Coronavirus vaccine

Abstract

Provided are non-natural 5' untranslated region (5'-UTR) and 3' untranslated region nucleotides and uses thereof.

Description

[Technical field]

[0001] The present invention relates to non-natural 5'-untranslated regions (5'-UTRs) and 3'-untranslated regions (3'-UTRs), and uses thereof. [Background technology]

[0002] Nucleic acid vaccines rely on antigens encoded by DNA or RNA. DNA vaccines generally contain an antigen-encoding gene inserted into a bacterial plasmid under the control of a eukaryotic promoter, whereas RNA vaccines utilize messenger RNA (mRNA) or other antigen-encoding RNA. Similar to protein vaccines, nucleic acid vaccines can be delivered via a variety of routes, such as intramuscular, subcutaneous, mucosal or transdermal delivery.

[0003] DNA vaccines are known to induce less immune response than peptide vaccines, cell vaccines, virus vector vaccines, and RNA vaccines. In addition, DNA vaccines not only have low immunogenicity, but also have the potential to induce oncogenesis by being inserted into the host genome. Therefore, there is a demand for RNA-based vaccines. Summary of the Invention [Problem to be solved by the invention]

[0004] One embodiment provides an isolated polynucleotide comprising a nucleotide sequence encoding a non-native 5' untranslated region (5'-UTR), a nucleotide sequence encoding a 3' untranslated region (3'-UTR), or a combination thereof.

[0005] Another aspect provides RNA obtained by transcription using as a template the polynucleotide of the first aspect above.

[0006] Another embodiment provides an isolated polynucleotide comprising a non-native 5' untranslated region (5'-UTR) nucleotide sequence, a 3' untranslated region (3'-UTR) nucleotide sequence, or a combination thereof.

[0007] Another embodiment provides a method for obtaining RNA comprising the step of using said polynucleotide as a template and transcribing RNA.

[0008] Another embodiment provides a method for obtaining a polypeptide comprising translating the RNA obtained by the method described above.

[0009] Another aspect provides a composition comprising the polynucleotide and for delivering the polynucleotide to an individual.

[0010] Another aspect provides a composition for inducing an immune response to said polynucleotide comprising said polynucleotide.

[0011] Another aspect provides a method of utilizing the polynucleotide comprising introducing the polynucleotide into a host cell. [Means for solving the problem]

[0012] As used herein, the term "identity" in the context of a polypeptide or polynucleotide refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. The identity refers to the degree of sequence relatedness, as determined by the number of matches between two or more strings of amino acid or nucleotide residues. The identity of related polypeptides or polynucleotides can be calculated by well-known methods. "% identity" as applied to polypeptides or polynucleotides is defined as the percentage of residues in a candidate sequence that are identical to the residues of a second sequence, after aligning and introducing gaps, if necessary, for a candidate amino acid or nucleotide sequence to obtain the maximum percent identity with the second sequence. Methods and programs for said alignment are well-known. Such programs are, for example, BLAST, the Smith-Waterman algorithm or the Needleman-Wunsch algorithm.

[0013] As used herein, "5' untranslated region (5'-UTR)" refers to the region of an mRNA that is directly upstream (i.e., 5') from the start codon, i.e., the first codon of an mRNA transcript that is translated by ribosomes, that does not code for a polypeptide.

[0014] As used herein, "3' untranslated region (3'-UTR)" refers to the region of an mRNA that is directly downstream (i.e., 3') from a stop codon that does not code for a polypeptide, i.e., a codon of the mRNA transcript that signals translation termination.

[0015] As used herein, "open reading frame (ORF)" refers to a contiguous region of DNA beginning with a start codon, e.g., a methionine codon (ATG), and ending with a stop codon, e.g., TAA, TAG, or TGA, that encodes a polypeptide.

[0016] As used herein, "polyadenylate sequence" or "polyA tail" refers to a region of an mRNA downstream, e.g., directly downstream (i.e., 3'), from the 3'UTR that contains multiple consecutive adenosine monophosphates. The polyA tail may contain 10 to 300 adenosine monophosphates. For example, the polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. The polyA tail may contain, for example, 50 to 250 adenosine monophosphates. In an organism, such as a cell or an individual, the poly(A) tail can play a role in protecting the mRNA from attack by enzymes, for example those in the cytoplasm, and in aiding in transcription termination, mRNA export from the nucleus, and translation.

[0017] As used herein, the term "operatively linked" refers to nucleotide sequences linked on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., an open reading frame (ORF)) if it can affect the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence is also operably linked to a regulatory sequence in a sense or antisense orientation.

[0018] In this specification, with respect to the position of a nucleotide sequence, unless otherwise specified, it indicates that it is linked or located in the direction from the 5' end to the 3' end.

[0019] As used herein, the term "vector" or "nucleic acid construct" refers to any nucleic acid capable of carrying a gene, or an open reading frame (ORF) or a DNA fragment into a cell. The vector may also be, for example, capable of replicating in a cell. The vector may also be a virus, a bacteriophage, a provirus, a plasmid, a phagemid, a transposon, or an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a plant artificial chromosome (PLAC).

[0020] A first aspect provides an isolated polynucleotide comprising a nucleotide sequence encoding a non-native 5' untranslated region (5'-UTR), a nucleotide sequence encoding a 3' untranslated region (3'-UTR), or a combination thereof.

[0021] The isolated polynucleotide may also include a nucleotide sequence encoding a non-natural 5' untranslated region (5'-UTR) and a nucleotide sequence encoding a non-natural 3' untranslated region (3'-UTR). The isolated polynucleotide may also include a nucleotide sequence encoding a non-natural 5' untranslated region (5'-UTR) and a nucleotide sequence encoding a natural 3' untranslated region (3'-UTR).

[0022] The aforementioned isolated polynucleotide may also be a polydeoxyribonucleotide.

[0023] The 5' untranslated region (5'-UTR) may also comprise a non-natural nucleotide sequence. The 5' untranslated region (5'-UTR) may also have 70% or more, for example 80% or more, 90% or more, or 95% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1. The 5' untranslated region (5'-UTR) may also comprise SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a combination thereof. These 5' untranslated regions (5'-UTR) may also be polyribonucleotides. The nucleotide sequence encoding the non-natural 5' untranslated region (5'-UTR) may also comprise SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or a combination thereof. These sequences may also be polydeoxyribonucleotides.

[0024] The 3' untranslated region (3'-UTR) may comprise a non-natural nucleotide sequence or a natural nucleotide sequence. The 3' untranslated region (3'-UTR) may have 70% or more, e.g., 80% or more, 90% or more, or 95% or more, sequence identity with the nucleotide sequence of SEQ ID NO: 15, or 70% or more, e.g., 80% or more, 90% or more, or 95% or more, sequence identity with the nucleotide sequence of SEQ ID NO: 20. The 3' untranslated region (3'-UTR) may comprise SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, or a combination thereof. The nucleotide sequence encoding the non-natural 3' untranslated region (3'-UTR) or the natural 3' untranslated region (3'-UTR) may comprise SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or a combination thereof.

[0025] The non-native untranslated region (3'-UTR) or the nucleotide sequence encoding the native 3' untranslated region (3'-UTR) may additionally comprise a restriction enzyme recognition sequence. The restriction enzyme may be XhoI, NheI, or XhoI and NheI.

[0026] The nucleotide sequence encoding the 5' untranslated region (5'-UTR) may further comprise an upstream promoter region nucleotide sequence operably linked thereto.

[0027] The nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises a transcribable nucleotide sequence or a nucleotide sequence for introducing the transcribable nucleotide sequence, and the transcribable nucleotide sequence or the nucleotide sequence for introducing the transcribable nucleotide sequence is also operably linked to the 5' untranslated region (5'-UTR) downstream of the nucleotides encoding the 5' untranslated region (5'-UTR).

[0028] The transcribable nucleotide sequence may be a sequence coding for a polypeptide or an RNA. The polypeptide may be an antigenic polypeptide or a therapeutic polypeptide. The antigenic polypeptide may be a viral antigenic polypeptide. The viral antigenic polypeptide may be one or more antigenic polypeptides of BetaCoV (Betacoronavirus), one or more antigenic polypeptides of RSV (respiratory syncytial virus), one or more antigenic polypeptides of MeV (measles virus), one or more antigenic polypeptides of hMPV (human Metapneumovirus), one or more antigenic polypeptides of PIV (human parainfluenza virus), antigenic fragments thereof, or combinations thereof. The BetaCoV may be MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, variants thereof, or combinations thereof. SARS-CoV-2 was identified in 2019 and is also known as COVID-19. The antigenic polypeptide can be the spike protein of BetaCoV or an immunogenic fragment thereof. The therapeutic polypeptide can be, for example, an antibody, a hormone, a cytokine, an enzyme, a derivative thereof, or a combination thereof.

[0029] The nucleotide sequence for introducing the transcribable nucleotide sequence is also a cloning site. The cloning site is also a multiple cloning site. The cloning site also includes a restriction enzyme recognition site, a cleavage site, or a combination thereof.

