Nucleic acid constructs utilizing SNARE

Abstract

To provide a nucleic acid construct that enhances an allergen-specific immune response.SOLUTION: A nucleic acid construct includes a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen, the SNARE protein being selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding an antigen. [Background technology]

[0002] Vaccines are a means of preventing infection and treating diseases by administering antigens and establishing acquired immunity against them. Traditionally, vaccines have been administered mainly by subcutaneous or intramuscular injection, including live vaccines that use weakened bacteria or viruses as vaccines, inactivated vaccines in which bacteria or viruses are treated with formalin or heat to eliminate their infectivity, and toxoids, which are toxins produced by bacteria that have been isolated, purified, and inactivated with formalin. In addition to these, development is underway to improve efficacy, safety, and convenience by developing mucosal vaccines, which are administered to mucous membranes, and nucleic acid vaccines, which use nucleic acids as antigenic components. Among these, the development of nucleic acid vaccines is progressing rapidly as a new modality that can be manufactured quickly and at low cost.

[0003] Improving the immunogenicity of antigens is one of the important challenges in enhancing the effectiveness of nucleic acid vaccines, and it is necessary to guide antigens to appropriate tissues or antigen-presenting cells and efficiently induce an immune response. Patent Document 1 discloses that the immune response to an antigen was enhanced by using nucleic acids encoding a fusion protein in which an allergen protein (antigen) is inserted between the organelle-stabilizing domain and the transmembrane domain of a lysosomal associated membrane protein (LAMP). On the other hand, it has been reported that when an antigen is added to the lumen of LAMP, antigen-specific antibody production may be significantly reduced (Non-Patent Document 1). Furthermore, Patent Document 2 discloses that the immunogenicity of an antigen was increased by using nucleic acids encoding a fusion molecule containing an antigen, a transmembrane domain, and the cytoplasmic domain of a major histocompatibility complex (MHC) molecular chain. Patent Document 2 also mentions a fusion molecule containing an antigen and the cytoplasmic domain of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, but it does not show at all how the immunogenicity of the antigen changes with this fusion molecule.

[0004] SNARE proteins are a protein family having a 20-30 kDa SNARE motif. Many SNARE proteins are anchored on lipid bilayers via a C-terminal transmembrane domain and are involved in the process by which vesicles fuse with target intracellular organelles (Non-Patent Documents 2 and 3). Membrane fusion mediated by SNARE proteins is essential for many important vital phenomena indispensable for cell function in eukaryotic cells, including vesicle transport, organelle membrane morphogenesis, endocytosis processes including recycling of extracellular receptors, and exocytosis processes including hormone secretion and release of synaptic neurotransmitters. Furthermore, the molecular mechanism of membrane fusion mediated by SNARE proteins is considered to be conserved across all eukaryotes, ranging from unicellular budding yeast to higher animals including humans. Based on the characteristics of the amino acid residues of the SNARE motif, SNARE proteins are further classified into four subfamilies: QA-SNARE, QB-SNARE, QC-SNARE, and R-SNARE. Each SNARE protein localizes to a specific intracellular membrane fraction (organelles such as the endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles / lysosomes, secretory vesicles, and cytoplasmic membrane) and is considered to function in the membrane fusion process along specific intracellular transport pathways. It is also known that SNARE proteins may be contained in exosomes.

Prior Art Literature

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

[0007] The present invention relates to providing a nucleic acid construct that enhances an antigen-specific immune response. [Means for Solving the Problem]

[0008] The present inventors have found that, by using a nucleic acid construct comprising a polynucleotide encoding a specific SNARE protein and a polynucleotide encoding an antigen, an antigen-specific Th1-type immune response can be induced or enhanced, and an antigen-specific cellular immune response can also be induced or enhanced, as compared with a nucleic acid construct comprising only the polynucleotide encoding the antigen.

[0009] Accordingly, the present invention provides the following 1) to 4). 1) A nucleic acid construct comprising a polynucleotide encoding any SNARE protein selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and a polynucleotide encoding an antigen. 2) An antigen-specific Th1-type immune response inducer or enhancer comprising the nucleic acid construct of 1) as an active ingredient. 3) An antigen-specific cellular immune response inducer or enhancer comprising the nucleic acid construct of 1) as an active ingredient. 4) A nucleic acid vaccine comprising the nucleic acid construct of 1) as an active ingredient. [Effects of the Invention]

[0010] The nucleic acid construct of the present invention can induce or enhance an antigen-specific Th1-type immune response, and can also induce or enhance an antigen-specific cellular immune response. Such a nucleic acid construct is useful as a nucleic acid vaccine. [Brief explanation of the drawing]

[0011] [Figure 1] Evaluation of immunogenicity of mRNA encoding SNARE protein-antigen fusion polypeptide. IFNγ production is indicated by the number of spot-forming cells (SFCs). Empty represents the control with transfection reagent only, Empty(NT) represents the control without antigen restimulation, and SNARE protein-OVA represents mRNA encoding SNARE protein-antigen (OVA) fusion polypeptide. [Figure 2] Antibody titers of IgG subclasses after administration of plasmid vectors encoding SNARE protein-antigen fusion polypeptides to mice. (A) shows the IgG antibody titer, (B) shows the IgG1 antibody titer, and (C) shows the IgG2a antibody titer. Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, and V7-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide. [Figure 3] Evaluation of the immune response when a plasmid vector encoding a SNARE protein-antigen fusion polypeptide was administered to mice. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, as measured by SFC. (C) shows the ratio of IFNγ production to IL-4 production (IFNγ / IL-4). Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, and V7-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide. [Figure 4]Antibody titers of IgG subclasses after administration of plasmid vectors encoding SNARE protein-antigen fusion polypeptides to mice. (A) shows the IgG antibody titer, (B) shows the IgG1 antibody titer, (C) shows the IgG2a antibody titer, and (D) shows the ratio of the IgG2a antibody titer to the IgG1 antibody titer (IgG2a / IgG1). Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, and V7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA indicate plasmid vectors that encode VAMP7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA fusion polypeptides, respectively. [Figure 5] Evaluation of the immune response when a plasmid vector encoding a SNARE protein-antigen fusion polypeptide was administered to mice. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, both measured by SFC. (C) shows the ratio of IL-4 production under antigen-stimulated conditions to unstimulated conditions (IL-4 ratio). Empty represents an empty plasmid vector that does not encode the antigen (OVA), OVA represents a plasmid vector that encodes OVA, and V7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA represent plasmid vectors that encode VAMP7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA fusion polypeptides, respectively. [Figure 6]Antibody titers over time after administration to mice with a plasmid vector encoding a SNARE protein-antigen fusion polypeptide or a fusion polypeptide containing an antigen within LAMP. (A) shows the IgG antibody titer, (B) shows the IgG1 antibody titer, (C) shows the IgG2a antibody titer, and (D) shows the ratio of the IgG2a antibody titer to the IgG1 antibody titer at week 5 (IgG2a / IgG1). Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, V7-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide, V7-pc-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence, and LAMP[OVA] indicates a plasmid vector that encodes a fusion polypeptide containing OVA within LAMP. [Figure 7] Evaluation of the immune response when a plasmid vector encoding a SNARE protein-antigen fusion polypeptide was administered to mice. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, both measured by SFC. (C) shows the ratio of IL-4 production under antigen-stimulated conditions to unstimulated conditions (IL-4 ratio). Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, V7-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide, and V7-pc-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence. [Figure 8]Antibody titers of IgG subclasses when mice were administered plasmid vectors encoding SNARE protein-antigen fusion polypeptides. (A) to (D) show the results when WT1 was used as the antigen, and (E) to (H) show the results when MOG35-55 was used as the antigen. (A) and (E) show the IgG antibody titer, (B) and (F) show the IgG1 antibody titer, (C) and (G) show the IgG2a antibody titer, and (D) and (H) show the ratio of the IgG2a antibody titer to the IgG1 antibody titer (IgG2a / IgG1). Empty indicates an empty plasmid vector that does not encode an antigen (WT1 or MOG35-55), WT1 and MOG35-55 indicate plasmid vectors encoding WT1 and MOG35-55, respectively, and V7-pc-WT1 and V7-pc-MOG35-55 indicate plasmid vectors encoding VAMP7-proprotein convertase recognition sequence-WT1 and VAMP7-proprotein convertase recognition sequence-MOG35-55 fusion polypeptides, respectively. [Figure 9] Evaluation of the immune response when a plasmid vector encoding a SNARE protein-antigen fusion polypeptide was administered to mice. (A) to (D) show the results when WT1 was used as the antigen, and (E) to (H) show the results when MOG35-55 was used as the antigen. (A) and (E) show the amount of IFNγ produced, and (B) and (F) show the amount of IL-4 produced, expressed as SFC. (C) and (G) show the ratio of IL-4 production under antigen-stimulated conditions to unstimulated conditions (IL-4 ratio), and (D) and (H) show the ratio of IFNγ production to IL-4 production (IFNγ / IL-4). Empty represents an empty plasmid vector that does not encode an antigen (WT1 or MOG35-55), WT1 and MOG35-55 represent plasmid vectors encoding WT1 and MOG35-55, respectively, and V7-pc-WT1 and V7-pc-MOG35-55 represent plasmid vectors encoding VAMP7-proprotein convertase recognition sequence-WT1 and VAMP7-proprotein convertase recognition sequence-MOG35-55 fusion polypeptides, respectively. [Figure 10]Evaluation of the immune response when mice were administered plasmid vectors encoding SNARE protein-antigen fusion polypeptides or fusion polypeptides containing an antigen within LAMP. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, expressed as SFC. (C) shows the ratio of IL-4 production under antigen-stimulated conditions to unstimulated conditions (IL-4 ratio), and (D) shows the ratio of IFNγ production to IL-4 production (IFNγ / IL-4). Empty indicates an empty plasmid vector that does not encode the antigen (OVA), OVA indicates a plasmid vector that encodes OVA, V7-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide, V7-pc-OVA indicates a plasmid vector that encodes a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence, and LAMP[OVA] indicates a plasmid vector that encodes a fusion polypeptide containing OVA within LAMP. [Modes for carrying out the invention]

[0012] In this specification, the terms “nucleic acid,” “nucleotide,” “oligonucleotide,” or “polynucleotide” are used interchangeably and mean DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, while RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA. mRNA, in particular, is synthesized by an in vitro transcription reaction for reasons such as initiating translation in vivo, stabilizing mRNA, and suppressing degradation. After synthesis, a 5' cap (methylated guanosine) is added by a cap-forming enzyme, and poly(A) polymerase is added. The poly(A) sequence may be incorporated into the template DNA used in the in vitro transcription reaction. This includes mRNA with a cap structure or polyA added, as well as RNA with some base modifications (for example, uridine being replaced with pseudouridine or 1-methylpseudridine).

[0013] In this specification, "gene" means a double-stranded DNA including genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA having a sequence complementary to the positive strand (complementary strand), and fragments thereof, in which the sequence information of the bases constituting the DNA contains some kind of biological information. Furthermore, the term "gene" in question encompasses not only genes represented by a specific nucleotide sequence, but also nucleic acids encoding their homologs (i.e., homologs or orthologs), variants such as genetic polymorphisms, and derivatives. The gene names and Gene IDs disclosed herein follow the Official Symbols and Gene IDs listed on the NCBI ([www.ncbi.nlm.nih.gov / ]).

[0014] In this specification, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably.

[0015] In this specification, "amino acid residue" means the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0016] In this specification, the identity of nucleotide or amino acid sequences is calculated using the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the homology analysis (Search homology) using the Genetyx-Win genetic information processing software with a Unit size to compare (ktup) of 2.

[0017] In this specification, "at least 80% identity" with respect to a nucleotide sequence or amino acid sequence means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0018] In this specification, unless otherwise defined, “one or several” as used with respect to the deletion, substitution, addition or insertion of nucleotides in a nucleotide sequence may preferably mean 1 to 15, more preferably 1 to 9, and even more preferably 1 to 6. Similarly, in this specification, unless otherwise defined, “one or several” as used with respect to the deletion, substitution, addition or insertion of amino acid residues in an amino acid sequence may preferably mean 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. In this specification, “addition” of a nucleotide or amino acid residue includes the addition of a nucleotide or amino acid residue to one end and both ends of a sequence.

[0019] In this specification, "stringent conditions" refer to the conditions for the Southern hybridization method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001), for example, conditions in which a solution containing 6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhart, and 100 mg / mL herring sperm DNA is incubated with the probe at 42°C for 8 to 16 hours to hybridize.

[0020] In this specification, "fragment" of a polynucleotide means a partial polynucleotide of the polynucleotide. The length of a partial polynucleotide is not particularly limited, as long as it encodes a polypeptide having the same function as the polypeptide encoded by the full-length polynucleotide. For example, a partial polynucleotide may mean a polynucleotide consisting of consecutive nucleotides that are preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and preferably 90% or less, more preferably 80% or less, even more preferably 70% or less, and even more preferably 60% or less in length relative to the full-length polynucleotide. Also in this specification, "fragment" of a polypeptide means a partial polypeptide of the polypeptide. The length of a partial polypeptide is not particularly limited. For example, a partial polypeptide may mean a polypeptide consisting of consecutive amino acid residues that are preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and preferably 90% or less, more preferably 80% or less, even more preferably 70% or less, and even more preferably 60% or less in length relative to the full-length polypeptide.

