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.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 allergen. [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 a nucleic acid encoding a fusion protein in which an allergen protein (antigen) was 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 a nucleic acid encoding a fusion molecule that includes 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 specify an allergen as the antigen, nor does it show how the immunogenicity of the antigen changes due to the fusion molecule. Since allergies are primarily caused by the excessive induction of an allergen-specific Th2 immune response, when applying nucleic acid vaccines to allergies, it is desirable to selectively induce a Th1 immune response that can suppress the Th2 immune response, rather than inducing a target allergen-specific Th2 immune response. However, the effect on the allergen-specific Th2 immune response when using nucleic acids that encode such a fusion molecule, which has a transmembrane domain and contains an allergen as the antigen, is unknown. Therefore, the effect of nucleic acids encoding such a fusion molecule using an allergen on the balance between Th1 and Th2, which are involved in the onset and suppression of allergic symptoms, is unknown.Furthermore, there is a possibility that allergens produced in the body by nucleic acid vaccines may leak into the bloodstream and bind to allergen-specific IgE antibodies present on the surface of mast cells and basophils, potentially causing type I allergic side effects. To date, no technology for preventing or improving allergies using nucleic acid constructs has been established.

[0004] SNARE proteins are a family of proteins possessing a 20-30 kDa SNARE motif. Many SNARE proteins are anchored to lipid bilayers via a C-terminal transmembrane domain and are involved in the process of vesicle fusion with target intracellular organelles (Non-Patent Literature 2 and 3). Membrane fusion involving SNARE proteins is essential for many important life phenomena indispensable to cellular function in eukaryotic cells, including endocytosis processes such as vesicle transport, organelle membrane morphogenesis, and extracellular receptor recycling, as well as exocytosis processes such as hormone secretion and synaptic neurotransmitter release. Furthermore, the molecular mechanism of membrane fusion by SNARE proteins is thought to be conserved across all eukaryotes, from single-celled 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 is thought to be localized to a specific intracellular membrane fraction (such as the endoplasmic reticulum (ER), Golgi apparatus, endosomes, organelles like vacuoles and lysosomes, secretory vesicles, and cytoplasmic membrane) and to function in membrane fusion processes in specific intracellular transport pathways. Furthermore, SNARE proteins are known to potentially be present in exosomes. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2017-79742 [Patent Document 2] Special Publication No. 2008-500014 [Non-patent literature]

[0006] [Non-Patent Document 1] Chen AC, et al. J Immunother Cancer. 2020; 8(1): e000258. [Non-Patent Document 2] Jahn R, Scheller RH. Nat Rev Mol Cell Biol. 2006; 7(9): 631-643. [Non-Patent Document 3] Hong W. Biochim Biophys Acta. 2005; 1744(2): 120-144. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention relates to providing nucleic acid constructs that enhance allergen-specific immune responses. [Means for solving the problem]

[0008] The inventors have found that by using a nucleic acid construct containing a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen, it is possible to induce or enhance an allergen-specific Th1 immune response, and also to induce or enhance an allergen-specific cellular immune response, compared to a nucleic acid construct containing only a polynucleotide encoding an allergen.

[0009] Therefore, the present invention provides the following 1) to 4). 1) A nucleic acid construct containing a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen. 2) An allergen-specific Th1-type immune response inducer or enhancer comprising the nucleic acid construct described in 1) as an active ingredient. 3) An allergen-specific cellular immune response inducer or enhancer comprising the nucleic acid construct described in 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] According to the nucleic acid construct of the present invention, an allergen-specific Th1-type immune response can be induced or enhanced, and an allergen-specific cellular immune response can be induced or enhanced. Such a nucleic acid construct is useful as a nucleic acid vaccine. [Brief Description of the Drawings]