[0030] The nucleotide sequence encoding the 5' untranslated region (5'-UTR) and the transcribable nucleotide sequence, or the nucleotide sequence for introducing the transcribable nucleotide sequence, are also linked to form a common transcript when transcribed in vitro or in vivo.

[0031] The above-mentioned promoter, the nucleotide sequence encoding the 5' untranslated region (5'-UTR), and the transcribable nucleotide sequence, or the nucleotide sequence for introducing the transcribable nucleotide sequence, are also linked to form a common transcript when transcribed in vitro or in vivo.

[0032] The nucleotide sequence encoding the 3' untranslated region (3'-UTR) may further comprise a downstream polyadenylate or poly(A) attachment signal nucleotide sequence operably linked thereto. The nucleotide sequence encoding the 3' untranslated region (3'-UTR) may comprise a transcribable nucleotide sequence or a nucleotide sequence for introducing the transcribable nucleotide sequence, and the transcribable nucleotide sequence or the nucleotide sequence for introducing the transcribable nucleotide sequence may be operably linked to the 3' untranslated region (3'-UTR) upstream of the nucleotide sequence encoding the 3' untranslated region (3'-UTR).

[0033] The transcribable nucleotide sequence, or a nucleotide sequence for introducing the transcribable nucleotide sequence, the nucleotide sequence encoding the 3' untranslated region (3'-UTR), and the polyadenylate or polyadenylate attachment signal, when transcribed in vitro or in vivo, form a common transcript or are linked to add a polyA tail downstream of the 3' untranslated region (3'-UTR) of the common transcript.

[0034] In the polynucleotide, the nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises a transcribable nucleotide sequence operably linked thereto, or a nucleotide sequence for introducing the transcribable nucleotide sequence, which is operably linked to the 5' untranslated region (5'-UTR) sequence downstream of the 5' untranslated region (5'-UTR) sequence, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) comprises a downstream polyadenylate or polyadenylate attachment signal nucleotide sequence operably linked thereto, and is also operably linked to the transcribable nucleotide sequence or the nucleotide sequence for introducing the transcribable nucleotide sequence upstream of the 3' untranslated region (3'-UTR) sequence. For example, the polynucleotide may be a nucleotide sequence encoding a 5' untranslated region (5'-UTR), a nucleotide sequence encoding a polypeptide or a cloning site nucleotide sequence, a nucleotide sequence encoding a 3' untranslated region (3'-UTR), and a polyadenylate or polyadenylate attachment signal nucleotide sequence linked in this order.

[0035] In the polynucleotide, the nucleotide sequence encoding the 5' untranslated region (5'-UTR) is selected from the nucleotide sequences of SEQ ID NOs: 22 to 33, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) is also selected from the sequences including the nucleotide sequences of SEQ ID NOs: 34 to 40. In this case, the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof also has any one of the nucleotide sequences of SEQ ID NOs: 49 to 55.

[0036] In the polynucleotide, the nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises the nucleotide sequence of SEQ ID NO: 22 or 31, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) also comprises the nucleotide sequence of SEQ ID NO: 40. In this case, the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof also has any one of the nucleotide sequences of SEQ ID NOs: 49 to 55. The polyadenylate nucleotide sequence also comprises the nucleotide sequence of SEQ ID NO: 41.

[0037] The polynucleotide also encodes any one of the nucleotide sequences set forth in SEQ ID NOs:63-76.

[0038] The nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises an upstream promoter region nucleotide sequence operably linked thereto, a transcribable nucleotide sequence operably linked to the promoter, or a nucleotide sequence for introducing the transcribable nucleotide sequence, which is operably linked to the 5' untranslated region (5'-UTR) sequence downstream of the 5' untranslated region (5'-UTR) sequence, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) comprises a downstream polyadenylate or polyadenylate attachment signal nucleotide sequence operably linked thereto, and is also operably linked to the transcribable nucleotide sequence, or the nucleotide sequence for introducing the transcribable nucleotide sequence, upstream of the 3' untranslated region (3'-UTR) sequence. For example, the polynucleotide may be one that is linked in the following order: a promoter, a nucleotide sequence encoding a 5' untranslated region (5'-UTR), a nucleotide sequence encoding a polypeptide or a cloning site nucleotide sequence, a nucleotide sequence encoding a 3' untranslated region (3'-UTR), and a polyadenylate or a polyadenylate attachment signal nucleotide sequence.

[0039] The nucleotide sequence encoding the 5' untranslated region (5'-UTR), the nucleotide sequence encoding a polypeptide or the cloning site nucleotide sequence, the nucleotide sequence encoding the 3' untranslated region (3'-UTR), and the polyadenylate or polyadenylate attachment signal, when transcribed in vitro or in vivo, form a common transcript or are linked to add a polyA tail downstream of the 3' untranslated region (3'-UTR) of the common transcript formed.

[0040] The above-mentioned promoter, nucleotide sequence encoding the 5' untranslated region (5'-UTR), nucleotide sequence encoding a polypeptide or cloning site nucleotide sequence, nucleotide sequence encoding the 3' untranslated region (3'-UTR), and polyadenylate or polyadenylate attachment signal, when transcribed in vitro or in vivo, form a common transcript or are linked so as to add a polyA tail downstream of the 3' untranslated region (3'-UTR) of the common transcript formed.

[0041] The polynucleotide may also be a nucleic acid construct or vector. As used herein, a "polyadenylate attachment signal" is also a nucleotide sequence that allows polyadenylate to be added to a nascent mRNA. The nucleic acid construct or vector may also include the regulatory sequences required to express the polynucleotide. The expression may be transcription, translation, or a combination thereof.

[0042] The second aspect provides an RNA obtained by transcription using the polynucleotide of the first aspect as a template. The RNA may be an mRNA. The mRNA may include a signal sequence. The mRNA may include any one of the nucleotide sequences of SEQ ID NOs: 63 to 76.

[0043] The mRNA may also include one or more nucleoside or nucleotide modifications in the sequence. The modified polynucleotide may exhibit reduced degradation in a cell or individual when introduced into the cell or individual compared to the unmodified polynucleotide. The modified polynucleotide may also exhibit reduced immunogenicity (e.g., reduced innate response) in a cell or individual compared to the unmodified polynucleotide when introduced into the cell or individual.

[0044] Nucleosides having modified cytosines also include ac4C (N4-acetyl-cytidine), m5C (5-methyl-cytidine), 5-halo-cytidine (e.g., 5-iodo-cytidine), hm5C (5-hydroxymethyl-cytidine), 1-methyl-pseudoisocytidine, s2C (2-thio-cytidine), 2-thio-5-methyl-cytidine, or combinations thereof.

[0045] Nucleosides having modified adenines also include 7-deaza-adenine, m1A (1-methyl-adenosine), m2A (2-methyl-adenine), m6A (N6-methyl-adenosine), or combinations thereof.

[0046] Nucleobases or nucleosides having modified guanosine also include I (inosine), m1I (1-methyl-inosine), imG (wyosine), mimG (methylwyosine), 7-deaza-guanosine, preQ0 (7-cyano-7-deaza-guanosine), preQ1 (7-aminomethyl-7-deaza-guanosine), m7G (7-methyl-guanosine), m1G (1-methyl-guanosine), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, or combinations thereof.

[0047] The nucleotides having the chemical modifications include pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio- 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, ), 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2'-O-methyluridine, or combinations thereof. The chemical modification may be at the 5-position of uracil. The chemical modification may also be a modification of uracil to N1-methylpseudouridine. The chemical modification is also the modification of uracil to N1-ethylpseudouridine.

[0048] The mRNA may also have one or more U's substituted with N1-methyl-pseudouridine in the sequence. The 5'-terminal GA in the mRNA may also have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA). The mRNA may also have all U's substituted with N1-methyl-pseudouridine. The mRNA may also have all U's substituted with N1-methyl-pseudouridine and a 5'-terminal GA may also have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA).

[0049] A third aspect provides an isolated polynucleotide comprising a non-native 5' untranslated region (5'-UTR) nucleotide sequence, a non-native 3' untranslated region (3'-UTR) nucleotide sequence, or a naturally occurring 3' untranslated region (3'-UTR) nucleotide sequence, or a combination thereof.

[0050] In the polynucleotide of the third aspect, the 5' untranslated region (5'-UTR) may have 70% or more, e.g., 80% or more, 90% or more, or 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 1. The 5' untranslated region (5'-UTR) may comprise SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a combination thereof. The nucleotide sequence encoding the non-native 5' untranslated region (5'-UTR) may comprise SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or a combination thereof.

[0051] The 3' untranslated region (3'-UTR) may have 70% or more, e.g., 80% or more, 90% or more, or 95% or more, sequence identity with the nucleotide sequence of SEQ ID NO: 15, or 70% or more, e.g., 80% or more, 90% or more, or 95% or more, sequence identity with the nucleotide sequence of SEQ ID NO: 20. The 3' untranslated region (3'-UTR) may also include SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, or a combination thereof. The non-native 3' untranslated region (3'-UTR) or a nucleotide sequence encoding the native 3' untranslated region (3'-UTR) may also include SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or a combination thereof.