[0021] In this specification, a “regulatory region” is a region that has the function of controlling the expression of a gene located downstream of it (for example, a protein-coding region). More specifically, a “regulatory region” can be defined as a region located upstream of the coding region of a gene that interacts with RNA polymerase to control the transcription of the coding region. A regulatory region includes a transcription initiation regulatory region and / or a translation initiation regulatory region, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation regulatory region is the region containing the promoter and the transcription start site, and the translation initiation regulatory region is the region corresponding to the Kozak sequence necessary for translation initiation, which is recognized by the ribosome along with the start codon.

[0022] In this specification, "operable linkage" between a regulatory region and a polynucleotide of a gene (e.g., a polynucleotide encoding a protein) means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0023] In this specification, "expressible linkage" between the polynucleotide of a first gene (e.g., a protein-coding polynucleotide) and the polynucleotide of a second gene (e.g., a protein-coding polynucleotide) means that the first gene and the second gene are linked such that, when inserted into a suitable expression vector and introduced into a suitable cell, the protein encoded by the first gene and the protein encoded by the second gene are produced as a fusion protein. Here, "linkage" includes cases where the first gene and the second gene are directly linked, as well as cases where they are linked via other nucleotide sequences. The procedure for "expressible linkage" between the first gene and the second gene is well known to those skilled in the art.

[0024] In this specification, "upstream" and "downstream" with respect to a gene or its nucleotide sequence refer to the upstream and downstream directions of the gene's transcription. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 5' and 3' ends of the gene in the DNA sense strand, respectively.

[0025] In this specification, "antigen" means a molecule that triggers an immune response in a living organism, such as antibody production or cellular immunity. In this specification, "immunogenicity" means the property of an antigen to induce antibody production or cellular immunity.

[0026] In this specification, "nucleic acid vaccine" refers to a vaccine that induces immunity by administering a polynucleotide (DNA or RNA) encoding an antigen to a living organism. Nucleic acid vaccines include DNA vaccines, mRNA vaccines, and viral vector vaccines, all of which are thought to induce both humoral and cellular immunity. DNA vaccines contain plasmids encoding antigens. Plasmids administered to living organisms are taken up by cells, transcribed into mRNA in the nucleus, and then translated into antigen proteins in the cytoplasm, inducing an antigen-specific immune response. mRNA vaccines contain mRNA encoding antigens. mRNA administered to living organisms is taken up by cells, translated into antigen proteins in the cytoplasm, inducing an antigen-specific immune response. Viral vector vaccines contain non-pathogenic or attenuated viral vectors incorporating polynucleotides encoding antigens. Viruses administered to living organisms invade cells, causing cells to synthesize antigen proteins, inducing an antigen-specific immune response.

[0027] In this specification, "cellular immunity" refers to the acquired immune response that works to eliminate foreign substances such as pathogens, virus-infected cells, and cancer cells that have entered the body, and which uses cytotoxic T cells, macrophages, NK cells, etc. as effectors. In this specification, "humoral immunity" refers to the acquired immune response that uses antibodies as effectors.

[0028] In this specification, "Th1-type immune response" refers to the immune response promoted by Th1 cells, a subset of helper T cells. Th1 cells primarily produce IFNγ as a cytokine and target M1 macrophages, cytotoxic T cells, and NK cells, mainly inducing cellular immunity. Th1 cells are known to activate B cells and induce IgG2a production. In this specification, "Th2-type immune response" refers to the immune response promoted by Th2 cells, a subset of helper T cells. Th2 cells primarily produce IL-4, IL-5, and IL-13 as cytokines and target mast cells, M2 macrophages, eosinophils, and basophils, mainly inducing humoral immunity. Th2 cells are known to activate B cells and induce IgG1 production.

[0029] The present invention provides a nucleic acid construct comprising a polynucleotide encoding any SNARE protein selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and a polynucleotide encoding an antigen.

[0030] In the present invention, a SNARE protein is a protein belonging to the protein family having a SNARE motif, and specifically, it is any protein selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B (hereinafter sometimes simply referred to as a SNARE protein). The SNARE protein is preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, and GOSR1, more preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, and GOSR1, even more preferably selected from the group consisting of VAMP7, GOSR2, STX10, and GOSR1, and even more preferably VAMP7. The SNARE protein has a transmembrane domain and is localized to intracellular vesicles.

[0031] VAMP7 (vesicle-associated membrane protein 7) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to late endosomes, lysosomes, and cell membranes. In a preferred example, VAMP7 is mammalian VAMP7. In a more preferred example, VAMP7 is human VAMP7, which is a protein consisting of the amino acid sequence of SEQ ID NO: 10, encoded by a gene (Gene ID: 6845) consisting of the nucleotide sequence of SEQ ID NO: 1. The VAMP7 used in this invention includes VAMP7 and polypeptides having equivalent function.

[0032] GOSR2 (golgi SNAP receptor complex member 2) is a type of SNARE protein that has a transmembrane domain and is expected to be localized in the endoplasmic reticulum-Golgi intermediate region and the Golgi apparatus. In a preferred example, GOSR2 is mammalian GOSR2. In a more preferred example, GOSR2 is human GOSR2, which is a protein consisting of the amino acid sequence of SEQ ID NO: 11, encoded by a gene (Gene ID: 9570) consisting of the nucleotide sequence of SEQ ID NO: 2. The GOSR2 used in this invention includes GOSR2 and polypeptides having equivalent function.

[0033] STX(syntaxin)10 is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the trans-Golgi network. In a preferred example, STX10 is mammalian STX10. In a more preferred example, STX10 is human STX10, which is a protein consisting of the amino acid sequence of SEQ ID NO: 12, encoded by a gene (Gene ID: 8677) consisting of the nucleotide sequence of SEQ ID NO: 3. The STX10 used in this invention includes STX10 and polypeptides having equivalent function.

[0034] STX18 is a type of SNARE protein that has a transmembrane domain and is expected to be localized in the endoplasmic reticulum. In a preferred example, STX18 is mammalian STX18. In a more preferred example, STX18 is human STX18, which is a protein consisting of the amino acid sequence of SEQ ID NO: 13, encoded by a gene (Gene ID: 53407) consisting of the nucleotide sequence of SEQ ID NO: 4. The STX18 used in this invention includes STX18 and polypeptides having equivalent function.

[0035] BNIP1 (BCL2 interacting protein 1) is a type of SNARE protein that has a transmembrane domain and is expected to be localized in the endoplasmic reticulum. In a preferred example, BNIP1 is mammalian BNIP1. In a more preferred example, BNIP1 is human BNIP1, which is a protein consisting of the amino acid sequence of SEQ ID NO: 14, encoded by a gene (Gene ID: 662) consisting of the nucleotide sequence of SEQ ID NO: 5. The BNIP1 used in this invention includes BNIP1 and polypeptides having equivalent function.

[0036] STX7 is a type of SNARE protein that has a transmembrane domain and is expected to localize to early and late endosomes. In a preferred example, STX7 is mammalian STX7. In a more preferred example, STX7 is human STX7, which is a protein consisting of the amino acid sequence of SEQ ID NO: 15, encoded by a gene (Gene ID: 8417) consisting of the nucleotide sequence of SEQ ID NO: 6. The STX7 used in this invention includes STX7 and polypeptides having equivalent function.

[0037] VTI1A (vesicle transport through interaction with t-SNAREs 1A) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the trans-Golgi network. In a preferred example, VTI1A is mammalian VTI1A. In a more preferred example, VTI1A is human VTI1A, which is a protein consisting of the amino acid sequence of SEQ ID NO: 16, encoded by a gene (Gene ID: 143187) consisting of the nucleotide sequence of SEQ ID NO: 7. The VTI1A used in this invention includes VTI1A and polypeptides having equivalent function.

[0038] STX16 is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the trans-Golgi network. In a preferred example, STX16 is mammalian STX16. In a more preferred example, STX16 is human STX16, which is a protein consisting of the amino acid sequence of SEQ ID NO: 17, encoded by a gene (Gene ID: 8675) consisting of the nucleotide sequence of SEQ ID NO: 8. The STX16 used in this invention includes STX16 and polypeptides having equivalent function.

[0039] STX5 is a type of SNARE protein that has a transmembrane domain and is expected to be localized in the Golgi apparatus. In a preferred example, STX5 is mammalian STX5. In a more preferred example, STX5 is human STX5, which is a protein consisting of the amino acid sequence of SEQ ID NO: 18, encoded by a gene (Gene ID: 6811) consisting of the nucleotide sequence of SEQ ID NO: 9. The STX5 used in this invention includes STX5 and polypeptides having equivalent function.

[0040] GOSR1 is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the Golgi apparatus and the trans-Golgi network. In a preferred example, GOSR1 is mammalian GOSR1. In a more preferred example, GOSR1 is human GOSR1, which is a protein consisting of the amino acid sequence of SEQ ID NO: 76, encoded by a gene (Gene ID: 9527) consisting of the nucleotide sequence of SEQ ID NO: 71. The GOSR1 used in this invention includes GOSR1 and polypeptides having equivalent function.

[0041] STX8 is a type of SNARE protein that possesses a transmembrane domain and is expected to localize to early endosomes, late endosomes, and the cell membrane. In a preferred example, STX8 is mammalian STX8. In a more preferred example, STX8 is human STX8, which is a protein consisting of the amino acid sequence of SEQ ID NO: 77, encoded by a gene (Gene ID: 9482) consisting of the nucleotide sequence of SEQ ID NO: 72. The STX8 used in this invention includes STX8 and polypeptides having equivalent function.

[0042] STX12 is a type of SNARE protein that has a transmembrane domain and is expected to localize to the Golgi apparatus, early endosomes, and recycling endosomes. In a preferred example, STX12 is mammalian STX12. In a more preferred example, STX12 is human STX12, which is a protein consisting of the amino acid sequence of SEQ ID NO: 78, encoded by a gene (Gene ID: 23673) consisting of the nucleotide sequence of SEQ ID NO: 73. The STX12 used in this invention includes STX12 and polypeptides having equivalent function.

[0043] VAMP8 is a type of SNARE protein that possesses a transmembrane domain and is expected to localize to lysosomes, early endosomes, late endosomes, and the cell membrane. In a preferred example, VAMP8 is mammalian VAMP8. In a more preferred example, VAMP8 is human VAMP8, which is a protein consisting of the amino acid sequence of SEQ ID NO: 79, encoded by a gene (Gene ID: 8673) consisting of the nucleotide sequence of SEQ ID NO: 74. The VAMP8 used in this invention includes VAMP8 and polypeptides having equivalent function.

[0044] SEC22B (SEC22 homolog B, vesicle trafficking protein) is a type of SNARE protein that has a transmembrane domain and is expected to localize to the endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate, the Golgi apparatus, the cis-Golgi network, and the trans-Golgi network. In a preferred example, SEC22B is mammalian SEC22B. In a more preferred example, SEC22B is human SEC22B, a protein consisting of the amino acid sequence of SEQ ID NO: 80, encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 75 (Gene ID: 9554). SEC22B as used in this invention includes SEC22B and polypeptides having equivalent function.

[0045] In the present invention, a polypeptide having equivalent function to a SNARE protein refers to a polypeptide having equivalent biological activity to the SNARE protein. Examples of such polypeptides include polypeptides having a transmembrane domain and capable of localizing to intracellular vesicles (preferably intracellular vesicles where the corresponding SNARE protein is localized) or exosomes. Specific examples include the polypeptides (b) to (e) below, or the polypeptides (b), (c), and (e) below.

[0046] Specifically, the following can be mentioned regarding VAMP7: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 10, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 10, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes. Splicing variants of the VAMP7 gene consisting of the nucleotide sequence of Sequence ID No. 1 include variants that are registered in NCBI's RefSeq (Reference Sequence) as NM_001185183.2 or NM_001145149.3 and encode proteins registered as NP_001172112.1 or NP_001138621.1, respectively.

[0047] Specifically, the following can be cited as examples of GOSR2: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 11; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 11, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 11, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the intermediate section between the endoplasmic reticulum and the Golgi apparatus or the Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes. Splicing variants of the GOSR2 gene consisting of the nucleotide sequence of SEQ ID NO: NM_054022.4, NM_004287.5, NM_001353114.2, NM_001012511.3, NM_001363851.2, NM_001330252.2, NM_001353116.2, NM_001353115.2, or NM_00132 Examples include variants encoding proteins registered as 1134.2, and registered as NP_473363.1, NP_004278.2, NP_001340043.1, NP_001012529.1, NP_001350780.1, NP_001317181.1, NP_001340045.1, NP_001340044.1, or NP_001308063.1, respectively.

[0048] Specifically, the following can be mentioned regarding the STX10: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 12; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 12, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 12, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes. Splicing variants of the STX10 gene consisting of the nucleotide sequence of Sequence ID No. 3 include variants that are registered in NCBI's RefSeq as NM_001271610.2, NM_1271609.2, or NM_001271611.2, and that encode proteins registered as NP_001258539.1, NP_001258538.1, or NP_001258540.1, respectively.

[0049] Specifically, the following can be mentioned regarding the STX18: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 13, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 13, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to an intracellular vesicle (e.g., endoplasmic reticulum) or exosome. Splicing variants of the STX18 gene consisting of the nucleotide sequence of Sequence ID No. 4 include variants that are registered in NCBI's RefSeq as NM_001346281.2, NM_001346282.2, or NM_001346300.2, and that encode proteins registered as NP_001333210.1, NP_001333211.1, or NP_001333229.1, respectively.