[0011] [Figure 1] Evaluation of immunogenicity of mRNA encoding a SNARE protein-allergen fusion polypeptide. The amount of IFN-γ produced is shown as the number of producing cells (spot forming cell; SFC). Empty represents a control with only transfection reagent, Empty (N.T.) represents a control that did not undergo restimulation with allergen, and SNARE protein-OVA represents mRNA encoding a SNARE protein-allergen (OVA) fusion polypeptide. [Figure 2] IgG subclass antibody titers obtained when a plasmid vector encoding a SNARE protein-allergen fusion polypeptide is administered to mice. (A) shows IgG antibody titers, (B) shows IgG1 antibody titers, and (C) shows IgG2a antibody titers. Empty represents an empty plasmid vector that does not encode allergen (OVA), OVA represents a plasmid vector that encodes OVA, and V7-OVA represents a plasmid vector that encodes a VAMP7-OVA fusion polypeptide. [Figure 3] Evaluation of immune response obtained when a plasmid vector encoding a SNARE protein-allergen fusion polypeptide is administered to mice. (A) shows the amount of IFN-γ produced, and (B) shows the amount of IL-4 produced in SFC. (C) shows the ratio of IFN-γ production amount to IL-4 production amount (IFN-γ / IL-4). Empty represents an empty plasmid vector that does not encode allergen (OVA), OVA represents a plasmid vector that encodes OVA, and V7-OVA represents a plasmid vector that encodes a VAMP7-OVA fusion polypeptide. [Figure 4] The antibody titers of IgG subclasses when plasmid vectors encoding SNARE protein-allergen fusion polypeptides are administered 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 IgG2a antibody titer to IgG1 antibody titer (IgG2a / IgG1). Empty indicates an empty plasmid vector that does not encode the allergen (OVA), OVA indicates a plasmid vector encoding OVA, and V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA respectively indicate plasmid vectors encoding VAMP7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA fusion polypeptides. [Figure 5] Evaluation of immune response when plasmid vectors encoding SNARE protein-allergen fusion polypeptides are administered to mice. (A) shows the production amount of IFNγ, (B) shows the production amount of IL-4 in SFC. (C) shows the ratio of IL-4 production under allergen stimulation conditions to that under non-stimulation conditions (IL-4 ratio). Empty indicates an empty plasmid vector that does not encode the allergen (OVA), OVA indicates a plasmid vector encoding OVA, and V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA respectively indicate plasmid vectors encoding VAMP7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA fusion polypeptides. [Figure 6]Antibody titers over time when mice were administered plasmid vectors encoding SNARE protein-allergen fusion polypeptides or fusion polypeptides containing an allergen 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 allergen (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-allergen 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 allergen-stimulated conditions to unstimulated conditions (IL-4 ratio). Empty indicates an empty plasmid vector that does not encode the allergen (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]Evaluation of the immune response when mice were administered plasmid vectors encoding SNARE protein-allergen fusion polypeptides or fusion polypeptides containing an allergen within LAMP. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, both in SFC. (C) shows the ratio of IL-4 production under allergen-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 allergen (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 9] Evaluation of allergic reactions in allergy model mice when administered plasmid vectors encoding SNARE protein-allergen fusion polypeptides or fusion polypeptides containing an allergen within LAMP. (A) shows the change in rectal temperature over time, and (B) shows the amount of allergen-specific IgE produced. Empty indicates an empty plasmid vector that does not encode the allergen (OVA), OVA indicates a plasmid vector that encodes OVA, V7-pc-OVA indicates a plasmid vector that encodes a VAMP7-proprotein convertase recognition sequence-OVA fusion polypeptide, and LAMP[OVA] indicates a plasmid vector that encodes a fusion polypeptide containing OVA within LAMP. (-) indicates no sensitization, and (+) indicates sensitization. [Figure 10]Evaluation of OVA blood concentrations in mice after administration of plasmid vectors encoding SNARE protein-allergen fusion polypeptides. Empty represents an empty plasmid vector that does not encode the allergen (OVA), OVA represents a plasmid vector that encodes OVA, V7-OVA represents a plasmid vector that encodes a VAMP7-OVA fusion polypeptide, and V7-pc-OVA represents a plasmid vector that encodes a VAMP7-proprotein convertase recognition sequence-OVA fusion polypeptide. [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" refers to a molecule that triggers an immune response in a living organism, such as antibody production or cellular immunity, and specifically refers to an allergen. Furthermore, in this specification, "immunogenicity" refers to 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 a SNARE protein and a polynucleotide encoding an allergen.

[0030] In the present invention, a SNARE protein is a protein belonging to the protein family having a SNARE motif. SNARE proteins are classified into four subfamilies—QA-SNARE, QB-SNARE, QC-SNARE, and R-SNARE—based on the characteristics of the amino acid residues of the SNARE motif, but the SNARE protein used in the present invention may belong to any of these subfamilies. The SNARE protein preferably has a transmembrane domain and is localized to intracellular vesicles. Specifically, examples include, but are not limited to, VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, SEC22B, STX6, VAMP3, VAMP4, VTI1B, BET1, BET1L, and USE1. As the SNARE protein, from the viewpoint of immune induction or enhancement, any of the following is preferred:

[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: 83, 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 has 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: 84, 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: 85, 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: 86, 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: 87, 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] STX6 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, STX6 is mammalian STX6. In a more preferred example, STX6 is human STX6, which is a protein consisting of the amino acid sequence of SEQ ID NO: 88, encoded by a gene (Gene ID: 10228) consisting of the nucleotide sequence of SEQ ID NO: 76. The STX6 used in this invention includes STX6 and polypeptides having equivalent function.

[0046] VAMP3 (Vesicle-associated membrane protein 3) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the cell membrane and Golgi apparatus. In a preferred example, VAMP3 is mammalian VAMP3. In a more preferred example, VAMP3 is human VAMP3, which is a protein consisting of the amino acid sequence of SEQ ID NO: 89, encoded by a gene (Gene ID: 9341) consisting of the nucleotide sequence of SEQ ID NO: 77. The VAMP3 used in this invention includes VAMP3 and polypeptides having equivalent functions.

[0047] VAMP4 (Vesicle-associated membrane protein 4) 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, VAMP4 is mammalian VAMP4. In a more preferred example, VAMP4 is human VAMP4, which is a protein consisting of the amino acid sequence of SEQ ID NO: 90, encoded by a gene (Gene ID: 8674) consisting of the nucleotide sequence of SEQ ID NO: 78. The VAMP4 used in this invention includes VAMP4 and polypeptides having equivalent function.

[0048] VTI1B (Vesicle transport through interaction with t-SNAREs homolog 1B) is a type of SNARE protein that has a transmembrane domain and is expected to localize to early endosomes, late endosomes, lysosomes, and recycling endosomes. In a preferred example, VTI1B is mammalian VTI1B. In a more preferred example, VTI1B is human VTI1B, which is a protein consisting of the amino acid sequence of SEQ ID NO: 91, encoded by a gene (Gene ID: 10490) consisting of the nucleotide sequence of SEQ ID NO: 79. The VTI1B used in this invention includes VTI1B and polypeptides having equivalent function.

[0049] BET1 (Bet1 golgi vesicular membrane trafficking protein) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the endoplasmic reticulum, Golgi apparatus, and cis-Golgi network. In a preferred example, BET1 is mammalian BET1. In a more preferred example, BET1 is human BET1, which is a protein consisting of the amino acid sequence of SEQ ID NO: 92, encoded by a gene (Gene ID: 10282) consisting of the nucleotide sequence of SEQ ID NO: 80. The BET1 used in this invention includes BET1 and polypeptides having equivalent function.

