Polynucleotide encoding virus-derived protein

Abstract

The purpose of the present invention is to provide a novel polynucleotide that efficiently expresses the HIV Tat protein or an antigenic fragment thereof and has an excellent ability to induce an anti-Tat antibody, and to provide a pharmaceutical composition containing the polynucleotide. Disclosed is a novel polynucleotide encoding a polypeptide comprising: (a) the HIV Tat protein or an antigenic fragment thereof; and (b) a signal peptide. The pharmaceutical composition containing the polynucleotide can be used for the treatment and / or prevention of HIV-related diseases.

Description

Polynucleotides encoding virus-derived proteins

[0001] The present invention relates to a novel polynucleotide encoding a polypeptide comprising (a) an HIV (human immunodeficiency virus) Tat (Trans-Activator of Transcription) protein or an antigenic fragment thereof and (b) a signal peptide, and to a pharmaceutical composition comprising the polynucleotide.

[0002] Human immunodeficiency virus (HIV), a type of retrovirus, is known to cause acquired immunodeficiency syndrome (AIDS). HIV consists of only 14 genes, and two types, HIV-1 and HIV-2, are known. HIV-1 and HIV-2 commonly encode structural proteins (Gag and Env), enzymes required for replication (Pol), and regulatory proteins essential for viral proliferation (Tat and Rev).

[0003] The main therapeutic agents for AIDS are nucleoside reverse transcriptase inhibitors (tenofovir alafenamide, emtricitabine, abacavir, lamivudine, etc.), non-nucleoside reverse transcriptase inhibitors (rilpivirine, efevirenz, doravirine, etc.), protease inhibitors (darunavir, ritonavir, etc.), and integrase inhibitors (dolutegravir, raltegravir, bictegravir, elvitegravir, etc.). In recent years, Combination Anti-Retroviral Therapy (cART), which combines these agents for treatment, has been established, and the average life expectancy of HIV-infected individuals has improved significantly.

[0004] On the other hand, with the aging of HIV patients, HIV-associated neurocognitive disorder (HAND), which had not previously received much attention as a research subject, is becoming more apparent. Non-Patent Documents 1 and 2 describe HAND as a condition characterized by cognitive impairment, motor impairment, and abnormal behavior despite successful HIV treatment, and are found in approximately 30% of HIV-infected individuals. One possible cause of HAND is thought to be the toxicity of Tat protein, which is essential for HIV gene transcription, to neurons, or alteration of the blood-brain barrier due to long-term HIV infection. It has been reported that Tat protein forms complexes with amyloid beta peptides to enhance adhesion to neurons, and larger complexes adhere to the neuronal surface. Furthermore, it has been shown that in individuals with Tat protein expression in the brain, there is an inverse correlation between the amount of anti-Tat antibodies and the severity of HAND, and the higher the anti-Tat antibody titer, the milder the HAND symptoms. Therefore, a vaccine that induces anti-Tat antibodies is expected to be effective as a therapeutic agent for HAND. Non-Patent Document 3 and Patent Document 6 describe Tat protein vaccines that induce anti-Tat antibodies, but state that at least three administrations are preferable for the induction of anti-Tat antibodies. Therefore, there is still a need for a Tat vaccine that has a better antibody induction ability with fewer administrations.

[0005] In recent years, mRNA vaccines have attracted attention in the field of vaccines. Patent documents 1, 2, and 5 describe mRNAs encoding Tat proteins characterized by containing a histone stem loop, but do not disclose the specific sequences of the mRNAs encoding Tat proteins or any biological data. Patent document 3 describes a composition containing mRNAs encoding two or more pathogenic antigens, and lists Tat antigen as an example of a pathogenic antigen. However, it does not disclose the specific sequences of the mRNAs encoding Tat antigens and other pathogenic antigens or any biological data. Patent document 4 describes a polynucleotide encoding a secreted protein mutant in which a secretory signal sequence and a self-penetrating peptide sequence derived from Tat protein (an 11-residue amino acid sequence represented by SEQ ID NO: 9 in Patent document 4) are added to a non-secreted protein normally present only within cells. Here, the Tat peptide is added to cooperate with the secretory signal sequence to allow the non-secreted protein to pass through cell membranes and the blood-brain barrier.

[0006] International Publication No. WO 2013 / 120628, International Publication No. WO 2012 / 019780, International Publication No. WO 2022 / 212659, International Publication No. WO 2023 / 115194, International Publication No. WO 2013 / 120499, International Publication No. WO 2011 / 113618, International Publication No. WO 2017 / 081082, International Publication No. WO 2023 / 218420

[0007] Hategan et al. , Nat Struct Mol Biol, 2017(24), 379-386Omeragic et al. , Fluids Barriers CNS (2020) 17:42 Cafaro et al. , Expert Opin Biol Ther. 2015;15 Suppl 1:S13-29Fabienne et al. , EMBO, 2010(29), 1348-1362 Annunziata et al. , J Cell Biochem, 2013(114), 510-513

[0008] An object of the present invention is to provide a novel polynucleotide encoding the HIV Tat protein or an antigenic fragment thereof, which can be used as a therapeutic and / or preventive agent for HIV-related diseases (e.g., HIV infection, HAND, etc.).

[0009] In the field of mRNA vaccines, one method for improving the antigenicity of proteins encoded by mRNA is to optimize the secretory signal peptide sequence at the end of the protein's conventional amino acid sequence, thereby increasing the amount of peptide transported to the endoplasmic reticulum and thereby enabling efficient antigen presentation. Tat protein does not have a secretory signal peptide sequence, and the arginine-rich domain is considered important for extracellular secretion of Tat protein. Tat protein is said to bind to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the cell membrane and be inserted into the cell membrane, thereby being secreted extracellularly without passing through the endoplasmic reticulum (Non-Patent Document 4). Therefore, adding a signal sequence to mRNA encoding Tat protein does not necessarily improve the amount of Tat protein transported to the endoplasmic reticulum, nor does it necessarily improve antigenicity. Furthermore, there is no example of adding a secretory signal sequence to the mRNA encoding Tat protein. In addition, it is generally believed that artificially introducing a secretory signal peptide into a protein that does not have one may disrupt functions such as protein folding, the secretory pathway, and regulation of secretion levels (Non-Patent Document 5).

[0010] Patent Document 8 was published on November 16, 2023, after the priority date of this application.

[0011] As a result of extensive research, the present inventors have discovered a novel polynucleotide (hereinafter also referred to as "polynucleotide of the present invention") encoding a polypeptide comprising (a) HIV Tat protein or an antigenic fragment thereof and (b) a signal peptide. As described in the Examples below, this polynucleotide efficiently expresses HIV Tat protein and has excellent anti-Tat antibody induction ability. Furthermore, the Examples below suggest that a pharmaceutical composition comprising the polynucleotide of the present invention has therapeutic and / or preventive effects on HIV-related diseases (e.g., HIV infection, HAND, etc.).