[0050] Specifically, the following are examples of BNIP1 features: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 14; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 14, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 14, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to an intracellular vesicle (e.g., endoplasmic reticulum) or exosome. Splicing variants of the BNIP1 gene consisting of the nucleotide sequence of Sequence ID No. 5 include variants registered in NCBI's RefSeq as NM_013980.3, NM_001205.3, or NM_013978.3, which encode proteins registered as NP_053583.2, NP_001196.2, or NP_053581.2, respectively.

[0051] Specifically, the following are examples of STX7: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 15, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 15, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes. Splicing variants of the STX7 gene consisting of the nucleotide sequence of Sequence ID No. 6 include variants that are registered in NCBI's RefSeq as NM_001326578.2, NM_001326579.2, or NM_001326580.2, and that encode proteins registered as NP_001313507.1, NP_001313508.1, or NP_001313509.1, respectively.

[0052] Specifically, the following are examples of VTI1A: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 16; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 16, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 16, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes. Splicing variants of the VTI1A gene consisting of the nucleotide sequence of Sequence ID No. 7 include variants that encode proteins registered in NCBI's RefSeq as NM_145206.4, NM_001365711.1, NM_001365710.2, NM_001365712.1, NM_001365713.1, NM_001365714.1, or NM_001318205.2, respectively, and registered as NP_660207.2, NP_001352640.1, NP_001352639.1, NP_001352641.1, NP_001352642.1, NP_001352643.1, or NP_001305134.1.

[0053] Specifically, the following are examples of STX16: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 17, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 17, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes. Splicing variants of the STX16 gene consisting of the nucleotide sequence of Sequence ID No. 8 include variants that are registered in NCBI's RefSeq as NM_001134772.3, NM_1134773.3, NM_003763.6, or NM_001204868.2, and that encode proteins registered as NP_001128244.1, NP_001128245.1, NP_003754.2, or NP_001191797.1, respectively.

[0054] Specifically, the following can be mentioned regarding the STX5: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 18, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 18, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes. Splicing variants of the STX5 gene consisting of the nucleotide sequence of Sequence ID No. 9 include variants that encode proteins registered in NCBI's RefSeq as NM_001244666.3 or NM_001330294.2, and registered as NP_001231595.1 or NP_001317223.1, respectively.

[0055] Specifically, the following are examples of GOSR1: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 76; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 76, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 76, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 71, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes. Splicing variants of the GOSR1 gene consisting of the nucleotide sequence of SEQ ID NO: 71 include variants that encode proteins registered in NCBI's RefSeq as NM_001007025.2 or NM_001007024.2, and registered as NP_001007026.1 or NP_001007025.1, respectively.

[0056] Specifically, the following can be mentioned regarding the STX8: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 77; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 77, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 77, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (c) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0057] Specifically, the following are examples of STX12: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 78; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 78, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 78, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (c) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes.

[0058] Specifically, the following can be mentioned regarding VAMP8: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 79; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 79, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 79, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (c) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes.

[0059] Specifically, the following are examples of SEC22B: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 80; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 80, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 80, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (c) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes.

[0060] The polynucleotides encoding SNARE proteins used in this invention include polynucleotides encoding SNARE proteins and polynucleotides encoding polypeptides having equivalent functionality to SNARE proteins. Specific examples of polynucleotides encoding polypeptides having equivalent functionality to SNARE proteins include the following (g) to (k) and (m) to (o), or the following (g), (h), (j), (k), and (m) to (o).

[0061] The following are specific examples of polynucleotides that encode VAMP7. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 1; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 1, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 1, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and which has a transmembrane domain and encodes a polypeptide that can localize to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, has a transmembrane domain, and can localize to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (m) A polynucleotide encoding a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 10, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 10, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes.

[0062] The following are examples of polynucleotides that encode GOSR2. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 2; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 2, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 2, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 11; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 11, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 11, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi intermediate section or the Golgi apparatus) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the intermediate section between the endoplasmic reticulum and the Golgi apparatus or the Golgi apparatus) or exosomes.

[0063] The following are specific examples of polynucleotides that encode STX10: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 3; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 3, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 3, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, and which has a transmembrane domain and encodes a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 12, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 12, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0064] The following are specific examples of polynucleotides that encode STX18: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 4; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 4, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 4, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, and has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, has a transmembrane domain, and can localize to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 13, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 13, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes.

[0065] The following are examples of polynucleotides that encode BNIP1. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 5; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 5, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 5, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 5, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, has a transmembrane domain, and can localize to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 14, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 14, and which can localize to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the endoplasmic reticulum) or exosomes.

[0066] The following are specific examples of polynucleotides that encode STX7: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 6; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 6, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 6, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (i) A polynucleotide encoding a polypeptide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 6, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 15, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 15, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0067] The following are specific examples of polynucleotides that encode VTI1A: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 7; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 7, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 7, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 7, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 16, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0068] The following are specific examples of polynucleotides that encode STX16: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 8; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 8, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 8, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 8, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 17, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 17, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0069] The following are specific examples of polynucleotides that encode STX5: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 9; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 9, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 9, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, and which has a transmembrane domain and encodes a polypeptide that can localize to intracellular vesicles (e.g., the Golgi apparatus) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, has a transmembrane domain, and can localize to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 18, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 18, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0070] The following are examples of polynucleotides that encode GOSR1. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 71; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 71, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 71, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (i) A polynucleotide encoding a polypeptide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 71, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 71, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 76, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 76, and which can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any polypeptide described in (l) to (n) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0071] The following are specific examples of polynucleotides that encode STX8: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 72; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 72, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 72, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (k) A polynucleotide that is a fragment of any of (f) to (h) and (j) above, and has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 77, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of Sequence ID No. 77, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0072] The following are specific examples of polynucleotides that encode STX12: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 73; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 73, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 73, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 73, has a transmembrane domain, and can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (k) A polynucleotide that is a fragment of any of (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 78; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 78, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of Sequence ID No. 78, and which can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes.

[0073] The following are specific examples of polynucleotides that encode VAMP8: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 74; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 74, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 74, and having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary strand of the polynucleotide sequence of SEQ ID NO: 74, has a transmembrane domain, and can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (k) A polynucleotide that is a fragment of any of (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 79; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 79, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 79, and capable of localizing to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes.

[0074] The following are specific examples of polynucleotides that encode SEC22B. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 75; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 75, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 75, and having a transmembrane domain, which can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 75, has a transmembrane domain, and can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 80; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 80, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 80, and which can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any polypeptide described in (l) to (n) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes.

[0075] The method for obtaining the polynucleotide encoding the SNARE protein of the present invention is not particularly limited and can be obtained by conventional chemical synthesis methods or genetic engineering techniques. For example, the polynucleotide encoding the SNARE protein can be artificially synthesized based on any of the nucleotide sequences of SEQ ID NOs: 1-9 and 71-75. For artificial synthesis, commercially available DNA synthesis services provided by companies such as GenScript can be used. Alternatively, for example, the nucleotide sequences of SEQ ID NOs: 1-9 and 71-75 can be cloned from a human-derived sample according to the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001).

[0076] The polynucleotide encoding the SNARE protein of the present invention can also be produced, for example, by introducing mutations into DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1-9 and 71-75. Examples of mutation introduction methods include ultraviolet irradiation and site-directed mutagenesis. Examples of site-directed mutagenesis methods include Splicing overlap extension (SOE) PCR (Horton et al., Gene, 77, 61-68, 1989), ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio), Transformer TMCommercially available site-directed mutagenesis kits, such as the Site-Directed Mutagenesis Kit (Clonetech) and the KOD-Plus-Mutagenesis Kit (Toyobo), can also be used. By selecting from the mutated DNA that possesses a transmembrane domain and can localize to intracellular vesicles, polynucleotides encoding the SNARE protein of the present invention can be obtained. Whether or not the polypeptide encoded by the mutated DNA has a transmembrane domain can be determined, for example, by using prediction tools such as SOSUI (Hirokawa et al., Bioinformatics, 14(4):378-379, 1998) or TMHMM (Krogh et al., J Mol Biol. 305(3):567-580, 2001) based on the amino acid sequence of the polypeptide encoded by the DNA.

[0077] Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into a nucleotide sequence are described, for example, by Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995).

[0078] Alternatively, the polynucleotide encoding the SNARE protein of the present invention can also be obtained, for example, by subjecting DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1-9 and 71-75 to genome editing using artificial DNA nucleases (or programmable nucleases).

[0079] In the present invention, any antigen can be used. Specifically, examples include cancer antigens, autoantigens, infectious disease antigens, and allergens.

[0080] In the present invention, a cancer antigen refers to a protein or peptide derived therefrom that is expressed in cancer cells but not in normal cells, or expressed in small amounts, and serves as a marker for distinguishing cancer cells from normal cells when the immune system attacks cancer. Cancer antigens are not particularly limited, but examples include proteins or peptides derived therefrom that are commonly found in cancer patients, such as MAGEA1-4, NY-ESO-1, PRAME, SSX2, CT83, CD19, GP100, MART1, PSA, PSMA, tyrosinase, WT1, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, MUC1T, LSP1, BCR-ABL1, HRAS, and KRAS, as well as neoantigens, which are cancer antigens newly arising from gene mutations in cancer cells and are thought to have high immunogenicity. Of these, WT1, neoantigens, or peptides derived therefrom are preferred as cancer antigens, and WT1 or peptides derived therefrom are more preferred.

[0081] In the present invention, an autoantigen means a protein or peptide derived therefrom that, despite being a normal bodily component, induces an autoimmune reaction in the host and causes an autoimmune disease, or an antigen that causes a specific disease in the host, and which is a target protein or peptide derived therefrom that can be expected to treat the disease by artificially inducing an immune response to the antigen. There are no particular restrictions on the autoantigens that cause autoimmune diseases, but for example, in type 1 diabetes, examples include proteins or peptides derived therefrom such as Carboxypeptidase H, Chromogranin A, Glutamate decarboxylase, Imogen-38, Insulin, Insulinoma antigen-2, Insulinoma antigen-2B, Islet-specific glucose-6-phosphatase catalytic subunit related protein, and Proinsulin; in multiple sclerosis, examples include proteins or peptides derived therefrom such as α-enolase, Aquaporin-4, β-arrestin, Myelin basic protein, Myelin oligodendrocytic glycoprotein, Proteolipid protein, and S100-β; in rheumatoid arthritis, examples include proteins or peptides derived therefrom such as Citrullinated protein, Collagen II, Heat shock proteins, and Human cartilage glycoprotein 39; and in systemic lupus erythematosus, examples include La antigen and histone. Examples of proteins or peptides derived therefrom include proteins such as ribonucleoproteins, phospholipid-β-2 glycoprotein I complex, poly (ADP-ribose) polymerase, and Sm antigens of UI small ribonucleoprotein complex. In Sjögren's syndrome, RNA-binding protein RO60, Lupus La protein, and muscarinic acetylcholine are also involved.Examples of proteins or peptides derived therefrom include receptor M3, Aquaporin-5, Salivary Gland Protein 1, Carbonic anhydrase 6, Parotid secretory protein, Vasoactive intestinal peptide, and Kallikrein-11. In polymyositis / dermatomyositis, examples include Aminoacyl-tRNA synthetase, Histidyl-tRNA synthetase, Threonyl-tRNA synthetase, Alanyl-tRNA synthetase, Isoleucyl-tRNA synthetase, Glycyl-tRNA synthetase, Asparaginyl-tRNA synthetase, Phenylalanyl-tRNA synthetase, Tyrosyl-tRNA synthetase, Signal recognition particle, Elongation factor 1α, NuRD helicase, transcriptional intermediary factor-1-γ, PMS1 protein, Nuclear matrix protein NXP2, and SUMO-1 activating enzyme. Examples include proteins such as A or peptides derived therefrom, and in Graves' disease, examples include proteins such as the thyrotropin receptor or peptides derived therefrom. Of these, myelin oligodendrocytic glycoprotein or peptides derived therefrom are preferred as autoantigens. Autoantigens that can be artificially targeted to induce an immune response include proteins such as Amyloid β and Tau protein or peptides derived therefrom in Alzheimer's disease, proteins such as α-sinuclein or peptides derived therefrom in Parkinson's disease, proteins such as Cholesterol ester transfer protein, ApoB-100, Proprotein convertase subtilisin / kexin type 9, and Heat shock protein 60 or peptides derived therefrom in arteriosclerosis, proteins such as Type-1 angiotensin II receptor and Alpha 1D-Adrenergic Receptor or peptides derived therefrom in hypertension, proteins such as Dipeptidyl peptidase 4 or peptides derived therefrom in type 2 diabetes, proteins such as Glucagon-like peptide-1, Ghrelin, and Peptide YY or peptides derived therefrom in obesity, and proteins such as receptor activator of NF-kappa B ligand (RANKL) or peptides derived therefrom in osteoporosis.

