronavirus infection and influenza virus infection, especially against various mutant strains.

[0185] Polynucleotides The present disclosure further provides isolated polynucleotides encoding one or more polypeptides or proteins, wherein the polypeptides or proteins are coronavirus antigens. 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 a spike protein (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 spike protein of any of the following viral strains: 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 (BA.4), etc. In some embodiments, the SARS-COV-2 spike protein comprises or is an amino acid sequence set forth in any one of SEQ ID NOs: 12, 14, 21, 23, 25, 80, or 136, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto.

[0186] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A virus or influenza B virus, and illustratively, the influenza virus is influenza A virus H1N1, influenza A virus H3N2, influenza A virus H3N8, influenza A virus H2N2, influenza A virus H5N1, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of an influenza virus, such as hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is an 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), or influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some specific embodiments, the influenza virus antigen is an NA protein of an influenza virus, such as the NA protein of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria (e.g., Influenza B / Washington / 02 / 2019), or influenza B virus Yamagata (e.g., Influenza B / Phuket / 3073 / 2013).In some embodiments, the HA protein of the influenza virus comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83 to 86, 98 to 101, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto, or is the above sequence.

[0187] In some embodiments, the ORF comprises a codon-optimized nucleotide sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to a wild-type nucleotide sequence encoding a SARS-COV-2 antigen (e.g., wild-type SARS-COV-2 mRNA, SEQ ID NO:82).

[0188] In some embodiments, the isolated polynucleotide comprises a nucleotide sequence set forth in SEQ ID NO:54-55, 59-61, 82, 126-134, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or is the above sequence.

[0189] In some embodiments, the polynucleotide is RNA, e.g., mRNA. In some embodiments, the polynucleotide is an ORF region of an mRNA.

[0190] In some embodiments, the disclosure provides for the use of any one of the above nucleic acid constructs or RNA molecules in any one of the following: (1) preparing a vaccine; (2) encoding a viral antigen in vivo or in vitro in a subject; (3) preparing a medicament encoding a viral antigen in vivo or in vitro in a subject; or (4) use in the preparation of a medicament.

[0191] In some embodiments, a nucleic acid construct or RNA molecule of the present disclosure encodes a target protein, wherein the open reading frame (ORF) comprises an enhanced expression of the target protein.

[0192] qualification To further improve the stability of protein expression of the RNA or polynucleotide molecule of the present disclosure, the RNA or polynucleotide molecule may further include one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, and / or lipid modifications. In some embodiments, the RNA or polynucleotide molecule is uniformly modified with a particular modification (e.g., completely modified throughout the entire sequence). For example, the RNA can be uniformly modified with pseudouridine, such that each U in the sequence is a pseudouridine.

[0193] The backbone modification of the present disclosure refers to a chemical modification of the backbone phosphate of the nucleotides contained in the RNA or polynucleotide molecules of the present disclosure. In some embodiments, the backbone modification includes, but is not limited to, the complete replacement of the unmodified phosphate moieties in the backbone with modified phosphate, for example, the backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the modified phosphate includes, but is not limited to, phosphorothioates, phosphite selenates, borane phosphates, borane phosphates, phosphonates, phosphoramidates, alkyl or aryl phosphates, and phosphate triesters.

[0194] Sugar modifications according to the present disclosure refer to chemical modifications of the sugars of nucleotides contained in an RNA or polynucleotide molecule of the present disclosure. In some embodiments, the sugar modifications include, but are not limited to, modifying or replacing the 2' hydroxyl (OH) group of an RNA molecule with a number of different "oxy" or "deoxy" substituents. In some embodiments, the "oxy" modifications include, but are not limited to, substitution modifications such as alkoxy, aryloxy, polyethylene glycol (PEG), etc. In some embodiments, the "deoxy" modifications include, but are not limited to, hydrogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid) modifications.

[0195] Base modification according to the present disclosure refers to chemical modification of the base portion of a nucleotide contained in an RNA or polynucleotide molecule of the present disclosure. In some embodiments, the base modification includes modifications to adenine, guanine, cytosine, and uracil in the nucleotide. For example, the nucleosides and nucleotides described herein may be chemically modified on the surface of the major groove. In some embodiments, the chemical modification of the major groove may include an amino group, a thiol group, an alkyl group, or a halogen group. In some embodiments, the base modification includes, but is not limited to, modifications with pseudouridine, 1-methyl-pseudouridine, 5-azacytidine, 5-methylcytosine-5'-triphosphate, or 2-methoxyadenine. In some embodiments, the base modification is a pseudouridine modification. In some specific embodiments, the RNA may be uniformly modified with pseudouridine such that each U in the sequence is a pseudouridine.

[0196] The lipid modification according to the present disclosure refers to the lipid modification contained in the RNA or polynucleotide molecule of the present disclosure.In some embodiments, the lipid modification includes, but is not limited to, the covalent bond of the RNA or polynucleotide molecule of the present disclosure to at least one linker, and the covalent bond of the corresponding linker to at least one lipid.In some embodiments, the lipid modification includes, but is not limited to, the covalent bond (without linker) of the RNA or polynucleotide molecule of the present disclosure to at least one lipid.

[0197] UTRs UTRs (5'UTR and / or 3'UTR) may be provided as flanking regions in the nucleic acid construct, RNA or polynucleotide molecule of the disclosure. The UTRs may be homologous or heterologous to the coding region in the nucleic acid construct, RNA or polynucleotide molecule of the disclosure. The flanking regions may comprise one or more 5'UTR and / or 3'UTR, said UTRs may be of the same or different sequences. Any portion of the flanking regions may also be codon optimized. Any portion of the flanking regions may also independently comprise one or more different structural or chemical modifications before and / or after codon optimization.

[0198] UTRs heterologous to the ORF of the present disclosure are introduced or engineered into the nucleic acid construct, RNA, or polynucleotide of the present disclosure to alter one or more properties of the nucleic acid construct, RNA, or polynucleotide of the present disclosure. The recombinant nucleic acid construct, RNA, or polynucleotide is then administered to a cell, tissue, or organism, and the beneficial effect of the heterologous UTR on the RNA or polynucleotide of the present disclosure is evaluated by measuring the results, such as protein level, localization, and / or half-life. In some embodiments, the UTR comprises a wild-type UTR or a mutant thereof, the UTR mutant comprising one or more nucleotides added or removed at the terminus, including A, T, C, or G. In some embodiments, the UTR mutant also comprises codon optimization or modification performed in any manner. In some embodiments, the UTR mutant also comprises a derived sequence of any embodiment of the present disclosure, for example, a point mutation of ATG to GTG based on the native UTR sequence to inhibit initiation of translation from the ATG within the UTR.

[0199] vector The present disclosure further provides a vector comprising any one of the above described nucleic acid constructs, RNA or polynucleotides. Wherein, the nucleic acid constructs, RNA or polynucleotides may be present in and / or part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may be an expression vector, i.e., the nucleic acid constructs, RNA or polynucleotides may provide a vector that codes for the expression of a polypeptide. Such an 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., promoter, terminator, etc.). It is common knowledge of the skilled artisan to select the above elements and their sequences for expression in a particular host. Regulatory elements and other elements useful or necessary for coding the expression of the polypeptide of the present disclosure are, for example, promoters, terminators, selection markers, leader sequences, reporter genes, etc.

[0200] The nucleic acid constructs of the present disclosure can be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on the nucleotide sequence information of the present disclosure, and / or can be isolated from a suitable natural source.

[0201] In some embodiments, the vector of the present disclosure further comprises a promoter, e.g., the promoter is at the 5' end of the nucleic acid construct 5'UTR, e.g., the promoter is a T7 promoter, a T7 lac promoter, a Tac promoter, a Lac promoter, a Trp promoter.

[0202] In some specific embodiments, the T7 promoter comprises or is the nucleotide sequence set forth in SEQ ID NO:17.

[0203] host cell The present disclosure includes any one of the nucleic acid constructs, RNA or polynucleotides described above. host Further provided is a cell. In some embodiments, the cell is capable of expressing one or more of the nucleic acid constructs, RNAs or polynucleotides encoded polypeptides of the present disclosure. In some embodiments, the cell is capable of expressing one or more of the nucleic acid constructs, RNAs or polynucleotides encoded polypeptides of the present disclosure. host The cell may be a bacterial cell, a fungal cell or a mammalian cell.

[0204] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas strains) and gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus strains).

[0205] 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.

[0206] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0207] However, the present disclosure may be used with amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0208] Production or preparation method The present disclosure provides methods for preparing the nucleic acid constructs, RNA or polynucleotides of the disclosure, and methods for preparing the encoded polypeptides.

[0209] Methods and reagents for preparing and producing nucleic acid constructs, RNA or polynucleotides and the encoded polypeptides are known in the art, such as specific appropriate vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques in methods applied to produce the encoded polypeptides of the present disclosure are known to those of skill in the art.

[0210] In some embodiments, the method of preparing the nucleic acid construct, RNA or polynucleotide comprises culturing the host cell and recovering the produced nucleic acid construct, RNA or polynucleotide from the culture. The nucleic acid construct, RNA or polynucleotide of the present disclosure and its encoded polypeptide may also be obtained by other production methods known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0211] In some embodiments, the method for preparing an RNA molecule includes preparing a nucleic acid construct or vector, and then performing reverse transcription using the nucleic acid construct or vector to obtain an RNA molecule. In some specific embodiments, the method further includes adding a 5' Cap to the 5' end of the RNA molecule.