[0052] The polynucleotide of the third aspect also comprises a non-naturally occurring 5' untranslated region (5'-UTR) and a naturally occurring 3' untranslated region (3'-UTR).

[0053] The 5' untranslated region (5'-UTR) nucleotide sequence comprises a nucleotide sequence encoding a polypeptide, the nucleotide sequence encoding the polypeptide being operably linked to the 5' untranslated region (5'-UTR) downstream of the 5' untranslated region (5'-UTR) nucleotides. The polypeptide is also an antigenic polypeptide or a therapeutic polypeptide. The antigenic polypeptide is also a viral antigenic polypeptide. The viral antigenic polypeptide is also one or more BetaCoV antigenic polypeptides, one or more RSV antigenic polypeptides, one or more MeV antigenic polypeptides, one or more hMPV antigenic polypeptides, one or more PIV antigenic polypeptides, antigenic fragments thereof, or a combination thereof. The BetaCoV is also MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, variants thereof, or a combination thereof. The antigenic polypeptide may be a spike protein of BetaCoV or an immunogenic fragment thereof. The spike protein of BetaCoV may be a natural spike protein or a variant of SARS-CoV-2. The variants include alpha (α), beta (β), gamma (γ), delta (δ), epsilon (ε) and omicron (ο) variants. The natural spike protein of SARS-CoV-2 or its alpha (α), beta (β), gamma (γ), delta (δ), epsilon (ε) and omicron (ο) variants may comprise the amino acid sequence of SEQ ID NOs: 42-48, respectively. The therapeutic polypeptide may be, for example, an antibody, a hormone, a cytokine, an enzyme, a derivative thereof, or a combination thereof.

[0054] The 5' untranslated region (5'-UTR) nucleotide sequence and the nucleotide sequence encoding the polypeptide are also linked as a common transcript.

[0055] The 3' untranslated region (3'-UTR) nucleotide sequence may further comprise a downstream polyadenylate nucleotide sequence operably linked thereto, the polyadenylate nucleotide sequence comprising, for example, 20 or more, 40 or more, 80 or more, or 100 or more adenyl nucleotides, and 500 or less, 400 or less, 300 or less, 200 or less, or 150 or less adenyl nucleotides.

[0056] The 3' untranslated region (3'-UTR) nucleotide sequence comprises a nucleotide sequence encoding a polypeptide, and the nucleotide sequence encoding the polypeptide is also operably linked to the 3' untranslated region (3'-UTR) upstream of the 3' untranslated region (3'-UTR) nucleotide sequence. The nucleotide sequence encoding the polypeptide may also be a ribonucleotide sequence, such as an mRNA.

[0057] The nucleotide sequence encoding said polypeptide and the 3' untranslated region (3'-UTR) nucleotide sequence are also linked as a common transcript.

[0058] The 5' untranslated region (5'-UTR) comprises a nucleotide sequence encoding a polypeptide, the nucleotide sequence encoding the polypeptide being operably linked to the 5' untranslated region (5'-UTR) sequence downstream of the 5' untranslated region (5'-UTR) sequence, and the 3' untranslated region (3'-UTR) comprises a downstream polyadenylate nucleotide sequence operably linked thereto, and is also operably linked to the nucleotide sequence encoding the polypeptide upstream of the 3' untranslated region (3'-UTR) sequence.

[0059] The 5' untranslated region (5'-UTR) nucleotide sequence, the nucleotide sequence encoding the polypeptide, the 3' untranslated region (3'-UTR) nucleotide sequence, and the polyA are also linked as a common transcript.

[0060] The 5' untranslated region (5'-UTR) or the 3' untranslated region (3'-UTR) may also have an activity that increases the translation efficiency, the stability of a nucleotide sequence encoding a polypeptide, or a combination thereof, compared to when a natural 5' untranslated region (5'-UTR) and / or a natural 3' untranslated region (3'-UTR) is used.

[0061] The polynucleotide may be an RNA, e.g., an mRNA. The polynucleotide may include a 5' cap. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule. The 5' cap may include a guanosine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate linkage. The guanosine may be methylated at the 7 position, e.g., m 7 G, or 3'-O-Me-m 7The term "conventional 5'-cap" refers to naturally occurring RNA 5' caps, such as m7G (7-methylguanosine). As used herein, the term "5' cap" includes 5' cap analogs that have been modified to resemble an RNA cap structure and have the ability to stabilize an RNA, e.g., in vivo and / or in a cell, when attached to an RNA. A 5' cap or 5' cap analog can be provided to an RNA by in vitro transcription of a DNA template in the presence of a 5' cap or 5' cap analog, with the 5' cap or 5' cap analog being co-transcriptionally inserted into the generated RNA strand, or the 5' cap or 5' cap analog can be generated post-transcriptionally using a capping enzyme, e.g., vaccinia virus capping enzyme. The 5' end cap structure can be, for example, 7mG(5')ppp(5')ImpNp, 3'-O-Me-m7G(5')ppp(5')G, m7(3'OMeG)(5')ppp(5')(2'OMeA) or m7G(5')ppp(5')(2'OMeA)pG.

[0062] The polynucleotide is an RNA, the 5' untranslated region (5'-UTR) is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 12, and the 3' untranslated region (3'-UTR) is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 14 to 20. The RNA, the 5' untranslated region (5'-UTR) is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 or 10, and the 3' untranslated region (3'-UTR) is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 20. The RNA, the 5' untranslated region (5'-UTR) nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1 or 10, the nucleotide sequence encoding the 3' untranslated region (3'-UTR) comprises the nucleotide sequence of SEQ ID NO: 20, and the polyadenylate nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 101. The 3' untranslated region (3'-UTR) additionally comprises a restriction enzyme recognition sequence. The restriction enzymes may also include XhoI, NheI, or XhoI and NheI.

[0063] One or more of the U's in the polynucleotide may be substituted with N1-methyl-pseudouridine.

[0064] The polynucleotide includes any one of the nucleotide sequences of SEQ ID NOs: 63 to 76, and one or more of U in the polynucleotide is substituted with N1-methyl-pseudouridine. The polynucleotide also includes all of U in the polynucleotide is substituted with N1-methyl-pseudouridine.

[0065] The polynucleotide also has an m7(3'OMeG)(5')ppp(5')(2'OMeA) cap structure at the 5' end.

[0066] The fourth aspect provides a method for obtaining RNA, comprising a step of transcribing RNA using the polynucleotide of the first aspect as a template. The transcription may include in vitro transcription, ex vivo transcription, or in vivo transcription. The in vivo transcription may include transcription within an individual. The RNA may be an mRNA. The mRNA may include any one of the nucleotide sequences of SEQ ID NOs: 63 to 76. The mRNA may include one or more nucleoside or nucleotide modifications in the sequence. The modified polynucleotide may exhibit reduced degradation in a cell or individual when introduced into a cell or individual, compared to an unmodified polynucleotide. The modified polynucleotide may also exhibit reduced immunogenicity (e.g., reduced innate response) in a cell or individual, compared to an unmodified polynucleotide when introduced into a cell or individual.

[0067] Nucleosides having modified cytosines also include ac4C (N4-acetyl-cytidine), m5C (5-methyl-cytidine), 5-halo-cytidine (e.g., 5-iodo-cytidine), hm5C (5-hydroxymethyl-cytidine), 1-methyl-pseudoisocytidine, s2C (2-thio-cytidine), 2-thio-5-methyl-cytidine, or combinations thereof.

[0068] Nucleosides having modified adenines also include 7-deaza-adenine, m1A (1-methyl-adenosine), m2A (2-methyl-adenine), m6A (N6-methyl-adenosine), or combinations thereof.

[0069] Nucleobases or nucleosides having modified guanosine also include I (inosine), m1I (1-methyl-inosine), imG (wyosine), mimG (methylwyosine), 7-deaza-guanosine, preQ0 (7-cyano-7-deaza-guanosine), preQ1 (7-aminomethyl-7-deaza-guanosine), m7G (7-methyl-guanosine), m1G (1-methyl-guanosine), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, or combinations thereof.

[0070] The nucleotides having the chemical modifications include pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio- 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, ), 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2'-O-methyluridine, or combinations thereof. The chemical modification may be at the 5-position of uracil. The chemical modification may also be a modification of uracil to N1-methylpseudouridine. The chemical modification is also the modification of uracil to N1-ethylpseudouridine.The variant also has U replaced with N1-methyl-pseudouridine. In the mRNA, the 5'-terminal GA has a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA). In the mRNA, all U's are replaced with N1-methyl-pseudouridine. In the mRNA, all U's are replaced with N1-methyl-pseudouridine, and the 5'-terminal GA has a cap structure of 5'-[1,2-[(3'-O-methyl)m7G-(5'→5')-ppp-Am]]. In the structure, Am represents 2'-O-methyladenosine. However, the 5' cap structure of the mRNA is not limited to this structure and may be replaced with any well-known natural 5' cap structure or 5' cap analog.