[0012] That is, the present invention relates to the following: (1-1) A polynucleotide encoding a polypeptide comprising the following (a) and (b): (a) an HIV Tat protein or an antigenic fragment thereof (b) a signal peptide (1-2) The polypeptide according to (1-1), wherein the signal peptide is a secretory signal peptide. (1-3) The polynucleotide according to (1-2), wherein the secretory signal peptide is an N-terminal secretory signal peptide. (2-1) The polynucleotide according to (1-2), wherein the secretory signal peptide is (i) or (ii). (i) A polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 161 to 167 and 183 to 187. (ii) A polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 161 to 167 and 183 to 187 in which one or more amino acids have been deleted, substituted, inserted, or added, and which has secretory signal activity. (2-2) The polynucleotide according to any of (1-1) to (2-1), further comprising a polynucleotide encoding a polypeptide comprising (c) a functional sequence. (2-3) The polynucleotide according to (2-2), wherein the functional sequence is (iii) or (iv). (iii) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 168 or 169. (iv) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 168 or 169 in which one or more amino acids have been deleted, substituted, inserted or added, and which has a function. (3) The polynucleotide according to any of (1-1) to (2-3), wherein (a) is an HIV Tat protein. (4) The polynucleotide according to any of (1-1) to (3), wherein the HIV Tat protein is an HIV-1 Tat protein. (5-1) The polynucleotide according to (4), wherein the HIV-1 Tat protein is a polypeptide consisting of an amino acid sequence identical to or having 90% or more identity to the amino acid sequence set forth in any of SEQ ID NOs: 131 to 148 and 182. (5-2) The polynucleotide according to (5-1), wherein the HIV-1 Tat protein is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 133 or 134.(6-1) The polynucleotide according to any of (1-1) to (5-2), wherein the polypeptide comprising (a) and (b) is a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 168 to 178 and 190 to 197. (6-2) The polynucleotide according to (6-1), wherein the polypeptide comprising (a) and (b) is a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 174 to 178. (6-3) The polynucleotide according to (6-2), wherein the polypeptide comprising (a) and (b) is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 174. (7-1) The polynucleotide according to any of (1-1) to (6-3), wherein the polynucleotide is an mRNA comprising an open reading frame encoding a polypeptide comprising the following (a) and (b) (hereinafter also referred to as "the mRNA of the present invention"; "the mRNA of the present invention" is included in "the polynucleotide of the present invention"). (a) an HIV Tat protein or an antigenic fragment thereof; and (b) a secretory signal peptide. (7-2) The polynucleotide according to any one of (1-1) to (7-1), which is an mRNA consisting of a 5' Cap structure, a 5' UTR, an open reading frame encoding a polypeptide comprising the following (a) and (b), a 3' UTR, and Poly(A): (a) an HIV Tat protein or an antigenic fragment thereof; and (b) a secretory signal peptide. (8-1) The polynucleotide according to (7-1) or (7-2), comprising a modified base selected from 5-methyluridine, pseudouridine, N1-methylpseudouridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudouridine, 4-thiouridine, 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-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-O-methyluridine, or a combination thereof.(8-2) The polynucleotide according to (8-1), wherein the modified base is selected from 5-methyluridine, N1-methylpseudouridine, 5-methylcytidine, or a combination thereof. (9) The polynucleotide according to (7-1) to (8-2), comprising a 5'Cap structure which is Cap1 or Cap2. (10) The polynucleotide according to (9), wherein the 5'Cap structure is Cap1. (11-1) The polynucleotide according to any of (7-1) to (10), comprising a 60- to 160-mer Poly(A). (11-2) The polynucleotide according to (11-1), wherein Poly(A) is 80-mer. (12-1) The polynucleotide according to any of (7-1) to (11-2), comprising a 5'UTR which is a polynucleotide consisting of a nucleotide sequence identical to or having 90% or more identity to a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 63 and 211 to 218. (12-2) The polynucleotide according to (12-1), wherein the 5'UTR is a polynucleotide consisting of a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 9. (12-3) The polynucleotide according to (12-2), wherein the 5'UTR is a polynucleotide consisting of a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 6. (13-1) The polynucleotide according to any of (7-1) to (12-3), comprising a 3'UTR that is a polynucleotide consisting of a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 64 to 112. (13-2) The polynucleotide according to (13-1), wherein the 3'UTR is a polynucleotide consisting of a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 64 to 69. (14-1) The polynucleotide according to any one of (7-1) to (13-2), wherein the open reading frame consists of a polynucleotide having a nucleotide sequence set forth in any one of SEQ ID NOs: 114 to 119, 121 to 126, and 198 to 210. (14-2) The polynucleotide according to (14-1), wherein the open reading frame consists of a polynucleotide having a nucleotide sequence set forth in any one of SEQ ID NOs: 114 to 119, 121 to 125, and 198 to 203.(15-1) The polynucleotide according to any one of (7-1) to (14-2), which is an mRNA defined as any one of SNG-299, 322 to 325, 327, 328, 331 to 336, 338 to 341, and 345. (That is, the polynucleotide described in any one of Tables 5, 6-1, and 6-2 in Example 7 below.) (15-2) The polynucleotide according to (15-1), which is an mRNA defined as any one of SNG-299, 322 to 325, 327, and 328. (That is, the polynucleotide described in Table 6-2 in Example 7 below.) (16) A pharmaceutical composition (hereinafter also referred to as the "pharmaceutical composition of the present invention"), comprising the polynucleotide according to any one of (1-1) to (15-2). (17-1) The pharmaceutical composition according to (16), which is an agent for treating and / or preventing HIV-related diseases. (17-2) The pharmaceutical composition according to (17-1), wherein the HIV-related disease is HIV infection or HAND.

[0013] (18) A method for treating and / or preventing an HIV-related disease, comprising administering the polynucleotide according to any one of (1-1) to (15-2). (19) The polynucleotide according to any one of (1-1) to (15-2) for producing an agent for treating and / or preventing an HIV-related disease. (20) The polynucleotide according to any one of (1-1) to (15-2) for treating and / or preventing an HIV-related disease. (21) Use of the polynucleotide according to any one of (1-1) to (15-2) for treating and / or preventing an HIV-related disease.

[0014] The polynucleotide of the present invention efficiently expresses HIV Tat protein and has excellent anti-Tat antibody induction ability, and therefore, a pharmaceutical composition containing the polynucleotide is highly useful as a medicine for treating and / or preventing HIV-related diseases (e.g., HIV infection, HAND, etc.).

[0015] The meaning of each term used in this specification is explained below. Unless otherwise specified, each term has the same meaning whether used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The term "comprise" means not being limited to the constituent elements and does not exclude unrecited elements. The present invention will be explained below with reference to embodiments. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to be used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0016] "Polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked by peptide bonds, and refers to a protein of any size, structure, or function. A polypeptide is referred to as a peptide if the encoded polypeptide is smaller than about 50 amino acids, and if the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing, and other equivalents, variants, and analogs. A polypeptide may be a single molecule or a multimolecular complex, such as a dimer, trimer, or tetramer.

[0017] The "HIV Tat protein" is present in both HIV-1 and HIV-2 and plays an important role in the regulation of gene expression and replication. The HIV Tat protein contains a proline-rich domain, a cysteine-rich domain, a core domain, an arginine-rich domain, and a glutamine-rich domain in exon 1, and an RGD motif in exon 2. The HIV-1 Tat protein, while varying depending on the isolate, consists of 99 to 106 residues, and is known to be classified into M, O, and N groups. The M group is primarily classified into subtypes A to D, CRF01 AE, F to H, J, and K. Meanwhile, the HIV-2 Tat protein, while varying depending on the isolate, is a protein consisting of approximately 130 residues, and is primarily classified into subtypes A to G. Both the HIV-1 Tat gene and the HIV-2 Tat gene consist of two exons that encode the protein. Examples of the amino acid sequence of the HIV-1 Tat protein include SEQ ID NOs: 129 to 130 (subtype A), 131 to 148 (subtype B), 149 to 153 (subtype C), 154 to 156 (subtype D), and 157 to 158 (subtype E). Examples of the amino acid sequence of the HIV-2 Tat protein include SEQ ID NO: 159. The HIV Tat protein is preferably the HIV-1 Tat subtype B, more preferably HIV-1 Tat Oyi or BH10. The amino acid sequence of the HIV Tat protein is preferably any of the amino acid sequences set forth in SEQ ID NOs: 131 to 148, more preferably the amino acid sequence set forth in SEQ ID NO: 133 (HIV-1 Tat Oyi) or SEQ ID NO: 134 (HIV-1 Tat BH10).

[0018] In one embodiment, the HIV Tat protein encoded by the polynucleotide of the present invention may be a mutant such as an HIV Tat chimeric protein, an HIV Tat protein having a mutation in the arginine-rich domain, an HIV Tat protein having a mutation in the RGD motif, a partially deleted mutant of the HIV Tat protein, or a conjugate of two or more valent HIV Tat proteins.

[0019] The term "HIV Tat chimeric protein" refers to a protein comprising / consisting of fragments derived from HIV Tat proteins of two or more different subtypes, or a chimeric protein of an HIV Tat protein and an SIV Tat protein. Examples include chimeric proteins comprising / consisting of a protein derived from HIV Tat exon 1 linked to the C-terminus of a protein derived from HIV Tat exon 2. Preferably, the chimeric protein is derived from HIV-1 Tat, comprising exon 2 of a protein derived from HIV-1 Tat subtype C. More preferably, the chimeric protein comprises a protein derived from exon 1 of HIV-1 Tat subtype B and a protein derived from exon 2 of HIV-1 Tat subtype C. One embodiment includes a chimeric protein of a protein derived from HIV-1 Tat subtype B and an SIV Tat protein. One embodiment is a chimeric protein of a protein derived from exon 1 of HIV Tat and exon 2 of HIV Tat or SIV Tat. The antigenic fragment of the chimeric protein may have about 1 to 10 amino acids inserted at the N-terminus, C-terminus, or between the protein derived from exon 1 and the protein derived from exon 2, to the extent that the antigenicity of the HIV Tat protein is not lost.

[0020] "HIV Tat protein having a mutation in the arginine-rich domain" refers to an HIV Tat protein in which a mutation has been introduced into the arginine-rich domain of a wild-type HIV Tat protein. The arginine-rich domain of the HIV Tat protein has been reported to be cleaved by furin, a cell-derived protease. HIV Tat protein having a mutation in the arginine-rich domain is expected to have increased stability as an antigen because it is no longer susceptible to cleavage by furin.

[0021] "HIV Tat protein with a mutation in the RGD motif" refers to an HIV Tat protein in which a mutation has been introduced into the RGD motif present in exon 2 of the wild-type HIV Tat protein. It has been reported that HIV Tat protein induces cell death in nervous system cells, immune system cells, etc. via the RGD motif in exon 2. HIV Tat proteins with a specific mutation in the RGD motif are expected to have lost or attenuated the activity of inducing cell death in nervous system cells, immune system cells, etc., and to be able to induce immunity with neutralizing activity equivalent to that of HIV Tat proteins without mutations. For example, a protein consisting of the amino acids of SEQ ID NO: 182 can be mentioned.