[0082] In the present invention, infectious disease antigens include antigens derived from viruses, bacteria, fungi, and protozoa that cause infectious diseases. Viral antigens refer to proteins that constitute a virus or peptides derived therefrom. Viral antigens are not particularly limited, but include constituent proteins or peptides derived therefrom of viruses such as influenza virus, coronavirus, RSV, adenovirus, poliovirus, coxsackievirus, echovirus, Japanese encephalitis virus, herpesvirus, mumps virus, measles virus, rubella virus, norovirus, rotavirus, Zika virus, cytomegalovirus, papillomavirus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and adult T-cell leukemia virus. A bacterial antigen refers to a protein that makes up a bacterium or a peptide derived therefrom. Examples of bacterial antigens, though not particularly limited, include the constituent proteins or peptides derived therefrom of bacteria such as Bordetella pertussis, Neisseria diphtheriae, Escherichia coli, Haemophilus influenzae, Helicobacter, Neisseria meningitidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus, Staphylococcus aureus, Neisseria tetanus, Legionella, Mycobacterium tuberculosis, and Mycoplasma. Fungal antigens refer to proteins that make up fungi or their spores, or peptides derived therefrom. Examples of fungal antigens include those of the Aspergillus genus (e.g., Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Aspergillus ustus, etc.), Blastomyces genus (e.g., Blastomyces dermatitidis, etc.), Candida genus (e.g., Candida albicans, etc.), Coccidioides genus (e.g., Coccidiodes immitis, etc.), Cryptococcus genus (e.g., Cryptococcus neoformans, Cryptococcus gattii, etc.), Histoplasma genus (e.g., Histoplasma capsulatum, etc.), Paracoccidioides genus (e.g., Paracoccidioides brasiliensis, etc.), and Sporothrix genus (e.g., Sporothrix). Examples include fungi such as Schenckii, and their spore constituent proteins or peptides derived therefrom. Protozoan antigens refer to proteins that constitute protozoa or peptides derived therefrom. Examples of protozoan antigens include the constituent proteins or peptides derived therefrom of protozoa such as malaria parasites, leishmania, cryptosporidiosis, gambian trypanosomes, rhodesia trypanosomes, cruzi trypanosomes, trichomoniasis, toxoplasma, babesiosis, dysentery amoeba, and giardia. Of these, bacterial antigens, fungal antigens, or protozoan antigens are preferred as infectious disease antigens.

[0083] In the present invention, an allergen means a protein or peptide derived therefrom that enters the body from the outside through inhalation, puncture, ingestion, or contact, and causes a hypersensitivity or allergic reaction. Allergens are not particularly limited, but include: grass pollen (such as reeds, timothy grass, giant hornbeam, orchard grass, buckwheat, wheat, dwarf barnyard grass, barnyard grass, sorghum, longleaf grass, sweet vernal grass, broadleaf ryegrass, and ryegrass); weed pollen (such as goldenrod, nettle, giant ragweed, Japanese hop, white dandelion, wormwood, dwarf sorrel, ragweed, false ragweed, oxeye daisy, and leaf stalk); and weed pollen (such as goldenrod, nettle, giant ragweed, Japanese hop, white dandelion, bitter wormwood, dwarf sorrel, ragweed, false ragweed, oxeye daisy, and leaf stalk). Plantain, mugwort, etc.); tree pollen (acacia, olive, maple, walnut, mulberry, sawtooth oak, white birch, elm, alder, cedar, cypress, juniper, beech, pine, willow, etc.); fungi or bacteria (Aspergillus, Alternaria, Staphylococcus aureus enterotoxin A, Staphylococcus aureus enterotoxin B, Candida, Cladosporium, Trichophyton, Pityrosporium, Penicillium, Helmintosporium, M Lassezia, Mucor, etc.); Animal skin (duck feathers, cat dander, dog dander, cow dander, horse dander, rabbit hide, hamster hide, guinea pig hide, sheep hide, pig hide, goat hide, chicken feathers, goose feathers, budgerigar feathers, budgerigar droppings, mice, rats, etc.); Insects (paper wasps, moths, cockroaches, hornets, honeybees, mosquitoes, midges (adults), etc.); Parasites (Anisakis, roundworms, etc.); Mites (thick-legged dust mites) Examples of allergens include: mites such as the long-haired flour mite, house dust mite, peanut mite, and black house dust mite; foods (eggs, milk, wheat, buckwheat, peanuts, shrimp, crab, almonds, abalone, squid, salmon roe, oranges, cashews, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, etc.); and proteins or peptides derived therefrom contained in human insulin, etc. Of these, proteins or peptides derived therefrom contained in food are preferred as allergens, and proteins or peptides derived therefrom contained in eggs are more preferred.

[0084] Other antigens include glutenin and gliadin proteins, or peptides derived from them, which make up gluten that causes celiac disease.

[0085] The method for obtaining the polynucleotide encoding the antigen of the present invention is not particularly limited and can be obtained by conventional chemical synthesis or genetic engineering methods, similar to the method for obtaining the polynucleotide encoding the SNARE protein described above. The polynucleotide encoding the antigen may be a polynucleotide encoding the full length of the antigen, or it may be a polynucleotide encoding a partial polypeptide of the antigen, insofar as it functions as an antigen. It may also be a polynucleotide obtained by linking multiple polynucleotides encoding one antigen in a sequence so as to be expressible, or a polynucleotide obtained by linking two or more polynucleotides encoding two or more antigens so as to be expressible. The antigen used in the nucleic acid construct of the present invention is at least one type, preferably five types or less, more preferably three types or less, and even more preferably two types or less. Furthermore, the antigen used in the nucleic acid construct of the present invention may be five types, four types, three types, two types, or one type.

[0086] Preferably, the nucleic acid construct of the present invention is a nucleic acid construct in which a polynucleotide encoding an antigen is expressably linked downstream of a polynucleotide encoding a SNARE protein; more preferably, the nucleic acid construct is a nucleic acid construct in which a polynucleotide encoding an antigen is expressably linked downstream of a polynucleotide encoding a SNARE protein via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence; and even more preferably, the nucleic acid construct is a nucleic acid construct in which a polynucleotide encoding an antigen is expressably linked downstream of a polynucleotide encoding a SNARE protein via a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence. When the nucleic acid construct of the present invention includes a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence, the linking order is not particularly limited, and the polynucleotide encoding the proprotein convertase recognition sequence may be linked downstream of the polynucleotide encoding the linker, or the polynucleotide encoding the linker may be linked downstream of the polynucleotide encoding the proprotein convertase recognition sequence. Furthermore, a sequence in which the proprotein convertase recognition sequence is repeated about 1 to 5 times may be linked, or the polynucleotide encoding the proprotein convertase recognition sequence may be linked in a manner that is sandwiched between a plurality of polynucleotides encoding linkers. Furthermore, any polynucleotide other than the linker-coding polynucleotide and the proprotein convertase recognition sequence-coding polynucleotide may be included between the polynucleotide encoding the SNARE protein and the polynucleotide encoding the antigen, as long as the expression of both is not impaired.

[0087] In the present invention, "linker" refers to a peptide linker that links two polypeptides. The linker is not particularly limited as long as it can enable the SNARE protein and antigen to function normally. The length of the linker is preferably 3 amino acid residues or more, more preferably 4 amino acid residues or more, even more preferably 5 amino acid residues or more, and preferably 30 amino acid residues or less, more preferably 25 amino acid residues or less, and even more preferably 20 amino acid residues or less. Alternatively, the length of the linker is preferably 3 to 30 amino acid residues, more preferably 4 to 25 amino acid residues, and even more preferably 5 to 20 amino acid residues. Examples of such linkers include linkers having G, GS, GGS, GGGS (SEQ ID NO: 92), GGGGS (SEQ ID NO: 93), EAAAK (SEQ ID NO: 94), or XP as constituent elements. Here, X represents any amino acid residue. Specific examples include linkers consisting of sequences in which each constituent element is repeated 1 to 5 times, preferably 3 or 4 times, and a preferred specific example is a linker consisting of a sequence in which GGGGS is repeated 3 times (SEQ ID NO: 20). If the sequence components are GGGGS, an amino acid residue S may be added before the repeat sequence, or if they are EAAAK, an amino acid residue A may be added before and after the repeat sequence.

[0088] In this invention, the proprotein convertase recognition sequence refers to the amino acid sequence recognized by proprotein convertase, a serine protease that converts proproteins into physiologically active proteins or peptides in the Golgi apparatus, and specifically refers to the amino acid sequence consisting of X-Arg-X-(Arg / Lys)-Arg (for example, Sequence ID No. 22). Here, X represents any amino acid residue. Examples of proprotein convertases include furin, PC2, PC4, PC5 / 6, PC7, and PACE4, and it is known that these enzymes commonly recognize the Arg-X-(Arg / Lys)-Arg motif (Remacle AG, et al. Journal of Biological Chemistry 2008, 283(30): 20897-20906). Therefore, the proprotein convertase recognition sequence can be Arg-X-(Arg / Lys)-Arg. Here, X represents any amino acid residue. The proprotein convertase recognizes the above amino acid sequence and cleaves the C-terminal arginine residue. In the present invention, preferably, the proprotein convertase is furin, and the furin recognition sequence is the same as the above proprotein convertase recognition sequence.

[0089] There are no particular limitations on the method for obtaining the polynucleotide encoding the linker or the polynucleotide encoding the proprotein convertase recognition sequence, and they can be obtained by conventional chemical synthesis methods or genetic engineering techniques.

[0090] The polynucleotides encoding SNARE proteins, antigens, linkers, or proprotein convertase recognition sequences contained in the nucleic acid construct of the present invention may be codon-optimized as needed to suit the target species of the nucleic acid construct. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0091] In a preferred example, the nucleic acid construct of the present invention is an expression cassette comprising a regulatory region for controlling the expression of a polynucleotide encoding a SNARE protein and a polynucleotide encoding an antigen. In the expression cassette, the polynucleotide encoding the SNARE protein and the polynucleotide encoding the antigen are operably ligated to the regulatory region. Examples of the regulatory region include promoters, terminators, and enhancers. Preferably, the expression cassette includes promoters ligated upstream of the polynucleotide encoding the SNARE protein and the polynucleotide encoding the antigen.

[0092] The nucleic acid construct of the present invention may have restriction enzyme recognition sites at one or both ends. The nucleic acid construct of the present invention can be introduced into a vector using these restriction enzyme recognition sites. For example, the nucleic acid construct can be introduced into a vector by cleaving a vector with a restriction enzyme and then adding the nucleic acid construct of the present invention having restriction enzyme recognition sites at its ends.

[0093] The type of vector is not particularly limited and may be any vector such as plasmid vectors, phages, phagemids, cosmids, or viral vectors. In one example, the vector into which the nucleic acid construct of the present invention is to be incorporated may be an expression vector, but on the other hand, if the nucleic acid construct to be incorporated is an expression cassette, it does not need to be an expression vector. In one example, the nucleic acid construct of the present invention is an expression cassette including a control region, and is incorporated into any vector to construct an expression vector. In another example, by incorporating the nucleic acid construct of the present invention into an expression vector including a control region, the expression cassette of the present invention is constructed on the expression vector.

[0094] In a preferred example, the nucleic acid construct of the present invention is a plasmid vector. Plasmid vectors include, but are not limited to, plasmid vectors such as pVAX1. Plasmid vectors generally have drug resistance genes incorporated into their sequence for the purpose of selectively retaining the plasmid vector in bacterial culture. However, because there is a risk of transmission of such genes to the bacterial flora in vivo, or activation and expression of such genes via mammalian promoters, plasmid vectors from which such gene sequences have been removed are more preferred.

[0095] In a preferred example, the nucleic acid construct of the present invention is a viral vector. Examples of viral vectors, but not limited to these, include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, Sendai virus vectors, and herpesvirus vectors.

[0096] In a preferred example, the nucleic acid construct of the present invention is an mRNA construct. For example, using DNA containing polynucleotides encoding SNARE proteins and polynucleotides encoding antigens as a template, HiScribe T7 ARCA mRNA Kit (New England Biolabs), T7 mScript TM The nucleic acid constructs of the present invention can be obtained as mRNA by using commercially available in vitro transcription kits such as the Standard mRNA Production System (CELLSCRIPT) and the High Yield T7 ARCA mRNA Synthesis Kit (Jena Bioscience).

[0097] As shown in the examples below, nucleic acid constructs containing a polynucleotide encoding the SNARE protein and a polynucleotide encoding the antigen exhibit higher immunogenicity compared to nucleic acid constructs containing only the polynucleotide encoding the antigen. They can induce or enhance antigen-specific Th1-type immune responses and antigen-specific cellular immune responses. This is presumed to be because, in the antigen presentation process where the nucleic acid construct taken up into the cell is translated or transcribed and expressed as an antigen protein, transported to intracellular vesicles where a portion of the antigen binds to major histocompatibility complex (MHC) molecules and is transported to the cell surface, the nucleic acid construct of the present invention expresses the antigen protein as a fusion polypeptide of the SNARE protein and the antigen. This targets the antigen protein to intracellular vesicles, increasing the probability of association with MHC molecules and thus improving the efficiency of antigen presentation. In addition, it has been reported that antigen-containing exosomes enhance antigen-specific Th1-type immune responses in vivo (Qazi KR, et al. Blood. 2009, 113(12): 2673-83). Therefore, it is possible that the expression of the antigen protein as a fusion polypeptide of the SNARE protein and the antigen facilitates the targeting of the antigen protein to exosomes, thereby improving its immunogenicity.