[0071] The term "in vitro transcription" or "RNA in vitro transcription" refers to the process of synthesizing RNA, including mRNA, in a cell-free system, i.e., in vitro. Cloning vector DNA, including plasmid DNA vectors, can be applied as templates for generating RNA transcripts. These cloning vectors are also commonly called transcription vectors. RNA is obtained by DNA-dependent in vitro transcription of a suitable DNA template. The DNA template can be a linearized plasmid DNA template. The promoter controlling the RNA in vitro transcription can be any promoter for DNA-dependent RNA polymerase. The DNA-dependent RNA polymerase can be T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or a combination thereof. The DNA template for the RNA in vitro transcription can be obtained by cloning a nucleic acid and introducing it into a vector for RNA in vitro transcription. The DNA can also include a cDNA corresponding to each RNA to be in vitro transcribed. The vector for the RNA in vitro transcription can be a circular plasmid DNA. The cDNA can be obtained by reverse transcription of mRNA or chemical synthesis.

[0072] The transcription vector also includes a promoter, a nucleotide sequence encoding a 5' untranslated region (5'-UTR), a sequence encoding a polypeptide, such as an open reading frame (ORF), a nucleotide sequence encoding a 3' untranslated region (3'-UTR), and polyA. The nucleotide sequence encoding the 5' untranslated region (5'-UTR) also includes one or more of the nucleotide sequences of SEQ ID NOs: 22 to 33. The nucleotide sequence encoding the 3' untranslated region (3'-UTR) also includes one or more of the nucleotide sequences of SEQ ID NOs: 34 to 40. The transcription vector also includes an E. coli replication origin ColE1 ori, a selection marker gene, or a combination thereof. The promoter can be a T7 promoter. The selection marker can be an antibiotic, such as a kanamycin resistance enzyme.

[0073] The in vitro transcription also includes a step of introducing the transcription vector into a host cell, for example, E. coli, to obtain a transformed host cell, culturing the obtained host cell, and growing the host cell containing the plasmid DNA serving as a template for mRNA. The in vitro transcription also includes a step of isolating the plasmid DNA from the grown host cell, for example, E. coli. The step of isolating the plasmid DNA also includes separating a cell layer from the culture and separating the plasmid DNA from the cells. The cell layer separation can be performed by centrifugation, separation, precipitation, or a combination thereof. The plasmid DNA separation from the cells can be performed by a well-known method, for example, alkaline extraction, affinity chromatography, HPLC, electrophoresis, or a combination thereof.

[0074] The method for obtaining the RNA also includes a step of culturing a cell containing the polynucleotide of the first aspect to produce an RNA transcript of the polynucleotide. The RNA transcript is also an mRNA. The mRNA also includes any one of the nucleotide sequences of SEQ ID NOs: 63 to 76. The mRNA also has the sequence in which one or more U's are replaced with N1-methyl-pseudouridine. In the mRNA, GA at the 5' end has a cap structure of 5'-[1,2-[(3'-O-methyl)m7G-(5'→5')-ppp-Am]]. In the mRNA, all U's are also replaced with N1-methyl-pseudouridine. In the mRNA, all U's are replaced with N1-methyl-pseudouridine, and the 5'-terminal GA has a cap structure of 5'-[1,2-[(3'-O-methyl)m7G-(5'→5')-ppp-Am]]. However, the 5' cap structure of the mRNA is not limited to this structure, and may be replaced with a well-known natural 5' cap structure or a 5' cap analog.

[0075] A fifth aspect provides a method for obtaining a polypeptide comprising translating an RNA of the third aspect, or an RNA obtained by the method of the fourth aspect. The RNA of the third aspect, or an RNA obtained by the method of the fourth aspect, may for example be an mRNA.

[0076] The method also includes culturing a cell containing the polynucleotide of the first aspect to produce the polypeptide.

[0077] A sixth aspect provides a composition for delivering a polypeptide to an individual comprising as an active ingredient a polynucleotide of the third aspect. The polynucleotide of the third aspect may also be RNA, for example mRNA. The polynucleotide of the third aspect may also be RNA obtained by the method of the fourth aspect.

[0078] A seventh aspect provides a composition for inducing an immune response against said polypeptide comprising as an active ingredient a polynucleotide of the third aspect. The polynucleotide of the third aspect may also be RNA, for example mRNA. The polynucleotide of the third aspect may also be RNA obtained by the method of the fourth aspect.

[0079] In the sixth and seventh aspects, the composition is also for treating an immunogenic or disease. The disease may vary depending on the polypeptide selected. The disease may be, for example, an infection caused by a virus or a foreign cell, a cancer, a metabolic disease, an anti-inflammatory, a gastrointestinal disease, an endocrine disease, a sepsis or an autoimmune disease. The disease may also be a disease caused by an enzyme deficiency and treatable by enzyme replacement therapy, such as Hunter syndrome, Gaucher disease, Fabry disease. In the disease, the foreign cell may be a prokaryotic or eukaryotic cell. The foreign cell may also be a bacterium.

[0080] The polynucleotide may also comprise a nucleotide sequence encoding the polypeptide. The polypeptide may also be an antigenic polypeptide or a therapeutic polypeptide. The antigenic polypeptide may also be a viral antigenic polypeptide. The viral antigenic polypeptide may also be one or more BetaCoV antigenic polypeptides, one or more RSV antigenic polypeptides, one or more MeV antigenic polypeptides, one or more hMPV antigenic polypeptides, one or more PIV antigenic polypeptides, antigenic fragments thereof, or a combination thereof. The BetaCoV may also be MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, variants thereof, or a combination thereof. The antigenic polypeptide may also be a spike protein of BetaCoV or an immunogenic fragment thereof. The therapeutic polypeptide may also be, for example, an antibody, a hormone, a cytokine, an enzyme, a derivative thereof, or a combination thereof. The composition may also be for preventing, treating, or diagnosing a condition or infection in an individual. The individual may be a mammal, including a human. The infection may be a bacterial or viral infection. The virus may be BetaCoV, RSV, MeV, hMPV, or PIV. The BetaCoV may be MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, a variant thereof, or a combination thereof. The composition may be used to prime immune effector cells. For example, the composition may be used to ex vivo activate peripheral blood mononuclear cells (PBMCs) and infuse them into an individual. The composition may be for use in inducing an immune response against SARS-CoV-2 or a variant thereof. The composition may be for use in preventing infection with SARS-CoV-2 or a variant thereof.

[0081] The composition is administered to an individual and the RNA polynucleotide (eg, mRNA) can be translated in vivo to produce the antigenic polypeptide.

[0082] An "effective amount" of the composition comprising a polynucleotide having one or more translatable regions encoding an antigenic polypeptide can be contacted with a cell, tissue or individual.

[0083] The effective amount can be determined based on the target tissue, the target cell, the means of administration, the physical characteristics of the polynucleotide (e.g., size and content of modified nucleosides, etc.), and other components of the composition. Generally, an effective amount of the composition can cause an immune response in a cell that is induced or boosted as a function of antigen production.

[0084] The composition may be administered with other prophylactic or therapeutic compounds. The prophylactic or therapeutic compounds may be, for example, adjuvants or boosters. As used herein, when referring to a prophylactic composition, such as a vaccine, the term "booster" refers to an additional administration of the prophylactic composition. A booster or booster vaccine may be given after an earlier administration of the prophylactic composition. The administration time between the initial administration of the prophylactic composition and the booster may be 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year. The composition may also be adjuvant-free.

[0085] The compositions may be administered intramuscularly, subcutaneously, intradermally, nasally or pulmonary.

[0086] The composition also includes one or more pharma- ceutically acceptable excipients, in which the polynucleotide is formulated or complexed with the excipient, and which are well known in the art.

[0087] As used herein, the term "active ingredient" refers to the composition or a polynucleotide contained therein, such as an RNA polynucleotide, including an mRNA, that may, for example, code for an antigenic or therapeutic polypeptide.

[0088] The relative amounts of active ingredient, pharma- ceutically acceptable excipients, and / or other additional ingredients contained in the compositions may vary depending on the identity, size and / or condition of the individual being treated, and the route of administration, For example, the compositions may contain from 0.1% to 100%, e.g., 0.5% to 50%, 1.0% to 30%, 5.0% to 80%, or even more than 80% (w / w) active ingredient.

[0089] In the composition, the polynucleotide may also be an mRNA. The mRNA may also include stabilizing elements including a 5' untranslated region (5'-UTR) and / or a 3' untranslated region (3'-UTR). The mRNA may also include additional structures such as a 5' cap structure or a 3' polyA tail. The 5' untranslated region (5'-UTR) and the 3' untranslated region (3'-UTR) are generally elements of the immature mRNA transcribed from genomic DNA. Structural features of mature mRNA such as the 5' cap and the 3' polyA tail are generally added to the transcribed (immature) mRNA during mRNA processing. The polyA tail is generally a stretch of adenine nucleotides added to the transcribed mRNA. The polyA tail may, for example, include up to 400 adenine nucleotides. In some embodiments, the length of the 3' polyA tail is also an essential element in the stability of the individual mRNA.