[0022] The term "HIV Tat protein partial deletion mutant" refers to an antigen lacking a partial functional domain of the HIV Tat protein. Examples of antigens lacking a partial functional domain include antigens lacking the cysteine-rich domain, arginine-rich domain, or a region containing the RGD motif from the wild-type amino acid sequence of the HIV Tat protein. The cysteine-rich domain is known to be involved in transcription activation of the HIV Tat protein, the arginine-rich domain in intracellular and extracellular transport, and the region containing the RGD motif (exon 2 region) inducing cell death. Therefore, a partial deletion mutant of the HIV Tat protein lacking the cysteine-rich domain, arginine-rich domain, or a region containing the RGD motif lacks the above-mentioned functions of the HIV Tat protein and is expected to function only as an antigen.

[0023] The term "conjugate of divalent or higher HIV Tat proteins" refers to a multimer in which divalent or higher HIV Tat proteins are linked in series, optionally via a linker of several amino acids. The conjugate preferably refers to the full-length of divalent to decavalent, more preferably divalent to hexavalent, and even more preferably trivalent or pentavalent HIV Tat proteins. Here, the HIV Tat protein may be not only a wild-type protein, but also a protein having a mutation in the arginine-rich domain and / or RGD motif. Even if the arginine-rich domain is cleaved by Furin during the extracellular transport of Tat proteins, a conjugate of divalent or higher HIV Tat proteins is expected to express a nearly full-length HIV Tat protein and maintain its antigenicity. Furthermore, improved antigenicity is also expected.

[0024] The "SIV Tat protein" is a protein present in simian immunodeficiency virus that has a function similar to that of the HIV Tat protein. Examples of the amino acid sequence include SEQ ID NO: 160.

[0025] The term "antigen" refers to a substance that can be recognized by the immune system, preferably by the adaptive immune response. As part of the adaptive immune response, antibodies and antigen-specific T cells are induced, thereby eliciting an antigen-specific immune response. Antigens include peptides or proteins that can be presented to T cells by MHC, and translation products of polypeptides that encode them. Examples of antigens include peptide and protein fragments containing epitopes, as well as mutants and derivatives of such fragments.

[0026] The term "antigenic fragment of HIV Tat protein" refers to a fragment of HIV Tat protein that contains a portion of the HIV Tat protein and that, like the full-length HIV Tat protein, is recognized by immune cells such as T cells and B cells and can induce immune responses such as antibody production against the HIV Tat protein. Examples of antigenic fragments of HIV Tat protein include fragments in which 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid residues are deleted from the N-terminus, central region, and / or C-terminus of the HIV Tat protein.

[0027] "Polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. This term includes triple-, double-, and single-stranded deoxyribonucleic acid (DNA) and triple-, double-, and single-stranded ribonucleic acid (RNA). It also includes unmodified and modified forms of polynucleotides and circular polynucleotides, such as polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides such as tRNA, rRNA, hRNA, siRNA, and mRNA containing D-ribose, other types of polynucleotides that are N- or C-glycosides of purine or pyrimidine bases, and other polymers containing non-nucleotide backbones. Polymers include polyamides, such as peptide nucleic acids (PNAs), polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers. mRNA includes IVT mRNA, self-replicating RNA, and replicon RNA. Self-replicating RNA and replicon RNA include ssRNA viruses, such as positive-strand ssRNA viruses.

[0028] The polynucleotide of the present invention is preferably a ribonucleotide or a deoxyribonucleotide, more preferably an mRNA. Here, the polynucleotide of the present invention refers to a non-natural polynucleotide, and the mRNA of the present invention refers to a non-natural mRNA. The non-natural mRNA refers to an mRNA that differs in at least one nucleotide from a wild-type sequence present in nature. The mRNA is produced by existing gene modification techniques. In one embodiment, the non-natural mRNA contains at least one modified base. In one embodiment, all nucleic acid bases are replaced with modified bases. In one embodiment, the polynucleotide (e.g., synthetic RNA or synthetic DNA) consists only of natural nucleic acid bases, i.e., A, C, G, and T bases in the case of synthetic DNA, and A, C, G, and U bases in the case of synthetic RNA.

[0029] The term "functional sequence" refers to a polypeptide of 3 to 60 amino acids located at the N-terminus, internal region, or C-terminus of a protein or a peptide fragment thereof, which can direct the secretory pathway and localization of the protein or peptide fragment. A functional sequence is synonymous with a heterologous peptide. Depending on the location of the functional sequence within a protein or a peptide fragment thereof, functional sequences are classified as N-terminal, central, or C-terminal functional sequences. Examples of functional sequences include signal peptides, linker peptides, immune-activating helper epitopes, TLR-activating peptides, and multimerization domain peptides such as SEQ ID NO: 189. Examples of signal peptides include secretory signal peptides, nuclear transport signal peptides, nuclear localization signal peptides, ubiquitination signal peptides, endoplasmic reticulum targeting signal peptides such as SEQ ID NO: 188, plasma membrane transport signal peptides, Golgi apparatus transport signal peptides, endosomal transport signal peptides, cytoskeleton transport signal peptides, and cytoplasmic transport signal peptides.

[0030] "Secretory signal peptide (SSP)" refers to a short peptide of about 15 to 30 amino acids that is located at the N-terminus, middle, or C-terminus of a protein or a peptide fragment thereof, promotes the transport of the protein or its peptide fragment into the endoplasmic reticulum, and has secretory function. The secretory signal peptide may be operably linked (fused) to the protein or its peptide fragment, or may be linked to a polypeptide in which methionine has been removed from the N-terminus of the protein or its peptide fragment.

[0031] Examples of the amino acid sequence of the secretory signal peptide encoded by the polynucleotide of the present invention include polypeptides consisting of the amino acid sequences set forth in SEQ ID NOS: 1 to 1115 and 1728 in Patent Document 7 and SEQ ID NOS: 161 to 167 and 183 to 187 herein, as well as polypeptides consisting of the amino acid sequences described above in which one or more amino acids have been deleted, substituted, inserted, or added, and which have secretory signal activity. "Addition" refers to one or more amino acids being bound to the N-terminus or C-terminus of the amino acid sequence. "One or more" refers to 1 to 10, preferably 1 to 5. The secretory signal peptide is preferably an N-terminal secretory signal peptide consisting of the amino acid sequence set forth in any of SEQ ID NOS: 161 to 167 herein in which one or more amino acids have been deleted, substituted, inserted, or added, and which has secretory signal activity. More preferred are IgE (SEQ ID NO: 161), Igk (SEQ ID NO: 162), JEV (SEQ ID NO: 163), SARS (SEQ ID NO: 164), IgG (SEQ ID NO: 167), tPA (SEQ ID NO: 165), albumin (SEQ ID NO: 166), Cystatin S (SEQ ID NO: 183), tPA (SEQ ID NO: 165), Mod-tPA (SEQ ID NO: 184), IL-6 (SEQ ID NO: 185), CTLA4 (SEQ ID NO: 186), or HSV / gDsec (SEQ ID NO: 187). More preferred are IgE, Igk, JEV, SARS, tPA, or IgG. Even more preferred are IgE, JEV, or SARS. Particularly preferred is IgE. In one embodiment, the polynucleotide of the present invention may encode an HIV Tat protein or an antigenic fragment thereof having a secretory signal peptide linked to the N-terminus and a functional sequence linked to the C-terminus.

[0032] "5'UTR" is synonymous with 5'-untranslated region and refers to the region of an mRNA located between the 5'Cap structure and the start codon on the mRNA that is translated by the ribosome. Typically, it refers to the region from the nucleotide adjacent to the 3' end of the 5'Cap structure to the nucleotide adjacent to the 5' end of the start codon on the open reading frame (ORF). The 5'UTR may contain a regulatory element for gene expression, a protein binding region, or the like. Examples of regulatory elements include a ribosome binding site, a miRNA binding site, or a 5'-terminal oligopyrimidine tract.

[0033] Examples of the 5'UTR of the mRNA of the present invention include polynucleotides consisting of a nucleotide sequence identical to or having 80%, 85%, 90%, or 95% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 63 and 211 to 218. Preferably, the polynucleotide is a polynucleotide containing a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 9, and more preferably a polynucleotide containing a nucleotide sequence identical to or having 90% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 6. Particularly preferably, the polynucleotide is a polynucleotide containing a nucleotide sequence identical to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 3.

[0034] "3'UTR" is synonymous with 3'-untranslated region and refers to the region of mRNA downstream (3' side) of the termination codon on the mRNA translated by the ribosome. The 3'UTR may contain a regulatory element for gene expression, a protein binding region, a PolyA addition signal sequence, or the like. Examples of regulatory elements include a ribosome binding site and a miRNA binding site.