[0050] BET1L (Bet1 golgi vesicular membrane trafficking protein like) 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, BET1L is mammalian BET1L. In a more preferred example, BET1L is human BET1L, which is a protein consisting of the amino acid sequence of SEQ ID NO: 93, encoded by a gene (Gene ID: 51272) consisting of the nucleotide sequence of SEQ ID NO: 81. The BET1L used in this invention includes BET1L and polypeptides having equivalent function.

[0051] USE1 (Unconventional SNARE in the ER 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, USE1 is mammalian USE1. In a more preferred example, USE1 is human USE1, which is a protein consisting of the amino acid sequence of SEQ ID NO: 94, encoded by the gene (Gene ID: 55850) consisting of the nucleotide sequence of SEQ ID NO: 82. USE1 as used in this invention includes USE1 and polypeptides having equivalent function.

[0052] 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.

[0053] 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 that encode proteins registered as NP_001172112.1 or NP_001138621.1, respectively.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 registered in NCBI's RefSeq as NM_003569.3, NM_001326578.2, NM_001326579.2, or NM_001326580.2, which encode proteins registered as NP_003560.2, NP_001313507.1, NP_001313508.1, or NP_001313509.1, respectively.

[0059] 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 Sequence 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., trans-Golgi compartments) 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.

[0060] 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.

[0061] 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.

[0062] Specifically, the following are examples of GOSR1: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 83; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 83, 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: 83, 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.

[0063] Specifically, the following can be mentioned regarding the STX8: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 84; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 84, 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: 84, 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.

[0064] Specifically, the following are examples of STX12: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 85; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 85, 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: 85, 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.

[0065] Specifically, the following can be mentioned regarding VAMP8: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 86; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 86, 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: 86, 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.

[0066] Specifically, the following are examples of SEC22B: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 87; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 87, 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: 87, 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.

[0067] Specifically, the following are examples of STX6: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 88; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 88, 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: 88, 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: 76, 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. An example of a splicing variant of the STX6 gene consisting of the nucleotide sequence of sequence number 76 is the variant encoding a protein registered in NCBI's RefSeq as NM_001286210.2 and NP_001273139.1.

[0068] Specifically, the following can be mentioned regarding VAMP3: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 89; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 89, 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: 89, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., Golgi apparatus) 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 an intracellular vesicle (e.g., the Golgi apparatus) or an exosome.

[0069] Specifically, the following can be mentioned regarding VAMP4: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 90; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 90, 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: 90, 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: 78, 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. An example of a splicing variant of the VAMP4 gene consisting of the nucleotide sequence of SEQ ID NO: 78 is the variant that codes for a protein registered in NCBI's RefSeq as NM_001185127.2 and NP_001172056.1.

[0070] Specifically, the following are examples of VTI1B: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 91; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 91, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, 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: 91, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, 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., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes.

[0071] Specifically, BET1 offers the following: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 92; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 92, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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: 92, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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: 80, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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., endoplasmic reticulum, Golgi apparatus, or cis-Golgi network) or exosomes. An example of a splicing variant of the BET1 gene consisting of the nucleotide sequence of SEQ ID NO: 80 is the variant that codes for a protein registered in NCBI's RefSeq as NM_001317739.2 and NP_001304668.1.

[0072] Specifically, the following can be cited for BET1L: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 93; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 93, 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: 93, 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: 81, 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 BET1L gene consisting of the nucleotide sequence of SEQ ID NO: 81 include variants that are registered in NCBI's RefSeq as NM_001098787.2 or NM_016526.5 and encode proteins registered as NP_001092257.1 or NP_057610.2, respectively.

[0073] USE1 specifically includes the following: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 94; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 94, 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: 94, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum) 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 an intracellular vesicle (e.g., endoplasmic reticulum) or exosome.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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., the 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 that is at least 80% identical to the amino acid sequence of Sequence ID No. 18, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., 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: 18, and which can localize 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, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0084] 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: 83; (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. 83, 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: 83, 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 of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0085] 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 the polynucleotides described in (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: 84; (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. 84, 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: 84, 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.

[0086] 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: 85; (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. 85, 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 SEQ ID NO: 85, and capable of localizing 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.

[0087] 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: 86; (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. 86, 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: 86, 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.

[0088] 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: 87; (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. 87, 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: 87, 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.

[0089] The following are specific examples of polynucleotides that encode STX6: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 76; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 76, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., Golgi apparatus) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain and comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 76, and which can localize to intracellular vesicles (e.g., the Golgi apparatus) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 76, 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: 76, 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., the Golgi apparatus) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 88; (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. 88, and 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: 88, and which can localize 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, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0090] The following are examples of polynucleotides that encode VAMP3. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 77; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 77, 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: 77, and capable of localizing 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: 77, 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 (f) to (h) and (j) above, and has a transmembrane domain, and encodes a polypeptide that can localize 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: 89; (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. 89, and 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: 89, and which can localize 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, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0091] The following are specific examples of polynucleotides that encode VAMP4. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 78; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 78, 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: 78, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 78, has a transmembrane domain, and encodes a polypeptide that 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: 78, 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: 90; (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. 90, 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: 90, and capable of localizing 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 of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0092] The following are specific examples of polynucleotides that encode VTI1B: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 79; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 79, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling 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 Sequence ID No. 79, and capable of localizing to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary strand of the nucleotide sequence of SEQ ID NO: 79, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling 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, late endosomes, lysosomes, or recycling endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 91; (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. 91, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, 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 SEQ ID NO: 91, and capable of localizing to intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, 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., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes.

[0093] The following are examples of polynucleotides that encode BET1. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 80; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 80, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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: 80, and having a transmembrane domain, encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 80, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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: 80, has a transmembrane domain, and can localize to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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, Golgi apparatus, or cis-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92; (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. 92, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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: 92, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-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., endoplasmic reticulum, Golgi apparatus, or cis-Golgi network) or exosomes.