[0090] The composition may be formulated as a nanoparticle. The nanoparticles are also known for delivering polynucleotides such as mRNA to cells. For example, the nanoparticles are also known nanoparticles used in vaccines, such as RNA vaccines, used to induce immune responses against COVID-19. The nanoparticles may be lipid nanoparticles (LNPs). The composition may be formulated as or bound to lipid nanoparticles (LNPs). The binding includes binding within or to the surface of lipid nanoparticles (LNPs). For example, the composition may be formulated in or bound to a lipid polycation complex. The lipid polycation complex may also be referred to as a cationic lipid nanoparticle. The polycation may include cationic polypeptides such as MC3, Lipid 319, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, SM-102, ALC-0315, Arcturus Lipid 2,2 (8,8) 4C CH3, Genevant CL1, polylysine, polyornithine, and / or polyarginine. The composition may also be formulated in or coupled to lipid nanoparticles that include sterols such as distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-Sn-glycerol-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol, or non-cationic lipids such as dioleoylphosphatidylethanolamine (DOPE).The compositions may also be formulated in or conjugated to lipid nanoparticles including PEG lipids such as PEG2000-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), and PEG-DMA (polyethylene glycol dimethacrylate).

[0091] The lipid nanoparticle formulation may include a cationic lipid, a phospholipid, a sterol such as cholesterol, a PEG lipid, or a combination thereof. The lipid nanoparticle formulation may include, for example, a cationic lipid, a phospholipid, a sterol such as cholesterol, and a PEG lipid. The lipid nanoparticle may also include a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable lipid, or a combination thereof. The lipid nanoparticle may include 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable lipid. The PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG2000-DMG), the non-cationic lipid is 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), the sterol is cholesterol, and the ionizable cationic lipid is also compound 1 of the following structure:

[0092] [ka]

[0093] The PEG-modified lipid is compound 2 (ALC-0159) of the following structure, the non-cationic lipid is 1,2-distearoyl-Sn-glycerol-3-phosphocholine (DSPC), the sterol is cholesterol, and the ionizable cationic lipid is also compound 3 (ALC-0315) of the following structure:

[0094] [ka]

[0095] [ka]

[0096] The composition is also a composition for inducing an immune response against SARS-CoV-2 or a variant thereof, comprising the polynucleotide and a lipid nanoparticle (LNP) as an active ingredient. The composition is also for preventing infection with SARS-CoV-2 or a variant thereof.

[0097] An eighth aspect provides a method of utilising a polynucleotide of the third aspect comprising introducing said polynucleotide into a host cell. The polynucleotide of the third aspect may also be RNA, for example mRNA. The polynucleotide of the third aspect may also be RNA obtained by the method of the fourth aspect.

[0098] In the method, the polynucleotide is also RNA, including mRNA. The host cell is also any cell from an individual. The individual is also a mammal, including a human. The cell is, for example, a stem cell, a germ cell, or a somatic cell. In the method, the polynucleotide is also RNA and the host cell is also an antigen-presenting cell, including a dendritic cell, a monocyte, or a macrophage.

[0099] The method may also include administering the polynucleotide of the third aspect to an individual. The administration may be parenteral or oral. The administration may be intramuscular, subcutaneous, intradermal, nasal or pulmonary. The individual may be a mammal, including a human.

[0100] The method also includes immunizing an individual against the polypeptide. The method also includes treating a disease in an individual. The polypeptide is an antigenic protein of a BetaCoV. The BetaCoV is MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, a mutant thereof, or a combination thereof. The method also includes inducing an immune response in an individual against a BetaCoV, e.g., a spike protein or a mutant thereof, or a subunit thereof. The method also includes preventing infection in an individual with a BetaCoV, e.g., MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1, a mutant thereof, or a combination thereof.

[0101] In the third to eighth aspects above, the polynucleotide also includes a 5' end cap, which may be 7mG(5')ppp(5')ImpNp, 3'-O-Me-m7G(5')ppp(5')G, or m7G(5')ppp(5')(2'OMeA)pG.

[0102] In the third to seventh aspects, the polynucleotide also comprises nucleotides having one or more chemical modifications. The modifications are also uniform across the nucleotides. As used herein, "chemical modification" and "chemically modified" refer to modifications in one or more of the position, pattern, percentage or population of adenosine (A), guanosine (G), uridine (U), thymine (T) or cytidine (C) ribonucleotides or dioxynucleotides. In general, the terms do not refer to ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties.

[0103] Polynucleotides, such as RNA, including mRNA, may also include one or more other modifications. Specific regions of the polynucleotide may also include one or more nucleoside or nucleotide modifications. Modified polynucleotides may exhibit reduced degradation in cells or individuals when introduced into cells or individuals compared to unmodified polynucleotides. Modified polynucleotides may also exhibit reduced immunogenicity (e.g., reduced innate response) in cells or individuals when introduced into cells or individuals compared to unmodified polynucleotides.

[0104] Nucleosides having modified cytosines also include ac4C (N4-acetyl-cytidine), m5C (5-methyl-cytidine), 5-halo-cytidine (e.g., 5-iodo-cytidine), hm5C (5-hydroxymethyl-cytidine), 1-methyl-pseudoisocytidine, s2C (2-thio-cytidine), 2-thio-5-methyl-cytidine, or combinations thereof.

[0105] Nucleosides having modified adenines also include 7-deaza-adenine, m1A (1-methyl-adenosine), m2A (2-methyl-adenine), m6A (N6-methyl-adenosine), or combinations thereof.

[0106] Nucleobases or nucleosides having modified guanosine also include I (inosine), m1I (1-methyl-inosine), imG (wyosine), mimG (methylwyosine), 7-deaza-guanosine, preQ0 (7-cyano-7-deaza-guanosine), preQ1 (7-aminomethyl-7-deaza-guanosine), m7G (7-methyl-guanosine), m1G (1-methyl-guanosine), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, or combinations thereof.

[0107] The nucleotides having the chemical modifications include pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio- 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, ), 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2'-O-methyluridine, or combinations thereof. The chemical modification may be at the 5-position of uracil. The chemical modification may also be a modification of uracil to N1-methylpseudouridine. The chemical modification is also the modification of uracil to N1-ethylpseudouridine. Effect of the Invention

[0108] An isolated polynucleotide comprising a nucleotide sequence encoding a non-native 5' untranslated region (5'-UTR), a non-native 3' untranslated region (3'-UTR) or a naturally occurring 3' untranslated region (3'-UTR), or a combination thereof, according to one embodiment, can be used to stabilize mRNA, thereby increasing translation efficiency.

[0109] RNA according to other embodiments is relatively stable and has high translation efficiency and can be used to efficiently translate RNA.

[0110] The isolated polynucleotides according to other embodiments are relatively stable and have high translation efficiency and can be used to efficiently translate RNA.

[0111] According to the method for obtaining RNA according to another embodiment, RNA can be obtained efficiently.

[0112] According to the method for obtaining a polypeptide according to another embodiment, the polypeptide can be obtained efficiently.

[0113] Immunogenic compositions according to other embodiments can be used to effectively immunize individuals.

[0114] Methods utilizing polynucleotides according to other embodiments can be used to effectively immunize individuals. [Brief description of the drawings]

[0115] [Figure 1] FIG. 1 shows a vector for producing mRNA encoding the engineered SARS-CoV-2 spike protein or its mutants. [Figure 2A] FIG. 1 shows the results of analyzing the produced mRNA via chromatography. [Figure 2B] FIG. 1 shows the results of analyzing the produced mRNA via chromatography. [Figure 2C] FIG. 1 shows the results of analyzing the produced mRNA via chromatography. [Diagram 3] FIG. 1 shows the results of Western blot analysis confirming that spike protein is expressed when the produced mRNA-LNP is introduced into cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0117] Example 1: 5' untranslated region (5'-UTR) sequences that increase expression of genes encoding mRNA A 5' untranslated region (5'-UTR) sequence was designed to increase expression of the mRNA coding gene.

[0118] As a result, 12 non-natural 5' untranslated region (5'-UTR) sequences having the nucleotide sequences of SEQ ID NO: 1 to 12 were obtained. The nucleotide sequence of SEQ ID NO: 2 is a sequence in which the position of the sequence (GUCCCUCUGA (SEQ ID NO: 13)) that forms a stable hairpin structure is shifted forward in the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 3 is a sequence in which some of the guanines in the nucleotide sequence of SEQ ID NO: 1 are replaced with uridines.

[0119] The nucleotide sequence of SEQ ID NO: 4 is a sequence in which a part of the nucleotide sequence of SEQ ID NO: 1 is replaced with consecutive uridines. The nucleotide sequences of SEQ ID NO: 5, 6, 7, and 8 are sequences in which a part of the nucleotide sequence of SEQ ID NO: 1 is replaced with an adenine-rich sequence. The nucleotide sequences of SEQ ID NO: 9, 10, and 11 are sequences in which a part of the nucleotide sequence of SEQ ID NO: 6 is replaced randomly with other nucleotides.