[0212] vaccine The present disclosure further provides a vaccine comprising any one of the nucleic acid constructs described above, any one of the RNAs described above, and / or any one of the polynucleotides described above. In some embodiments, the nucleic acid constructs, RNAs, or polynucleotides encode one or more antigens of one or more virus strains. The vaccines provided in the present disclosure may be monovalent, multivalent, or combination vaccines.

[0213] Monovalent vaccine The present disclosure provides a monovalent vaccine comprising one antigen encoding one organism. In some embodiments, the monovalent vaccine comprises one antigen encoding one virus strain.

[0214] Multivalent / Combination Vaccines In some embodiments, the vaccine may comprise a nucleic acid construct, RNA or polynucleotide molecule, or a plurality of nucleic acid constructs, RNA or polynucleotide molecules, encoding two or more antigens of the same or different species. In some embodiments, the vaccine comprises an RNA or a plurality of 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.

[0215] In some embodiments, in the monovalent vaccine and the multivalent / combination vaccine, the antigen is a coronavirus antigen, for example, 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, for example, 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, for example, a SARS-COV-2 spike protein. In some embodiments, the SARS-COV-2 spike protein is selected from the spike protein of any viral strain, such as 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), or SARS-COV-2 Omicron (BA.4). In some embodiments, the SARS-COV-2 spike protein comprises or is an amino acid sequence set forth in any one of SEQ ID NOs: 12, 14, 21, 23, 25, 80, or 136, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto.In some embodiments, the DNA sequence encoding the SARS-COV-2 spike protein comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 13, 15, 20, 22, 24, 81, 97, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto. In some embodiments, the RNA sequence encoding the SARS-COV-2 spike protein comprises or is a nucleotide sequence set forth in SEQ ID NOs: 54-55, 59-61, 82, 130, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.

[0216] In some embodiments, the ORF encodes an influenza virus antigen. In some embodiments, the influenza virus is selected from influenza A virus or influenza B virus, and illustratively, the influenza virus is influenza A virus H1N1, influenza A virus H3N2, influenza A virus H3N8, influenza A virus H2N2, influenza A virus H5N1, influenza A virus H9N2, influenza A virus H7N7, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the influenza virus antigen is a structural protein of an influenza virus, such as hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein, matrix protein M1, nucleoprotein NP, etc. In some specific embodiments, the influenza virus antigen is an 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), or influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013). In some embodiments, the HA protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83-86, 98-101, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity thereto, or is any of the above sequences.In some embodiments, the DNA sequence encoding the HA protein comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 87-90, 93-97, 102-105, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In some embodiments, the RNA sequence encoding the HA protein comprises or is a nucleotide sequence set forth in any one of SEQ ID NOs: 126-129, 131-134, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0217] In some embodiments, two or more different RNAs (e.g., mRNAs) can be prepared in the same lipid nanoparticle. In other embodiments, two or more different RNAs can each be prepared in a single lipid nanoparticle, and the lipid nanoparticles can then be combined as a single vaccine composition (e.g., containing multiple RNAs encoding multiple antigens) or administered alone.

[0218] The present disclosure further provides multivalent / combination vaccines comprising RNA encoding one or more coronaviruses or antigens of one or more different organisms, i.e., the vaccine of the present disclosure may be a multivalent / combination vaccine targeting one or more antigens of the same strain / species or one or more antigens of different strains / species.

[0219] Delivery System The present disclosure further provides a delivery system comprising any one of the nucleic acid constructs described above, or any one of the RNA molecules described above, wherein the delivery vehicle is a cationic lipid delivery particle. In some embodiments, wherein the particle is a nanoparticle. In some embodiments, the delivery vehicle is a nanolipid particle. The RNA molecules of the present disclosure can be delivered intracellularly and / or in vivo using any type of nanolipid particle in the art, including, but not limited to, the lipid particles disclosed in WO2017075531, WO2018081480A1, WO2017049245A2, WO2017099823A1, WO2022245888A1, WO2022150717A1, CN101291653A, CN102119217A, WO2011000107A1, CN107028886A, the above patents are incorporated herein by reference in their entirety.

[0220] Pharmaceutical Compositions The present disclosure further provides a pharmaceutical composition comprising any one of the nucleic acid constructs described above, any one of the polynucleotides described above, any one of the vaccines described above, any one of the vectors described above, and / or any one of the delivery vehicles described above, and a pharma- ceutically acceptable carrier, diluent or excipient; specifically, the pharmaceutical composition is a solid formulation, an injectable formulation, an external formulation, a spray, a liquid formulation, or a combination formulation.

[0221] Product or Reagent Kit The present disclosure provides an article of manufacture or a reagent kit comprising any one of the nucleic acid constructs described above, any one of the polynucleotides described above, any one of the vaccines described above, any one of the vectors described above, any one of the delivery vehicles described above, and / or any one of the pharmaceutical compositions described above, which can be used to provide a related detection or diagnostic use.

[0222] Methods and pharmaceutical uses for treating and / or preventing diseases The present disclosure further provides a use in the preparation of a medicament for treating and / or preventing a disease, using a therapeutically and / or prophylactically effective amount of any one of the nucleic acid constructs described above, any one of the polynucleotides described above, any one of the vaccines described above, any one of the vectors described above, any one of the delivery vehicles described above, any one of the pharmaceutical compositions described above, and / or any one of the products or reagent kits described above, in a subject in need thereof. In some embodiments, the disease comprises a viral infectious disease or a respiratory disease associated with a viral infection. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a coronavirus that infects humans, 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 an influenza virus that infects humans, such as influenza A virus or influenza B virus. Exemplary influenza viruses include influenza A virus H1N1, influenza A virus H3N2, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the respiratory disease associated with the viral infection (e.g., respiratory disease associated with SARS-CoV-2 infection) includes uncomplicated infections such as fever, cough and sore throat, headache, rhinitis, 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.

[0223] The present disclosure further provides a method for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of any one of the nucleic acid constructs described above, the polynucleotide described above, the vaccine described above, the vector described above, the delivery vehicle described above, the pharmaceutical composition described above, and / or the product or reagent kit described above. In some embodiments, the disease comprises a viral infectious disease or a respiratory disease associated with a viral infection. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a coronavirus that infects humans, 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 an influenza virus that infects humans, such as influenza A virus or influenza B virus. Exemplary influenza viruses include influenza A virus H1N1, influenza A virus H3N2, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), etc. In some embodiments, the respiratory disease associated with the viral infection (e.g., respiratory disease associated with SARS-CoV-2 infection) includes uncomplicated infections such as fever, cough and sore throat, headache, rhinitis, 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.

[0224] The present disclosure further provides a method comprising administering to a subject in need thereof an effective amount of any one of the nucleic acid constructs described above, any one of the polynucleotides described above, any one of the vaccines described above, any one of the vectors described above, any one of the delivery vehicles described above, any one of the pharmaceutical compositions described above, and / or any one of the products or reagent kits described above, which can induce a neutralizing antibody response and / or a T cell immune response in the subject, for example, a neutralizing antibody response against a viral antigen, for example, a CD4+ and / or CD8+ T cell immune response against a viral antigen, wherein the antigen antibody titer of the subject is increased after vaccination, relative to the antigen antibody titer of the subject vaccinated with a prophylactically effective dose of a conventional vaccine against the antigen. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a coronavirus that infects humans, 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 an influenza virus that infects humans, such as an influenza A virus or an influenza B virus. Exemplary influenza viruses include influenza A virus H1N1, influenza A virus H3N2, influenza B virus / Victoria (e.g., Influenza B / Washington / 02 / 2019), influenza B virus / Yamagata (e.g., Influenza B / Phuket / 3073 / 2013), and the like.

[0225] In some embodiments, the subject has been immunized. In some embodiments, the subject has a pulmonary disease. In some embodiments, the subject is 5 years of age or younger, or 65 years of age or older.

[0226] In some embodiments, the method includes administering to a subject at least one, two, three, four, or more of the nucleic acid constructs described in any one of the above, the polynucleotides described in any one of the above, the vaccines described in any one of the above, the vectors described in any one of the above, the delivery vehicles described in any one of the above, the pharmaceutical compositions described in any one of the above, or the products or reagent kits described in any one of the above.

[0227] In some embodiments, a detectable level of a viral antigen (e.g., a coronavirus antigen) is produced in the serum of a subject 1 to 72 hours after administration of the nucleic acid construct, the polynucleotide, the vaccine, the vector, the delivery vehicle, the pharmaceutical composition, or the product or reagent kit of any one of the above. In some embodiments, a neutralizing antibody titer of at least 100 NU / mL, 200 NU / mL, 300 NU / mL, 400 NU / mL, 500 NU / mL, 600 NU / mL, 700 NU / mL, 800 NU / mL, 900 NU / mL, or 1000 NU / mL is produced in the serum of the subject 1 to 72 hours after administration.

[0228] In some embodiments, the antibody titer generated in the subject is increased by at least 1 log relative to the control. For example, the antibody titer generated in the subject can be increased by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 logs relative to the control.

[0229] In some embodiments, the antibody titer produced in the subject increases by at least 2-fold compared to the control. For example, the antibody titer produced in the subject increases by at least 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold compared to the control. In some embodiments, the geometric mean is the nth power of the product of n numbers and is commonly used to represent proportional growth. In some embodiments, the geometric mean is used to characterize the antibody titer produced in the subject. In some embodiments, the control may be an unvaccinated subject or a subject administered an attenuated live virus vaccine, an inactivated virus vaccine, or a protein subunit vaccine.