[0098] Furthermore, the production levels of cytokines such as IFNγ and IL-4, which can serve as indicators for determining whether the immune response is Th1 or Th2 type, or whether it is a cellular or humoral immune response, can be measured by conventionally known methods. Such methods include, for example, ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunosorbent spot) assay, immunohistochemistry, in situ hybridization, RT-PCR, microarrays, and flow cytometry. Reagents and kits for measuring cytokine production, such as the ELISPOT assay kit (CTL) used in the examples below, are commercially available and may be used for measurement. Furthermore, the production levels of different IgG subclasses, which can serve as another indicator for determining whether the immune response is Th1 or Th2 type, or whether it is a cellular or humoral immune response, can be measured by conventionally known methods. Such methods include ELISA and immunoturbidimetry. Th1 immune responses and cellular immune responses can be evaluated using cytokine production and / or IgG subclass production as indicators. For example, an increase in IFNγ production or the ratio of IFNγ production to IL-4 (IFNγ / IL-4) can be evaluated as an induction or enhancement of Th1 immune responses and cellular immune responses, while a decrease in IFNγ production or IFNγ / IL-4 can be evaluated as a weakening of Th1 immune responses and cellular immune responses. Alternatively, an increase in IgG2a production and / or the ratio of IgG2a production to IgG1 (IgG2a / IgG1) can be evaluated as an induction or enhancement of Th1 immune responses and cellular immune responses, while a decrease in IgG2a production and / or IgG2a / IgG1 can be evaluated as a weakening of Th1 immune responses and cellular immune responses.

[0099] Therefore, the nucleic acid constructs of the present invention can serve as antigen-specific IFNγ production enhancers, IgG2a production enhancers, Th1-type immune response inducers or enhancers, and cellular immune response inducers or enhancers (hereinafter referred to as cellular immune response inducers or enhancers, etc.), and these nucleic acid constructs can be used to manufacture cellular immune response inducers or enhancers, etc. Furthermore, the nucleic acid constructs of the present invention can be used to enhance antigen-specific IFNγ production, enhance IgG2a production, induce or enhance Th1-type immune responses, and induce or enhance cellular immune responses. Here, such use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, treatment, or diagnosis of humans, and more specifically, methods of surgery, treatment, or diagnosis performed on humans by a physician or a person under the direction of a physician.

[0100] The cellular immune response inducer or enhancer of the present invention may itself be a pharmaceutical or quasi-drug for enhancing antigen-specific IFNγ production, enhancing IgG2a production, inducing or enhancing Th1-type immune responses, or inducing or enhancing cellular immune responses, or it may be a material or formulation used in combination with such pharmaceutical or quasi-drug.

[0101] When the cellular immune response inducer or enhancer of the present invention is used as a pharmaceutical product (including quasi-drugs), the pharmaceutical product may be administered in any dosage form. Examples of dosage forms include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., or parenteral administration in the form of injections, suppositories, inhalants, transdermal agents, topical agents, etc. Parenteral administration is preferred, and parenteral administration by injection is more preferred. Such various dosage forms of pharmaceutical preparations can be prepared by appropriately combining the nucleic acid construct of the present invention with other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, diluents, thickeners, emulsifiers, lubricants, dispersants, coating agents, surfactants, coating agents, osmotic pressure regulators, buffers, pH adjusters, preservatives, stabilizers, antioxidants, colorants, flavoring agents, deodorizers, fragrances, etc.

[0102] The content of the nucleic acid construct of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target antigen, the recipient, and the route of administration, and is therefore not particularly limited and can be appropriately selected over a wide range. For example, the nucleic acid construct may be contained in an amount between 0.00001% and 100% by mass of the total composition.

[0103] The dosage of the cellular immune response inducer or enhancer of the present invention may vary depending on the species, weight, sex, age, condition, or other factors of the subject. The dosage, route, and interval of administration can be appropriately determined by those skilled in the art. For example, the dosage is determined as the amount of nucleic acid construct of the present invention, between 1 ng and 10 mg per day for one adult (weighing 60 kg). If the nucleic acid construct is a viral vector, the dosage is, for example, 10 to 1 × 10 per day for one adult (weighing 60 kg). 15 It could be a virus particle.

[0104] The cellular immune response inducer or enhancer of the present invention can be administered to either humans or non-human animals. Examples of non-human animals include non-human mammals, such as great apes, other primates, mice, rats, horses, cattle, pigs, sheep, dogs, cattle, hamsters, and companion animals. Preferably, the cellular immune response inducer or enhancer of the present invention is administered to humans. More preferably, the cellular immune response inducer or enhancer of the present invention is administered to patients with cancer, autoimmune diseases, or allergies, or to humans at risk of developing cancer, autoimmune diseases, or allergies.

[0105] Furthermore, the nucleic acid construct of the present invention can serve as a nucleic acid vaccine, and the nucleic acid construct can be used to manufacture a nucleic acid vaccine. Furthermore, the nucleic acid constructs of the present invention can be used to induce or enhance antigen-specific Th1 immune responses and / or antigen-specific cellular immune responses. Herein, such use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic.

[0106] The nucleic acid vaccine of the present invention may, on its own, be a pharmaceutical product for inducing or enhancing an antigen-specific Th1 immune response and / or for inducing or enhancing an antigen-specific cellular immune response, or it may be a material or formulation used in combination with such pharmaceutical product.

[0107] In one embodiment, the nucleic acid vaccine is a DNA vaccine. The DNA vaccine includes the nucleic acid construct of the present invention, which is a plasmid vector. The plasmid vector is not particularly limited, but examples include the pVAX1 vector. Among these, plasmid vectors that do not contain drug resistance genes are preferred from the viewpoint of safety.

[0108] In another embodiment, the nucleic acid vaccine is an mRNA vaccine. The mRNA vaccine comprises the nucleic acid construct of the present invention, which is mRNA, and the nucleic acid construct may be mRNA that has been treated to add a Cap structure or polyA to stabilize the mRNA, improve translation efficiency or prevent an excessive immune response, and / or mRNA in which some bases have been modified (for example, uridine is replaced with pseudouridine or 1-methylpseudridine). It may also be a self-amplifying RNA comprising the nucleic acid construct of the present invention and containing the sequence of a virus-derived RNA-dependent RNA polymerase (RdRP) complex and its replication origins (5'CSE, 3'CSE), or a trans-amplifying RNA obtained by mixing RNA containing the nucleic acid construct of the present invention and the replication origins of the RdRP complex with mRNA containing the sequence of the RdRP complex. Preferably, the mRNA vaccine further comprises a construct responsible for drug delivery, such as liposomes or lipid nanoparticles composed of lipids, or polymer nanoparticles such as PLGA nanoparticles composed of high molecular weight polymers, as a carrier for stabilizing and delivering mRNA, and more preferably the mRNA is encapsulated in the construct.

[0109] In yet another embodiment, the nucleic acid vaccine is a viral vector vaccine. The viral vector vaccine comprises the nucleic acid construct of the present invention, which is a viral vector. The viral vector is not particularly limited, but examples include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, Sendai virus vectors, herpesvirus vectors, and the like.

[0110] The nucleic acid vaccine of the present invention may contain, in addition to the nucleic acid construct described above, a pharmaceutically acceptable carrier as appropriate, and may be formulated in a predetermined form. Here, examples of carriers include carriers commonly used in vaccine production, specifically buffers, emulsifiers, preservatives (e.g., thimerosal), isotonic agents, pH adjusters, viscosity modifiers, adjuvants, or immunostimulants. An adjuvant is a substance that enhances the immune response to an antigen when administered together with that antigen. However, since the nucleic acid vaccine of the present invention can function as an adjuvant itself, the addition of an adjuvant is not necessarily required, and the composition may not contain an adjuvant.

[0111] The nucleic acid vaccine of the present invention is preferably in liquid form and is appropriately formulated to suit the intended route of administration. Routes of administration include oral and parenteral administration, such as intramuscular, intradermal, subcutaneous, transdermal, intranasal, sublingual, oral, and inhalation, but intramuscular, intradermal, or subcutaneous administration is preferred. Injectable formulations include, for example, liquid formulations, emulsion formulations, water-soluble or hydrophobic suspension formulations, and dry powder formulations that are dissolved or suspended by adding a liquid. Alternatively, the nucleic acid vaccine of the present invention can also be administered as dendritic cells into which the nucleic acid vaccine of the present invention has been introduced. Specifically, such administration can be carried out by collecting peripheral blood from a target organism, separating dendritic cell progenitor cells, differentiating the progenitor cells into dendritic cells in the presence of appropriate cytokines, introducing the nucleic acid vaccine of the present invention to the dendritic cells to present antigens, and administering the dendritic cells to the target organism. The administered dendritic cells are referred to as a dendritic cell vaccine. The dendritic cell vaccine can improve the efficiency of antigen presentation and enhance immune induction.

[0112] The content of the nucleic acid construct of the present invention in the nucleic acid vaccine of the present invention varies depending on the target antigen, the recipient, and the route of administration, and is therefore not particularly limited and can be appropriately selected over a wide range. For example, the nucleic acid construct may be contained in an amount between 0.00001% and 100% by mass of the total nucleic acid vaccine.

[0113] The dosage of the nucleic acid vaccine of the present invention may vary depending on the species, weight, sex, age, condition, or other factors of the target. The dosage, route, and interval of administration may be appropriately determined by those skilled in the art. For example, the dosage is determined as the amount of nucleic acid construct of the present invention, between 1 ng and 10 mg per dose unit. If the nucleic acid construct is a viral vector, the dosage is, for example, 10 to 1 × 10 per dose unit. 15 It could be a virus particle.

[0114] The nucleic acid vaccine of the present invention can be administered to either humans or non-human animals. Examples of non-human animals are those described above. Preferably, the nucleic acid vaccine of the present invention is administered to humans. More preferably, the nucleic acid vaccine of the present invention is administered to patients with cancer, autoimmune diseases, or allergies, or to humans at risk of developing cancer, autoimmune diseases, or allergies.

[0115] The number of doses of the nucleic acid vaccine of the present invention may be set appropriately depending on the application, and is at least once, but may be two or more times from the viewpoint of effectiveness. Further administration is sometimes called booster immunization, and this can provide a more effective infection prevention or therapeutic effect. An interval of at least one week is recommended for booster immunization, and an interval of 1 to 4 weeks is preferred.

[0116] Nucleic acid vaccines, unlike bacteria or viruses themselves, are non-pathogenic and are considered safer than live vaccines or inactivated vaccines. Furthermore, by changing the nucleic acid encoding the antigen, they can be adapted to a wide variety of antigens, and because they are nucleic acid-based, they can be manufactured quickly and at low cost.

[0117] In a preferred embodiment, the nucleic acid vaccine of the present invention is a cancer vaccine. Specifically, the cancer vaccine comprises a nucleic acid construct containing a polynucleotide encoding the SNARE protein of the present invention and a polynucleotide encoding a cancer antigen as an antigen. The cancer vaccine can be administered directly to the body or as a dendritic cell vaccine. Such a cancer vaccine is thought to induce or enhance a cancer antigen-specific cellular immune response and exert an antitumor effect.

[0118] In another preferred embodiment, the nucleic acid vaccine of the present invention is an infectious disease vaccine. Specifically, the infectious disease vaccine comprises a nucleic acid construct containing a polynucleotide encoding the SNARE protein of the present invention and a polynucleotide encoding a viral antigen, bacterial antigen, fungal antigen, or protozoan antigen as an antigen. The infectious disease vaccine can be administered directly to the body. Such an infectious disease vaccine is thought to exert a preventive or therapeutic effect against infectious diseases by inducing or enhancing a cellular immune response specific to viral antigens, bacterial antigens, fungal antigens, or protozoan antigens. In particular, it is expected to be a preventive or therapeutic vaccine for chronic infectious diseases caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), tuberculosis, etc., for which conventional vaccines do not provide sufficient preventive or therapeutic effects.

[0119] In another preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the prevention or treatment of allergies, preferably a vaccine for allergy immunotherapy. Specifically, the vaccine for the prevention or treatment of allergies comprises a nucleic acid construct comprising a polynucleotide encoding the SNARE protein of the present invention and a polynucleotide encoding an allergen as an antigen. The vaccine for the prevention or treatment of allergies can be administered directly to the body. Such a vaccine for the prevention or treatment of allergies is thought to exert an inhibitory effect against allergic responses caused by excessive activation of the Th2 immune response by inducing or enhancing allergen-specific Th1 immune responses and cellular immune responses.

[0120] In another preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the treatment of autoimmune diseases. Specifically, the vaccine for the treatment of autoimmune diseases comprises a nucleic acid construct containing a polynucleotide encoding the SNARE protein of the present invention and a polynucleotide encoding an autoantigen as an antigen. The vaccine for the treatment of autoimmune diseases can be administered directly to the body. Such a vaccine for the treatment of autoimmune diseases is thought to exert an inhibitory effect on autoimmune responses by inducing immune tolerance to the encoded autoantigen.

[0121] Exemplary embodiments of the present invention are further disclosed herein, including the following substances, manufacturing methods, uses, and methods. However, the present invention is not limited to these embodiments.