[0094] The following are specific examples of polynucleotides that encode BET1L: (f) A polynucleotide consisting of the nucleotide sequence of sequence number 81; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 81, 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: 81, 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: 81, 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: 81, 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: 93; (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. 93, 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: 93, and capable of localizing 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 of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0095] The following are specific examples of polynucleotides that encode USE1. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 82; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 82, 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: 82, and capable of localizing to intracellular vesicles (e.g., 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: 82, 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 (h) and (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 94; (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. 94, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the 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: 94, 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.

[0096] 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 71-82. For artificial synthesis, commercially available DNA synthesis services provided by companies such as GenScript can be used. Alternatively, for example, any of the nucleotide sequences of SEQ ID NOs: 1-9 and 71-82 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).

[0097] 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 71-82. 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 TM Commercially 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.

[0098] 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).

[0099] 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 71-82 to genome editing using artificial DNA nucleases (or programmable nucleases).

[0100] In the present invention, any allergen can be used. 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 induces a hypersensitivity or allergic reaction. The allergen is not particularly limited, but includes grass pollen (reeds, timothy grass, giant hornbeam, orchard grass, buckwheat, wheat, dwarf barnyard grass, barnyard grass, sorghum, longleaf grass, sweet vernal grass, broadleaf ryegrass, ryegrass, etc.); weed pollen (solida virgaurea, nettle, giant ragweed, Japanese hop, white dandelion, wormwood, dwarf sorrel, ragweed, false ragweed, oxeye daisy, leopard plant, etc.); and weed pollen (solida virgaurea, nettle, giant ragweed, Japanese hop, white dandelion, wormwood, dwarf sorrel, ragweed, false ragweed, oxeye daisy, leopard plant). 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.

[0101] The method for obtaining the polynucleotide encoding the allergen of the present invention is not particularly limited and can be obtained by conventional chemical synthesis methods or genetic engineering techniques, similar to the method for obtaining the polynucleotide encoding the SNARE protein described above. The polynucleotide encoding the allergen may be a polynucleotide encoding the full length of the allergen, or it may be a polynucleotide encoding a partial polypeptide of the allergen, insofar as it functions as an allergen. It may also be a polynucleotide obtained by linking multiple polynucleotides encoding one allergen in a manner that allows for expression, or a polynucleotide obtained by linking two or more polynucleotides encoding allergens in a manner that allows for expression. The allergen 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 allergen used in the nucleic acid construct of the present invention may be five types, four types, three types, two types, or one type.

[0102] Preferably, the nucleic acid construct of the present invention is a nucleic acid construct in which a polynucleotide encoding an allergen is expressably linked downstream of a polynucleotide encoding a SNARE protein; more preferably, the nucleic acid construct is in which a polynucleotide encoding an allergen 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 in which a polynucleotide encoding an allergen 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 approximately 1 to 5 times may be linked, or a polynucleotide encoding the proprotein convertase recognition sequence may be linked between multiple polynucleotides encoding the linker. In addition, any polynucleotide other than the polynucleotide encoding the linker and the polynucleotide encoding the proprotein convertase recognition sequence 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.

[0103] 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 the allergen 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: 96), GGGGS (SEQ ID NO: 97), EAAAK (SEQ ID NO: 98), 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.

[0104] 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.

[0105] 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.

[0106] The polynucleotides encoding SNARE proteins, allergens, 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 / ]).

[0107] 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 allergen. In the expression cassette, the polynucleotide encoding the SNARE protein and the polynucleotide encoding the allergen 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 allergen.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 allergens as a template, the 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).

[0113] As shown in the examples below, the nucleic acid construct of the present invention, which includes a polynucleotide encoding the SNARE protein and a polynucleotide encoding the allergen, exhibits higher immunogenicity and can induce or enhance allergen-specific Th1-type immune responses and allergen-specific cellular immune responses compared to a nucleic acid construct containing only the polynucleotide encoding the allergen (hereinafter referred to as the control). This is presumed to be because, in the antigen presentation process in which the nucleic acid construct taken up into cells 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 allergen protein as a fusion polypeptide of the SNARE protein and the allergen, thereby targeting the allergen protein to intracellular vesicles and increasing the probability of association with MHC molecules, 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 allergen proteins as fusion polypeptides of SNARE proteins and allergens leads to targeting of exosomes by allergen proteins, thereby improving immunogenicity. Furthermore, as shown in the examples below, the nucleic acid construct of the present invention can improve the decrease in body temperature associated with anaphylaxis and reduce the concentration of allergen-specific IgE antibodies in the blood compared to the control in an allergy model. This result indicates that the nucleic acid construct of the present invention has an excellent allergy-suppressing effect. Moreover, in conventional allergen immunotherapy, it is known that some allergens ingested from outside the body are released into the bloodstream and captured by allergen-specific IgE antibodies, causing adverse reactions classified as type I allergies such as anaphylaxis. However, as shown in the examples below, the nucleic acid construct of the present invention suppresses the release of allergens into the bloodstream after administration. This result indicates the potential of the nucleic acid construct of the present invention to reduce the risk of anaphylaxis.

[0114] 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.

[0115] Therefore, the nucleic acid constructs of the present invention can serve as allergen-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 allergen-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.

[0116] The cellular immune response inducers or enhancers of the present invention may themselves serve as pharmaceuticals or quasi-drugs for enhancing allergen-specific IFNγ production, enhancing IgG2a production, inducing or enhancing Th1-type immune responses, or for inducing or enhancing cellular immune responses. They may also be materials or formulations used in combination with such pharmaceuticals or quasi-drugs.

[0117] 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.

[0118] The content of the nucleic acid construct of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target allergen, 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.

[0119] 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.

[0120] 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 allergy sufferers or humans at risk of developing allergies.

[0121] 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 an allergen-specific Th1 immune response and / or to induce or enhance an allergen-specific cellular immune response. 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.