[0230] Therapeutic efficacy of vaccines In some embodiments, the antigen-specific immune response is characterized by measuring the titer of anti-(corona)virus antigen antibodies produced in the subject after administration of the above-described embodiment. The titer is a measurement of the amount of antibodies in the subject, e.g., antibodies specific to a particular antigen or epitope of the antigen. The titer is usually expressed as the reciprocal of the highest dilution that gives a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers.

[0231] In some embodiments, antibody titers are used to determine whether a subject is infected or needs immunization. In some embodiments, antibody titers are used to determine the strength of autoimmune responses, to determine whether booster vaccinations are required, to determine whether previous vaccines are effective, and to identify any recent or previous infections. According to the present disclosure, antibody titers can be used to determine the strength of the immune response induced in a subject from an immune composition (e.g., an RNA vaccine).

[0232] In some embodiments, the titer of anti-(coronavirus) antigen antibodies produced in a subject is increased by at least 1 log relative to a control. For example, the titer of antibodies produced in a subject can be increased by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 logs relative to a control.

[0233] In some embodiments, the titer of antibodies produced in a subject is increased by at least 2-fold relative to a control. For example, the titer of antibodies produced in a subject is increased by at least 3, 4, 5, 6, 7, 8, 9, or 10-fold relative to a control.

[0234] In some embodiments, the antigen-specific immune response is measured by the ratio of serum neutralizing antibody titers to the coronavirus geometric mean titer (GMT), referred to as the geometric mean ratio (GMR). The geometric mean titer (GMT) is the average antibody titer of a group of subjects, and is calculated by multiplying all the values ​​and taking the n-th root, where n is the number of subjects with useful data.

[0235] In some embodiments, the control may be an unvaccinated subject or a subject administered a live attenuated virus vaccine, an inactivated virus vaccine, or a protein subunit vaccine.

[0236] In some embodiments, vaccine efficacy can be assessed by standard analytical methods (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1, 201(11):1607-10). For example, vaccine efficacy can be measured by double-blind, randomized, clinically controlled trials. Vaccine efficacy can be expressed as the reduction in the proportion of disease incidence (AR) between unvaccinated (ARU) and vaccinated (ARV) study cohorts, and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formula:

[0237]

number

[0238] Effectiveness = (1-OR) x 100. [Brief description of the drawings]

[0239] [Figure 1] Schematic diagram of a plasmid template containing a T7 promoter, 5'UTR, 3'UTR, polyA tail and a foreign gene CDS sequence. [Diagram 2] This is a chip electrophoresis diagram of mRNA transcribed in vitro using a linearized plasmid as a template, in which L is an RNA molecular weight marker (RNA ladder) and 1 is a purified mRNA sample. [Diagram 3] Figure 1 shows the difference in the expression level of SAS-Cov-2 B.1.351 spike protein with different 5'UTR and 3'UTR combinations detected by ELISA, of which the abbreviations of 5'UTR and 3'UTR are shown in Table 3. [Figure 4]Figure 1 shows the difference in expression levels of SAS-Cov-2 B.1.1.7 spike protein with different 5'UTR and 3'UTR combinations detected by ELISA, of which the abbreviations of 5'UTR and 3'UTR are shown in Table 3. [Diagram 5] Schematic diagram of point mutation of 5'UTR I to 5'UTR I'. [Figure 6] A diagram showing the difference in expression level of SAS-Cov-2 B.1.617.2 spike protein before and after point mutation of 5'UTR I detected by Western Blot. Among them, the 5'UTR used in samples 1 and 3 is I, the 5'UTR used in samples 2 and 4 is I', the mRNA synthesis template of samples 1 and 2 is derived from a plasmid miniprep, the mRNA synthesis template of samples 3 and 4 is derived from a plasmid maxiprep, sample 5 is a non-transfected cell supernatant control, and M is a molecular weight marker. [Figure 6A] Western blot detection diagram. [Figure 6B] Fig. 7 is a graph showing the effect of 5'UTR I' on the expression levels of different heterologous genes detected by Western blot. [Figure 7A] is a Western blot detection diagram, [Figure 7B] 4 shows the relative values ​​of samples after quantification of Western blot detection data. [Figure 8] Luciferase expression mediated by 5'UTR I'-3'UTR B and 5'UTR I-3'UTR B compared to the BNT162b2 UTR combinations. [Figure 9] FIG. 1 shows the results of regulating luciferase expression by 5'UTR I' truncations and 5'UTR I' mutants. [Figure 10] This is a result of the difference in expression levels of HA mRNA under the regulation of 5'UTR I' and the control 5'UTR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0240] <Terminology> In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0241] The official classification name of the 2019 novel coronavirus (2019-nCoV) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0242] The official name of the disease caused by the 2019 novel coronavirus (2019-nCoV) is COVID-19.

[0243] "Nucleic acid" or "nucleotide" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleotide sequences that code for a given protein can be produced. The term nucleic acid may be used interchangeably with the term "polynucleotide". An "oligonucleotide" is a short-stranded nucleic acid molecule. A "primer" is an oligonucleotide, whether naturally occurring in a restriction enzyme digestion or produced synthetically, that can act as a synthesis initiator when placed under conditions that induce the synthesis of a primer extension product that is complementary to the nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at the appropriate temperature and pH). To maximize amplification efficiency, the primer is preferably single-stranded, but may also optionally be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is a deoxyribonucleotide. The primer must be sufficiently long to trigger the synthesis of an extension product in the presence of the inducing agent. The exact length of the primer is determined by many factors, including the temperature, the source of the primer and the method used.

[0244] "Vector" or "expression vector" refers to a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, that can be linked to another DNA segment, i.e., an "insert," to effect replication of the linked segment in a cell. A vector may be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral in origin and / or final form, such as the PUC57 DNA vector used herein. The term "vector" covers any genetic element that is capable of replicating and transferring genetic sequences to a cell when associated with the appropriate control elements. In some embodiments, a vector may be an expression vector or a recombinant vector.

[0245] "Promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter may be constitutive, inducible, repressible, tissue specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled.

[0246] "Gene" refers to a segment of DNA involved in the production of a polypeptide chain, which may or may not include preceding and following coding regions, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "untranscribed tail region" sequences, as well as inserted sequences (introns) between each coding segment (exon).

[0247] "Recombinant" refers to a polynucleotide that is the product of various combinations of cloning, restriction, or ligation steps, as well as other steps that result in a difference and / or different construction from naturally occurring polynucleotides.

[0248] "Introducing" means "transfection," "transformation," or "transduction" in the context of inserting a nucleic acid sequence into a cell, and includes the incorporation of a reference nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence may be integrated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), introduced into autonomous replication, or expressed transiently (e.g., transfection of mRNA).

[0249] "Nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule, such as a DNA fragment, that is modified or synthesized to contain a nucleic acid segment in a non-naturally occurring manner, said nucleic acid molecule comprising one or more control sequences or regulatory elements. In the context of the present disclosure, a nucleic acid construct comprises a recombinant nucleotide sequence, which consists essentially of, and optionally comprises, one, two, three or more isolated nucleotide sequences: a 5'UTR, an open reading frame (ORF) and a 3'UTR. In embodiments relating to a construct comprising two or more sequences, the sequences are operably linked to each other in the construct.

[0250] "Derived sequence" refers to a nucleotide sequence that is highly homologous to the UTR sequence of the present disclosure (e.g., has at least 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 100% identity to the UTR sequence of the present disclosure) and still retains the same or similar functional activity as the UTR sequence of the present disclosure. In some embodiments, the derived sequence is a nucleotide sequence based on the native UTR sequence, obtained by substitution, deletion, or addition of one or more nucleotides. In some embodiments, the derived sequence is a nucleotide sequence based on the native UTR sequence, obtained by truncation. In some embodiments, the derived sequence is a point mutation of ATG to GTG, CTG, or TTG based on the native UTR sequence to inhibit initiation of translation from the ATG within the UTR.

[0251] "Operably linked" is defined herein as the following structure: the control sequences, a promoter sequence and / or a 5'UTR sequence, are appropriately positioned relative to a coding DNA sequence such that the control sequences direct the transcription of the coding sequence and the translation of mRNA into a polypeptide sequence encoded by the coding DNA.

[0252] "Open reading frame" is abbreviated as "ORF" and refers to a segment or region of an mRNA molecule that encodes a polypeptide. An ORF contains consecutive, non-overlapping, in-frame codons, beginning with an initiation codon and ending with a termination codon, and is translated by the ribosome.

[0253] "Endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0254] "Exogenous" refers to any substance introduced or produced from outside an organism, cell, tissue, or system.

[0255] "Identical sequence" or "sequence identity" refers to sequence identity at the nucleotide or amino acid level between genes or proteins, respectively. "Identical sequence" or "sequence identity" is a measure of identity at the amino acid level between proteins and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates the percentage of sequence identity and statistically analyzes the similarity between two sequences. Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0256] "Homology" or "homology" is defined as the percentage of nucleotide residues that match the nucleotide residues in the corresponding sequence in the target chromosome after aligning the sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage. To determine the alignment of the homology percentage of nucleotide sequences, various methods within the skill of the art can be implemented, for example, by publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. In some specific embodiments, the present disclosure calculates the sequence homology percentage based on BLAST. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of a homologous arm is considered to be "homologous" if it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0257] A "substitution" is defined as a change in amino acid or nucleotide sequence that occurs by replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of a reference polypeptide. If the substitution is conservative, the amino acid substituted in the polypeptide has similar structure or chemical properties (e.g., charge, polarity, hydrophobicity, etc.) as the substituted amino acid. In some embodiments, a polypeptide variant may have "non-conservative" changes, in which the substituted amino acid differs in structure and / or chemical properties.