[0035] Examples of the 3'UTR of the mRNA of the present invention include polynucleotides consisting of a nucleotide sequence identical to or having 80%, 85%, 90%, or 95% or more identity with the nucleotide sequence set forth in any of SEQ ID NOs: 64 to 112. Polynucleotides containing a nucleotide sequence identical to or having 90% or more identity with the nucleotide sequence set forth in any of SEQ ID NOs: 64 to 69 are preferred, and polynucleotides containing a nucleotide sequence identical to or having 90% or more identity with the nucleotide sequence set forth in SEQ ID NOs: 64 to 66 are particularly preferred. The 3'UTR of the mRNA of the present invention may contain a short nucleotide sequence at the 3' end, such as a restriction enzyme cleavage sequence (GCUAGC) derived from a cloning site such as NheI. Each of the 3'UTRs represented by SEQ ID NOs: 64 to 66 is a polynucleotide in which an NheI cleavage sequence has been added to each of the 3'UTRs represented by SEQ ID NOs: 67 to 69.

[0036] "Identity" refers to the percentage of nucleotides or amino acids that match the same nucleotides or amino acids in a reference sequence at that sequence position. Two or more sequences are said to be identical if they have the same length and order of nucleotides or amino acids. When assessing identity, the compared sequences are considered to have the same length, i.e., the length of the longest sequence among the sequences being compared. This means that a first sequence consisting of 9 nucleotides or amino acids is 90% identical to a second sequence consisting of 10 nucleotides or amino acids that includes the first sequence. The identity of amino acid sequences or nucleotide sequences can be determined using software such as the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7) or ClustalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI).

[0037] "Poly(A)" is also called a PolyA tail or a 3'-PolyA tail and refers to a stretch of approximately 10 to 500 mer adenosine nucleotides located directly downstream (i.e., on the 3' side) of the 3' UTR of an mRNA. In one embodiment, the mRNA of the present invention may have an amino terminal enhancer of split gene (AES) sequence, a mitochondrial-encoded 12S ribosomal RNA sequence, an A30LA70 sequence, or the like inserted between the ORF and Poly(A). In one embodiment, the mRNA of the present invention may have a mask structure made of nucleotides other than adenyl (A) residues, INVERTED-dT, 2'-modified RNA, or the like inserted 3' closer to the Poly(A) than the Poly(A). The phosphodiester moiety of Poly(A) may have phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate bonds to improve mRNA stability. In one embodiment, the mRNA of the present invention may have Poly(A) and / or a histone stem-loop at the 3'-end, or may be present in combination with other elements such as a purine-rich region (histone downstream element, or HDE).

[0038] Poly(A) of the mRNA of the present invention is preferably a 20- to 250-mer, more preferably a 60- to 160-mer, particularly preferably a 75- to 85-mer, or 80-mer adenosine nucleotide.

[0039] "Open reading frame (ORF)" refers to the nucleotide sequence from the start codon to the stop codon. The stop codon may be one or more, and may contain any number of UAA, UAG, or UGA. The ORF may have all available codons converted, or may be optimized using an optimization algorithm to ensure that the mRNA forms a stable secondary structure. Codon optimization tools, algorithms, and services are known in the art, and include GeneArt's service (Life Technologies), DNA2.0 (Menlo Park, CA), ViennaRNA Package 2.0 (University of Vienna), and LinearDesign (Baidu).

[0040] The ORF nucleotide sequences of the polynucleotides of the present invention include nucleotide sequences that have an increased G / C content or codon optimization without altering the amino acid sequence from the wild-type sequence, thereby increasing the expression level of the protein encoded by the ORF. In some embodiments of the polynucleotides of the present invention, the G / C content of the ORF is increased by, for example, 5% or more, 10% or more, 15% or more, or 20% or more compared to the G / C content of the ORF of a wild-type Tat polynucleotide.

[0041] The ORF of the polynucleotide of the present invention may encode a polypeptide comprising an HIV-1 Tat protein and a secretory signal peptide. For example, Example 3 lists an mRNA encoding a polypeptide in which a secretory signal peptide is linked to an HIV-1 Tat protein. Examples of ORF sequences in an ORF encoding a protein comprising an HIV-1 Tat protein and a secretory signal peptide include the nucleotide sequences set forth in SEQ ID NOs: 114 to 119, 121 to 126, and 198 to 210 described in Example 3. Preferred are SEQ ID NOs: 121 to 126 and 202 to 210. More preferred are the nucleotide sequences set forth in any of SEQ ID NOs: 121 and 198 to 203.

[0042] 5'Cap refers to both natural 5'Cap and modified 5'Cap. Natural 5'Cap refers to a ribose-guanosine residue (m7G) in which the 7' position of the guanine base is methylated, which is present in all eukaryotes. m7G is linked to the 5'-terminal nucleotide of mRNA via a 5'-5'-triphosphate bond (hereinafter referred to as ppp). 5'Cap protects mRNA from degradation by exonucleases, promotes transport of mRNA from the nucleus to the cytoplasm, and plays an important role in the assembly of the translation initiation complex, thereby contributing to the efficient translation of proteins encoded by mRNA. Modified 5'Cap refers to a 5'Cap that has been chemically modified to have the same or greater function as a natural 5'Cap in terms of protein translation.

[0043] Examples of modified 5'Cap include modified guanosine (hereinafter referred to as [G]), as well as the structures described in WO 2017 / 066793, WO 2023 / 007019, CN116987137, and WO 2023 / 246860. Nucleosides in [G] include guanosine methylated at the 7th position of the guanine and the 3' position of the ribose (m7G-3'OMe), guanosine methylated at the 7th position of the guanine residue and the 2' position of the ribose (m7G-2'OMe), as well as other guanine modifications, sugar modifications, and the like. Examples of guanine modifications include hypoxanthine, N1-methyl-guanine, 7-deaza-guanine, and 8-oxo-guanine. Examples of sugar modifications include riboses modified at the 2'- and / or 3'-positions, such as 2'-fluororibose, 2'-aminoribose, and 2'-azidoribose, bridged nucleic acids (BNAs) having a bridged structure, and modified riboses described in WO 2014 / 081507.

[0044] The natural 5'Cap m7G and the modified 5'Caps m7G-3'OMe and m7G-2'OMe are specifically represented by the following structures:

[0045] As used herein, the term "5' Cap structure" refers to a 5'-[G]pppN structure present at the 5' end of an mRNA. １ N ２ N ３ (N １ , N ２ , N ３ is any nucleoside.) The 5' Cap structure includes Cap0, Cap1 (N １ is methylated at the 2' position, and 5'-[m7G]-ppp-N １ mN ２ N ３ ), Cap2(N １ and N ２ is methylated at the 2' position, and 5'-[m7G]-ppp-N １ mN ２ mN ３ ), Cap3 (N １ , N ２ and N ３ is methylated at the 2' position, and 5'-[m7G]-ppp-N １ mN ２ mN ３ m). Specifically, Cap1 is represented by the following structure: Base in the chemical formula refers to any base. For example, it can be GGG, AGG, AUA, etc. depending on the capping method. It has been confirmed that mRNA having a 5'Cap structure represented by 5'-[m7G]-ppp-GmGG, 5'-[m7G]-ppp-AmGG, or 5'-[m7G]-ppp-AmUA is expressed at equivalent levels in HEK293 cells and mice.

[0046] Cap2 is represented by the following structure: In the chemical formula, Base refers to any base.

[0047] Cap3 is represented by the following structure: Base in the chemical formula refers to any base.

[0048] The 5'-5'-triphosphate bond (ppp) linking the 5' Cap and the 5'-terminal nucleotide of the mRNA can use modified phosphate groups such as phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester.

[0049] The 5' Cap structure of the mRNA of the present invention is preferably Cap1 or Cap2, and particularly preferably Cap1. The 5' Cap structure of the mRNA in one embodiment of the present invention can be 5'-[G]pppN depending on the reagent used for capping. １ N ２ N ３ N in １ N ２ N ３ can be GGG, AGG, AGA, AUG, GAA, or AUA. Thus, the base sequence of an mRNA in one embodiment of the present invention starts with 5'-GGGG, 5'-GAGG, 5'-GAGA, 5'-GAUG, 5'-GGAA, or 5'-GAUA, followed by a 5' UTR sequence.

[0050] The mRNA of the present invention may have an internal ribosome entry site (IRES) instead of having a 5' Cap structure. Preferred IRES include IRES derived from picornavirus, plague virus, poliovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, hog cholera virus, murine leukemia virus, simian immunodeficiency virus, and cricket paralysis virus.

[0051] The polynucleotides of the present invention may contain at least one chemical modification, such as modified phosphate groups, modified bases, and sugar modifications, as long as the modification is known in the art.

[0052] Modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Phosphate linkers may also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0053] Examples of modified bases include 5-methyluridine, pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudouridine, 4'-thiouridine, 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, and dihydropseudouridine. Further, WO 2001 / 79502, WO 2002 / 98443, WO 2009 / 127230, US 20100249219, WO 2012 / 019168, WO 2012 / 138453, WO 2013 / 143698, WO 2015 / 089511, US 20150056253, WO 2015 Modified bases described in International Publication Nos. 2016 / 209966, 2017 / 070613, US20180126003, International Publication Nos. 2020 / 243002, 2021 / 198258, 2021 / 251453, 2022 / 233880, and 2023 / 006999 can also be used. The polynucleotide of the present invention may contain multiple types of modified bases.The modified bases in the mRNA of the present invention are preferably selected from 5-methyluridine, pseudouridine, N1-methylpseudouridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudouridine, 4-thiouridine, 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-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-O-methyluridine, or combinations thereof. More preferred is a modified base selected from 5-methyluridine, 5-methylcytidine, and N1-methylpseudouridine, or a combination thereof, and particularly preferred is N1-methylpseudouridine.