[0120] The nucleotide sequence of SEQ ID NO:12 is the same as the nucleotide sequence of SEQ ID NO:5 except that a guanine has been added.

[0121] In the nucleotide sequences of SEQ ID NOs: 1 to 12, each nucleotide may be modified from its natural form. For example, in the sequences, one or more U's may be substituted with N1-methyl-pseudouridine. In the sequences, all U's may be substituted with N1-methyl-pseudouridine.

[0122] Example 2: 3' untranslated region (3'-UTR) sequences that increase gene expression coding mRNA A 3' untranslated region (3'-UTR) sequence was designed to increase the expression of the mRNA coding gene.

[0123] As a result, seven 3' untranslated region (3'-UTR) sequences having the nucleotide sequences of SEQ ID NOs: 14 to 20 were obtained.

[0124] The nucleotide sequence of SEQ ID NO: 14 is also a sequence that does not contain a predicted miRNA binding site. The nucleotide sequences of SEQ ID NOs: 15 and 18 also contain a CU-rich element (UCCACCCCCCCAUCUCC (SEQ ID NO: 21)).

[0125] The nucleotide sequences of SEQ ID NOs: 16 and 19 also contain an antigen R binding element (UUGGUUU).

[0126] In the nucleotide sequences of SEQ ID NOs: 14 to 20, each nucleotide may be modified from its natural form. For example, in the sequences, one or more U's may be substituted with N1-methyl-pseudouridine. In the sequences, all U's may be substituted with N1-methyl-pseudouridine.

[0127] Example 3: Preparation of template vector using non-natural 5'-untranslated region (5'-UTR) and 3'-untranslated region (3'-UTR) sequences A vector was prepared by operably linking one or more of the sequences encoding the non-natural 5'-untranslated region (5'-UTR) nucleotide sequences including the nucleotide sequences of SEQ ID NOs: 1 to 12 obtained in Examples 1 and 2, one or more of the sequences encoding the 3'-untranslated region (3'-UTR) nucleotide sequences including the nucleotide sequences of SEQ ID NOs: 14 to 20, and a gene encoding a polypeptide.

[0128] Specifically, the vector is a combination of a promoter, a nucleotide sequence encoding a 5'-untranslated region (5'-UTR), an open reading frame (ORF) encoding a polypeptide, a nucleotide sequence encoding a 3'-untranslated region (3'-UTR), and polyA. The nucleotide sequence encoding the 5'-untranslated region (5'-UTR) is one or more of the nucleotide sequences of SEQ ID NOs: 22 to 33. The nucleotide sequence encoding the 3'-untranslated region (3'-UTR) is one or more of the nucleotide sequences of SEQ ID NOs: 109 to 115. The nucleotide sequences of SEQ ID NOs: 109 to 115 correspond to the nucleotide sequences of SEQ ID NOs: 34 to 40, which contain an XhoI enzyme recognition sequence at the 5' end and an NheI enzyme recognition sequence at the 3' end, respectively.

[0129] The nucleotide sequence encoding the polyA has the nucleotide sequence of SEQ ID NO: 41. The vector also contains an E. coli origin of replication ColE1 ori and a selection marker gene. The promoter is also a T7 promoter. The selection marker is also a kanamycin resistance enzyme.

[0130] When the selected non-native 5' untranslated region (5'-UTR)-encoding nucleotide sequence and / or 3' untranslated region (3'-UTR)-encoding nucleotide sequence is linked to a polypeptide-encoding sequence, not only is the production of the polypeptide increased, but the stability of the mRNA comprising the non-native 5' untranslated region (5'-UTR) sequence, the polypeptide-encoding sequence, and the 3' untranslated region (3'-UTR) is significantly increased. The polypeptide is a SARS-CoV-2 spike protein having the amino acid sequence of SEQ ID NOs: 42-48, respectively, or an alpha (α), beta (β), gamma (γ), delta (δ), epsilon (ε) or omicron (ο) mutant thereof. The polynucleotides encoding the SARS-CoV-2 spike protein or its alpha (α), beta (β), gamma (γ), delta (δ), epsilon (ε) and omicron (ο) variants have the nucleotide sequences of SEQ ID NOs: 49 to 55, and the mRNAs thereof also have the nucleotide sequences of SEQ ID NOs: 56 to 62. The cloning process of the polypeptides, e.g., mRNAs encoding the SARS-CoV-2 spike protein or variants thereof, is as follows:

[0131] (1) Preparation of template vector for production of SARS-CoV-2 spike protein and its mutant mRNA A vector for producing the mRNA of SARS-CoV-2 spike protein or its mutant, and / or its protein product, was prepared, which contains one of the nucleotide sequences encoding the 12 designed 5' untranslated region (5'-UTR) sequences, one of the nucleotide sequences encoding the SARS-CoV-2 spike protein or its mutant, one of the 7 3' untranslated region (3'-UTR) sequences encoding, and polyA, and the vector was introduced into a microbial cell, and the resulting microbial cell was cultured to produce a cloning vector. The mRNA was produced through in vitro transcription using the produced vector as a template. The mutants include alpha (α) mutant, beta (β) mutant, gamma (γ) mutant, delta (δ) mutant, epsilon (ε) mutant, and omicron (ο) mutant.

[0132] Specifically, first, pUC57-Amp R The antibiotic resistance gene was replaced with the kanamycin resistance gene from the vector pUC57-Kan (Addgene). R The pUC57-Kan R The vector was digested with the restriction enzymes HindIII and EcoRI, and the digested vector was then ligated with a synthetic polynucleotide encoding the SARS-CoV-2 spike protein or a mutant thereof using a ligase to create pUC57-Kan containing the sequence encoding the SARS-CoV-2 spike protein or a mutant thereof. RA vector was obtained. The polynucleotide encoding the SARS-CoV-2 spike protein or a mutant thereof has a nucleotide sequence of SEQ ID NO: 49 to 55. The inserted polynucleotide encoding the spike protein or a mutant thereof includes a nucleotide sequence encoding a 5' untranslated region (5'-UTR) and a sequence encoding a SARS-CoV-2 spike protein or a mutant thereof, and the nucleotide sequence of the SARS-CoV-2 spike protein from the 5' to the 879th nucleotide has a BamHI restriction enzyme recognition sequence, which was then cleaved and used to introduce the nucleotide sequence encoding the designed 5' untranslated region (5'-UTR).

[0133] (2) a link between the 5' untranslated region (5'-UTR) sequence and a nucleotide sequence encoding the SARS-CoV-2 spike protein or a variant thereof; In order to replace the 5' untranslated region (5'-UTR) sequence of the prepared template vector with a nucleotide sequence encoding the 5' untranslated region (5'-UTR) sequence of SEQ ID NO: 1 to 12, a primer containing the sequence and a primer containing the BamHI restriction enzyme recognition site sequence of the SARS-CoV-2 spike protein were used as a primer set, and a DNA fragment containing a nucleotide sequence encoding the 5' untranslated region (5'-UTR) was obtained through a PCR reaction using the template vector as a template. In order to more stably secure the DNA fragment containing a nucleotide sequence encoding the 5' untranslated region (5'-UTR), a PCR reaction was carried out in two stages for each sequence. After carrying out PCR using a primary primer, a second PCR was carried out using the obtained gene as a template and a secondary primer to obtain a DNA fragment containing a nucleotide sequence encoding the 5' untranslated region (5'-UTR). The primer sequences used therein were polynucleotides of SEQ ID NO: 77 to 100.

[0134] Each of the DNA fragments containing nucleotide sequences encoding the 12 5'-untranslated regions (5'-UTR) obtained through the PCR reaction and the template vector prepared in (1) were digested with the restriction enzymes HindIII and BamHI, and the digested sequences were ligated using a ligase. As a result, vectors encoding the SARS-CoV-2 spike protein or mutants thereof in which the nucleotide sequences encoding the 5'-untranslated regions (5'-UTR) were replaced with the nucleotide sequences encoding the 5'-untranslated regions (5'-UTR) of SEQ ID NOs: 1 to 12 were prepared.

[0135] (3) Linkage of nucleotide sequences encoding the 3' untranslated region (3'-UTR) In the vector encoding the SARS-CoV-2 spike protein or a mutant thereof in which the nucleotide sequence encoding the 5'-untranslated region (5'-UTR) prepared in (2) is replaced with the nucleotide sequence encoding the 5'-untranslated region (5'-UTR) of each of SEQ ID NOs: 22 to 33, nucleotides encoding the 3'-untranslated region (3'-UTR) of each of SEQ ID NOs: 109 to 115 are linked to the 3' end of the sequence encoding the SARS-CoV-2 spike protein or a mutant thereof. Each of the nucleotide sequences of SEQ ID NOs: 109 to 115 corresponds to the nucleotide sequences of SEQ ID NOs: 34 to 40, which contain an XhoI enzyme recognition sequence at the 5' end and an NheI enzyme recognition sequence at the 3' end.

[0136] Specifically, the nucleotide sequences encoding the 3' untranslated regions (3'-UTRs) of SEQ ID NOs: 109 to 115 were obtained through synthesis. The sequences of SEQ ID NOs: 102 to 108 are RNA sequences corresponding to the sequences of SEQ ID NOs: 109 to 115.

