Nucleic acid construct 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 disease by administering antigens and establishing acquired immunity against them. Traditionally, vaccines have been administered primarily via subcutaneous or intramuscular injection. These include live vaccines, which use attenuated bacteria or viruses as vaccines; inactivated vaccines, in which bacteria or viruses are treated with formalin or heat to render them infective; and toxoid vaccines, in which bacterial toxins are separated and purified and then inactivated by treatment with formalin. In addition to these, development is underway to improve efficacy, safety, and convenience, including mucosal vaccines, which are administered via the mucosa, and nucleic acid vaccines, which use nucleic acids as antigenic components. Among these, nucleic acid vaccines are seeing rapid development as a new modality that can be produced quickly and at low cost.

[0003] In improving the effectiveness of nucleic acid vaccines, improving the immunogenicity of antigens is one of the key challenges, and it is necessary to target antigens to appropriate tissues and antigen-presenting cells and efficiently induce immune responses. Patent Document 1 discloses that immune responses to antigens are enhanced by using nucleic acids encoding fusion proteins in which an allergen protein (antigen) is inserted between the intraorganelle stabilizing domain and transmembrane domain of a lysosomal associated membrane protein (LAMP). On the other hand, it has been reported that adding an antigen to the LAMP lumen can significantly attenuate antigen-specific antibody production (Non-Patent Document 1). Patent Document 2 also discloses that the immunogenicity of an antigen is increased by using nucleic acids encoding fusion molecules containing an antigen, a transmembrane domain, and the cytoplasmic domain of a major histocompatibility complex (MHC) molecular chain. Patent Document 2 also mentions a fusion molecule containing an antigen and the cytoplasmic region of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, but does not specifically mention an allergen as the antigen, nor does it disclose how the fusion molecule alters the immunogenicity of the antigen. Because allergies are primarily pathologically caused by the excessive induction of an allergen-specific Th2 immune response, applying a nucleic acid vaccine to treat allergies is desirable to selectively induce a Th1 immune response that can suppress the Th2 immune response, rather than inducing a targeted allergen-specific Th2 immune response. However, the effect of using a nucleic acid encoding such a fusion molecule that has a transmembrane domain and contains an allergen as the antigen on the allergen-specific Th2 immune response remains unknown. Therefore, the effect of a nucleic acid encoding such an allergen-based fusion molecule on the balance between Th1 and Th2, which are involved in the development and suppression of allergic symptoms, remains unknown.In addition, 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 has been established to prevent or improve allergies using nucleic acid constructs.

[0004] SNARE proteins are a family of proteins containing a 20-30 kDa SNARE motif. Many SNARE proteins are anchored to lipid bilayers via their C-terminal transmembrane domains and are involved in the process of vesicle fusion with target intracellular organelles (Non-Patent Documents 2 and 3). SNARE-mediated membrane fusion is essential for many important biological phenomena in eukaryotic cells, including vesicle transport, organelle membrane morphogenesis, endocytosis (including extracellular receptor recycling), and exocytosis (including hormone secretion and synaptic neurotransmitter release). Furthermore, the molecular mechanism of membrane fusion by SNARE proteins is thought to be conserved in all eukaryotes, from unicellular budding yeast to higher animals, including humans. SNARE proteins are further classified into four subfamilies: QA-SNARE, QB-SNARE, QC-SNARE, and R-SNARE, based on the characteristics of the amino acid residues in the SNARE motif. Each SNARE protein is localized to a specific intracellular membrane compartment (such as the endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, lysosomes, secretory vesicles, or the plasma membrane) and is thought to function in the membrane fusion process of specific intracellular transport pathways. SNARE proteins are also known to potentially be contained in exosomes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open 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. Summary of the Invention [Problem to be solved by the invention]

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

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

[0009] Therefore, the present invention provides the following 1) to 4). 1) A nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen. 2) An agent for inducing or enhancing an allergen-specific Th1-type immune response, which comprises the nucleic acid construct of 1) as an active ingredient. 3) An agent for inducing or enhancing an allergen-specific cellular immune response, which comprises the nucleic acid construct of 1) as an active ingredient. 4) A nucleic acid vaccine containing the nucleic acid construct of 1) as an active ingredient. [Effects of the Invention]