[0258] A "deletion" is defined as a change in an amino acid or nucleotide sequence such that one or more amino acids or nucleotide residues, respectively, are deleted compared to a reference polypeptide amino acid or nucleotide sequence. In the case of a polypeptide or polynucleotide sequence, a deletion may affect the deletion of 2, 5, 10, up to 20, up to 30, or up to 50 or more amino acids or nucleotide residues, taking into account the length of the modified polypeptide or polynucleotide sequence.

[0259] An "insertion" or "addition" refers to a change in an amino acid or nucleotide sequence, which change results in the addition of one or more amino acid or nucleotide residues, respectively, compared to the amino acid or nucleotide sequence of a reference polypeptide. An "insertion" usually refers to the addition of one or more amino acid residues (or nucleotide residues in a polynucleotide) to the amino acid sequence of a polypeptide, although an "addition" can also refer to an insertion or an amino acid residue added to the N- or C-terminus of a polypeptide (or a nucleotide residue added to the 5' or 3' terminus of a polynucleotide). In the case of a polypeptide or polynucleotide sequence, an insertion or addition can be up to 10, up to 20, up to 30, up to 50 or more amino acids (or nucleotide residues).

[0260] "Codon optimization" refers to the replacement of codons present in a target sequence that are generally rare in highly expressed genes of a given species with codons that are generally found in highly expressed genes of such species, but the codons before and after the replacement code for the same amino acid. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is usually associated with the translation efficiency of messenger RNA (mRNA), which is believed to depend, inter alia, on the properties of the translated codons and the availability of specific transport RNA (tRNA) molecules. The dominance of a selected tRNA in a cell is usually a reflection of the codons most commonly used in peptide synthesis. Thus, based on codon optimization, a gene can be modified for optimal gene expression in a given organism. Thus, the selection of optimal codons depends on the codon usage preferences of the host genome.

[0261] "Antigen-antibody" refers to a serum antibody that specifically binds to an antigen.

[0262] A "cell" or "host cell" includes any cell type susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or vector of the present disclosure. As non-limiting examples, a host cell may be an isolated primary cell, a pluripotent stem cell, a CD34+ cell, an induced pluripotent stem cell, or any one of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, a host cell may be an in situ or in vivo cell in a tissue, organ, or organism.

[0263] "Treatment" refers to, for example, administering an oral or topical therapeutic agent comprising a composition of any one of the nucleic acid constructs disclosed herein to a patient, the patient having one or more disease symptoms, the therapeutic agent being known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is administered an amount of therapeutic agent that effectively relieves one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, including the disease state, age and weight of the patient, and the ability of the drug to produce the required therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition.

[0264] An "effective amount" or "pharmaceutical effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as, for example, the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. In some embodiments, an "effective amount" is a dose of RNA effective to generate an antigen-specific immune response.

[0265] A "prophylactically effective dose" refers to a dose that is effective at a clinically acceptable level to prevent viral infection. In some embodiments, the effective dose is the dose listed in the instructions on the vaccine's packaging. As used herein, a conventional vaccine refers to a vaccine other than the mRNA vaccine of the present disclosure. For example, a conventional vaccine includes, but is not limited to, a live microbial vaccine, an inactivated microbial vaccine, a subunit vaccine, a protein antigen vaccine, a DNA vaccine, a virus-like particle (VLP) vaccine, and the like. In an exemplary embodiment, a conventional vaccine is a vaccine that has already been approved by a regulatory agency and / or registered by a national drug regulatory agency, such as the China Food and Drug Administration.

[0266] The term "pharmacologically acceptable" means that the therapeutic agents, materials, compositions and / or dosage forms are, within the scope of reasonable medical judgment, suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response or other problem or complication, and are effective for the desired use, having a reasonable benefit / risk ratio.

[0267] "Monovalent vaccine" refers to a vaccine product for immunization made with one serotype antigen of a pathogenic organism, for example, a vaccine that includes only one antigen of the SARS-CoV-2 strain of the present disclosure is a monovalent vaccine.

[0268] "Multivalent vaccine" refers to a vaccine product for immunization made with multiple serotype antigens of the same pathogenic organism. For example, a vaccine made with a mixture containing an antigen of a SARS-CoV-2 variant and one or more antigens of other different subtypes of SARS-CoV-2 variants is a multivalent vaccine.

[0269] A "mixed vaccine" refers to a vaccine product for immunization made with multiple serotype antigens of different pathogenic organisms. For example, a vaccine made by mixing several pathogenic microorganisms capable of preventing and / or treating SARS-CoV-2 strain antigens and other viruses disclosed herein is a mixed vaccine. A mixed vaccine is generally prepared by preparing a monovalent vaccine by the same preparation method and then mixing them into the same injection for immunization, or by mixing antigens of several pathogenic microorganisms and then preparing them as a vaccine product. In some specific embodiments, the present disclosure first mixes antigens of pathogenic microorganisms and then prepares them as a vaccine product. Mixed, multivalent vaccines are a future development trend, and their advantages include (1) reducing the cost of vaccination, (2) reducing the cost of packaging, logistics and assembly of each of multiple single vaccines, (3) reducing injury to recipients, especially infants, (4) increasing the coverage rate of vaccination, (5) saving the time of vaccination, and (6) reducing the storage space of vaccines. Combination or polyvalent vaccines can reduce direct and indirect costs, including the need for multiple visits and missed doses. For example, the currently used polyvalent combination vaccine contains pneumococcal, meningococcal, poliovirus, rotavirus, influenza, and HPV vaccines.

[0270] An "immune response" of the vaccine of this disclosure refers to a humoral and / or cellular immune response that a subject mounts against the viral protein(s) (e.g., coronavirus) present in the vaccine. In the context of this disclosure, a "humoral" immune response refers to an immune response mediated by antibody molecules, including, for example, secretory IgA or IgG molecules, while a "cellular" immune response refers to an immune response mediated by T lymphocytes (e.g., CD4+ helper and / or CD8+ T cells (e.g., CTLs) and / or other white blood cells. One important aspect of cellular immunity relates to the antigen-specific response of cytolytic T cells (CTLs). CTLs are specific for peptide antigens, which bind to proteins encoded by the major histocompatibility complex (MHC). Together, these CTLs are expressed on the cell surface and help induce and promote the destruction of intracellular microorganisms and the lysis of cells infected with these microorganisms. Another aspect of cellular immunity relates to the antigen-specific response of helper T cells, whose role is to stimulate the function and focus the activity of non-specific effector cells on cells that have peptide antigens associated with MHC molecules. The cellular immune response also results in the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes.

[0271] <Example> The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as cell culture manuals, molecular cloning manuals, or conditions proposed by raw material or product manufacturers. Reagents for which a specific source is not specified are standard commercially available reagents.

[0272] The following typical experimental procedures 1) to 4) are given as examples: 1) Production of mRNA To produce in vitro transcribed mRNA, the plasmid was linearized downstream of the polyadenylic acid tail by BspQ 1 (Vazyme, DD4302-PC, China) and purified by PCR purification reagent kit (QIAGEN, 28106, Germany). The purified linearized plasmid was used as a template to perform in vitro transcription by T7 in vitro transcription reagent kit (Invitrogen, AM1333, USA). The mRNA synthesized by in vitro transcription was purified by MEGAclear® Kit (Invitrogen, AM1908, USA) to obtain relatively high purity mRNA for subsequent experiments such as in vitro cell transfection.

[0273] 2) Chip electrophoresis To verify the integrity and purity of the mRNA obtained by in vitro transcription, the mRNA was analyzed using a chip electrophoresis instrument (Agilent 2100, USA) and an RNA 6000 Nano reagent kit (Agilent, 5067-1511, USA). The experimental method was specifically as follows: the mRNA to be measured was denatured (incubated at 70°C for 2 min, then quickly placed on ice), and the treated samples were spotted in order into the corresponding wells of the chip, after which the instrument was operated and the analysis results were waited for.

[0274] 3) Cell culture Materials: FBS (Gibco), DMEM medium (Gibco), Opti-MEM (Gibco), PBS (Gibco), Trypsin-EDTA (Gibco), double antibodies (Pen / Strep, Gibco) and Lipofectamine. TM 2000 (Invitrogen, 52887). Human embryonic kidney cells (293T, ATCC) were grown in DMEM medium supplemented with 10% FBS and 1% double antibody and in a humidified gas environment of 5% CO2.

[0275] 4) In vitro transfection Eight hours before transfection, 8 × 105 Cells were seeded into 12-well cell culture plates at 100 cells / well using the commercially available transfection reagent Lipofectamine. TM 2.5 µL of Lipofectamine 2000 per 1 µg of mRNA TM The cells were transfected with a mixture ratio of Lipofectamine 2000 and 2000, and the transfection dose of mRNA in each well was 2 μg. The experimental method is specifically as follows: TM 2000 and mRNA were each diluted in Opti-MEM medium to a final total volume of 100 μL each, and then lysed with Lipofectamine TM The 2000 solution and the mRNA solution were mixed and incubated under room temperature conditions for 10-15 minutes to form liposome complexes containing mRNA. After removing the DMEM medium in the cell culture well plate, the liposome complexes containing different mRNA were added to the corresponding well plate, and 400 μL of Opti-MEM medium containing 10% FBS was supplemented to each well, and the cells were cultured at 37 °C (5% CO2 level) for 4-6 hours. Then, the transfection medium was removed, and 1 mL of DMEM medium supplemented with 10% FBS and 1% double antibody was added to each cell culture well plate, and the cells were cultured at 37 °C (5% CO2 level) for 48 hours. At the same time, the liposome complexes containing no mRNA were added according to the above method. TM The 2000 solution was transfected into some cells as a negative control.