[0122] The nucleic acid vaccine of the present invention may itself be a pharmaceutical product for inducing or enhancing an allergen-specific Th1 immune response and / or for inducing or enhancing an allergen-specific cellular immune response, or it may be a material or formulation used in combination with such pharmaceutical product.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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 allergen, 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.

[0129] 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.

[0130] 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 allergy sufferers or humans at risk of developing allergies.

[0131] 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.

[0132] Nucleic acid vaccines can address a wide variety of allergens by changing the nucleic acid that codes for the allergen, and because they are nucleic acids, they can be manufactured quickly and at low cost.

[0133] In a 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 containing a polynucleotide encoding the SNARE protein of the present invention and a polynucleotide encoding an allergen. 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.

[0134] 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.

[0135] [1] A nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen. [2] The nucleic acid construct according to [1], wherein a polynucleotide encoding the allergen 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 allergen 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 SNARE protein is preferably a mammalian SNARE protein, and more preferably a human SNARE protein. [7] The nucleic acid construct according to any one of [1] to [6], 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 82 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 82. [8] The SNARE protein is preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, SEC22B, STX6, VAMP3, VAMP4, VTI1B, BET1, BET1L, and USE1, and more preferably selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B. A nucleic acid construct according to any one of items [1] to [7], which is any of the following, more 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, more preferably selected from the group consisting of VAMP7, GOSR2, STX10, and GOSR1, and more preferably VAMP7.

[0136] A pharmaceutical composition comprising a nucleic acid construct as described in any one of items [9], [1], to [8]. A nucleic acid vaccine containing a nucleic acid construct described in any one of items

[10] , [1], to [8] as an active ingredient.

[11] A nucleic acid vaccine as described in

[10] that induces or enhances an allergen-specific Th1 immune response.

[12] A nucleic acid vaccine as described in

[10] , which induces or enhances an allergen-specific cellular immune response.

[13] A nucleic acid vaccine described in any one of items

[10] to

[12] , which is a vaccine for the prevention or treatment of allergies.

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

[10] to

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

[0137]

[15] Use of any one of the nucleic acid constructs described in [1] to [8] for the manufacture of nucleic acid vaccines.

[16] The use described in

[15] , wherein the nucleic acid vaccine is a vaccine for the prevention or treatment of allergies.

[17] The use according to

[15] or

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

[0138]

[18] Use of any one of the nucleic acid constructs described in [1] to [8] for induction or enhancement of an allergen-specific Th1 immune response and / or induction or enhancement of an allergen-specific cellular immune response.

[19] A nucleic acid construct according to any one of items [1] to [8] for use in inducing or enhancing an allergen-specific Th1 immune response and / or inducing or enhancing an allergen-specific cellular immune response. A method for inducing or enhancing an allergen-specific Th1 immune response and / or an allergen-specific cellular immune response, comprising administering a nucleic acid construct described in any one of items

[20] [1] to [8] to a subject requiring it in an effective amount.

[0139]

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

[10] to

[14] for the induction or enhancement of an allergen-specific Th1 immune response and / or the induction or enhancement of an allergen-specific cellular immune response.

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

[10] to

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

[23] ,

[10] to

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

[24]

[13] to a person in need of it in an effective dose.

[25] 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 according to

[23] or

[24] , wherein the particles are virus particles.

[0140] An allergen-specific IFNγ production enhancer comprising a nucleic acid construct described in any one of items

[26] [1] to [8] as an active ingredient. An allergen-specific IgG2a production enhancer comprising a nucleic acid construct described in any one of items

[27] [1] to [8] as an active ingredient. An allergen-specific Th1-type immune response inducer or enhancer comprising a nucleic acid construct described in any one of items

[28] [1] to [8] as an active ingredient. An allergen-specific cellular immune response inducer or enhancer comprising a nucleic acid construct described in any one of items

[29] [1] to [8] as an active ingredient.

[30] The agent according to any one of

[26] to

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

[0141]

[31] Use of any one of the nucleic acid constructs described in [1] to [8] for the production of an allergen-specific IFNγ production enhancer.

[32] Use of any one of the nucleic acid constructs described in [1] to [8] for the production of an allergen-specific IgG2a production enhancer.

[33] Use of any one of the nucleic acid constructs described in [1] to [8] for the production of an allergen-specific Th1 immune response inducer or enhancer.

[34] Use of any one of the nucleic acid constructs described in [1] to [8] for the production of an allergen-specific cellular immune response inducer or enhancer.

[35] The use according to any one of

[31] to

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

[0142]

[36] Use of any one of the nucleic acid constructs described in [1] to [8] for the enhancement of allergen-specific IFNγ production.

[37] Use of any one of the nucleic acid constructs described in [1] to [8] for the enhancement of allergen-specific IgG2a production.

[38] Use of any one of the nucleic acid constructs described in [1] to [8] for the induction or enhancement of an allergen-specific Th1 immune response.

[39] Use of any one of the nucleic acid constructs described in [1] to [8] for the induction or enhancement of an allergen-specific cellular immune response.

[0143]

[40] A nucleic acid construct according to any one of items [1] to [8] for use in enhancing allergen-specific IFNγ production.

[41] A nucleic acid construct according to any one of items [1] to [8] for use in enhancing allergen-specific IgG2a production.

[42] A nucleic acid construct according to any one of items [1] to [8] for use in inducing or enhancing an allergen-specific Th1 immune response.

[43] A nucleic acid construct according to any one of items [1] to [8] for use in inducing or enhancing an allergen-specific cellular immune response.