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

[0011] [Figure 1] Evaluation of the immunogenicity of mRNA encoding a SNARE protein-allergen fusion polypeptide. IFNγ production is shown as the number of producing cells (spot-forming cells; SFC). Empty refers to a control containing only the transfection reagent; Empty (NT) refers to a control without restimulation with allergen; SNARE protein-OVA refers to mRNA encoding a SNARE protein-allergen (OVA) fusion polypeptide. [Figure 2] IgG subclass antibody titers after mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide. (A) shows the IgG antibody titer, (B) shows the IgG1 antibody titer, and (C) shows the IgG2a antibody titer. Empty indicates an empty plasmid vector that does not encode an allergen (OVA), OVA indicates a plasmid vector encoding OVA, and V7-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide. [Figure 3] Evaluation of immune responses when mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, measured by SFC. (C) shows the ratio of IFNγ production to IL-4 production (IFNγ / IL-4). Empty indicates an empty plasmid vector that does not encode an allergen (OVA), OVA indicates a plasmid vector encoding OVA, and V7-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide. [Figure 4] IgG subclass antibody titers after mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide. (A) shows the IgG antibody titer, (B) the IgG1 antibody titer, (C) the IgG2a antibody titer, and (D) the ratio of the IgG2a antibody titer to the IgG1 antibody titer (IgG2a / IgG1). Empty indicates an empty plasmid vector that does not encode an allergen (OVA), OVA indicates a plasmid vector encoding OVA, and V7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA indicate plasmid vectors encoding the VAMP7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA fusion polypeptides, respectively. [Figure 5] Evaluation of immune responses when mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, as measured by SFC. (C) shows the ratio of IL-4 production under allergen-stimulated conditions to unstimulated conditions (IL-4 ratio). Empty indicates an empty plasmid vector that does not encode an allergen (OVA). OVA indicates a plasmid vector encoding OVA. V7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA indicate plasmid vectors encoding VAMP7-OVA, STX10-OVA, STX18-OVA, and GOSR1-OVA fusion polypeptides, respectively. [Figure 6]Antibody titers over time after mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide or a fusion polypeptide containing an allergen within a LAMP. (A) shows the IgG antibody titer, (B) the IgG1 antibody titer, (C) the IgG2a antibody titer, and (D) the ratio of the IgG2a antibody titer to the IgG1 antibody titer (IgG2a / IgG1) at week 5. Empty indicates an empty plasmid vector not encoding an allergen (OVA), OVA indicates a plasmid vector encoding OVA, V7-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide, V7-pc-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence, and LAMP[OVA] indicates a plasmid vector encoding a fusion polypeptide containing OVA within a LAMP. [Figure 7] Evaluation of immune responses when mice were administered a plasmid vector encoding a SNARE protein-allergen fusion polypeptide. (A) shows the amount of IFNγ produced, and (B) shows the amount of IL-4 produced, 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 an allergen (OVA), OVA indicates a plasmid vector encoding OVA, V7-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide, and V7-pc-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence. [Figure 8]Evaluation of immune responses after administration of a plasmid vector encoding a SNARE protein-allergen fusion polypeptide or a fusion polypeptide containing an allergen within a LAMP. (A) shows the amount of IFNγ production, and (B) shows the amount of IL-4 production, measured by SFC. (C) shows the ratio of IL-4 production under allergen-stimulated conditions to that under unstimulated conditions (IL-4 ratio). (D) shows the ratio of IFNγ production to IL-4 production (IFNγ / IL-4). "Empty" indicates an empty plasmid vector that does not encode an allergen (OVA). "OVA" indicates a plasmid vector encoding OVA. "V7-OVA" indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide. "V7-pc-OVA" indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide containing a proprotein convertase recognition sequence. "LAMP[OVA]" indicates a plasmid vector encoding a fusion polypeptide containing OVA within a LAMP. [Figure 9] Allergic reactions were evaluated when a plasmid vector encoding a SNARE protein-allergen fusion polypeptide or a fusion polypeptide containing an allergen within LAMP was administered to allergy model mice. (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 an allergen (OVA), OVA indicates a plasmid vector encoding OVA, V7-pc-OVA indicates a plasmid vector encoding a VAMP7-proprotein convertase recognition sequence-OVA fusion polypeptide, and LAMP[OVA] indicates a plasmid vector encoding a fusion polypeptide containing OVA within LAMP. (-) indicates no sensitization, and (+) indicates sensitization. [Figure 10]Evaluation of OVA blood concentrations after administration of a plasmid vector encoding a SNARE protein-allergen fusion polypeptide to mice. Empty indicates an empty plasmid vector that does not encode an allergen (OVA), OVA indicates a plasmid vector encoding OVA, V7-OVA indicates a plasmid vector encoding a VAMP7-OVA fusion polypeptide, and V7-pc-OVA indicates a plasmid vector encoding a VAMP7-proprotein convertase recognition sequence-OVA fusion polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the terms "nucleic acid," "nucleotide," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to 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. For the purposes of initiating translation reactions in vivo, stabilizing mRNA, and preventing degradation, mRNA is synthesized by an in vitro transcription reaction, followed by the addition of a 5' Cap (methylated guanosine) by a capping enzyme and poly(A) by a poly(A) polymerase. 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 poly(A) added, as well as RNA with some base modifications (e.g., uridine replaced with pseudouridine or 1-methylpseudouridine).