[0122] [1] A nucleic acid construct comprising a polynucleotide encoding any SNARE protein selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and a polynucleotide encoding an antigen. [2] The nucleic acid construct according to [1], wherein a polynucleotide encoding the antigen is linked downstream of a polynucleotide encoding the SNARE protein. [3] The nucleic acid construct according to [1] or [2], wherein the polynucleotide encoding the SNARE protein and the polynucleotide encoding the antigen are linked via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence, preferably a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence. [4] The nucleic acid construct according to [3], wherein the proprotein convertase recognition sequence is an amino acid sequence consisting of Arg-X-(Arg / Lys)-Arg (where X represents any amino acid residue), preferably an amino acid sequence consisting of X-Arg-X-(Arg / Lys)-Arg (where X represents any amino acid residue), and more preferably the amino acid sequence shown in Sequence ID No. 22. [5] A nucleic acid construct described in any one of items [1] to [4], which is a plasmid vector, mRNA, or viral vector. [6] The nucleic acid construct according to any one of [1] to [5], wherein the antigen is at least one selected from the group consisting of cancer antigens, autoantigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, and allergens, preferably at least one selected from the group consisting of cancer antigens, autoantigens, viral antigens, bacterial antigens, fungal antigens, and protozoan antigens, and more preferably at least one selected from the group consisting of cancer antigens, autoantigens, bacterial antigens, fungal antigens, and protozoan antigens. [7] The nucleic acid construct according to any one of [1] to [6], wherein the SNARE protein is preferably a mammalian SNARE protein, and more preferably a human SNARE protein. [8] The nucleic acid construct according to any one of [1] to [7], wherein the polynucleotide encoding the SNARE protein is preferably a polynucleotide selected from the group consisting of polynucleotides comprising the nucleotide sequences of SEQ ID NOs. 1 to 9 and 71 to 75 and polynucleotides having equivalent function thereto, and more preferably a polynucleotide comprising any of the nucleotide sequences of SEQ ID NOs. 1 to 9 and 71 to 75. [9] The nucleic acid construct according to any one of [1] to [8], wherein the SNARE protein is preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, and GOSR1, more preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, and GOSR1, even more preferably selected from the group consisting of VAMP7, GOSR2, STX10, and GOSR1, and even more preferably VAMP7.

[0123] A pharmaceutical composition comprising a nucleic acid construct as described in any one of items

[10] , [1], to [9]. A nucleic acid vaccine containing a nucleic acid construct described in any one of items

[11] , [1], to [9] as an active ingredient.

[12] A nucleic acid vaccine as described in

[11] , which induces or enhances an antigen-specific Th1 immune response.

[13] A nucleic acid vaccine as described in

[11] , which induces or enhances an antigen-specific cellular immune response.

[14] A cancer vaccine, wherein the antigen is at least one selected from the group consisting of cancer antigens, according to any one of the items

[11] to

[13] .

[15] An infectious disease vaccine, wherein the antigen is at least one selected from the group consisting of viral antigens, bacterial antigens, fungal antigens and protozoan antigens, according to any one of the claims

[11] to

[13] .

[16] A nucleic acid vaccine according to any one of

[11] to

[13] , wherein the antigen is at least one selected from the group consisting of allergens, for the prevention or treatment of allergies.

[17] A nucleic acid vaccine according to any one of

[11] to

[13] , wherein the antigen is selected from the group consisting of autoantigens, wherein the antigen is at least one of the group consisting of autoantigens.

[18] The nucleic acid vaccine according to any one of

[11] to

[17] , wherein the nucleic acid vaccine preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0124]

[19] Use of any one of the nucleic acid constructs described in [1] to [9] for the manufacture of nucleic acid vaccines.

[20] The use according to

[19] , wherein the nucleic acid vaccine is a cancer vaccine and the antigen is at least one selected from the group consisting of cancer antigens.

[21] The use according to

[19] , wherein the nucleic acid vaccine is an infectious disease vaccine and the antigen is at least one selected from the group consisting of viral antigens, bacterial antigens, fungal antigens and protozoan antigens.

[22] The use according to

[19] , wherein the nucleic acid vaccine is a vaccine for the prevention or treatment of allergies, and the antigen is at least one selected from the group consisting of allergens.

[23] The use according to

[19] , wherein the nucleic acid vaccine is a vaccine for the treatment of an autoimmune disease, and the antigen is at least one selected from the group consisting of autoantigens.

[24] The use according to any one of

[19] to

[23] , wherein the nucleic acid vaccine preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0125]

[25] Use of any one of the nucleic acid constructs described in [1] to [9] for induction or enhancement of an antigen-specific Th1 immune response and / or induction or enhancement of an antigen-specific cellular immune response.

[26] A nucleic acid construct according to any one of items [1] to [9] for use in inducing or enhancing an antigen-specific Th1 immune response and / or inducing or enhancing an antigen-specific cellular immune response. A method for inducing or enhancing an antigen-specific Th1 immune response and / or an antigen-specific cellular immune response, comprising administering an effective amount of a nucleic acid construct described in any one of items [1] to [9] to a subject requiring it.

[0126]

[28] Use of any one of the nucleic acid vaccines described in

[11] to

[18] for induction or enhancement of an antigen-specific Th1 immune response and / or induction or enhancement of an antigen-specific cellular immune response.

[29] A nucleic acid vaccine according to any one of items

[11] to

[18] , for use in inducing or enhancing an antigen-specific Th1 immune response and / or inducing or enhancing an antigen-specific cellular immune response. A method for inducing or enhancing an antigen-specific Th1 immune response and / or an antigen-specific cellular immune response, comprising administering an effective dose of a nucleic acid vaccine described in any one of items

[30] ,

[11] , to a subject in need thereof. A method of treating cancer, comprising administering a nucleic acid vaccine as described in

[31] and

[14] to a target in need in an effective dose. A method for preventing or treating an infectious disease, comprising administering the nucleic acid vaccine described in

[32]

[15] to a target in need in an effective dose. A method for preventing or treating allergies, comprising administering the nucleic acid vaccine described in

[33]

[16] to a person in need of it in an effective dose. A method for treating autoimmune diseases, comprising administering the nucleic acid vaccine described in

[34]

[17] to a target in an effective dose.

[35] The dosage of the nucleic acid vaccine is preferably 1 ng to 10 mg per dose unit if the nucleic acid vaccine is a DNA vaccine or an mRNA vaccine, and preferably 10 to 1 × 10 per dose unit if the nucleic acid vaccine is a viral vector vaccine. 15 The method described in any one of the items

[30] to

[34] , wherein the particles are virus particles.

[0127] An antigen-specific IFNγ production enhancer comprising a nucleic acid construct described in any one of items

[36] [1] to [9] as an active ingredient. An antigen-specific IgG2a production enhancer comprising a nucleic acid construct described in any one of items

[37] [1] to [9] as an active ingredient. An antigen-specific Th1 immune response inducer or enhancer comprising a nucleic acid construct described in any one of items

[38] [1] to [9] as an active ingredient. An antigen-specific cellular immune response inducer or enhancer comprising a nucleic acid construct described in any one of items

[39] , [1], to [9] as an active ingredient.

[40] The agent according to any one of

[36] to

[39] , wherein the agent preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0128]

[41] Use of any one of the nucleic acid constructs described in [1] to [9] for the production of an antigen-specific IFNγ production enhancer.

[42] Use of any one of the nucleic acid constructs described in [1] to [9] for the production of an antigen-specific IgG2a production enhancer.

[43] Use of any one of the nucleic acid constructs described in [1] to [9] for the production of antigen-specific Th1 immune response inducers or enhancers.

[44] Use of any one of the nucleic acid constructs described in [1] to [9] for the production of antigen-specific cellular immune response inducers or enhancers.

[45] The use according to any one of

[41] to

[44] , wherein the agent preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0129]

[46] Use of any one of the nucleic acid constructs described in [1] to [9] for the enhancement of antigen-specific IFNγ production.

[47] Use of any one of the nucleic acid constructs described in [1] to [9] for the enhancement of antigen-specific IgG2a production.

[48] ​​Use of any one of the nucleic acid constructs described in [1] to [9] for the induction or enhancement of an antigen-specific Th1 immune response.

[49] Use of any one of the nucleic acid constructs described in [1] to [9] for the induction or enhancement of an antigen-specific cellular immune response.

[0130]

[50] A nucleic acid construct according to any one of items [1] to [9] for use in enhancing antigen-specific IFNγ production.

[51] A nucleic acid construct according to any one of items [1] to [9] for use in enhancing antigen-specific IgG2a production.

[52] A nucleic acid construct according to any one of items [1] to [9] for use in inducing or enhancing an antigen-specific Th1 immune response.

[53] A nucleic acid construct according to any one of items [1] to [9] for use in inducing or enhancing an antigen-specific cellular immune response.

[0131] A method for enhancing antigen-specific IFNγ production, comprising administering an effective amount of a nucleic acid construct described in any one of items

[54] [1] to [9] to a target that requires it. A method for enhancing antigen-specific IgG2a production, comprising administering an effective amount of a nucleic acid construct described in any one of items

[55] [1] to [9] to a target that requires it. A method for inducing or enhancing an antigen-specific Th1 immune response, comprising administering an effective amount of a nucleic acid construct described in any one of items

[56] [1] to [9] to a subject requiring it. A method for inducing or enhancing an antigen-specific cellular immune response, comprising administering a nucleic acid construct described in any one of items [1] to [9] to a subject requiring it in an effective amount.

[58] The dose of the nucleic acid construct is preferably 1 ng to 10 mg / 60 kg body weight per day, and when the nucleic acid construct is a viral vector, preferably 10 to 1 × 10 per day. 15 The method described in any one of items

[54] to

[57] , wherein the virus particle is equal to 60 kg of body weight.

[0132] In

[59] ,

[11] to

[24] , and

[28] to

[35] , nucleic acid vaccines are administered orally or parenterally, preferably parenterally. In paragraphs

[60] ,

[27] ,

[30] ,

[35] , and

[54] -

[58] , the subjects are patients with cancer, autoimmune disease, or allergies, or people at risk of developing cancer, autoimmune disease, or allergies. In

[61] and

[31] , the subjects are cancer patients or people at risk of developing cancer. In

[62] and

[33] , the subjects are allergy patients or people at risk of developing allergies. In

[63] and

[34] , the subjects are patients with autoimmune diseases or people at risk of developing autoimmune diseases. [Examples]

[0133] Hereinafter, the present invention will be described in further detail based on examples, but the present invention is not limited thereto.

[0134] Example 1 Study on SNARE proteins mRNAs encoding the amino acid sequences (SEQ ID NOs: 25 to 45) of fusion polypeptides or the amino acid sequence of OVA alone were prepared, wherein the fusion polypeptide was obtained by linking ovalbumin (OVA, an allergen, amino acid sequence: SEQ ID NO: 24) to the C-terminal side of each of 21 types of human SNARE proteins via a linker (nucleotide sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20). At this step, the nucleotide sequences encoding the antigen or the fusion polypeptide were subjected to codon optimization and then inserted into a plasmid DNA (pVAX1 vector, Thermo Fisher Scientific). The nucleotide sequence of OVA inserted into the plasmid DNA is set forth in SEQ ID NO: 81, and the nucleotide sequences of the fusion polypeptides are set forth in SEQ ID NOs: 48 to 68. In addition, the nucleotide sequence of OVA before codon optimization is set forth in SEQ ID NO: 23. The HiScribe T7 ARCA mRNA Kit (New England Biolabs) was used for mRNA preparation. Specifically, after linearizing the plasmid DNA inserted with each sequence using a restriction enzyme, in vitro mRNA synthesis was carried out with T7 RNA polymerase using the DNA as a template, and 5'-end capping was carried out with ARCA. Subsequently, after decomposing the template DNA by adding DNase, a poly(A) tail was added by Poly(A) Polymerase. The obtained mRNA was purified using an RNeasy kit (QIAGEN) and used for the subsequent operations. Using TransIT-mRNA Transfection Reagent (Mirus Bio), the purified mRNA was applied to mature human CD14 + dendritic cells (Lonza) for transfection. As a control, only the transfection reagent without mRNA was added to the dendritic cells. From the day after transfection, human CD4 derived from the same donor + T cells (Lonza) 1.0×10 62.5 × 10⁶ dendritic cells transfected with cells 5 The cells were co-cultured for a total of 7 days, and antigen presentation of mRNA-derived OVA was performed. Subsequently, CD4 cells were co-cultured on an ELISPOT plate. + T cells 1.0×10 6 cells derived from the same donor human CD14 + Dendritic cells 1.0 × 10 5 The cells were co-cultured again, and antigen stimulation was performed by adding 200 μg / mL of OVA protein. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol, and IFNγ-producing CD4 + T cells were detected.

[0135] The results are shown in Figure 1. By expressing a fusion protein of a specific SNARE protein (VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, or SEC22B) with OVA in dendritic cells, CD4 cells that produce OVA-specific IFNγ were produced. + The number of T cells increased significantly. Generally, CD4 + When T cells receive antigen presentation, they differentiate into various cell types such as Th1, Th2, Th17, and Treg, and release specific cytokines, with IFNγ being a cytokine characteristically secreted by Th1 cells. Therefore, it was demonstrated that introducing a specific SNARE-OVA fusion protein according to the present invention into antigen-presenting cells such as dendritic cells can induce or enhance the Th1 immune response. This experiment was conducted with n=3 in each group. Using the control group (Empty) that underwent restimulation with the OVA protein as a reference, groups that achieved p<0.05 in Dunnett's test were considered statistically significant. Similarly, groups that achieved p<0.1 in Student's t-test (using Empty as a reference) were also considered statistically significant. The Empty (NT) group was the control group (Empty) that did not undergo restimulation with the OVA protein.