[0276] Among them, the target gene sequences used in the present disclosure are summarized as shown in Table 1.

[0277] [Table 3-1] [Table 3-2]

[0278] The mutation of the amino acids KV to PP in the SARS-COV-2 spike protein changes the spike protein from an unstable prefusion conformation to a stable postfusion conformation (Structure-based design of prefusion-stabilized SARS-CoV-2 spikes, Science, VOL.369, NO.6510), making the spike protein conformationally stable, which is advantageous for designing and producing vaccines using the spike protein as an immunogen.

[0279] [Example 1, Screening and preparation of 5'UTR sequences] 1. UTR screening In order to obtain a regulatory element capable of highly expressing a target protein in dendritic cells (DCs), this example obtained a UTR with a new structure through a four-stage screening.

[0280] The method includes: First, a high protein expression gene library was established, including genes with high protein expression in monocytes, genes with higher protein expression in DCs than in monocytes, genes with high protein levels, and genes with UTRs that increase protein expression according to the test, and 2140 genes were obtained. Second, a UTR library was established, and all sequences in the gene library with high protein expression were downloaded from the Genbank database (https: / / www.ncbi.nlm.nih.gov / genbank / ), and then the completeness was first analyzed, and then the merging was repeated, and the UTR data was obtained by methods such as CDS (coding sequence) truncation. 940 UTRs were obtained. 3: Establish a UTR selection library, analyze the UTR library, and screen for UTR sequences with suitable sequence length and free energy, among which the 5'UTR sequence length is between 40 and 70, the 3'UTR length is between 100 and 150 or between 300 and 400, predict the free energy for each sequence, select sequences with relatively high 5'UTR free energy, and select sequences with relatively low 3'UTR free energy, and obtain 64 UTRs from the selection library. 4: Determine the final candidate sequences, and use the screening strategy of 5'UTR free energy above -10 and 3'UTR free energy very low, and finally determine 20 5'UTR and 3'UTR for screening and intracellular validation.

[0281] Based on the above, it is believed that the 5'UTR sequence derived from human Rho GTPase activating protein 15 (abbreviated as ARHGAP15) (designated 5'UTR I, the DNA sequence of which is shown in SEQ ID NO: 1 and the RNA sequence of which is shown in SEQ ID NO: 45) may efficiently express target proteins in human cells (particularly DCs).

[0282] 2. Preparation of UTR This example illustrates the process of preparing mRNA containing 5'UTR I. First, a sequence containing 5'UTR, 3'UTR, polyA tail and foreign target gene CDS is artificially synthesized, and then inserted into a pUC57 vector to prepare a template vector, the schematic diagram of which is shown in Figure 1. Sequencing shows that the target gene sequence inserted into the vector is accurate.

[0283] As shown in the pUC57 Delta13 vector in Figure 1, the 5'UTR is sequence number 1, the 3'UTR is the human hemoglobin β subunit (HBB) 3'UTR sequence (sequence number 7, designated as 3'UTR B), the CDS sequence is a codon-optimized base sequence encoding the B.1.617.2 (Delta) spike protein (sequence number 13), the polyA tail is a polyadenylic acid sequence (the DNA sequence is shown in sequence number 16, and the RNA sequence is shown in sequence number 56), and the T7 promoter (sequence number 17).

[0284] To produce in vitro transcribed mRNA, the plasmid was linearized downstream of the polyadenylic acid tail by BspQ I (Vazyme, DD4302-PC, China) and purified by PCR purification reagent kit (QIAGEN, 28106, Germany). Using the purified linearized plasmid as a template, in vitro transcription was performed to synthesize mRNA according to the following steps. A 100 μL mRNA reaction system was synthesized, and the reaction system is shown in Table 2. In the reaction system, the linearized template was 2 μg, and the reaction was supplemented with nucleosidase-free water to 100 μL, reacted at 37 ° C for 4 hours, and digested with DNase I for 30 min. Detection showed that the synthesized mRNA had less by-product dsRNA and excellent production. The capping process was achieved in the in vitro transcription synthesis process by adopting a chemical capping method, and the cap structure was m7G(5')ppp(5')(2'OMeA)pG·NH4 (Hongene, ON~134). It was purified with MEGAclear® Kit (Invitrogen, AM1908, USA) to obtain relatively high purity mRNA for subsequent experiments such as in vitro cell transfection.

[0285] [Table 4-1] [Table 4-2]

[0286] After repeated screening and functional validation, the present disclosure has obtained a 5'UTR I that has the potential to efficiently express a target protein, the sequence of which is SEQ ID NO: 1, and a construct containing 5'UTR I can efficiently transcribe the corresponding mRNA, which was identified by chip electrophoresis to be consistent with the theoretical size (shown in FIG. 2).

[0287] [Example 2, Screening of different combinations of 5'UTR and 3'UTR for efficient expression of heterologous proteins] In this example, combinations of different 5'UTRs and different 3'UTRs were screened to obtain combinations of UTRs that can efficiently express a target gene.

[0288] The method was to artificially synthesize a plasmid containing a combination of different UTRs of the CDS of the same target gene. A basic plasmid was constructed according to the method of Example 1, and the target gene CDS sequence was recombined and replaced by artificial synthesis and HindIII / XhoI enzyme cleavage. The newly constructed plasmid was linearized according to the method of Example 1, and mRNA was synthesized and purified. Lipofectamine TM The mRNA was transfected into 293T cells using 2000, and 48 h later, the 293T supernatant was collected and subjected to Western blot and ELISA detection, respectively, to detect the level of spike protein in the secreted cell supernatant.

[0289] The target gene used is a gene encoding the novel coronavirus B.1.351 spike protein (CDS is shown in SEQ ID NO: 20) or a gene encoding the novel coronavirus B.1.1.7 spike protein (CDS is shown in SEQ ID NO: 22). The information of the different 5'UTR and 3'UTR used and their combination methods are shown in Tables 3 and 4.

[0290] Western blot method: To detect the in vitro expression level of mRNA obtained from each construct, the cell supernatant in the well plate was collected after transfection was completed, and then subjected to polyacrylamide gel electrophoresis, membrane transfer, incubation with primary and secondary antibodies for color development, and finally the in vitro expression level of mRNA obtained from each construct was obtained. Among them, the primary antibody was SARS-CoV-2 (2019-nCoV) spike RBD antibody (Sino Biology, 40592-T62), and the secondary antibody was HRP-linked anti-rabbit IgG (Transgene, HS101).

[0291] ELISA method: After transfection was completed, the corresponding cell supernatants were collected and the cell supernatants were diluted at a ratio of 1:50 to 1:100, then an ELISA reagent kit (SARS-CoV-2 (2019-nCoV) Spike Detection ELISA Reagent Kit, KIT40591, Sino Biology) was used to detect the in vitro expression level of mRNA of each construct, and the absorbance value at 450 nm was read to calculate the concentration of the relevant antigen.

[0292] [Table 5] [Table 6-1] [Table 6-2]

[0293] In Table 4, A, G, I and J of the 5'UTR are represented by SEQ ID NOs: 4, 3, 1 and 5, respectively, and B, E, D and F of the 3'UTR are represented by SEQ ID NOs: 7, 9, 10 and 8, respectively.

[0294] The effects of different 5'UTR and 3'UTR combinations on the expression of COVID-19 B.1.351 spike protein and COVID-19 B.1.1.7 spike protein are shown in Figure 3 and Table 5, and Figure 4 and Table 6, respectively.

[0295] [Table 7] [Table 8]

[0296] As can be seen from Figure 3 and Table 5, the comparison of the expression level of B.1.351 spike protein mediated by different UTR combinations is as follows: IB>AB>GB, IB>IE. For 5'UTR, I is superior to A and G, and for the related combination of I, IB is superior to IE. As can be seen from Figure 4 and Table 6, the comparison of the expression level of B.1.1.7 spike protein mediated by different UTR combinations is as follows: IF>IB>ID>IE. The results of the expression levels of B.1.351 spike and B.1.1.7 spike proteins were analyzed together, and the result showed that the UTR combination of IB has a more significant advantage and can mediate efficient expression of target genes, and the combination of IF also shows the advantage of high-level expression of target genes.

[0297] Furthermore, the inventors prepared B.1.617.2 spike mRNA containing IB or IF regulation, and performed cell transfection and ELISA detection. The detection results for the expression level of spike protein are shown in Table 7.

[0298] [Table 9]

[0299] Unlike the 5'UTR and 3'UTR in the IB combination, which are derived from different genes, the 5'UTR and 3'UTR in the IF combination are derived from the same gene, ARHGAP15. The results in Table 7 show that the combinations of IB (SEQ ID NO: 1 and SEQ ID NO: 7) and IF (SEQ ID NO: 1 and SEQ ID NO: 8) have comparable levels of regulation of target gene expression, further demonstrating the versatility of 5'UTR I, which can be combined with different 3'UTRs to mediate efficient expression of target genes (e.g., genes encoding spike proteins).