[0137] The obtained sequence and the template vector prepared in (2) were cleaved with XhoI and NheI restriction enzymes, and the product was ligated using DNA ligase. As a result, a vector was prepared containing a structure of a sequence encoding a 5' untranslated region (5'-UTR)-a sequence encoding a SARS-CoV-2 spike protein or a mutant thereof-a sequence encoding a 3' untranslated region (3'-UTR). Here, the sequence encoding the 5' untranslated region (5'-UTR) represents the sequences encoding the 5' untranslated region (5'-UTR) of SEQ ID NOs: 22 to 33, respectively. The sequence encoding the 3' untranslated region (3'-UTR) represents the sequences encoding the 3' untranslated region (3'-UTR) of SEQ ID NOs: 109 to 115, respectively. The sequence encoding the SARS-CoV-2 spike protein or a mutant thereof represents the amino acid sequence of the SARS-CoV-2 spike protein or a mutant thereof of SEQ ID NOs: 42 to 48, respectively.

[0138] (4) Linkage of consecutive adenine nucleotides In a vector containing the structure of a sequence encoding the 5'-untranslated region (5'-UTR) prepared in (3)-a sequence encoding the SARS-CoV-2 spike protein or a mutant thereof-a sequence encoding the 3'-untranslated region (3'-UTR), a consecutive adenine nucleotide sequence was linked to the 3' end portion of the sequence encoding the 3'-untranslated region (3'-UTR).

[0139] A polyadenylate was obtained by synthesis, in which an NheI sequence and an EcoRI sequence were added to the 5'-end and 3'-end of the consecutive adenylate sequence of SEQ ID NO: 41. The obtained sequence and the template vector were cleaved with NheI restriction enzyme and EcoRI restriction enzyme, respectively, and the cleaved products were ligated using DNA ligase.

[0140] As a result, a vector was prepared that includes a structure of a sequence encoding a 5' untranslated region (5'-UTR)-a sequence encoding a SARS-CoV-2 spike protein or a variant thereof-a sequence encoding a 3' untranslated region (3'-UTR)-polyA. Here, the sequence encoding the 5' untranslated region (5'-UTR), the sequence encoding the 3' untranslated region (3'-UTR), and the sequence encoding the SARS-CoV-2 spike protein or a variant thereof are as described above. The polyA has the nucleotide sequence of SEQ ID NO:41.

[0141] FIG. 1 is a diagram showing a vector for producing mRNA encoding the engineered SARS-CoV-2 spike protein or its mutants.

[0142] Example 4: Production of gene products using non-native 5'-untranslated region (5'-UTR) and 3'-untranslated region (3'-UTR) sequences The vector containing the structure of the 5' untranslated region (5'-UTR) coding sequence-SARS-CoV-2 spike protein or a variant thereof-3' untranslated region (3'-UTR) coding sequence-polyA prepared in Example 3 was introduced into E. coli to obtain transformed E. coli, which was then cultured to grow E. coli containing the plasmid DNA serving as an mRNA template. The grown E. coli was separated from the culture medium through high-speed centrifugation, and the cell-derived template DNA was obtained using a known alkaline extraction method. The obtained template DNA was linearized using a restriction enzyme recognition sequence located at the polyA end. The linearized cell-derived template DNA was used to produce mRNA having the nucleotide sequence of 5' untranslated region (5'-UTR)-SARS-CoV-2 spike protein or a variant thereof-3' untranslated region (3'-UTR)-polyA through in vitro transcription. Here, the 5' untranslated region (5'-UTR), the 3' untranslated region (3'-UTR), the polyA, and the sequence encoding the SARS-CoV-2 spike protein or a mutant thereof are as described above.

[0143] Specifically, 5U / μl of T7 RNA polymerase was added to an aqueous solution containing 10ng / μl of linearized plasmid DNA, 4mM of CleanCap® Reagent AG, 5mM of NTP, 5mM of N1-methylpseudouridine, 20mM of magnesium ion salt, 10mM of DTT, 0.01U / μl of inorganic pyrophosphate degrading enzyme (pyrophosphatase), and 1U / μl of RNAse inhibitor, and the resulting reaction mixture was incubated at 37°C for 1 hour or more to allow the in vitro transcription reaction to proceed. CleanCap® Reagent AG is a reagent for co-transcriptional capping with mRNA transcription, and allows the m7G(5')ppp(5')(2'OMeA)pG cap structure to be formed. The reaction mixture was treated with DNase, and RNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs). As a result, mRNAs having the nucleotide sequences of SEQ ID NOs: 63 to 76 were produced. The nucleotide sequence configurations of the 5'-untranslated region (5'-UTR), SARS-CoV-2 spike protein or its mutant sequence, 3'-untranslated region (3'-UTR), and polyA contained in these mRNAs are as shown in Table 1 below.

[0144] [Table 1]

[0145] Figures 2A, 2B and 2C are diagrams showing the results of analyzing, via chromatography, mRNAs having the nucleotide sequences of SEQ ID NOs: 67, 70 and 76 in the produced mRNA. In Figures 2A, 2B and 2C, the maximum peaks (main) indicate mRNAs having the nucleotide sequences of SEQ ID NOs: 67, 70 and 76, respectively. As shown in Figures 2A, 2B and 2C, the purity of the mRNAs having the nucleotide sequences of SEQ ID NOs: 67, 70 and 76 was 83.0%, 81.9% and 83.1%, respectively.

[0146] Example 5: Preparation of lipid nanoparticles loaded with gene product mRNA A lipid complex solution was prepared by dissolving the ionizable lipid ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (BLDpharm Ltd., China), the phospholipid distearoylphosphatidylcholine (DSPC) (Avanti, USA), the cholesterol Synthechol (Sigma, USA), and the lipid-PEG conjugate ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide) (SINOPEG, China) in ethanol in a molar ratio of 46.3:9.4:42.7:1.6.

[0147] 0.3 mg of the mRNA prepared in Example 4 was diluted with 100 mM sodium acetate to 0.08 mg / mL to obtain an mRNA-containing solution. The obtained mRNA solution and the lipid complex solution were injected into each microchannel of a microfluidic device (Ignite Nanoassembly (PNI (Canada))) at a flow rate of 12 mL / min in a volume ratio of 5:1 so that the weight ratio of the mRNA to lipid complex was 1:25.4, and the mRNA solution and the lipid complex solution were mixed. By the mixing, ethanol in the lipid complex solution is diluted to a critical ethanol concentration, and lipid nanoparticles loaded with mRNA are formed. The microfluidic device includes a microchannel formed by the convergence of two microchannels, and is a device in which an mRNA solution is flowed into one microchannel and a lipid complex solution is flowed into the other microchannel, so that the two solutions are mixed while flowing. The mixture eluted through the microchannels of the microfluidic device is mRNA loaded lipid nanoparticles (LNPs).

[0148] The prepared mRNA-loaded lipid nanoparticles (LNP) were loaded onto a 10,000 Da centrifugal filter and buffer-exchanged with a buffer containing 0.45 mM potassium chloride, 0.24 mM monobasic potassium phosphate, 20.53 mM sodium chloride, 1.31 mM dibasic sodium phosphate, and 2% sucrose (pH 6.9-7.9).Then, the mRNA-loaded lipid nanoparticle (LNP) solution in the buffer was filtered through a 0.2 μm filter to prepare the final mRNA-loaded lipid nanoparticles (LNP).

[0149] Example 6: Characterization of lipid nanoparticles (1) Encapsulation efficiency of lipid nanoparticles The loading efficiency or encapsulation efficiency (%) of the mRNA-loaded lipid nanoparticles (LNP) (hereinafter also referred to as "mRNA-LNP") prepared in Example 5 was confirmed using the Quant-iT RiboGreen RNA Kit (Invitrogen, USA) according to the manufacturer's guidelines. Specifically, Triton X in the kit was mixed with the mRNA-loaded lipid nanoparticles (LNP) to denature the lipid nanoparticles (LNP) and release the mRNA. The mixed solution was added to a well containing Ribogreen reagent, and the released mRNA was detected by detecting the fluorescence emitted from the Ribogreen reagent. The encapsulation efficiency was calculated according to the following formula.

[0150]

number

[0151] [Table 2]

[0152] As shown in Table 2, wild-type S, delta (δ)S and omicron (ο)S mRNA-LNPs were confirmed to load mRNA with high encapsulation efficiency.

[0153] (2) Size confirmation of lipid nanoparticles The particle size and polydispersity index (PDI) of the mRNA-LNP prepared in Example 5 were confirmed using a Zetasizer NanoZS (Malvern Instruments, UK). The Zetasizer is an instrument that uses dynamic light scattering technology to determine particle size and particle size distribution. Table 3 shows the measurement results of the particle size and polydispersity index.

[0154] [Table 3]

[0155] As shown in Table 3, the particle sizes of wild-type S, delta (δ) S, and omicron (ο) S mRNA-LNPs ranged from 80 nm to 100 nm.