[0136] Example 2: IgG, IgG1, and IgG2a antibody titers In Example 1, VAMP7 was selected from the SNARE proteins that increased IFNγ production. A plasmid vector (pVAX1 vector, Thermo Fisher Scientific) was constructed encoding either the amino acid sequence of a fusion polypeptide (V7-OVA) (SEQ ID NO: 25) with OVA linked to the C-terminus of VAMP7 via a linker, or only the amino acid sequence of OVA (SEQ ID NO: 24). An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21, Neppageen Co., Ltd.). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 35 and blood was collected. Serum was separated from the collected blood and antibody titers were measured. For antibody titer measurement, 100 μL / well of DPBS solution containing 10 μg / mL of OVA (Merck) was added to a 96-well ELISA plate (IWAKI) to immobilize the OVA. The following day, after washing the plate, blocking was performed for 1 hour with 100 μL / well of 1% BSA-containing DPBS. Subsequently, after washing the plate, the separated serum was added in 100 μL / well in stepwise dilutions. After 2 hours, the serum was washed away, and 100 μL / well of 1 μg / mL of anti-Mouse IgG, IgG1, or IgG2a antibody (abcam) was added and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (abcam) was added and the color reaction was performed. After 10 minutes, 50 μL / well of Stop solution (abcam) was added to stop the color reaction, and absorbance measurement (450 nm) was performed. At this time, the antibody titer of each sample was defined as the value at the highest dilution ratio in which the absorbance was more than twice that of the control sample to which DPBS was added instead of serum.

[0137] The results are shown in Figure 2. Compared to OVA alone, VAMP7-OVA (V7-OVA) showed a significant decrease in OVA-specific IgG1 antibody titers. On the other hand, for OVA-specific IgG2a, a significant increase in antibody titers was observed only with VAMP7-OVA compared to the control group (Empty). There was no significant difference in the total antibody titer of OVA-specific IgG between OVA alone and VAMP7-OVA. Generally, it is known that IgG2a antibodies are produced from B cells in a Th1 immune response, while IgG1 antibodies are produced from B cells in a Th2 immune response. Therefore, by measuring the antibody titers of antigen-specific IgG1 and IgG2a antibodies, the Th1 / Th2 balance of the immune response occurring in vivo can be estimated. In this result, since VAMP7-OVA showed a decrease in OVA-specific IgG1 antibody titers and an increase in IgG2a antibody titers compared to OVA alone, it was shown that VAMP7-OVA can induce or enhance a Th1 immune response in vivo. This experiment was conducted with n=10 in each group. A statistically significant difference was determined if p<0.05 was obtained when performing the Tukey test in each group.

[0138] Example 3: ELISPOT Assay A fusion polypeptide (V7-OVA) was created by linking OVA to the C-terminus of VAMP7 with a linker. Alternatively, a plasmid vector encoding only the amino acid sequence of OVA (pVAX1 vector, Thermo Fisher Scientific) was constructed. An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each vector was intramuscularly administered into the thigh muscle of female BALB / c mice. Gene transfer was then performed by applying voltage using an electroporator (NEPA21). This procedure was performed on days 0, 7, and 14 from the start of the experiment. On day 35, the mice were euthanized and their spleens were collected. The collected spleens were ground on a 40 μm cell strainer while adding 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, and after removing the supernatant, Pharm Lyse was used. TMThe solution (BD Biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells in the suspension. The sample was then centrifuged again at 200 x g for 5 minutes, the supernatant was removed, and the sample was washed twice with DPBS before performing a CTL-Test. TM Splenocyte suspension was obtained by adding Medium (CTL). 1.0 × 10 7 Splenocytes with a cell / mL count were seeded at 100 μL / well on an ELISPOT plate and stimulated with the antigen at a final concentration of 50 μg / mL by mixing with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck). A negative control group (NT) without OVA protein supplementation was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol to detect cells that specifically produce IFNγ and IL-4 against the antigen (OVA). The spleen is a secondary lymphoid tissue, and its constituent cells, splenocytes, are rich in T cells; therefore, splenocytes were used in this experiment to detect T cells that exhibit an antigen-specific immune response.

[0139] The results are shown in Figure 3. When VAMP7-OVA (V7-OVA) was used, the number of T cells secreting IL-4 specifically for OVA remained unchanged compared to OVA alone, while the number of T cells secreting IFNγ specifically for OVA significantly increased. Generally, IL-4 is secreted by Th2 cells, and IFNγ is secreted by Th1 cells, or cytotoxic T cells (CD8) involved in cell-mediated immunity. + Since VAMP7 is known to be secreted by T cells, these results indicate that fusing VAMP7 with an antigen does not induce an antigen-specific Th2 immune response, but selectively induces or enhances an antigen-specific Th1 immune response and cellular immunity. In this experiment, n=10 was performed in each group, and a p<0.05 result in the Tukey test in each group under unstimulated conditions (NT) or OVA-stimulated conditions (OVA) was considered statistically significant.

[0140] Example 4: IgG, IgG1, and IgG2a antibody titers Plasmid vectors (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequences (SEQ ID NOs. 25, 27, 28, 41) of fusion polypeptides (V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA) formed by linking OVA to the C-terminus of VAMP7, STX10, STX18, or GOSR1 with a linker, or only the amino acid sequence of OVA (SEQ ID NO. 24), were constructed. An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each constructed vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 35 and blood was collected. Serum was separated from the collected blood and antibody titers were measured. For antibody titer measurement, 100 μL / well of DPBS solution containing 10 μg / mL of OVA (Merck) was added to a 96-well ELISA plate (IWAKI) to immobilize the OVA. The following day, after washing the plate, blocking was performed for 1 hour with 100 μL / well of 1% BSA-containing DPBS. Subsequently, after washing the plate, the separated serum was added in 100 μL / well in stepwise dilutions. After 2 hours, the serum was washed away, and 100 μL / well of 1 μg / mL of anti-Mouse IgG, IgG1, or IgG2a antibody (abcam) was added and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (abcam) was added and a color reaction was performed. After 10 minutes, 50 μL / well of Stop solution (abcam) was added to stop the color reaction, and absorbance measurement (450 nm) was performed. At this time, the antibody titer of each sample was defined as the value at the highest dilution ratio in which the absorbance was more than twice that of the control sample to which DPBS was added instead of serum.

[0141] The results are shown in Figure 4. At 5 weeks after vector administration, a significant increase in OVA-specific IgG antibody titers was observed in all groups, including OVA alone and groups with each SNARE sequence linked to OVA, compared to the group administered with an empty vector. In particular, while VAMP7-OVA (V7-OVA) and GOSR1-OVA showed no change in OVA-specific IgG antibody titers compared to OVA alone, STX10-OVA and STX18-OVA showed a significant decrease in OVA-specific IgG antibody titers compared to OVA alone. For OVA-specific IgG1 antibody titers, a significant decrease was observed in all groups with each SNARE sequence linked to OVA compared to OVA alone. Furthermore, for OVA-specific IgG2a antibody titers, only V7-OVA showed a significant increase compared to the group administered with an empty vector. Regarding the IgG2a / IgG1 ratio, calculated by determining the ratio of OVA-specific IgG1 and IgG2a antibody titers in each individual, no significant difference was observed, but it was elevated in all groups with each SNARE sequence linked to OVA compared to OVA alone. In the IgG2a / IgG1 graph, the mean value is shown above the dot plot for each group. These results indicate that, in addition to VAMP7, other SNARE family members such as STX10, STX18, and GOSR1 also show a decrease in OVA-specific IgG1 antibody titers and an increase in the IgG2a / IgG1 ratio. This suggests that when these SNARE sequences are linked to an antigen, they can induce or enhance an antigen-specific Th1 immune response in vivo. This experiment was conducted with n=5 in each group, and a p<0.05 result in Dunnett's test was considered statistically significant. Significant differences are indicated by * when comparing with the Empty group and † when comparing with the OVA group.

[0142] Example 5: ELISPOT assay Plasmid vectors (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequences (SEQ ID NOs. 25, 27, 28, 41) of fusion polypeptides (V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA) in which OVA was linked to the C-terminus of VAMP7, STX10, STX18, or GOSR1 with a linker, or only the amino acid sequence of OVA (SEQ ID NO. 24), were constructed. An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each constructed vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 35 and their spleens were collected. The collected spleens were ground on a 40 μm cell strainer while adding 2% FBS-containing DPBS. The obtained suspension was centrifuged at 200 × g for 5 minutes, and after removing the supernatant, Pharm Lyse solution (BD biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells in the suspension. The suspension was again centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the suspension was washed twice with DPBS. CTL-Test Medium (CTL) was then added to obtain a splenocyte suspension. 1.0 × 10 7 Splenocytes from cells were seeded at a rate of 100 μL / well on an ELISPOT plate and stimulated with the antigen at a final concentration of 50 μg / mL by mixing with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck). A negative control group (NT) without OVA protein supplementation was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol to detect cells that specifically produce IFNγ and IL-4 in response to the antigen (OVA). The spleen is a secondary lymphoid tissue, and its constituent cells, splenocytes, are rich in T cells. Therefore, splenocytes were used in this experiment to detect T cells that exhibit an antigen-specific immune response.

[0143] The results are shown in Figure 5. Compared to OVA alone, the number of T cells secreting OVA-specific IFNγ significantly increased in all groups where each SNARE sequence was linked to OVA. On the other hand, regarding the number of T cells secreting OVA-specific IL-4, significant differences were observed between groups under both the unstimulated (NT) and OVA-stimulated (OVA) conditions. However, no change was observed in the IL-4 ratio, calculated for each individual under both NT and OVA conditions, across all groups. These results indicate that linking other SNARE family members such as STX10, STX18, and GOSR1 to antigens does not induce an antigen-specific Th2 immune response, but rather selectively induces or enhances an antigen-specific Th1 immune response and cellular immunity. This experiment was conducted with n=5 in each group, and a p<0.05 result in Dunnett's test in each group under either the unstimulated or OVA-stimulated conditions was considered statistically significant. Significant differences are indicated by * when compared with the Empty group and † when compared with the OVA group.

[0144] Example 6: Antibody titers over time We constructed a plasmid vector encoding either the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 with a linker, the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide (V7-pc-OVA) in which a proprotein convertase recognition sequence (nucleotide sequence: SEQ ID NO: 21, amino acid sequence: SEQ ID NO: 22) was added to the C-terminus of the linker in the fusion polypeptide, the amino acid sequence (SEQ ID NO: 47) of a fusion polypeptide (LAMP[OVA]) in which the amino acid sequence of OVA was inserted into the lysosomal binding protein LAMP (Patent Document 1), or the amino acid sequence of OVA only (SEQ ID NO: 24). The nucleotide sequence of V7-pc-OVA incorporated into plasmid DNA is shown in SEQ ID NO: 69, and the nucleotide sequence of LAMP[OVA] is shown in SEQ ID NO: 70. An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each constructed vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). The above procedures were performed on days 0, 7, and 14 from the start of the experiment. During the experiment, blood was collected from the tail vein every week, and on day 35, the animals were euthanized and blood was collected. Serum was separated from the collected blood, and antibody titer was measured. For antibody titer measurement, 100 μL / well of DPBS solution containing 10 μg / mL OVA (Merck) was added to a 96-well ELISA plate (IWAKI) to immobilize the OVA. The following day, after washing the plate, blocking was performed for 1 hour with 100 μL / well of 1% BSA-containing DPBS. Subsequently, after washing the plate, the separated serum was added in 100 μL / well in stepwise dilutions. After 2 hours, the serum was washed away, and 100 μL / well of 1 μg / mL anti-Mouse IgG, IgG1, or IgG2a antibody (abcam) was added and allowed to stand for 1 hour. After that, 50 μL / well of TMB solution (abcam) was added and the color reaction was performed. After 10 minutes, 50 μL / well of Stop solution (abcam) was added to stop the color reaction, and absorbance was measured (450 nm).At this time, the antibody titer of each sample was defined as the value at the highest dilution ratio in which the absorbance was more than twice that of the control sample to which DPBS was added instead of serum.

[0145] The results are shown in Figure 6. Similar to Example 2, a significant decrease in OVA-specific IgG1 antibody titers was observed in VAMP7-OVA (V7-OVA) and VAMP7-pc-OVA (V7-pc-OVA) compared to OVA alone. On the other hand, a greater increase in OVA-specific IgG2a antibody titers was observed in VAMP7-pc-OVA. Regarding overall OVA-specific IgG, while OVA alone tended to show a greater increase in antibody titers at weeks 3 and 4, at week 5, VAMP7-pc-OVA showed an increase in antibody titers comparable to OVA alone. Furthermore, LAMP[OVA] showed almost no increase in OVA-specific antibody titers. Regarding the IgG2a / IgG1 ratio, calculated by determining the ratio of OVA-specific IgG1 and IgG2a antibody titers in each individual at week 5, no significant difference was observed, but it was higher in VAMP7-OVA and VAMP7-pc-OVA compared to OVA alone and LAMP[OVA]. These results indicate that introducing a proprotein convertase recognition sequence further promotes IgG2a antibody production, inducing or enhancing an antigen-specific Th1 immune response in vivo. Furthermore, compared to LAMP[OVA], VAMP7-OVA and VAMP7-pc-OVA demonstrate superior antigen-specific IgG antibody production and enhanced antigen-specific Th1 immune response. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Tukey test in each group at each week was considered statistically significant. * indicates significance when comparing with the Empty group, and † indicates significance when comparing with the OVA group.