[0300] [Example 3, sequence modification of 5'UTR I and initial function verification] 1) Bioinformatics analysis and mutation design 5'UTR I (SEQ ID NO: 1) is a naturally occurring human Rho GTPase activating protein family promoter. The experimental data in Examples 1-2 show that 5'UTR I assists in the efficient expression of the novel coronavirus spike protein, but because 5'UTR I contains one ATG, it may express one 27aa short peptide, affecting the expression of normal target gene proteins. In order to avoid the phenomenon in which ribosomes start translation from the ATG inside the UTR, increase the probability of translation from the ATG in the CDS region, and increase the expression level, the present disclosure performed a point mutation (A → G) to obtain a mutant 5'UTRI' (the DNA sequence is shown in SEQ ID NO: 2 and the RNA sequence is shown in SEQ ID NO: 46). The mutation position is shown in FIG. 5.

[0301] 2) Point mutation of 5'UTR I to 5'UTR I' can effectively increase the expression of the target gene. Using the method of Example 1, a plasmid containing 5'UTR IB and 5'UTR I'-B was prepared to regulate the expression of a target gene, the target gene being a gene encoding B.1.617.2 spike protein (SEQ ID NO: 13), the plasmids were named Delta13 and Delta24, transcribed and synthesized by PCR, and purified to obtain mRNA (the uncapped I'-Delta24-B mRNA sequence is shown in SEQ ID NO: 57), and the prepared mRNA was transfected into 293T cells, and the Western blot method in Example 2 was used to detect the expression of B.1.617.2 spike protein. In addition, a grayscale scan was performed using Image software to calculate the grayscale value, and the expression amount of sample 1 (Delta13) was set as 1, and the ratio in Table 8 was obtained after normalization.

[0302] The results are shown in Figures 6A and 6B and Table 8. As can be seen from the results, the expression level of spike protein in the construct of 5'UTR I' after point mutation is higher than that of the construct containing 5'UTR I, whether the mRNA was prepared using the plasmid miniprep (endotoxin not removed) as a template or the plasmid maxiprep (endotoxin removed) as a template. Therefore, 5'UTR I effectively improved the expression of heterologous protein by the point mutation modification in Example 3.

[0303] [Table 10]

[0304] [Example 4, Detection of Different Target Gene mRNA Expressions upon Regulation of 5'UTR I'] The target gene was replaced by enzyme cleavage to construct a plasmid encoding the B.1.617.2 (Delta) spike protein gene (SEQ ID NO: 13) and the B.1.1.529 (Omicron) spike protein gene (SEQ ID NO: 15), respectively, while retaining the 5'UTR I' and 3'UTR B. The target gene sequences contained in the above plasmids are SEQ ID NO: 13 and SEQ ID NO: 15, respectively, which are codon-optimized sequences and encode the target protein sequences shown in SEQ ID NO: 12 and SEQ ID NO: 14. The mRNA was prepared and purified using the method of Example 1, and then transfected with Lipofectamine. TM2 μg of mRNA was transfected into 293T cells by 2000, and the cell supernatant was collected after 2 days. The Western blotting method in Example 2 was adopted to detect the expression status of B.1.617.2 spike protein mRNA (named Delta13) under the regulation of IB, B.1.617.2 spike protein mRNA (named Delta24) under the regulation of I'-B, and B.1.1.529 spike protein mRNA (named Omicron24) under the regulation of I'-B, among which the sequences of Delta24 mRNA and Omicron24 mRNA without cap structure are shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively. Grayscale scan was performed by Image software, grayscale value was calculated, and the expression amount of Delta13 was set as 1, and normalization was performed, and the ratio in Table 9 was obtained.

[0305] Figures 7A and 7B show that the novel coronavirus genes B.1.617.2 and B.1.1.529 can both be efficiently expressed by regulating the combination of I'-B UTRs. This suggests that 5'UTR I' has the versatility to initiate efficient expression of different target genes. Comparing 5'UTR I and I', the expression level of Delta24 spike protein is obviously higher than that of Delta13 spike protein, suggesting that the point mutation modification of 5'UTR I' effectively improves the protein expression efficiency, thereby further confirming the conclusion of Example 3.

[0306] [Table 11]

[0307] [Example 5, Detection of luciferase expression level upon regulation of I'-B UTR combination] A vector containing a luciferase CDS sequence (SEQ ID NO: 26) was constructed, and it contained the same T7 promoter (SEQ ID NO: 17) and polyA tail as in the above-mentioned COVID-19 gene-containing vector. Three vectors with different UTRs, Luc, Luc13 and Luc24, were constructed, and the UTRs contained in the vectors are shown in Table 10. Among them, the combination of 5'UTR and 3'UTR of BNT162b2 in Luc was used as a control, which is derived from CN113521269A. In the same manner as in Example 1, in vitro transcription was performed to synthesize mRNA containing a cap structure, and in vitro transfection was performed, and the supernatant was collected, and then luciferase substrate was added for detection, and the plate was read, and the results are shown in Table 11 and Figure 8.

[0308] [Table 12] [Table 13]

[0309] As a result, the expression level of luciferase protein in the regulation of the point mutated 5'UTR I' is higher than that of 5'UTR I. Compared with the combination of 5'UTR and 3'UTR of BNT162b2 in the Luc control, the combination of IB of the present disclosure increases the expression level of the target gene, and the combination of I'-B significantly improves the expression level of the target gene. In addition, the above detection results of luciferase as the target gene suggest that either 5'UTR I or 5'UTR I' can universally regulate the efficient expression of any target protein.

[0310] [Example 6: Detection of luciferase expression level under regulation of 5'UTR I' truncations and mutants] 6.1 The 5' end of 5'UTR I' was sequentially truncated, and the regulatory efficiency of 5'UTR I' truncations of different lengths on protein expression was analyzed by bioinformatics methods (Nat Biotechnol. 2019 Jul, 37(7):803-809.). The relative MRL value of the 5'UTR I' truncation to 5'UTR I' was calculated, and the results are shown in Table 12 below. As can be seen from the results in Table 12, the sequential truncation of the 5'-end nucleotide sequence in 5'UTR I' did not cause a significant decrease in the MRL value, indicating that the 5'UTR I' truncation can retain the functional activity of regulating the expression of the target protein. The 3'-end nucleotide sequence of the 5'UTR I' truncation shown in SEQ ID NO: 153 was sequentially truncated, and the relative MRL value of the truncation after 3'-end truncation to the sequence shown in SEQ ID NO: 153 was calculated, and the results are shown in Table 13. As can be seen from Table 13, truncation of the 3' end did not significantly affect the regulatory efficiency of the 5'UTR I' truncation.

[0311] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 15-1] [Table 15-2]

[0312] 6.2 16 5'UTR I' truncations of different lengths (SEQ ID NO:28-40, CTATAAT) and 2 A→C, A→T point mutations (SEQ ID NO:42-43) different from A→G point mutation were constructed and synthesized by Suzhou GENEWIZ Company. The 16 synthetic sequences were then enzymatically cleaved with HindIII and ScaI and ligated into Luc 24 in Example 5 to obtain the corresponding luciferase 5'UTR I' truncations and point mutation constructs, which were named LUC1-16. The corresponding 5'UTR I' truncations (1-I'-14-I') and mutants (15-I'-16-I') sequences are shown in SEQ ID NO:63-78. The mRNA containing the cap structure was synthesized by in vitro transcription in the same manner as in Example 1, and after in vitro transfection into 293T cells for 24 hours, luciferase substrate was added for detection, and the plate was read. The results are shown in Figure 9. The expression levels of luciferase mediated by Luc24 (I'-B combination) and its 5'UTR I' truncations and mutants are all significantly higher than Luc (5' and 3'UTR of BNT162b2). The 5'UTR I' truncations can retain the activity of 5'UTR I' regulating target protein expression, and some of the 5'UTR I' truncations (1-I', 2-I', 3-I', 4-I', 5-I', 6-I', 7-I', 9-I', 10-I', 11-I', 13-I') have improved activity of regulating target protein expression. At the same time, it has been shown that 5'UTR I' mutants 15-I' and 16-I' have superior target gene expression levels to 5'UTR I', suggesting that truncation of the 5' end of 5'UTR I' or point mutations within it can further enhance the functional activity of 5'UTR regulating target gene expression.

[0313] Example 7: Differences in influenza HA mRNA expression levels with control 5'UTR and 5'UTR I' regulation The target gene was replaced by enzymatic cleavage, and plasmids encoding the influenza A virus H1N1 (A / Wisconsin / 588 / 2019) HA protein gene (sequence number 83), the influenza A virus H3N2 (A / Cambodia / e0826360 / 2020) HA protein gene (sequence number 84), the influenza B virus (Washington / 02 / 2019) HA protein gene (sequence number 85), and the influenza B virus (Phuket / 3073 / 2013) HA protein gene (sequence number 86) were constructed while retaining the 5'UTR I' and 3'UTR B. The target gene sequences contained in the above plasmids are SEQ ID NOs: 87 to 90, respectively, and all of them are codon-optimized sequences. By referring to the 5' and 3' UTR sequences (SEQ ID NOs: 91-92) of patents WO2022 / 245888A1 and WO2022 / 150717A1, four HA CDS sequences, SEQ ID NOs: 93-96, were synthesized, which encode SEQ ID NOs: 83-86. The method of Example 1 was used to prepare and purify mRNA, and the mRNA was then purified using Lipofectamine. TM 2000, 1μg of mRNA was transfected into 293T cells, and cell lysates were collected after 1d. Western blotting in Example 2 was used to detect the expression status of the four influenza HA protein mRNAs under the regulation of I'-B, in which influenza A virus Hemagglutinin / HA antibody (Sino Biology, 86001-RM01) and influenza B virus Hemagglutinin / HA antibody (Sino Biology, 11053-R004) were used as the primary antibody, and HRP-linked anti-rabbit IgG (Transgene, HS101) was used as the secondary antibody. As a result, as shown in Figures 10 and 11, the expression levels of the four influenza HAs under the regulation of 5'UTR-I' are obviously higher than those of the control 5'UTR.