[0156] Example 7: Confirmation of mRNA expression in HEK293T cells The mRNA-containing lipid nanoparticles prepared in Example 5 were delivered to HEK293T cells, and mRNA expression was confirmed. To this end, an in vitro experiment was carried out as follows.

[0157] 6x10 HEK293T cells in a 6-well plate 5 After seeding with cells / 2mL / well, 24 hours later, 0.5μg or 2μg of lipid nanoparticles were added and incubated for 24 hours. The incubation was carried out in a medium at 37℃. After incubation, lysis buffer, M-PER™ (mammalian protein extraction reagent) (Cat No. 78501 (Thermo Scientific™)) was added to the well at 160μl / well to rupture the cells, extract the proteins, and then perform BCA protein quantification using Pierce™ BCA Protein Assay Kit (Cat No. 23225 (Thermo Scientific™)). The obtained samples were diluted in burst buffer and 5x reducing dye (10% SDS, 0.5% bromophenol blue, 50% glycerol, 0.5% 2-mercaptoethanol, 250 mM tris (pH 6.8)) to equalize the protein concentration. Western blot experiments were performed on the diluted samples using Anti-SARS-CoV-2 Spike S1 Subunit monoclonal antibody (Cat No. MAB105403 (R&D systems)).

[0158] Figure 3 shows the results of confirming via Western blot that spike protein is expressed when the produced mRNA-LNP is introduced into cells. As shown in Figure 3, the expression of S protein increased with increasing concentration of mRNA-LNP treatment.

Claims

1. An isolated polynucleotide comprising a nucleotide sequence encoding a non-naturally occurring 5' untranslated region (5'-UTR), a nucleotide sequence encoding a 3' untranslated region (3'-UTR), or a combination thereof, said polynucleotide being a polydeoxyribonucleotide; the 5' untranslated region (5'-UTR) comprises an upstream promoter region nucleotide sequence operably linked thereto, a transcribable nucleotide sequence operably linked to the promoter, the transcribable nucleotide sequence being operably linked to the 5' untranslated region (5'-UTR) sequence downstream of the 5' untranslated region (5'-UTR) sequence; the 3' untranslated region (3'-UTR) comprises a downstream polyadenylate or polyadenylate attachment signal nucleotide sequence operably linked thereto, the transcribable nucleotide sequence being operably linked upstream of the 3' untranslated region (3'-UTR) sequence; The transcribable nucleotide sequence is a polynucleotide comprising a nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof, the nucleotide sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

42.

2. The polynucleotide of claim 1, wherein the 5' untranslated region (5'-UTR) has 70% or more sequence identity with the nucleotide sequence of SEQ ID NO:

1.

3. The polynucleotide of claim 1, wherein the 3' untranslated region (3'-UTR) has 80% or more sequence identity with a nucleotide sequence of SEQ ID NO: 15 or 20.

4. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22-33.

5. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the 3' untranslated region (3'-UTR) is selected from the group consisting of nucleotide sequences of SEQ ID NOs: 34 to 40 and nucleotide sequences of SEQ ID NOs: 109 to 115.

6. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 22 to 33, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) comprises a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 34 to 40.

7. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the 5' untranslated region (5'-UTR) comprises the nucleotide sequence of SEQ ID NO: 22 or 31, and the nucleotide sequence encoding the 3' untranslated region (3'-UTR) comprises the nucleotide sequence of SEQ ID NO:

40.

8. The polynucleotide of claim 7, wherein the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof comprises any one of the nucleotide sequences of SEQ ID NOs: 49 to 55.

9. The polynucleotide of claim 1, wherein the spike protein of SARS-CoV-2 or a variant thereof further comprises any one of the amino acid sequences of SEQ ID NOs: 42 to 48.

10. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof has any one of the nucleotide sequences of SEQ ID NOs: 49 to 55.

11. The polynucleotide of claim 1, which encodes any one of the nucleotide sequences of SEQ ID NOs: 63 to 76.

12. The polynucleotide of any one of claims 1 to 11, which is an expression construct or vector.

13. RNA obtained by transcription using as a template a polynucleotide according to any one of claims 1 to 12.

14. an isolated polynucleotide comprising a non-naturally occurring 5' untranslated region (5'-UTR) nucleotide sequence, a 3' untranslated region (3'-UTR) nucleotide sequence, or a combination thereof; the polynucleotide is a polyribonucleotide, the 5' untranslated region (5'-UTR) nucleotide sequence comprises a nucleotide sequence encoding a polypeptide, the nucleotide sequence encoding the polypeptide being operably linked to the 5' untranslated region (5'-UTR) downstream of the 5' untranslated region (5'-UTR) nucleotides; the 3' untranslated region (3'-UTR) nucleotide sequence is downstream of and operably linked to a nucleotide sequence encoding the polypeptide; A polynucleotide comprising a nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof, the nucleotide sequence encoding the polypeptide having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

42.

15. The polynucleotide of claim 14, wherein the 5' untranslated region (5'-UTR) comprises a nucleotide sequence having 70% or more sequence identity with SEQ ID NO:

1.

16. The polynucleotide of claim 15, wherein the 5' untranslated region (5'-UTR) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12.

17. The polynucleotide of claim 14, wherein the 3' untranslated region (3'-UTR) has 80% or more sequence identity with a nucleotide sequence of SEQ ID NO: 15 or 20.

18. The polynucleotide of claim 17, wherein the 3' untranslated region (3'-UTR) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-20 and 102-108.

19. The polynucleotide of claim 14, wherein the 5' untranslated region (5'-UTR) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, and the 3' untranslated region (3'-UTR) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-20, and SEQ ID NOs: 102-108.

20. The polynucleotide of claim 19, wherein the 5' untranslated region (5'-UTR) comprises the nucleotide sequence of SEQ ID NO: 1 or 10, and the 3' untranslated region (3'-UTR) comprises the nucleotide sequence of SEQ ID NO:

20.

21. The polynucleotide of claim 20, wherein one or more U in the polynucleotide is replaced with N1-methyl-pseudouridine.

22. The polynucleotide of claim 20, wherein the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof comprises any one of the nucleotide sequences of SEQ ID NOs: 56-62.

23. The polynucleotide of claim 14, wherein the spike protein of SARS-CoV-2 or a mutant thereof has an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 48.

24. The polynucleotide of claim 14, wherein the nucleotide sequence encoding the spike protein of SARS-CoV-2 or a variant thereof has a nucleotide sequence of any one of SEQ ID NOs: 56 to 62.

25. The polynucleotide according to claim 14, having a nucleotide sequence of any one of SEQ ID NOs: 63 to 76.

26. 15. The polynucleotide of claim 14, wherein the 3' untranslated region (3'-UTR) nucleotide sequence further comprises a downstream polyadenylate nucleotide sequence operably linked thereto.

27. The polynucleotide of claim 14, wherein the 5' end has a 5' cap structure.

28. 15. The polynucleotide of claim 14, comprising one or more modified nucleotides.

29. 29. The polynucleotide of claim 28, wherein the modified nucleotide comprises an N1-methyl-pseudouridine in place of U.

30. A method for obtaining RNA, comprising the step of using the polynucleotide according to any one of claims 1 to 13 as a template and transcribing RNA.

31. 31. A method according to claim 30, comprising culturing a cell containing a polynucleotide according to any one of claims 1 to 13, and producing an RNA transcript of said polynucleotide.

32. A method for obtaining spike protein of SARS-CoV-2 or a variant thereof, comprising translating RNA obtained by the method of claim 30.

33. 33. The method of claim 32, comprising culturing a cell comprising the polynucleotide of any one of claims 1 to 13 to produce the spike protein of SARS-CoV-2 or a variant thereof.

34. A composition for delivering the spike protein of SARS-CoV-2 or a variant thereof to an individual, comprising as an active ingredient a polynucleotide according to any one of claims 14 to 29.

35. The composition of claim 34, for inducing an immune response against SARS-CoV-2.

36. The composition of claim 34, for preventing SARS-CoV-2 infection.

37. A composition for inducing an immune response against SARS-CoV-2, comprising as an active ingredient a polynucleotide according to any one of claims 14 to 29.

38. The composition of claim 37, for preventing SARS-CoV-2 infection.

39. A composition for inducing an immune response against SARS-CoV-2, comprising as active ingredients a polynucleotide according to any one of claims 14 to 29 and lipid nanoparticles (LNPs).

40. The composition of claim 39, for preventing SARS-CoV-2 infection.

41. A method of utilizing a polynucleotide according to any one of claims 14 to 29, comprising the step of introducing said polynucleotide into a host cell.

42. 42. The method of claim 41, wherein the polynucleotide is RNA and the host cell is an antigen-presenting cell, including a dendritic cell, a monocyte or a macrophage.

43. 42. The method of claim 41, comprising administering to an individual a polynucleotide according to any one of claims 14 to 29.

44. 44. The method of claim 43, wherein the individual is immunized against a spike protein of SARS-CoV-2 or a variant thereof.

45. 44. The method of claim 43, for preventing SARS-CoV-2 infection in the individual.

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