[0013] As used herein, the term "gene" encompasses double-stranded DNA including genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, and refers to DNA in which some biological information is contained in the sequence information of the bases that make up the DNA. Furthermore, the "gene" in question includes not only "genes" represented by a specific nucleotide sequence, but also nucleic acids encoding their homologues (i.e., homologs or orthologs), variants such as genetic polymorphisms, and derivatives. The names and Gene IDs of the genes disclosed herein are in accordance with the Official Symbols and Gene IDs listed in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0014] As used herein, the terms "peptide," "polypeptide," or "protein" are used interchangeably.

[0015] As used herein, the term "amino acid residue" refers to 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] Herein, the identity of a nucleotide sequence or an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win, with the unit size to compare (ktup) set to 2.

[0017] As used herein, "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, still more preferably 98% or more, and even more preferably 99% or more.

[0018] Unless otherwise defined herein, "one or several" when used with respect to deletion, substitution, addition, or insertion of nucleotides in a nucleotide sequence preferably means 1 to 15, more preferably 1 to 9, and even more preferably 1 to 6. Furthermore, unless otherwise defined herein, "one or several" when used with respect to deletion, substitution, addition, or insertion of amino acid residues in an amino acid sequence preferably means 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. As used herein, "addition" of a nucleotide or amino acid residue includes addition of a nucleotide or amino acid residue to one or both ends of a sequence.

[0019] As used herein, "stringent conditions" refers 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), such as conditions in which a solution containing 6x SSC (1x SSC has the composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's, and 100 mg / mL herring sperm DNA is incubated with a probe at 42°C for 8 to 16 hours for hybridization.

[0020] As used herein, a "fragment" of a polynucleotide refers to a partial polynucleotide of the polynucleotide. The length of the 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 refer to a polynucleotide consisting of consecutive nucleotides that is 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. Furthermore, as used herein, a "fragment" of a polypeptide refers to a partial polypeptide of the polypeptide. The length of the partial polypeptide is not particularly limited. For example, a partial polypeptide may refer to a polypeptide consisting of consecutive amino acid residues that is 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] As used herein, a "control region" refers to a region that functions to control the expression of a gene (e.g., a region encoding a protein) located downstream of it. More specifically, a "control region" can be defined as a region that is located upstream of the coding region of a gene and that functions to control the transcription of the coding region by interacting with RNA polymerase. The control region includes a transcription initiation control region and / or a translation initiation control region, or the region from the transcription initiation control region to the translation initiation control region. The transcription initiation control region is a region that includes a promoter and a transcription start point, and the translation initiation control region is a site that corresponds to a Kozak sequence that is recognized by ribosomes together with an initiation codon and is necessary for translation initiation.

[0022] As used herein, the term "operably linked" between a regulatory region and a polynucleotide of a gene (e.g., a polynucleotide encoding a protein) means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene and a regulatory region are well known to those skilled in the art.

[0023] As used herein, "expressibly linked" between a polynucleotide of a first gene (e.g., a polynucleotide encoding a protein) and a polynucleotide of a second gene (e.g., a polynucleotide encoding a protein) means that when the first gene and the second gene are inserted into an appropriate expression vector and the expression vector is introduced into an appropriate cell, the protein encoded by the first gene and the protein encoded by the second gene are produced as a fusion protein. Here, "linkage" is a concept that includes both cases where the first gene and the second gene are directly linked and cases where they are linked via another nucleotide sequence. Procedures for "expressibly linked" between a first gene and a second gene are well known to those skilled in the art.

[0024] As used herein, "upstream" and "downstream" with respect to a gene or its nucleotide sequence refer to the upstream and downstream of the transcription direction of the gene. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 5' and 3' sides of the gene on the DNA sense strand, respectively.