[0054] Sugar modifications include nucleosides having a substituent at the 2'-position of the sugar and / or a bridged structure between the 4' and 2'-positions of the sugar. Substituents for the 2'-hydroxy group of the sugar include oxy or deoxy substituents. Examples of "oxy" modifications include alkoxy groups or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), and aminoalkoxy. "Deoxy" modifications include hydrogen and amino, halogen (e.g., fluorine), and the like. Examples of amino include alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, and amino acids. The amino may be linked to the sugar via a linker, which may include one or more of the atoms C, N, and O. Examples of such bridged structures include bridged nucleic acids (BNAs). Examples of BNA include AmNA (Amido-bridged nucleic acid, see International Publication No. 2011 / 052436), TrNA (Triazole-bridged nucleic acid, see International Publication No. 2014 / 126229), 4'-(CH ２ ) ｎ -O-2' (n is an integer of 1 to 4), 4'-(CH ２ ) ｍ —C(═O)—NR １ -2' (m is an integer from 0 to 4, R １ is a hydrogen atom or alkyl. Specific examples and preparation methods thereof are described in WO 98 / 39352, WO 2003 / 068795, WO 2005 / 021570, WO 2011 / 052436, WO 2013 / 052523, etc.

[0055] Nucleotide modifications and modification methods known in the art are also disclosed in, for example, the following patent documents: WO 98 / 39352, WO 99 / 014226, WO 2000 / 056748, WO 2003 / 068795, WO 2004 / 016749, WO 2005 / 021570, WO 2005 / 083124, WO 2007 / 143315, WO 2009 / 071680, WO 2011 / 052436, WO 2014 / 112463, WO 2014 / 126229, etc.

[0056] The polynucleotides of the present invention can be synthesized by any conventional method, including, but not limited to, chemical synthesis, enzymatic synthesis commonly referred to as in vitro transcription (IVT), or enzymatic or chemical cleavage of a longer precursor. See, for example, the present specification and Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984, and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005), etc.

[0057] The mRNA of the present invention can be synthesized by conventional methods. For example, the mRNA of the present invention can be synthesized by IVT using template DNA encoding the mRNA sequence of the present invention. When synthesizing mRNA by IVT, template DNA is required. Examples of template DNA include plasmids linearized with restriction enzymes or DNA fragments prepared by PCR. The template DNA for the mRNA of the present invention comprises, from the 5' end, an RNA polymerase promoter sequence (e.g., a T7 promoter sequence, an SP6 promoter sequence, a T3 promoter sequence, etc.), a 5'-UTR, an ORF region, and a 3'-UTR. The template DNA may further contain a portion corresponding to Poly(A). When the DNA template contains a portion corresponding to Poly(A), the portion corresponding to Poly(A) is synthesized in the RNA synthesis reaction catalyzed by RNA polymerase. Another method for adding Poly(A) to mRNA involves polyadenylation of the 3' end of the mRNA using an enzyme such as Escherichia coli Poly(A)-polymerase after IVT.

[0058] The RNA polymerase to be added to the IVT reaction solution can be appropriately selected depending on the promoter sequence of the template DNA. For example, if the template DNA has a T7 promoter sequence, it is preferable to use T7 RNA polymerase, which recognizes that sequence. Furthermore, if the transcription template has an SP6 or T3 promoter sequence, it is preferable to use SP6 RNA polymerase or T3 RNA polymerase, respectively.

[0059] The following is an example of a general IVT reaction method for synthesizing the mRNA of the present invention, but the method is not limited to the following: 1.0 μg of template DNA, 2.0 μL of 10x transcription buffer (400 mM Tris-HCl (pH 8.0), 190 mM MgCl ２ , 50 mM DTT, 10 mM spermidine), 0.2 μL custom NTP (25 mM each), 20 U RNase inhibitor, 3000 U T7 RNA polymerase and up to 20.0 μL dH ２The mixture is mixed with 0 and incubated at 37°C for 2 to 5 hours. The crude IVT mixture obtained after incubation is subjected to template DNA digestion using 1 U of RNase-free DNase. After 15 minutes of incubation at 37°C, mRNA is purified using Ambion's MEGAclear® Kit (Austin, TX) or similar, according to the manufacturer's instructions. After purification, RNA polynucleotides are quantified using NANODROP® or similar, and analyzed by agarose gel electrophoresis to confirm that the RNA polynucleotides are of the appropriate size and that no RNA degradation has occurred.

[0060] The mRNA of the present invention may have a Cap at its 5' end. In IVT, a method for adding a 5'-Cap to mRNA includes incorporating a cap dinucleotide, such as N7-methyl-guanosine-5'-triphosphate-5'-guanosine, into the IVT reaction mixture. Furthermore, the use of cap dinucleotide analogs known as Anti-Reverse Cap Analogues (ARCAs) (e.g., 3'-O-Me-m7G(5')ppp(5')G) can ensure that a Cap structure is added to mRNA in the correct orientation with a high probability. In another embodiment, CleanCap® Reagent (Trilink) can be used to add Cap1, in which the 2' hydroxyl group of the first ribose at the 5' end of the mRNA is methylated. Alternatively, a 5'-cap can be added to the IVT reaction product after the IVT reaction using a capping enzyme, for example, ScriptCap® m7G Capping System (Cellscript, Madison, WI, USA).

[0061] If the DNA template does not have a polyT, a Poly(A) tailing reaction may be performed before purifying the final product. If Poly(A) is already present in the transcription product, the Poly(A) tailing reaction may be skipped and one may proceed directly to purifying the mRNA.

[0062] The Poly(A) tailing reaction does not always result in adenine nucleotides of the exact desired length, and the length may vary by about 10 mer. For example, if a Poly(A) is described herein as an 80-mer, Poly(A)s of about 60 to 100 mer, preferably about 70 to 90 mer, 75 mer, 76 mer, 77 mer, 78 mer, 79 mer, 80 mer, 81 mer, 82 mer, 83 mer, 84 mer, or 85 mer are within the scope of the mRNA of the present invention.

[0063] A method for introducing modified bases into mRNA prepared by IVT includes adding a modified NTP instead of the NTP added in the above-described IVT. For example, to replace uridine in mRNA with pseudouridine (Ψ), pseudouridine (Ψ)-5'-triphosphate can be added instead of UTP to prepare mRNA containing pseudouridine (Ψ).

[0064] The polynucleotides of the present invention may be attached with a known ligand. Ligands known in the art can be used to enable tracking of the polynucleotides of the present invention, improve the pharmacokinetics or pharmacodynamics of the polynucleotides of the present invention, increase the stability or binding affinity of the polynucleotides of the present invention, improve the intracellular dynamics, including cellular uptake, of the polynucleotides of the present invention, or serve as components of transport carriers (e.g., liposomes, lipid nanoparticles (LNPs), polymers, micelles, virus particles) consisting of a single molecule or multiple molecules. Examples of such ligands include nucleic acids, reporter molecules, lipids (e.g., fatty acids, lipid chains, cholesterol, phospholipids), sugars (e.g., N-acetyl-galactosamine), vitamins, peptides (e.g., membrane-permeable peptides, cell-targeting peptides, receptor-binding peptides, endosomal escape-promoting peptides, RGD peptides, peptides with high affinity for blood components, tissue-targeting peptides), polyethylene glycol (PEG), dyes, fluorescent molecules, and the like.

[0065] The polynucleotides of the present invention encompass any pharmaceutically acceptable salts, esters, salts of such esters, or any other equivalents that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. That is, they encompass prodrugs and pharmaceutically acceptable salts of the polynucleotides of the present invention, pharmaceutically acceptable salts of the prodrugs, and other biological equivalents.

[0066] A "prodrug" is a derivative of an inactive or less active form that is converted into an active form (i.e., a drug) in the body or cells by the action and / or conditions of endogenous enzymes or other chemicals. For example, this is described in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0067] "Pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the polynucleotides of the present invention, i.e., salts that retain the desired biological activity of the polynucleotide and do not impart undesired toxicological effects thereto.

[0068] Pharmaceutically acceptable salts include salts with alkali metals (e.g., lithium, sodium, potassium, etc.), alkaline earth metals (e.g., calcium, barium, magnesium, etc.), transition metals (e.g., zinc, iron, etc.), ammonia, organic bases (e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, diethanolamine, ethylenediamine, pyridine, picoline, quinoline, etc.) and amino acids, or salts with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, etc.), etc. In particular, sodium salts and potassium salts are included. These salts can be formed by conventional methods.