[0146] Example 7 ELISPOT assay Plasmid vectors were constructed encoding either the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 with a linker, the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide (V7-pc-OVA) in which a proprotein convertase recognition sequence was added to the C-terminus of the linker in the fusion polypeptide, or only the amino acid sequence of OVA (SEQ ID NO: 24). An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each constructed vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 35 and their spleens were collected. The collected spleens were ground on a 40 μm cell strainer while adding 2% FBS-containing DPBS. The obtained suspension was centrifuged at 200 × g for 5 minutes, and after removing the supernatant, Pharm Lyse solution (BD biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells in the suspension. The suspension was again centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the suspension was washed twice with DPBS. CTL-Test Medium (CTL) was then added to obtain a splenocyte suspension. 1.0 × 10 7 Splenocytes from cells were seeded at 100 μL / well on an ELISPOT plate and stimulated with antigen at a final concentration of 50 μg / mL by mixing with 100 μL of CTL-Tes Medium supplemented with OVA protein (Merck). A negative control group (NT) without OVA protein supplementation was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol to detect cells that specifically produce IFNγ and IL-4 in response to the antigen (OVA). The spleen is a secondary lymphoid tissue, and its constituent cells, splenocytes, are rich in T cells; therefore, splenocytes were used in this experiment to detect T cells that exhibit an antigen-specific immune response.

[0147] The results are shown in Figure 7. Introducing the proprotein convertase recognition sequence further increased the number of T cells secreting OVA-specific IFNγ. On the other hand, while significant differences were observed between groups in the number of T cells secreting OVA-specific IL-4 under both the unstimulated (NT) and OVA-stimulated (OVA) conditions, no change was observed in the IL-4 ratio, calculated for each individual under each NT and OVA condition. These results suggest that introducing the proprotein convertase recognition sequence can further enhance antigen-specific Th1 immune responses and cellular immunity. Proprotein convertase is a type of protease that cleaves precursor and immature proteins within cells and converts them into mature and active forms. Because proprotein convertase is localized in the Golgi apparatus and endosomes, it is thought that by cleaving the VAMP7-pc-OVA fusion protein transported to intracellular vesicles in vivo, it releases OVA into the vesicles, thereby more efficiently inducing association with MHC molecules. In addition, it is possible that the encapsulation of released OVA within exosomes and subsequent secretion induced an enhanced immune response. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Tukey test in each group under either the unstimulated (NT) or OVA-stimulated conditions was considered statistically significant.

[0148] Example 8: IgG, IgG1, and IgG2a antibody titers The C-terminal side of VAMP7 contains a proprotein convertase recognition sequence and a repeating sequence of the cancer antigen human Wilm Tumor 1 (WT1, amino acid sequence: SEQ ID NO: 84) or the autoantigen mouse myelin oligodendrocyte glycoprotein peptide (MOG 35-55 A fusion polypeptide (V7-pc-WT1, V7-pc-MOG) is formed by linking amino acid sequences (SEQ ID NO: 87) with a linker. 35-55 ) Amino acid sequence (SEQ ID NOs. 89, 91), or WT1 or MOG 35-55A plasmid vector encoding only the amino acid sequence was constructed. At this time, the nucleotide sequence encoding the antigen or fusion polypeptide was codon-optimized and then incorporated into plasmid DNA (pVAX1 vector, Thermo Fisher Scientific). The WT1 nucleotide sequence incorporated into the plasmid DNA was named SEQ ID NO: 83, MOG 35-55 The nucleotide sequence of V7-pc-WT1 is set to SEQ ID NO: 86, the nucleotide sequence of V7-pc-MOG is set to SEQ ID NO: 88, and V7-pc-MOG 35-55 The nucleotide sequence is shown in SEQ ID NO: 90. Also, the nucleotide sequence of WT1 before codon optimization is shown in SEQ ID NO: 82, MOG 35-55 The nucleotide sequence before codon optimization is shown in SEQ ID NO: 85. Here, MOG 35-55 This is a peptide in which the 35-55 residue portion of mouse MOG is repeated seven times. The control is the antigen (WT1 or MOG). 35-55 An empty vector that does not encode () was used. 50 μg of each prepared vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 28 and blood was collected. Serum was separated from the collected blood and antibody titer was measured. For antibody titer measurement, 1 μg / mL human WT1 (Cusabio Technology LLC) or 10 μg / mL mouse MOG 35-55A DPBS solution containing a Merck peptide was added to a 96-well ELISA plate (Iwaki) at a rate of 100 μL / well to immobilize each antigen. The following day, after washing the plate, blocking was performed with 100 μL / well of 1% BSA-containing DPBS for 1 hour. Subsequently, after washing the plate, the separated serum was added in 100 μL / well in stepwise dilutions. After 2 hours, the serum was washed off, and 100 μL / well of 1 μg / mL anti-Mouse IgG, IgG1, or IgG2a antibody (abcam) was added and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (abcam) was added and a color reaction was performed. After 10 minutes, 50 μL / well of Stop solution (abcam) was added to stop the color reaction, and absorbance measurement (450 nm) was performed. At this time, the antibody titer of each sample was defined as the value at the highest dilution ratio in which the absorbance was more than twice that of the control sample to which DPBS was added instead of serum.

[0149] The results are shown in Figure 8. At four weeks after vector administration, while no significant difference was observed compared to the group administered with the Empty vector, an increase in WT1-specific IgG antibody titers was observed in both the WT1-only and VAMP7-pc-WT1 (V7-pc-WT1) groups. On the other hand, while no significant difference was observed in WT1-specific IgG1 and IgG2a antibody titers, an increase was observed in the V7-pc-WT1 group compared to Empty and WT1 alone. Regarding the IgG2a / IgG1 ratio, calculated from the ratio of WT1-specific IgG1 and IgG2a antibody titers in each individual, no change was observed between WT1 alone and the V7-pc-WT1 group. Furthermore, in experiments measuring MOG-specific IgG antibody titers, MOG showed an increase compared to Empty. 35-55 Standalone and VAMP7-pc-MOG 35-55 (V7-pc-MOG 35-55 No changes were observed in either group. On the other hand, regarding MOG-specific IgG1 and IgG2a antibody titers, Empty and MOG 35-55 V7-pc-MOG compared to standalone 35-55An increase was observed, and only the IgG1 antibody titer showed a significant change. 35-55 For IgG2a / IgG1, which is calculated as the ratio of specific IgG1 and IgG2a antibody titers in each individual, MOG 35-55 Standalone and V7-pc-MOG 35-55 No significant changes were observed during this period. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Tukey test was considered statistically significant.

[0150] Example 9: ELISPOT assay VAMP7 has a proprotein convertase recognition sequence at its C-terminus and human WT1 or mouse MOG 35-55 Fusion polypeptides linked by a linker (V7-pc-WT1, V7-pc-MOG 35-55 ) Amino acid sequence (SEQ ID NOs. 89, 91), or WT1 or MOG 35-55 A plasmid vector (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequences (SEQ ID NOs. 84, 87) was constructed. Antigens (WT1 or MOG) were used as controls. 35-55 An empty vector that does not encode ) was used. 50 μg of each prepared vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 28 and the spleen was collected. The collected spleen was ground on a 40 μm cell strainer while adding 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, the supernatant was removed, and Pharm Lyse solution (BD biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells in the suspension. The suspension was centrifuged again at 200 × g for 5 minutes, the supernatant was removed, and after washing twice with DPBS, CTL-Test Medium (CTL) was added to obtain a splenocyte suspension. 1.0 × 10 7Splenocytes from cells were seeded at 100 μL / well on an ELISPOT plate and treated with human WT1 peptide mix (Miltenyi Biotec) or mouse MOG. 35-55 Antigen stimulation was performed by mixing 100 μL of CTL-Test Medium containing peptide (Merck) with the antigen to achieve final concentrations of 1.2 nmol / mL or 5 μg / mL. A negative control group (NT) without antigen stimulation was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol, and the antigen (WT1 or MOG) was evaluated. 35-55 Cells specifically producing IFNγ and IL-4 were detected. The spleen is a secondary lymphoid tissue, and its constituent cells, spleenocytes, are rich in T cells. Therefore, spleenocytes were used in this experiment to detect T cells that exhibit antigen-specific immune responses.

[0151] The results are shown in Figure 9. Compared to WT1 alone, the number of T cells secreting IFNγ specifically in WT1 was significantly increased in V7-pc-WT1. Similarly, MOG 35-55 Compared to V7-pc-MOG alone, 35-55 So MOG 35-55 The number of T cells specifically secreting IFNγ increased significantly. On the other hand, regarding the number of T cells secreting WT1-specific IL-4, a significant difference was observed between groups under WT1 peptide mix stimulation (WT1), but no change was observed in the IL-4 ratio, which was calculated for each individual under unstimulated (NT) and WT1 conditions, across all groups. Similarly, MOG 35-55 The number of T cells secreting specific IL-4 was also investigated under unstimulated conditions (NT) and MOG. 35-55 Under peptide stimulation conditions (MOG 35-55 Although significant differences were observed between groups under each of the conditions, NT and MOG 35-55Under each of the conditions, the IL-4 ratio calculated for each individual showed no change in any of the groups. Furthermore, when the ratio of IFNγ to the number of T cells producing IL-4 (IFNγ / IL-4) was calculated for each individual, the V7-pc-WT1 or V7-pc-MOG groups were compared to each antigen alone at each antigen stimulation. 35-55 In this study, IFNγ / IL-4 levels were significantly increased. Although no IgG2a-dominant Th1-type antibody response was observed in Example 8, considering that this result was obtained using ELISPOT analysis, which can directly detect T-cell antigen-specific immune responses, it suggests that even when OVA is linked to a different antigen than OVA against certain SNARE families, including VAMP7, antigen-specific Th2-type immune responses are not induced, and antigen-specific Th1-type immune responses and cellular immunity can be selectively induced or enhanced. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Tukey test in each group under unstimulated or antigen-stimulated conditions indicated a statistically significant difference.

[0152] Example 10: ELISPOT assay Plasmid vectors were constructed encoding the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 with a linker, the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide (V7-pc-OVA) in which a proprotein convertase recognition sequence was added to the C-terminus of the linker in the fusion polypeptide, the amino acid sequence (SEQ ID NO: 47) of a fusion polypeptide (LAMP[OVA]) in which the amino acid sequence of OVA was inserted into the lysosomal binding protein LAMP (Patent Document 1), or the amino acid sequence of OVA alone (SEQ ID NO: 24). An empty vector not encoding the antigen (OVA) was used as a control. 50 μg of each constructed vector was intramuscularly administered into the thigh muscle of female BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). Seven days after the above procedure, the mice were euthanized and their spleens were collected. The collected spleens were ground on a 40 μm cell strainer while adding 2% FBS-containing DPBS. The obtained suspension was centrifuged at 200 × g for 5 minutes, and after removing the supernatant, Pharm Lyse solution (BD biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells in the suspension. The suspension was again centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the suspension was washed twice with DPBS. CTL-Test Medium (CTL) was then added to obtain a splenocyte suspension. 1.0 × 10 7Splenocytes from cells were seeded at a rate of 100 μL / well on an ELISPOT plate and stimulated with the antigen at a final concentration of 50 μg / mL by mixing with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck). A negative control group (NT) without OVA protein supplementation was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit (CTL) according to the attached protocol to detect cells that specifically produce IFNγ and IL-4 in response to the antigen (OVA). The spleen is a secondary lymphoid tissue, and its constituent cells, splenocytes, are rich in T cells. Therefore, splenocytes were used in this experiment to detect T cells that exhibit an antigen-specific immune response.

[0153] The results are shown in Figure 10. Even just one week after vector administration, VAMP7-pc-OVA showed a significant increase in the number of T cells secreting OVA-specific IFNγ compared to VAMP7-OVA and LAMP[OVA]. On the other hand, regarding the number of T cells secreting OVA-specific IL-4, significant differences were observed between groups under both the unstimulated (NT) and OVA-stimulated (OVA) conditions. However, no change was observed in the IL-4 ratio, calculated for each individual under both NT and OVA conditions, across all groups. Furthermore, when the ratio of T cells producing IFNγ and IL-4 (IFNγ / IL-4) was calculated for each individual, V7-pc-OVA showed a significant increase in IFNγ / IL-4 compared to V7-OVA and LAMP[OVA] during OVA stimulation. These results indicate that VAMP7-pc-OVA, from a relatively early stage (week 1), does not induce an antigen-specific Th2 immune response, but selectively induces antigen-specific Th1 immunity and cellular immunity, demonstrating superior immune induction compared to conventional techniques. In this experiment, n=5 was conducted in each group, and a p<0.05 result in the Tukey test in each group under either unstimulated (NT) or OVA-stimulated conditions was considered statistically significant.

Claims

1. An antigen-specific Th1 immune response inducer or enhancer comprising a nucleic acid construct as an active ingredient, which includes a polynucleotide encoding one of the SNARE proteins selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and a polynucleotide encoding an antigen.

2. A nucleic acid vaccine used for inducing or enhancing an antigen-specific Th1 immune response, comprising a nucleic acid construct as an active ingredient, which includes a polynucleotide encoding any SNARE protein selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and a polynucleotide encoding an antigen.

3. A nucleic acid vaccine according to claim 2, wherein the antigen is at least one selected from the group consisting of autoantigens, for the treatment of autoimmune diseases.

4. A nucleic acid vaccine according to claim 2 or 3, administered parenterally.

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