[0025] As used herein, the term "antigen" refers to a molecule that induces immune responses such as antibody production and cellular immunity in the body, specifically an allergen. Furthermore, as used herein, the term "immunogenicity" refers to the property of an antigen to induce antibody production and cellular immunity.

[0026] As used herein, the term "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 believed to induce both humoral and cellular immunity. DNA vaccines contain a plasmid encoding an antigen. The plasmid administered to a living organism is taken up by cells and transcribed into mRNA in the nucleus, and the mRNA is translated into antigen protein in the cytoplasm, thereby inducing an antigen-specific immune response. mRNA vaccines contain mRNA encoding an antigen. The mRNA administered to a living organism is taken up by cells and translated into antigen protein in the cytoplasm, thereby inducing an antigen-specific immune response. Viral vector vaccines include apathogenic or attenuated viral vectors incorporating a polynucleotide encoding an antigen. The virus administered to a living organism invades cells and induces the cells to synthesize antigen protein, thereby inducing an antigen-specific immune response.

[0027] As used herein, "cellular immunity" refers to an adaptive immune response that uses cytotoxic T cells, macrophages, NK cells, etc. as effectors, among adaptive immune responses that work to eliminate foreign substances such as pathogens that have invaded the body, virus-infected cells, and cancer cells. Also, as used herein, "humoral immunity" refers to an adaptive immune response that uses antibodies as effectors.

[0028] As used herein, the term "Th1-type immune response" refers to an immune response promoted by Th1 cells, a subset of helper T cells. Th1 cells primarily produce IFNγ as a cytokine, and primarily target M1 macrophages, cytotoxic T cells, and NK cells, thereby inducing cellular immunity. Th1 cells are known to activate B cells and induce IgG2a production. Furthermore, as used herein, the term "Th2-type immune response" refers to an 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 primarily target mast cells, M2 macrophages, eosinophils, and basophils, thereby 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 a 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 in the SNARE motif. 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 in intracellular vesicles. Specific 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. From the viewpoint of immune induction or enhancement, the SNARE protein is preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B, more preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, and GOSR1, even more preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, and GOSR1, even more preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, and GOSR1, with VAMP7 being even more preferred.

[0031] VAMP7 (vesicle-associated membrane protein 7) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in late endosomes, lysosomes, and the plasma membrane. In a preferred example, VAMP7 is mammalian VAMP7. In a more preferred example, VAMP7 is human VAMP7, 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. VAMP7 used in the present invention encompasses VAMP7 and polypeptides having equivalent functions thereto.

[0032] GOSR2 (golgi SNAP receptor complex member 2) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the endoplasmic reticulum-Golgi intermediate section 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. GOSR2 used in the present invention encompasses GOSR2 and polypeptides having equivalent functions thereto.

[0033] STX (syntaxin) 10 is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the trans-Golgi network. In a preferred example, STX10 is mammalian STX10. In a more preferred example, STX10 is human STX10, 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. STX10 used in the present invention includes STX10 and polypeptides having equivalent functions thereto.

[0034] STX18 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in the endoplasmic reticulum. In a preferred example, STX18 is mammalian STX18. In a more preferred example, STX18 is human STX18, 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. STX18 used in the present invention includes STX18 and polypeptides having equivalent functions thereto.

[0035] BNIP1 (BCL2 interacting protein 1) is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in the endoplasmic reticulum. In a preferred example, BNIP1 is mammalian BNIP1. In a more preferred example, BNIP1 is human BNIP1, 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. BNIP1 used in the present invention includes BNIP1 and polypeptides having equivalent functions thereto.

[0036] STX7 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in 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. STX7 used in the present invention includes STX7 and polypeptides having equivalent functions thereto.

[0037] VTI1A (vesicle transport through interaction with t-SNAREs 1A) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the trans-Golgi network. In a preferred example, VTI1A is mammalian VTI1A. In a more preferred example, VTI1A is human VTI1A, 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. VTI1A used in the present invention encompasses VTI1A and polypeptides having equivalent functions thereto.

[0038] STX16 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in 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. STX16 used in the present invention includes STX16 and polypeptides having equivalent functions thereto.

[0039] STX5 is a type of SNARE protein, has a transmembrane domain, and is predicted 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. STX5 used in the present invention includes STX5 and polypeptides having equivalent functions thereto.