[0069] The polynucleotides of the present invention not only have HIV Tat expression activity but also have pharmaceutical utility, and have any or all of the following excellent characteristics: a) weak inhibitory effect on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.); b) high chemical stability; c) non-mutagenicity; d) low risk of affecting the cardiovascular system; e) high solubility; f) induction of Tat antibodies with high neutralizing activity; g) exertion of superior antibody titers with fewer administrations than Tat protein (peptide). Specifically, two administrations of the polynucleotides of the present invention (particularly mRNA) are more effective at inducing antibodies than three administrations of Tat protein (peptide).

[0070] The polynucleotides of the present invention may be modified. Appropriately modified polynucleotides have any or all of the following characteristics compared to unmodified polynucleotides: a) sustained activity; b) high resistance to nucleases; c) improved pharmacokinetics; d) high tissue distribution; e) low immunostimulatory activity; f) high solubility; and g) high chemical stability.

[0071] The polynucleotides of the present invention can be used in conjunction with an appropriate transporter. As a result, they have any or all of the following characteristics: a) They are highly selectively delivered to target organs; b) They are efficiently delivered into target cells and exert their effects at low doses; c) They exhibit good pharmacokinetics; d) They have high metabolic stability; e) They are not mutagenic; f) They have a low risk of affecting the cardiovascular system; g) They exhibit high solubility; and h) They have high chemical stability.

[0072] The present invention also encompasses pharmaceutical compositions containing the polynucleotides of the present invention. In addition to the above-described modification methods and ligand-addition methods, any administration method and formulation known in the art can be used for the pharmaceutical compositions of the present invention. For example, a single or multiple-molecule transport carrier (liposome, LNP, polymer, micelle, virus particle, etc.) can be used. Other examples include polysomes and lipoplexes.

[0073] An example of synthesizing the pharmaceutical composition of the present invention is shown below. A lipid solution can be obtained by dissolving a cationic lipid, a neutral lipid, a sterol, and a polyethylene glycol-modified lipid in a polar organic solvent. A nucleic acid-lipid mixed solution can be obtained by mixing the mRNA solution of the present invention prepared in a buffer solution with a lipid solution. The pharmaceutical composition of the present invention can be obtained by replacing the polar organic solvent in the polar organic solvent with an aqueous solution such as a buffer solution.

[0074] A general description of the manufacturing method of the pharmaceutical composition of the present invention is described in Remington: The Science and Practice of Pharmacy, Twenty Third Edition, 2020, etc.

[0075] The pharmaceutical compositions of the present invention can be administered by various methods depending on whether local or systemic treatment is desired or the area to be treated. Administration methods may be, for example, topical (including ophthalmic, intravaginal, rectal, intranasal, and transdermal), oral, or parenteral. Parenteral administration includes intramuscular, intraperitoneal, or subcutaneous injection, intravenous injection or infusion, pulmonary administration by aspiration or inhalation, intrathecal administration, intraventricular administration, and the like. Intramuscular injection is preferred for parenteral administration.

[0076] When the pharmaceutical composition of the present invention is administered locally, it can be used in the form of a transdermal patch, ointment, lotion, cream, gel, drop, suppository, spray, liquid, powder, or other formulation. Compositions for oral administration include powders, granules, suspensions or solutions in water or non-aqueous media, capsules, powders, tablets, and the like. Compositions for parenteral, subdural, or intraventricular administration include sterile aqueous solutions containing buffers, diluents, and other suitable additives.

[0077] The pharmaceutical composition of the present invention can be obtained by mixing an effective amount of the polynucleotide of the present invention with various pharmaceutical additives, such as stabilizers, solubilizers, excipients, isotonicity agents, or pH adjusters, as needed, that are suitable for the dosage form. In the case of an injection, the polynucleotide may be sterilized together with an appropriate carrier to form a formulation.

[0078] Examples of stabilizers include refined sucrose, trehalose, maltose, glucose, sodium edetate, sodium chloride, and amino acids. Examples of solubilizers include polysorbate 20, polysorbate 80, polyoxyethylene hydrogenated castor oil, and poloxamer. Examples of excipients include maltose, xylitol, sodium chloride, amino acids, refined sucrose, trehalose, and maltose. Examples of isotonicity agents include sodium chloride, glycerin, glucose, and xylitol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, sodium hydrogen phosphate, sodium dihydrogen phosphate, citric acid, sodium citrate, acetic acid, sodium acetate, trometamol, and trometamol hydrochloride. Furthermore, when preparing a liquid, emulsion, or suspension-type injection, commonly used solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents may be added as appropriate. For oral administration, flavoring agents, fragrances, and the like may also be added.

[0079] Administration depends on the severity and responsiveness of the condition being treated, with the course of treatment lasting from several days to several months, or until a cure is achieved or a diminution of the disease symptoms is achieved. Optimal administration schedules can be calculated from measurements of drug accumulation in the body. Those skilled in the art can determine optimal doses, administration methods, and repetition frequencies. The optimal dose varies depending on the relative potency of individual nucleic acids, but can generally be calculated based on antibody titers, IC50, or EC50 in in vitro and in vivo animal experiments. For example, given the molecular weight of a polynucleotide (derived from the nucleic acid sequence and chemical structure) and an effective dose (derived experimentally), such as antibody titers, the dose expressed in mg / human is routinely calculated. When the pharmaceutical composition of the present invention is administered to a human, it can be administered once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous drip injection, or intravenous injection at a dose of about 0.001 to 1 mg, preferably 0.01 to 0.2 mg, in terms of the amount of mRNA per dose for an adult. However, the dose and the number of administrations can be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0080] The pharmaceutical composition of the present invention can be used as a therapeutic and / or preventive agent for HIV-related diseases, including HIV infection, candidiasis, coccidioidomycosis, cryptococcosis, cryptosporidiosis, cytomegalovirus, herpes simplex virus, shingles, histoplasmosis, isosporosis, and Mycobacterium avium complex (mycobacterium avium complex). complex), Pneumocystis pneumonia, bacterial pneumonia, progressive multifocal leukoencephalopathy, opportunistic infections selected from salmonella, toxoplasmosis, tuberculosis, AIDS-related cancers selected from cervical cancer, Kaposi's sarcoma, and lymphoma, candidiasis of the esophagus, bronchus, trachea, or lung, invasive cervical cancer, disseminated or extrapulmonary coccidioidomycosis, extrapulmonary cryptococcosis, chronic intestinal cryptosporidiosis, cytomegalovirus disease (other than liver, spleen, or nodules), cytomegalovirus retinitis with vision loss, HIV-associated encephalopathy, herpes simplex (associated with chronic ulcers, bronchitis, interstitial pneumonia, or esophagitis), disseminated or extrapulmonary histoplasmosis, chronic intestinal isosporosis, Kaposi's sarcoma, Burkitt's disease Lymphoma, immunoblastic lymphoma, primary cerebral lymphoma, disseminated or extrapulmonary Mycobacterium avium complex or Mycobacterium kansasii, pulmonary or extrapulmonary Mycobacterium tuberculosis, disseminated or extrapulmonary Mycobacterium species, Pneumocystis jirovecii pneumonia, recurrent pneumonia, progressive multifocal leukoencephalopathy, recurrent Salmonella septicemia, cerebral toxoplasmosis, and HIV-induced wasting syndrome, AIDS dementia complex, AIDS-induced encephalopathy, HIV encephalopathy, HIV-associated progressive encephalopathy, HIV-associated neurocognitive disorder (HAND), asymptomatic neurocognitive disorder (ANI), mild neurocognitive disorder (MND), HIV-associated dementia (HAD), mild cognitive and motor disorder (MCMD), vacuolar myelopathy, peripheral neuropathy, polymyositis, etc. Particularly preferred is HIV infection.

[0081] The present invention will be explained in more detail below by way of examples and reference examples of the present invention, but the present invention is not limited to these examples.

[0082] Example 1: Preparation of template DNA for IVT of Tat mRNA Plasmid DNA was amplified by PCR and purified to prepare template DNA for IVT. A DNA fragment containing a sequence in which the T7 promoter sequence, 5'-UTR sequence, ORF, and 3'-UTR sequence were linked in order was introduced into a plasmid. The plasmid was dissolved in nuclease-free water, to which 2x Prime STAR® MAX DNA polymerase Premix (Takara catalog #R045), 10 μM sense primer (SEQ ID NO: 180), and 10 μM antisense primer (SEQ ID NO: 181) were added. After incubation at 98°C for 2 minutes, the DNA was amplified by 35 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 10 seconds. After the reaction, the template DNA was purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (Takara catalog #740609.250).