[0314] Some of the sequences of this disclosure are as follows:

[0315] >5'UTR I(63bp) [ka] >5'UTR I'(63bp) [ka] >5'UTR G [ka] >5'UTR A [ka] >5'UTR J [ka] >5'UTR BNT162b2 [ka] >3'UTR B [ka] >3'UTR F [ka] >3'UTR E [ka] >3'UTR D [ka] >3'UTR BNT162b2 [ka] >B.1.617.2(Delta) spike protein amino acid sequence [ka] >B.1.617.2(Delta) spike protein CDS [ka] [ka] [ka] >B.1.1.529 (Omicron) spike protein amino acid sequence [ka] >B.1.1.529 (Omicron) spike protein CDS [ka] [ka] [ka] >poly A [ka] >T7 promoter [ka] >I'-Delta24-B [ka] [ka] [ka] >I'-OC24-B [ka] [ka] [ka] >B.1.351 spike protein CDS [ka] [ka] [ka] >B.1.351 spike protein amino acid sequence [ka] >B.1.1.7 spike protein CDS [ka] [ka] [ka] >B.1.1.7 Spike protein amino acid sequence [ka] >SARS-COV-2 spike protein CDS [ka] [ka] [ka] >SARS-COV-2 spike protein amino acid sequence [ka] >Luciferase CDS [ka] [ka] >Luciferase amino acid sequence

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[0316] >5'UTR I(63bp) [ka] >5'UTR I'(63bp) [ka] >5'UTR G [ka] >5'UTR A [ka] >5'UTR J [ka] >3'UTR B [ka] >3'UTR F [ka] >3'UTR E [ka] >3'UTR D [ka] >B.1.617.2 (Delta) spike protein mRNA [ka] [ka] [ka] >B.1.1.529 (Omicron) spike protein mRNA [ka] [ka] [ka] >poly A [ka] >I'-Delta24-B mRNA [ka] [ka] [ka] >I'-OC24-B mRNA [ka] [ka] [ka] >B.1.351 spike protein mRNA [ka] [ka] [ka] >B.1.1.7 spike protein mRNA [ka] [ka] [ka] >SARS-COV-2 spike protein mRNA [ka] [ka] [ka] >Luciferase mRNA [ka] [ka] >LUC1-I' truncation [ka] >LUC2-I' truncation [ka] >LUC3-I' truncation [ka] >LUC4-I' truncation [ka] >LUC5-I' truncation [ka] >LUC6-I' truncated form [ka] >LUC7-I' truncated form [ka] >LUC8-I' truncation [ka] >LUC9-I' truncation [ka] >LUC10-I' truncated body [ka] >LUC11-I' truncation [ka] >LUC12-I' truncated form [ka] >LUC13-I' truncation [ka] >LUC14-I' truncation [ka] >LUC15-I' mutant [ka] >LUC16-I' mutant [ka] >5'UTR I general formula [ka] > Wild-type SARS-COV-2 spike amino acid sequence [ka] > Wild-type SARS-COV-2 spike protein CDS (NC_045512.2) [ka] [ka] [ka] >Wild type SARS-COV-2 spike protein mRNA [ka] [ka] [ka] >Influenza A virus H1N1 (A / Wisconsin / 588 / 2019) HA amino acid sequence [ka] >Influenza A virus H3N2 (A / Cambodia / e0826360 / 2020) HA amino acid sequence [ka] >Influenza B virus Victoria lineage (B / Washington / 02 / 2019) HA amino acid sequence [ka] >Influenza B virus Yamagata lineage (B / Phuket / 3073 / 2013) HA amino acid sequence [ka] > Influenza A virus H1N1 (A / Wisconsin / 588 / 2019) HA CDS [ka] [ka] > Influenza A virus H3N2 (A / Cambodia / e0826360 / 2020) HA CDS [ka] [ka] >Influenza B virus Victoria lineage (B / Washington / 02 / 2019) HA CDS [ka] [ka] > Influenza B virus / Yamagata lineage (B / Phuket / 3073 / 2013) HA CDS [ka] [ka] >5'UTR-Moderna [ka] >3'UTR-Moderna [ka] > Influenza A virus H1N1 (A / Wisconsin / 588 / 2019) HA Moderna [ka] [ka] > Influenza A virus H3N2 (A / Cambodia / e0826360 / 2020) HA Moderna [ka] [ka] >Influenza B virus Victoria lineage (B / Washington / 02 / 2019) HA CDS Moderna [ka] [ka] >Influenza B virus Yamagata lineage (B / Phuket / 3073 / 2013) HA CDS Moderna [ka] [ka] >SARS-COV-2 BA.4 spike protein CDS [ka] [ka] [ka] >Amino acid sequence of influenza A virus H1N1 HA0 variant [ka] >Amino acid sequence of influenza A virus H3N2 HA0 mutant [ka] >Influenza B virus / Victoria HA0 variant amino acid sequence [ka] >Influenza B virus / Yamagata HA0 mutant amino acid sequence [ka] > Influenza A virus H1N1 HA0 mutant CDS [ka] [ka] > Influenza A virus H3N2 HA0 mutant CDS [ka] [ka] >Influenza B virus / Victoria HA0 variant CDS [ka] [ka] >Influenza B virus / Yamagata HA0 mutant CDS [ka] [ka] >I'-Delta24-B-120A mRNA

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Claims

1. A nucleic acid construct comprising: (a) an open reading frame (ORF); (b) a 5' untranslated region element (5'UTR); wherein the 5'UTR is derived from the ARHGAP gene; Preferably, the ARHGAP is ARHGAP15, More preferably, the 5'UTR comprises the nucleotide sequence set forth in SEQ ID NO: 44 or a truncation thereof. Nucleic acid constructs.

2. the 5'UTR comprises at least one point mutation that can be used to inhibit initiation of translation from an ATG within the UTR; Preferably, the point mutation is selected from any one or more of A, T, or G of the ATG sequence in the 5'UTR; More preferably, the mutation is an A to G, C or T. The nucleic acid construct of claim 1.

3. the 5'UTR truncation still retains a function of regulating expression of a protein encoded by the ORF, or the 5'UTR truncation has an enhanced function of regulating expression of a protein encoded by the ORF compared to the native 5'UTR; Preferably, the cleavage method of the 5'UTR truncate includes deleting a consecutive nucleotide sequence at the 5' end in the sequence direction from the 5' to the 3' end, and / or deleting a consecutive nucleotide sequence at the 3' end in the sequence direction from the 3' to the 5' end. A nucleic acid construct according to claim 1 or 2.

4. The 5'UTR truncation product is formed by deleting a consecutive nucleotide sequence at the 5' end in the 5' to 3' direction and / or by deleting a consecutive nucleotide sequence at the 3' end in the 3' to 5' direction, Preferably, the 5'UTR truncation comprises at least 5 consecutive nucleotides of the sequence shown in SEQ ID NO: 44, more preferably at least 7 consecutive nucleotides of the sequence shown in SEQ ID NO: 2; Preferably, the 5'-terminal nucleotide of the 5'UTR truncation is a nucleotide at any one of positions 1 to 57 in the sequence shown in SEQ ID NO:44, and more preferably a nucleotide at any one of positions 1 to 13 or 17 to 29 in the sequence shown in SEQ ID NO:2, in terms of natural counting; Preferably, the 5'UTR truncation comprises CTATAAT or the nucleotide sequence set forth in SEQ ID NO:

193. A nucleic acid construct according to any one of claims 1 to 3.

5. The 5'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 2, 28-43, 76, 137-196, or comprises CTATAAT, or comprises a nucleotide sequence having at least 80% identity to any one of the sequences; A nucleic acid construct according to any one of claims 1 to 3.

6. (c) further comprising a 3' untranslated region element (3'UTR); Preferably, the 3'UTR comprises a 3'UTR derived from any one of the HBB, ARHGAP, CORO1A or HPX genes; More preferably, the 3'UTR comprises a 3'UTR derived from ARHGAP15. A nucleic acid construct according to any one of claims 1 to 5.

7. The 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 7 to 10, or the 3'UTR comprises a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 7 to 10; The nucleic acid construct of claim 6.

8. (d) further comprising a polyadenylic acid (poly-A) tail; A nucleic acid construct according to any one of claims 1 to 7.

9. the poly-A tail is selected from HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40late polyA; Preferably, the poly-A tail comprises a nucleotide sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 135, or having at least 80% identity thereto. The nucleic acid construct of claim 8.

10. the ORF encodes a viral antigen, preferably the ORF encodes a coronavirus antigen or an influenza virus antigen; The coronavirus is preferably SARS-COV-2 and the coronavirus antigen is preferably the spike protein; 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; The influenza virus is preferably an influenza A virus or an influenza B virus, and the antigen is a hemagglutinin protein (HA) and / or a ceramidase (NA); More preferably, the influenza virus antigen is selected from the hemagglutinin protein and / or neuraminidase of any of the following influenza virus strains: influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, and influenza B virus Yamagata. A nucleic acid construct according to any one of claims 1 to 9.