[0040] GOSR1 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in the Golgi apparatus and 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. GOSR1 used in the present invention includes GOSR1 and polypeptides having equivalent functions thereto.

[0041] STX8 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in early endosomes, late endosomes, and the plasma 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. STX8 used in the present invention includes STX8 and polypeptides having equivalent functions thereto.

[0042] STX12 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in 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, 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. STX12 used in the present invention includes STX12 and polypeptides having equivalent functions thereto.

[0043] VAMP8 is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in lysosomes, early endosomes, late endosomes, and the plasma membrane. In a preferred example, VAMP8 is mammalian VAMP8. In a more preferred example, VAMP8 is human VAMP8, 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. VAMP8 used in the present invention includes VAMP8 and polypeptides having equivalent functions thereto.

[0044] SEC22B (SEC22 homolog B, vesicle trafficking protein) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate zone, 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 a gene (Gene ID: 9554) consisting of the nucleotide sequence of SEQ ID NO: 75. SEC22B used in the present invention encompasses SEC22B and polypeptides having equivalent functions thereto.

[0045] STX6 is a type of SNARE protein, has a transmembrane domain, and is predicted 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. STX6 used in the present invention includes STX6 and polypeptides having equivalent functions thereto.

[0046] VAMP3 (vesicle-associated membrane protein 3) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the cell membrane and Golgi apparatus. In a preferred example, VAMP3 is mammalian VAMP3. In a more preferred example, VAMP3 is human VAMP3, 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. VAMP3 used in the present invention encompasses VAMP3 and polypeptides having equivalent functions thereto.

[0047] VAMP4 (vesicle-associated membrane protein 4) is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in the Golgi apparatus and trans-Golgi network. In a preferred example, VAMP4 is mammalian VAMP4. In a more preferred example, VAMP4 is human VAMP4, 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. VAMP4 used in the present invention includes VAMP4 and polypeptides having equivalent functions thereto.

[0048] VTI1B (Vesicle transport through interaction with t-SNAREs homolog 1B) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in 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, 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. VTI1B used in the present invention encompasses VTI1B and polypeptides having equivalent functions thereto.

[0049] BET1 (Bet1 golgi vesicular membrane trafficking protein) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in 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. BET1 used in the present invention encompasses BET1 and polypeptides having equivalent functions thereto.

[0050] BET1L (Bet1 golgi vesicular membrane trafficking protein like) is a type of SNARE protein, has a transmembrane domain, and is predicted to be localized in the Golgi apparatus and 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. BET1L used in the present invention encompasses BET1L and polypeptides having equivalent functions thereto.

[0051] USE1 (Unconventional SNARE in the ER 1) is a type of SNARE protein that has a transmembrane domain and is predicted to be localized in the endoplasmic reticulum. In a preferred example, USE1 is mammalian USE1. In a more preferred example, USE1 is human USE1, a protein consisting of the amino acid sequence of SEQ ID NO: 94, encoded by a gene (Gene ID: 55850) consisting of the nucleotide sequence of SEQ ID NO: 82. USE1 used in the present invention encompasses USE1 and polypeptides having equivalent functions thereto.

[0052] In the present invention, a polypeptide having a function equivalent to a SNARE protein refers to a polypeptide having a biological activity equivalent to that of the SNARE protein. Examples of such polypeptides include polypeptides that have a transmembrane domain and can be localized in intracellular vesicles (preferably intracellular vesicles in which the corresponding SNARE protein is localized) or exosomes. Specific examples include the following polypeptides (b) to (e), or the following polypeptides (b), (c), and (e):

[0053] Specific examples of VAMP7 include: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 10, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes. Splicing variants of the VAMP7 gene consisting of the nucleotide sequence of SEQ ID NO: 1 include variants registered in NCBI RefSeq (Reference Sequence) as NM_001185183.2 or NM_001145149.3, and encoding proteins registered as NP_001172112.1 or NP_001138621.1, respectively.

[0054] Specifically, GOSR2 includes the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 11; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 11, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 11, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., endoplasmic reticulum-Golgi apparatus intermediate section or Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes. Splicing variants of the GOSR2 gene consisting of the nucleotide sequence of SEQ ID NO: 2 include NM_054022.4, NM_004287.5, NM_001353114.2, NM_001012511.3, NM_001363851.2, NM_001330252.2, NM_001353116.2, NM_001353115.2, and NM_00132 1134.2, and variants encoding proteins 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] Specific examples of STX10 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ 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 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 12, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes. Splicing variants of the STX10 gene consisting of the nucleotide sequence of SEQ ID NO: 3 include variants registered in NCBI RefSeq as NM_001271610.2, NM_1271609.2, or NM_001271611.2, and encoding proteins registered as NP_001258539.1, NP_001258538.1, or NP_001258540.1, respectively.