[0144] A method for enhancing allergen-specific IFNγ production, comprising administering an effective amount of a nucleic acid construct described in any one of items

[44] [1] to [8] to a target that requires it. A method for enhancing allergen-specific IgG2a production, comprising administering an effective amount of a nucleic acid construct described in any one of items

[45] [1] to [8] to a subject requiring it. A method for inducing or enhancing an allergen-specific Th1 immune response, comprising administering a nucleic acid construct described in any one of items

[46] [1] to [8] to a subject requiring it in an effective amount. A method for inducing or enhancing an allergen-specific cellular immune response, comprising administering a nucleic acid construct described in any one of items

[47] [1] to [8] to a subject requiring it in an effective amount.

[48] ​​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

[44] to

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

[0145] In

[49] ,

[10] to

[17] , and

[21] to

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

[50] ,

[20] ,

[23] -

[25] , and

[44] -

[48] , the subjects are allergy patients or people at risk of allergies. [Examples]

[0146] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0147] Example 1: Investigation of SNARE proteins mRNA encoding the amino acid sequence (SEQ ID NOs: 25 to 45) of a fusion polypeptide, in which ovalbumin (OVA, amino acid sequence: SEQ ID NO: 24), which is an allergen, is linked via a linker (nucleotide sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20) to the C-terminal side of each of 21 types of human SNARE proteins, or mRNA encoding the amino acid sequence of OVA alone was prepared. At this time, the nucleotide sequence encoding the antigen or the fusion polypeptide was subjected to codon optimization and then inserted into a plasmid DNA (pVAX1 vector, Thermo Fisher Scientific). The nucleotide sequence of OVA inserted into plasmid DNA is shown in SEQ ID NO: 95, and the nucleotide sequences of the fusion polypeptides are shown in SEQ ID NOs: 48 to 68. Also, the nucleotide sequence of OVA before codon optimization is shown 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 incorporating each sequence using a restriction enzyme, in vitro mRNA synthesis was performed by T7 RNA polymerase using the DNA as a template, and 5'-terminal capping was performed by ARCA. Subsequently, after degrading the template DNA by adding DNase, a poly(A) chain was added by Poly(A) Polymerase. The obtained mRNA was purified using an RNeasy kit (QIAGEN) and used in the following procedures. Using TransIT-mRNA Transfection Reagent (Mirus Bio), the purified mRNA was introduced into mature human CD14 + dendritic cells (Lonza) according to the attached protocol. As a control, only transfection reagent containing no mRNA was added to dendritic cells. From the day after transfection, human CD4 derived from the same donor + T cells (Lonza), 1.0 × 10 6 cells, and 2.5 × 10 5 transfected dendritic cells were co-cultured for a total of 7 days to achieve antigen presentation of mRNA-derived OVA. Thereafter, the co-cultured CD4 + T cells, 1.0 × 106 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.

[0148] 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.

[0149] 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 allergen (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.

[0150] The results are shown in Figure 2. Compared with OVA alone, VAMP7-OVA (V7-OVA) showed a significant decrease in OVA-specific IgG1 antibody titer. On the other hand, for OVA-specific IgG2a, only VAMP7-OVA showed a significant increase in antibody titer compared with the control group (Empty). No significant difference in OVA-specific total IgG antibody titer was observed between OVA alone and VAMP7-OVA. It is generally known that a Th1-type immune response induces IgG2a antibody production from B cells, while a Th2-type immune response induces IgG1 antibody production from B cells. Therefore, the Th1 / Th2 balance of the immune response induced in vivo can be estimated by measuring the antibody titers of allergen-specific IgG1 and IgG2a antibodies. The present results showed that compared with OVA alone, VAMP7-OVA decreased OVA-specific IgG1 antibody titer and increased IgG2a antibody titer, demonstrating that VAMP7-OVA can induce or enhance a Th1-type immune response even in vivo. This experiment was performed with n=10 for each group, and a significant difference was determined when p<0.05 was obtained after Tukey's test for each group.

[0151] Example 3 ELISPOT assay A plasmid vector (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequence of the fusion polypeptide (V7-OVA) formed by linking OVA to the C-terminal side of VAMP7 via a linker, or only the amino acid sequence of OVA, was prepared. An empty vector not encoding the allergen (OVA) was used as a control. After 50 µg of each prepared vector was intramuscularly administered into the femoral muscle of female BALB / c mice, a voltage was applied using an electroporator (NEPA21) to perform gene transfer. After the above treatment was performed on days 0, 7, and 14 from the start of the experiment, the mice were euthanized on day 35, and the spleens were collected. The collected spleens were ground on a 40 µm cell strainer while adding DPBS containing 2% FBS. The obtained suspension was centrifuged at 200×g for 5 minutes, the supernatant was removed, and then Pharm Lyse TMThe red blood cells in the suspension were hemolyzed by adding solution (BD Biosciences) and letting it stand for 3 minutes. The suspension was then centrifuged again at 200 x g for 5 minutes, the supernatant was removed, and the suspension was washed twice with DPBS. 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 mixed with 100 μL of CTL-Test Medium containing OVA protein (Merck) to stimulate the cells with an antigen at a final concentration of 50 μg / mL. A negative control group (NT) without OVA protein 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 allergen (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 allergen-specific immune response.

[0152] 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 allergen does not induce an allergen-specific Th2 immune response, but rather selectively induces or enhances an allergen-specific Th1 immune response and cellular immunity. In this experiment, n=10 was conducted in each group, and a p<0.05 result in the Tukey test performed in each group under either unstimulated conditions (NT) or OVA-stimulated conditions (OVA) was considered statistically significant.