[0083] Example 2 Preparation of Tat mRNA by IVT The template DNA obtained in Example 1, 75 mM ATP, 75 mM GTP, 75 mM CTP or 100 mM 5-methyl-CTP, 100 mM N1-methylpseudo-UTP or 100 mM 5-methyl-UTP, nuclease-free water, 10X Reaction Buffer (Thermo Fisher catalog #AMB13345), and Enzyme mix T7 RNA Polymerase (Thermo Fisher catalog #AMB13345) were mixed and incubated at 37°C for 2 hours. TURBO DNase (Thermo Fisher catalog #AM2239) was mixed and incubated at 37°C for 15 minutes. 7.5 M LiCl solution was mixed and left to stand overnight at -20°C. After centrifugation, the supernatant was discarded. 70% ethanol was added thereto, and after centrifugation, the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water, and a portion of the resulting solution was mixed with nuclease-free water, 10 mM GTP, 20 mM SAM, 10X Capping Buffer (New England Biolab catalog #M2080), Vaccinia Capping Enzyme (10 U / μL, New England Biolab catalog #M2080), and mRNA Cap 2'-O-Methyltransferase (50 U / μL, New England Biolab catalog #M2080). #M0366) was mixed and incubated at 37°C for 2 hours. 7.5 M LiCl solution was added and the mixture was left standing overnight at -20°C. After centrifugation, the supernatant was discarded, 70% ethanol was added, and after centrifugation, the supernatant was discarded and the mixture was air-dried.The resulting residue was dissolved in nuclease-free water, and the resulting solution was mixed with 10X Antarctic Phosphatase Reaction Buffer (New England Biolab catalog #B0289) and Antarctic Phosphatase (10 μL, New England Biolab catalog #M0289). The mixture was incubated at 37°C for 30 minutes, and then purified using Monarch RNA Cleanup Kit (New England Biolab catalog #T2050) according to the attached manual to obtain the target mRNA. The obtained mRNA was analyzed using TapeStation (Agilent) to confirm that it was of the desired length.

[0084] Example 3: Synthesis of Tat mRNA Using the methods of Examples 1 and 2, HIV Tat mRNA was synthesized having the 5'UTR, ORF, 3'UTR, Poly(A), and modified bases shown in Tables 1, 2-1, and 2-2. The 5'Cap structure of each Tat mRNA is Cap1, which is represented as 5'-[m7G]-ppp-GmGG, where Gm represents a guanosine methylated at the 2' position. Specifically, it is represented by the following structure: In addition, by using CleanCap (registered trademark) Reagent AG and CleanCap (registered trademark) Reagent AU (both TriLink), HIV Tat mRNA having Cap1 represented by 5'-[m7G]-ppp-AmGG or 5'-[m7G]-ppp-AmUA can be synthesized in a similar manner.

[0085] In all Tat mRNA sequences, all uridine bases are replaced with N1-methylpseudouridine (m1Ψ) or 5-methyluridine (5MeU) bases, and in some sequences, all cytidine bases are replaced with 5-methylcytidine (5MeC) bases. The replaced modified bases are listed in the "Modified base" column in the table. All mRNAs except SNG-330 and SNG-337 encode secretory signal peptides and Tat proteins, and the peptide names and their amino acid sequence numbers are listed in the "peptide" column in the table. Note that the nucleic acid sequences of mRNAs other than SNG-299 have been optimized for the secretory signal peptide codons and Tat codons, respectively. For example, SNG-331 represents an mRNA encoding the Igk HIV-Tat BH10 protein (SEQ ID NO: 168) in which the secretory signal peptide Igk (SEQ ID NO: 162) is linked to the N-terminus of the HIV-Tat BH10 protein (SEQ ID NO: 134). SNG-331 is an mRNA consisting, from 5' to 3', of a 5'Cap structure consisting of 5'-[m7G]-ppp-GmGG, a 5'UTR represented by SEQ ID NO: 1, an ORF represented by SEQ ID NO: 114, a 3'UTR represented by SEQ ID NO: 64, and an approximately 80-mer Poly(A), in which all uridine bases are m1Ψ. SNG-346 and SNG-347 represent mRNAs encoding IgE HIV-Tat Oyi MITD (SEQ ID NO: 191) protein or IgE HIV-Tat Oyi Foldon (SEQ ID NO: 192) protein, in which the secretory signal peptide IgE (SEQ ID NO: 161) is linked to the N-terminus of the HIV-Tat Oyi protein (SEQ ID NO: 133) and the secretory signal peptide MITD (SEQ ID NO: 188) or Foldon (SEQ ID NO: 189) is linked to the C-terminus. The other tables are similarly described.

[0086]

[0087]

[0088]

[0089] Furthermore, SIV Tat mRNA having the structure shown in Table 3, in which all uridine bases in the sequence have been replaced with N1-methylpseudouridine (m1Ψ) bases, was purchased from Trilink.

[0090]

[0091] Example 4: Construction of an ELISA system for measuring HIV-1 Tat protein. An anti-HIV-1 Tat monoclonal antibody (Abcam) was immobilized on a 384-well flat-bottom ELISA plate to prepare an anti-Tat antibody solid-phase plate. An ELISA system for measuring Tat protein was constructed by combining a biotin-labeled anti-HIV-1 Tat polyclonal antibody (Abcam), horseradish peroxidase-labeled streptavidin (Thermo Fisher Scientific), and SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

[0092] Example 5 Evaluation of Tat Expression in Cultured Cell Lines Using Tat mRNA The mRNA synthesized in Example 3 was used to evaluate the ability of a cultured cell line to express Tat protein. A human cervical cancer-derived cell line, HeLa, was suspended in DMEM medium containing 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 0.10 mg / ml streptomycin at a concentration of 10,000 cells / 90 μL. 90 μL of the suspension was seeded into a 96-well flat-bottom plate and incubated at 37°C, 95-98% humidity, and 5% CO. ２The cells were cultured at 37°C for 1 day. The next day, 100 ng of each mRNA listed in Table 3 was diluted in 5 μL of OPTI-MEM (Thermo Fisher Scientific). Lipofectamine MessengerMAX Transfection Reagent (Thermo Fisher Scientific) was used as the transfection reagent and diluted in 5 μL of OPTI-MEM. The amount of transfection reagent added was adjusted to 0.2 μL per 100 ng of mRNA. After incubation at room temperature for 10 minutes, both solutions were mixed and incubated for an additional 5 minutes. The mixed solution was diluted with OPTI-MEM to a concentration of 25 ng / 10 μL of mRNA, and 10 μL was added to a cell culture plate. Culture was then continued at 37°C. The test was performed with N = 2 or 3 for each condition. After washing with PBS, HeLa cells were solubilized with 100 μL of a solution containing RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) and a protease inhibitor cocktail (Merck). The Tat protein concentration (ng / ml) in the collected cell lysate was quantified using the ELISA system described in Example 4 (Tables 4-1 to 4-4). The leftmost columns in Tables 4-1 to 4-4 indicate the numbers of the control and mRNA used. All mRNAs were confirmed to express Tat protein.

[0093]

[0094]

[0095]

[0096]

[0097] Example 6 Preparation of mRNA-Encapsulated Nucleic Acid Lipid Particles Using Tat mRNA A cationic lipid described in WO 2022 / 168884 (hereinafter referred to as Lipid), distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, NIPPON FINE CHEMICAL CO., LTD.), and 1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol (hereinafter referred to as PEG-DMG, NOF) having a molecular weight of approximately 2,000 were used to prepare mRNA-encapsulated nucleic acid lipid particles. CHOCOLATE (DIPOLAR CORP.) was dissolved in ethanol at a molar ratio of DSPC:Chol:Lipid:PEG-DMG = 8.4:36:54:1.6 to a total lipid concentration of 33 mM to obtain a lipid solution. Meanwhile, the Tat mRNA synthesized in Example 3 was adjusted to 292 μg / mL in citrate buffer (pH 4.0) to obtain an mRNA solution. The lipid solution and mRNA solution were mixed in a microchannel using a NanoAssembler IGNITE (Precision Nanosystems Inc.) so that the volume ratio was 1:3, and a crude dispersion of nucleic acid-lipid particles was obtained. The crude dispersion of nucleic acid-lipid particles described above was diluted in-line with phosphate buffer (pH 7.5) at a volume ratio of 2:1 (the microchannel used was an NxGen mixer with dilution for the IGNITE microfluidic cartridge) to obtain a dispersion of nucleic acid-lipid particles.The nucleic acid-lipid particle dispersion was dialyzed against approximately 100 times the volume of 20 mM Tris-HCl buffer for 3 hours, and then dialyzed against approximately 100 times the volume of 20 mM Tris-HCl solution for a further 12 to 18 hours (Slide-A-Lyzer Dialysis Cassette G2, MWCO: 10 kDa, Spectra / Por) to remove ethanol. The dispersion was then concentrated using an Amicon (Amicon Ultra-15 PLHK Ultracel-PL membrane, MWCO 100 kDa, Merck). The concentration was adjusted with 20 mM Tris-HCl buffer, and the dispersion was then mixed with an additive stock (dispersion: 20 mM Tris-HCl buffer and 1.05 M Sucrose mixed at a volume ratio of 1:2) to obtain a 20 mM A dispersion of purified mRNA-encapsulating nucleic acid-lipid particles was obtained in Tris-HCl buffer and 350 mM sucrose solution.