11. The polypeptide sequence encoded by the ORF comprises any one of the amino acid sequences set forth in SEQ ID NOs: 12, 14, 21, 23, 25, 80, 83-86, 98-101, and 136; Preferably, the ORF comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 13, 15, 20, 22, 24, 81, 87-90, 97, 102-105, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 13, 15, 20, 22, 24, 81, 87-90, 97, 102-105; A nucleic acid construct according to any one of claims 1 to 10.

12. Contains the nucleotide sequence shown in SEQ ID NO: 18-19 or a nucleotide sequence having at least 80% identity thereto; A nucleic acid construct described in any one of the preceding claims.

13. 1. An RNA molecule comprising: (a) an open reading frame (ORF); (b) a 5' untranslated region element (5'UTR); and (c) a 3' untranslated region element (3'UTR); wherein the 5'UTR is selected from the 5'UTR derived from any one of the genes ARHGAP, HSPB1, HBB, and CCL13; and the 3'UTR is selected from the 3'UTR derived from any one of the genes HBB, ARHGAP, CORO1A, and HPX; The ARHGAP is preferably ARHGAP15, and the 5'UTR preferably comprises the nucleotide sequence set forth in SEQ ID NO: 79 or a truncation thereof; RNA molecule.

14. The 5'UTR and 3'UTR are selected from any one of the following combinations: 1) the 5'UTR is derived from the 5'UTR of ARHGAP, and the 3'UTR is derived from the 3'UTR of any one of HBB, ARHGAP, CORO1A, or HPX; 2) the 5'UTR is derived from the 5'UTR of HSPB1, and the 3'UTR is derived from the 3'UTR of any one of HBB, ARHGAP, CORO1A, or HPX; 3) the 5'UTR is derived from the 5'UTR of HBB, and the 3'UTR is derived from the 3'UTR of any one of HBB, ARHGAP, CORO1A, or HPX; 4) The 5'UTR is derived from the 5'UTR of CCL13, and the 3'UTR is derived from the 3'UTR of any one of HBB, ARHGAP, CORO1A, or HPX; Preferably, The 5'UTR of the ARHGAP comprises a nucleotide sequence represented by any one of SEQ ID NOs: 45-46, 63-75, 77-78, and CUAUAAU, or comprises CUAUAAU, or comprises a nucleotide sequence having at least 80% identity to any one of the above sequences; The 5'UTR derived from HSPB1 comprises a nucleotide sequence set forth in SEQ ID NO: 47 or having at least 80% identity thereto; The 5'UTR derived from HBB comprises a nucleotide sequence set forth in SEQ ID NO:48 or having at least 80% identity thereto; The 5'UTR derived from CCL13 comprises a nucleotide sequence set forth in SEQ ID NO:49 or having at least 80% identity thereto; The 3'UTR derived from HBB comprises a nucleotide sequence set forth in SEQ ID NO:50 or having at least 80% identity thereto; The 3'UTR derived from the ARHGAP comprises a nucleotide sequence set forth in SEQ ID NO:51 or having at least 80% identity thereto; The 3'UTR derived from CORO1A comprises a nucleotide sequence set forth in SEQ ID NO:52 or having at least 80% identity thereto; The 3'UTR derived from HPX comprises a nucleotide sequence set forth in SEQ ID NO:53 or having at least 80% identity thereto; The RNA molecule of claim 13.

15. the ORF encodes a viral antigen, preferably the ORF encodes a coronavirus antigen or an influenza virus antigen; The coronavirus is preferably SARS-COV-2 and the coronavirus antigen is preferably the spike protein; 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; or The influenza virus is selected from influenza A virus or influenza B virus, and the influenza virus antigen is hemagglutinin protein (HA) and / or neuraminidase (NA); More preferably, the influenza virus antigen is selected from the hemagglutinin protein and / or neuraminidase of any of the following influenza virus strains: influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, and influenza B virus Yamagata. An RNA molecule according to any one of claims 13 to 14.

16. The polypeptide sequence encoded by the ORF comprises any one of the amino acid sequences set forth in SEQ ID NOs: 12, 14, 21, 23, 25, 80, 83-86, 98-101, and 136; Preferably, the nucleotide sequence of the ORF is set forth in any one of SEQ ID NOs: 54-55, 59-61, 82 and 126-134, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 54-55, 59-61, 82, 126-134; The RNA molecule of claim 15.

17. (d) further comprising a polyadenylic acid (poly-A) tail; An RNA molecule according to any one of claims 13 to 16.

18. the poly-A tail is selected from HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40late polyA; Preferably, the poly-A tail comprises a nucleotide sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 135, or a nucleotide sequence having at least 80% identity thereto.

18. The RNA molecule of claim 17.

19. (a) an open reading frame (ORF); (b) a 5' untranslated region element (5'UTR); (c) a 3' untranslated region element (3'UTR); and (d) a polyadenylic acid (poly-A) tail; Preferably, the RNA molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 57-58, 106-125; An RNA molecule according to any one of claims 13 to 18.

20. (e) further comprising a 5' cap structure (5'Cap); An RNA molecule according to any one of claims 13 to 19.

21. said 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine; Preferably, the 5'Cap is selected from ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG; Preferably, the 5'Cap is m7G(5')ppp(5')(2'OMeA)pG; 21. The RNA molecule of claim 20.

22. The RNA molecule is mRNA. An RNA molecule according to any one of claims 13 to 21.

23. further comprising one or more modifications, Preferably, the modifications include backbone modifications, sugar modifications, base modifications and / or lipid modifications; More preferably, the base modification is a pseudouridine modification. An RNA molecule according to any one of claims 13 to 22.

24. An RNA molecule comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 54-55, 59-61, 82, 126-134, or a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 54-55, 59-61, 82, 126-134; Preferably, the RNA molecule further comprises one or more modifications, more preferably, the modifications are pseudouridine modifications. RNA molecule.

25. Use of the nucleic acid construct of any one of claims 1 to 12 or the RNA molecule of any one of claims 13 to 24 in any one of the following: (1) preparation of a vaccine; (2) encoding a viral antigen in vivo or in vitro in a subject; (3) preparation of a medicament encoding a viral antigen in vivo or in vitro in a subject; and (4) preparation of a medicament.

26. A vaccine comprising: The RNA molecule according to any one of claims 13 to 24, preferably encoding one or more antigens of one or more virus strains. vaccine.

27. A vector comprising: A nucleic acid construct according to any one of claims 1 to 12 or an RNA molecule according to any one of claims 13 to 24, vector.

28. A host cell comprising:

28. The vector of claim 27, host cell.

29. A method for preparing an RNA molecule according to any one of claims 13 to 24, comprising reverse transcribing a nucleic acid construct according to any one of claims 1 to 12 or a vector according to claim 27 to obtain an RNA molecule, preferably said method further comprising adding a 5'Cap to the 5' end of said RNA molecule. Preparation method.

30. A delivery system comprising a nucleic acid construct according to any one of claims 1 to 12 or an RNA molecule according to any one of claims 13 to 24, wherein the delivery vehicle of said delivery system is a cationic lipid delivery particle, preferably said delivery vehicle is a nanolipid particle. Delivery system.

31. 1. A pharmaceutical composition comprising: A pharma- ceutically acceptable carrier, diluent or excipient; and any one selected from the group consisting of a nucleic acid construct according to any one of claims 1 to 12, an RNA molecule according to any one of claims 13 to 24, a vaccine according to claim 26, and / or a delivery system according to claim 30. Pharmaceutical compositions.

32. A product or reagent kit comprising a nucleic acid construct according to any one of claims 1 to 12, an RNA molecule according to any one of claims 13 to 24, a vaccine according to claim 26, a delivery system according to claim 30 and / or a pharmaceutical composition according to claim 31. Products or reagent kits.

33. 20. Use in the preparation of a medicament for treating and / or preventing a disease comprising administering to a subject in need thereof an effective amount of a nucleic acid construct according to any one of claims 1 to 12, an RNA molecule according to any one of claims 13 to 24, a vaccine according to claim 26, a delivery system according to claim 30, a pharmaceutical composition according to claim 31 and / or a product or reagent kit according to claim 32, wherein the disease is a viral infectious disease or a respiratory disease associated with a viral infection, Preferably, the virus is a coronavirus or an influenza virus, more preferably, the virus is SARS-CoV-2, influenza A virus or influenza B virus; Preferably, the respiratory illness associated with a viral infection includes uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), use.

34. A method for treating and / or preventing a disease, comprising administering to a subject in need thereof an effective amount of a nucleic acid construct according to any one of claims 1 to 12, an RNA molecule according to any one of claims 13 to 24, a vaccine according to claim 26, a delivery system according to claim 30, a pharmaceutical composition according to claim 31 and / or a product or reagent kit according to claim 32, wherein the disease is a viral infectious disease or a respiratory disease associated with a viral infection, Preferably, the virus is a coronavirus or an influenza virus, more preferably, the virus is SARS-CoV-2, influenza A virus or influenza B virus; Preferably, the respiratory illness associated with a viral infection includes uncomplicated infection, fever, cough, sore throat, rhinitis, headache, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), method.

35. 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 a nucleic acid construct according to any one of claims 1 to 12, an RNA molecule according to any one of claims 13 to 24, a vaccine according to claim 26, a delivery system according to claim 30, a pharmaceutical composition according to claim 31 and / or a product or reagent kit according to claim 32, Preferably, 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; Preferably, the virus is a coronavirus or an influenza virus, more preferably, the virus is SARS-CoV-2, influenza A virus or influenza B virus; method.

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