[0153] Example 4 IgG, IgG1, IgG2a antibody titer A plasmid vector (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequences (SEQ ID NOs: 25, 27, 28, 41) of a fusion polypeptide (V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA) obtained by linking OVA via a linker to the C-terminal side of VAMP7, STX10, STX18, or GOSR1, or only the amino acid sequence of OVA (SEQ ID NO: 24) was prepared. An empty vector that does not encode the allergen (OVA) was used as a control. After 50 µg of each prepared vector was intramuscularly administered into the femoral muscle of female BALB / c mice, gene transfer was performed by applying voltage using an electroporator (NEPA21). The above treatment was performed on days 0, 7, and 14 from the start of the experiment, after which the mice were euthanized on day 35 and blood was collected. Serum was separated from the collected blood, and antibody titer measurement was performed. For antibody titer measurement, a DPBS solution in which 10 µg / mL OVA (Merck) was dissolved was added at 100 µL / well to a 96-well ELISA plate (IWAKI) to immobilize OVA on the solid phase. On the next day, after washing the plate, blocking was performed with 100 µL / well of DPBS containing 1% BSA for 1 hour. Subsequently, after washing the plate, the separated serum was added at 100 µL / well while being serially 2-fold diluted. After 2 hours, the serum was washed away, 1 µg / mL of anti-Mouse IgG, IgG1, or IgG2a antibody (abcam) was added at 100 µL / well, and the plate was allowed to stand for 1 hour. Thereafter, a TMB solution (abcam) was added at 50 µL / well to perform a color development reaction. After 10 minutes, a Stop solution (abcam) was added at 50 µL / well to stop the color development reaction, and absorbance was measured at 450 nm. At this time, the value of the maximum dilution factor at which an absorbance value 2 times or more that of a control supplemented with DPBS instead of serum was measured was defined as the antibody titer of each sample.

[0154] 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 allergens, they can induce or enhance allergen-specific Th1 immune responses 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.

[0155] 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 via a linker, or only the amino acid sequence of OVA (SEQ ID NO. 24), were constructed. An empty vector not encoding the allergen (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 mixed with 100 μL of CTL-Test Medium containing OVA protein (Merck) to stimulate antigen stimulation at a final concentration of 50 μg / mL. A negative control group (NT) without OVA protein 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 allergen (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 allergen-specific immune response.

[0156] 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 in which 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, in addition to VAMP7, other SNARE family members such as STX10, STX18, and GOSR1 can also be linked to allergens without inducing an allergen-specific Th2 immune response, and can selectively induce or enhance an allergen-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 comparing with the Empty group and † when comparing with the OVA group.

[0157] 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 allergen (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.

[0158] 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 allergen-specific Th1 immune response in vivo. Furthermore, compared to LAMP[OVA], VAMP7-OVA and VAMP7-pc-OVA demonstrate superior allergen-specific IgG antibody production and enhanced allergen-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.

[0159] 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 allergen (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 allergen (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 allergen-specific immune response.

[0160] 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 allergen-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. Since 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.

[0161] Example 8: 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 allergen (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 100 μL / well on an ELISPOT plate and mixed with 100 μL of CTL-Test Medium containing OVA protein (Merck) to stimulate antigen stimulation at a final concentration of 50 μg / mL. A negative control group (NT) without OVA protein 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 allergen (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 allergen-specific immune response.

[0162] The results are shown in Figure 8. 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 allergen-specific Th2 immune response, but selectively induces allergen-specific Th1 immunity and cellular immunity, and furthermore, demonstrates 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 performed in each group under unstimulated conditions (NT) or under OVA stimulation conditions was considered statistically significant.

[0163] Example 9: Efficacy of the OVA food allergy model A sensitizing mixture was prepared by mixing 100 μg OVA and 1 mg Alum adjuvant (Thermo Fisher Scientific) in 200 μL DPBS and inverting the mixture for 30 minutes. The prepared sensitizing mixture was administered intraperitoneally to female BALB / c mice on days 0 and 14 to induce sensitization. Plasmid vectors were also prepared encoding only the amino acid sequence of OVA (SEQ ID NO: 24), the amino acid sequence of a fusion polypeptide (V7-pc-OVA) in which OVA was linked to the C-terminus of VAMP7 with a linker and a proprotein convertase recognition sequence (SEQ ID NO: 46), or the amino acid sequence of a fusion polypeptide (LAMP[OVA]) in which the amino acid sequence of OVA was inserted into LAMP (SEQ ID NO: 47). An empty vector (Empty) that did not encode the allergen (OVA) was prepared as a control. On days 21, 28, and 35 of the experiment, 50 μg of each prepared vector was intramuscularly administered into the thigh muscle of BALB / c mice, and then gene transfer was performed by applying voltage using an electroporator (NEPA21). On days 42, 43, 44, 45, and 46 of the experiment, a challenge mixture of 50 mg of OVA dissolved in 200 μL of DPBS was orally administered once a day to induce food allergy symptoms. On day 46 of the experiment, rectal temperature was measured using a thermometer probe (Natsume Seisakusho Co., Ltd.) before administration of the challenge mixture and 15, 30, 45, and 60 minutes after administration. Subsequently, the mice were euthanized and blood was collected. Serum was separated from the collected blood, and OVA-specific IgE concentration was measured. The LBIS Mouse anti-OVA-IgE ELISA Kit (Fujifilm Wako Shibayagi Co., Ltd.) was used for OVA-specific IgE concentration measurement. 50 μL of biotin-conjugated anti-IgE antibody and 10 μL of diluted serum or standard solution were added to a 96-well plate immobilized with OVA protein, and the mixture was allowed to stand at room temperature for 1 hour. Subsequently, the plate was washed three times with washing solution, and then 100 μL of peroxidase-avidin conjugate was added, and the mixture was allowed to stand at room temperature for 30 minutes. Subsequently, the plate was washed three more times, and then 100 μL of TMB chromogenic solution was added, and the color reaction was carried out at room temperature.After 20 minutes, 100 μL of 1 M H2SO4 was added to stop the color reaction, and absorbance measurements were performed (primary wavelength 450 nm, secondary wavelength 620 nm). The OVA-specific IgE concentration in each serum sample was calculated using the calibration curve obtained from the absorbance values ​​of the standard solution.