[0098] Example 7: Evaluation of the antibody induction ability of Tat mRNA in mice. Mice were inoculated twice, three weeks apart, into the hind thigh muscle with an RNA vaccine containing 25 μg of mRNA per mouse. Two weeks after the second immunization, whole blood was collected from the abdominal vena cava using a syringe with a needle containing 10 μL of heparin, and then centrifuged in a refrigerated centrifuge (20,400 g, 5 minutes, 4°C) to obtain plasma. The anti-Tat antibody titer in the plasma was measured by ELISA. Tat Oyi-Peptide 101aa (Peptide Institute, Inc.) was prepared at 1 μg / mL with PBS, pH 7.4. 25 μL of the prepared solution was dispensed onto a plate, sealed, and allowed to stand overnight at 4°C. The next day, the antigen solution was removed from the plate, and 90 μL of wash buffer (PBS-T) was added per well, followed by two washes. 50 μL of blocking buffer (1% BSA / PBS) was added per well to the washed plate and allowed to stand at room temperature. After 1 hour, the blocking buffer was removed from the plate, and 90 μL of washing solution was added per well, followed by three washes. Plasma was diluted with dilution buffer (1% BSA / PBS) and added to the washed plate at 25 μL per well (N=2) and allowed to stand at room temperature. After 2 hours, the plasma sample was removed from the plate, and 90 μL of washing solution was added per well, followed by three washes. 25 μL of diluted HRP-labeled goat anti-mouse IgG Fc antibody (Jackson Immuno Research) was added per well as a secondary antibody to the washed plate and allowed to stand at room temperature. After 1 hour, the secondary antibody solution was removed from the plate, and 90 μL of washing solution was added per well, followed by three washes. 25 μL of TMB solution was added per well to the washed plate and allowed to stand at room temperature. 25 μL of 2N sulfuric acid was added per well for 10 minutes to stop the reaction. The absorbance at 450 nm was measured using an absorbance meter. The anti-Tat antibody titer was calculated using a program with equivalent calculation processing power, such as XLfit®, as follows. The absorbance of wells containing only dilution buffer was used as the blank, and the cutoff value was set by adding 0.5 to the average blank value.The dilution factor at which the mean absorbance of N=2 in the plasma sample dilution series reached the cutoff value was calculated as the anti-Tat antibody titer, and the Geometric Mean Titer (GMT) for each group was calculated. The results are shown in Tables 5, 6-1, and 6-2. When the absorbance was below the cutoff value at all dilutions, the lowest dilution factor was used as the antibody titer. Compared to mRNA encoding only HIV Tat protein, the mRNA of the present invention exhibited higher anti-Tat antibody titers (Tables 5 and 6-1). Furthermore, all of the mRNA of the present invention exhibited excellent anti-Tat antibody titers (Table 6-2). Using a similar method, mice were inoculated into the hind thigh muscle three times at 4-week intervals with 30 μg of Tat BH10 protein (SEQ ID NO: 134) or Tat Oyi protein (SEQ ID NO: 133) per mouse, and the anti-Tat antibody titers in plasma were measured two weeks after the second and third immunizations (Table 6-3). It was suggested that two administrations of the mRNA of the present invention exhibited superior anti-Tat antibody induction ability to three administrations of Tat protein (Tables 5, 6-1, 6-2 and 6-3).

[0099]

[0100]

[0101]

[0102]

[0103] Reference Example 1: Evaluation of the antibody induction ability of SIV-Tat mRNA in mice. Mice were inoculated twice, three weeks apart, into the hind thigh muscle with an RNA vaccine containing 25 μg of mRNA per mouse. Two weeks after the second immunization, blood was collected from the tail vein using a heparinized capillary tube and centrifuged in a refrigerated centrifuge (20,400 g, 5 minutes, 4°C) to obtain plasma. Alternatively, mice under isoflurane anesthesia underwent laparotomy, and blood was collected from the abdominal vena cava using a syringe with a syringe needle. After storing at room temperature for 4 to 5 hours, blood was centrifuged in a refrigerated centrifuge (200 g, 15 minutes, 0°C) to obtain serum. The anti-SIV-Tat antibody titer in the plasma or serum was measured by ELISA. SIV-Tat Protein (Creative Diagnostics) was adjusted to 1 μg / mL with PBS, pH 7.4. The prepared solution was dispensed into the plate in 25 μL aliquots, the plate was sealed, and the plate was left standing overnight at 4°C. The next day, the antigen solution was removed from the plate, and 80 μL of wash buffer (0.05% Tween-20 / 1% BSA / PBS) was added per well, followed by washing three times. 75 μL of blocking buffer (1% BSA / PBS) was added per well to the washed plate, and the plate was left standing at room temperature. After 1 hour, the blocking buffer was removed from the plate, and 80 μL of wash buffer was added per well, followed by washing three times. Blood samples were diluted with dilution buffer (1% BSA / PBS) and added to the washed plate at 25 μL per well (N=2), followed by leaving the plate standing at room temperature. After 2 hours, the blood samples were removed from the plate, and 80 μL of wash buffer was added per well, followed by washing three times. A diluted HRP-labeled goat anti-mouse IgG Fc antibody (Jackson Immuno Research) was added to the washed plate as a secondary antibody at 25 μL per well and allowed to stand at room temperature. After 1 hour, the secondary antibody solution was removed from the plate, and 80 μL of wash buffer was added per well, followed by washing three times. 25 μL of ELISA Pico Chemiluminescent Substrate solution (Thermo Scientific) was added to the washed plate at room temperature. After 5 minutes, the luminescence signal was measured using a luminometer.Anti-Tat antibody titers were calculated using a program with equivalent computational power, such as XL_fit, using the following method. The luminescence signal from wells containing only dilution buffer was used as the blank, and the cutoff value was determined by adding 100,000 to the average blank value. The dilution factor at which the average luminescence signal from N=2 in the blood sample dilution series reached the cutoff value was calculated as the anti-SIV-Tat antibody titer, and the GMT for each group was calculated. The results are shown in Table 7. If the luminescence signal was below the cutoff value at all dilution factors, the lowest dilution factor was used as the antibody titer. Compared to mRNA encoding only SIV Tat protein, mRNA encoding SIV Tat protein and secretory signal peptide showed higher anti-Tat antibody titers (Table 7). This suggests that mRNA encoding Tat protein and secretory signal peptide, such as HIV-2 Tat, which has an amino acid sequence similar to SIV Tat, also has a similar effect.

[0104]

[0105] As is clear from the above, the polynucleotide of the present invention encoding (a) an HIV Tat protein or an antigenic fragment thereof and (b) a polypeptide comprising a signal peptide can efficiently express the HIV Tat protein or an antigenic fragment thereof and has excellent anti-Tat antibody induction ability. Therefore, the pharmaceutical composition of the present invention is highly useful as a medicament for treating and / or preventing HIV-related diseases (e.g., HIV infection, HAND, etc.).

Claims

1. A polynucleotide encoding a polypeptide comprising the following: (a) an HIV Tat protein or an antigenic fragment thereof; and (b) a secretory signal peptide.

2. The polynucleotide according to claim 1, wherein (b) is (i) or (ii): (i) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 161 to 167 and 183 to 187; or (ii) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 161 to 167 and 183 to 187, and having secretion signal activity.

3. The polynucleotide of claim 1 or 2, wherein (a) is the HIV Tat protein.

4. The polynucleotide according to any one of claims 1 to 3, wherein the HIV Tat protein is the Tat protein of HIV-1.

5. The polynucleotide according to claim 4, wherein the HIV-1 Tat protein is a polypeptide consisting of an amino acid sequence that is identical to or has 90% or more identity to any of the amino acid sequences set forth in SEQ ID NOs: 131 to 148 and 182.

6. A polynucleotide according to any one of claims 1 to 5, wherein the polypeptide comprising (a) and (b) is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 168 to 178 and 190 to 197.

7. The polynucleotide according to any one of claims 1 to 6, which is an mRNA comprising an open reading frame encoding a polypeptide comprising the following: (a) an HIV Tat protein or an antigenic fragment thereof; and (b) a secretory signal peptide.

8. The polynucleotide of claim 7, comprising modified bases selected from 5-methyluridine, pseudouridine, N1-methylpseudouridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudouridine, 4-thiouridine, 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-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-O-methyluridine, or combinations thereof.

9. The polynucleotide of claim 7 or 8, which comprises a 5'Cap structure which is Cap1 or Cap2.

10. The polynucleotide of claim 9, wherein the 5'Cap structure is Cap1.

11. The polynucleotide according to any one of claims 7 to 10, comprising a 60-160 mer Poly(A).

12. A polynucleotide according to any one of claims 7 to 11, comprising a 5'UTR which is a polynucleotide consisting of a base sequence which is identical or has 90% or more identity to the base sequence set forth in any one of SEQ ID NOs: 1 to 6.

13. A polynucleotide according to any one of claims 7 to 12, comprising a 3'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity to any one of the base sequences set forth in SEQ ID NOs: 64 to 69.

14. A polynucleotide according to any one of claims 7 to 13, wherein the open reading frame consists of a polynucleotide having a base sequence set forth in any one of SEQ ID NOs: 114 to 119, 121 to 126, and 198 to 210.

15. The polynucleotide according to any one of claims 7 to 14, which is an mRNA defined as any one of SNG-299, 322 to 325, 327, 328, 331 to 336, 338 to 341 and 345.

16. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 15.

17. The pharmaceutical composition according to claim 16, which is a therapeutic and / or prophylactic agent for HIV infection.