[0164] The results are shown in Figure 9. Compared to the OVA-unsensitized Empty group (Empty(-)), the OVA-sensitized Empty group (Empty(+)), the OVA-alone group (OVA(+)), and the LAMP[OVA] group (LAMP[OVA](+)) showed a significantly greater decrease in body temperature associated with anaphylaxis after oral OVA sensitization. On the other hand, the OVA-sensitized VAMP7-pc-OVA group (V7-pc-OVA(+)) did not show a decrease in body temperature compared to Empty(-), and the decrease in body temperature was significantly suppressed compared to Empty(+), OVA(+), and LAMP[OVA](+). Furthermore, OVA-specific IgE antibody concentrations were significantly higher in the OVA(+) group compared to Empty(-), while OVA-specific IgE antibody concentrations were significantly lower in the V7-pc-OVA(+) and LAMP[OVA] groups compared to OVA(+). Considering that hypothermia associated with anaphylaxis is a common phenotype in animal models of allergies, and that allergen-specific IgE concentration in the blood is a common marker indicating the severity of allergies, these results indicate that VAMP7-pc-OVA has a superior allergy-suppressing effect compared to OVA alone or conventional techniques. Rectal temperature measurements were performed with n=8 in each group, and a statistically significant difference was considered to exist if p<0.05 was determined in Dunnett's test for each group at each measurement time. Significant differences are indicated by * when comparing with the Empty(-) group and † when comparing with the V7-pc-OVA(+) group. OVA-specific IgE concentration measurements were performed with n=7 only in the Empty(+) group, and n=8 in all other groups, and a statistically significant difference was considered to exist if p<0.05 was determined in Tukey's test for each group. Mean values ​​are shown on the dot plots for each group in the V7-pc-OVA and LAMP[OVA] groups.

[0165] Example 10: OVA blood concentration Plasmid vectors were constructed encoding either the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide (V7-OVA) linked with a linker, the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide (V7-pc-OVA) with a proprotein convertase recognition sequence added to the linker portion of the fusion polypeptide, or only the amino acid sequence of OVA (SEQ ID NO: 24). An empty vector (Empty) not encoding the allergen (OVA) was prepared as a control. 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). Seven days after the above procedure, the mice were euthanized and blood was collected. Serum was separated from the collected blood, and the OVA concentration was measured. The ITEA ovalbumin (OVA) ELISA kit (ITEA Corporation) was used for OVA concentration measurement. 100 μL / well of serum or standard solution was added to a 96-well plate immobilized with anti-OVA antibody, and the mixture was allowed to stand at room temperature for 1 hour. Next, the samples were washed three times with washing solution, and then 100 μL / well of enzyme-labeled anti-OVA antibody was added. The mixture was allowed to stand at room temperature for 1 hour. After three more washes, 100 μL / well of TMB chromogenic substrate solution was added, and the color reaction was carried out at room temperature. After 15 minutes, 100 μL / well of stop solution was added to stop the color reaction, and absorbance measurements were performed (primary wavelength 450 nm, secondary wavelength 620 nm). The OVA concentration in each serum sample was calculated using a calibration curve obtained from the absorbance values ​​of the standard solution.

[0166] The results are shown in Figure 10. Compared to the group administered the Empty vector (Empty), it was confirmed that OVA protein was detectable in the blood one week after vector administration when OVA was administered alone. On the other hand, compared to OVA alone, the blood concentration of OVA protein in VAMP7-OVA (V7-OVA) and VAMP7-pc-OVA (V7-pc-OVA) was reduced to almost the same level as in the Empty group. This result means that when OVA is administered alone in vivo, some of the OVA protein expressed in the body is released into the bloodstream. On the other hand, since the blood concentration of OVA protein in V7-OVA and V7-pc-OVA was reduced to about the same level as in the Empty group, where OVA protein was not expressed at all, it is thought that there is almost no OVA protein in the blood at the one-week mark. In conventional allergen immunotherapy, it is known that allergens ingested from outside the body are partially released into the bloodstream and captured by allergen-specific IgE antibodies, causing adverse reactions classified as type I allergies such as anaphylaxis. As these results suggest, linking VAMP7, or VAMP7 to a proprotein convertase recognition sequence, rather than the allergen alone, may suppress allergen release into the bloodstream and potentially reduce the adverse reactions classified as type I allergies associated with the aforementioned treatment. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Tukey test was considered statistically significant.

Claims

1. A nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen, wherein the SNARE protein is selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, and the allergen is a protein or peptide derived therefrom that elicits a hypersensitivity or allergic reaction.

2. The nucleic acid construct according to claim 1, wherein a polynucleotide encoding the allergen is linked downstream of a polynucleotide encoding the SNARE protein.

3. The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the SNARE protein and the polynucleotide encoding the allergen are linked via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence.

4. The nucleic acid construct according to claim 1, which is a plasmid vector, mRNA, or viral vector.

5. An allergen-specific Th1 immune response inducer or enhancer comprising a nucleic acid construct according to any one of claims 1 to 4 as an active ingredient.

6. An allergen-specific cellular immune response inducer or enhancer comprising a nucleic acid construct according to any one of claims 1 to 4 as an active ingredient.

7. A nucleic acid vaccine comprising a nucleic acid construct according to any one of claims 1 to 4 as an active ingredient.

8. The nucleic acid vaccine according to claim 7, which induces or enhances an allergen-specific Th1 type immune response.

9. The nucleic acid vaccine according to claim 7, which induces or enhances an allergen-specific cellular immune response.

10. The nucleic acid vaccine according to claim 7, which is a vaccine for the prevention or treatment of allergies.

11. The nucleic acid vaccine according to claim 7, which is administered parenterally.

Images