[0056] Specific examples of STX18 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 13, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 13, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes. Splicing variants of the STX18 gene consisting of the nucleotide sequence of SEQ ID NO: 4 include variants registered in NCBI RefSeq as NM_001346281.2, NM_001346282.2, or NM_001346300.2, and encoding proteins registered as NP_001333210.1, NP_001333211.1, or NP_001333229.1, respectively.

[0057] Specific examples of BNIP1 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 14, and having a transmembrane domain and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes. Splicing variants of the BNIP1 gene consisting of the nucleotide sequence of SEQ ID NO: 5 include variants registered in NCBI RefSeq as NM_013980.3, NM_001205.3, or NM_013978.3, and encoding proteins registered as NP_053583.2, NP_001196.2, or NP_053581.2, respectively.

[0058] Specific examples of STX7 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 15, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 15, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes. Splicing variants of the STX7 gene consisting of the nucleotide sequence of SEQ ID NO: 6 include variants registered in NCBI RefSeq as NM_003569.3, NM_001326578.2, NM_001326579.2, or NM_001326580.2, and encoding proteins registered as NP_003560.2, NP_001313507.1, NP_001313508.1, or NP_001313509.1, respectively.

[0059] Specific examples of VTI1A include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 16, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., trans-Golgi compartments) or exosomes. Splicing variants of the VTI1A gene consisting of the nucleotide sequence of SEQ ID NO: 7 include variants registered in NCBI RefSeq as NM_145206.4, NM_001365711.1, NM_001365710.2, NM_001365712.1, NM_001365713.1, NM_001365714.1, or NM_001318205.2, and variants encoding proteins registered as NP_660207.2, NP_001352640.1, NP_001352639.1, NP_001352641.1, NP_001352642.1, NP_001352643.1, or NP_001305134.1, respectively.

[0060] Specific examples of STX16 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ 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 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 17, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes. Splicing variants of the STX16 gene consisting of the nucleotide sequence of SEQ ID NO: 8 include variants registered in NCBI RefSeq as NM_001134772.3, NM_1134773.3, NM_003763.6, or NM_001204868.2, and encoding proteins registered as NP_001128244.1, NP_001128245.1, NP_003754.2, or NP_001191797.1, respectively.

[0061] Specific examples of STX5 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 18, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 18, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes. Splicing variants of the STX5 gene consisting of the nucleotide sequence of SEQ ID NO: 9 include variants registered in NCBI RefSeq as NM_001244666.3 or NM_001330294.2, and encoding proteins registered as NP_001231595.1 or NP_001317223.1, respectively.

[0062] Specifically, GOSR1 includes the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 83; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 83, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 83, and having a transmembrane domain and capable of localizing in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in 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 registered in NCBI RefSeq as NM_001007025.2 or NM_001007024.2 and encoding proteins registered as NP_001007026.1 or NP_001007025.1, respectively.

[0063] Specific examples of STX8 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 84; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 84, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 84, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0064] Specific examples of STX12 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 85; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ 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 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 85, having a transmembrane domain, and capable of localizing in 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 (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes.

[0065] Specifically, VAMP8 includes the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 86, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 86, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes.

[0066] Specific examples of SEC22B include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 87; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 87, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 87, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes.

[0067] Specific examples of STX6 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 88; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 88, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 88, and having a transmembrane domain and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes. Splicing variants of the STX6 gene consisting of the nucleotide sequence of SEQ ID NO: 76 include variants encoding proteins registered in NCBI RefSeq as NM_001286210.2 and NP_001273139.1.

[0068] Specific examples of VAMP3 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 89; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 89, and having a transmembrane domain and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0069] Specific examples of VAMP4 include: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 90; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 90, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 90, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes. Splicing variants of the VAMP4 gene consisting of the nucleotide sequence of SEQ ID NO: 78 include variants encoding proteins registered in NCBI RefSeq as NM_001185127.2 and NP_001172056.1.

[0070] Specific examples of VTI1B include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 91; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 91, having a transmembrane domain, and capable of localizing in 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 (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes.

[0071] Specific examples of BET1 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 92, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 92, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum, Golgi apparatus, or cis-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes. Splicing variants of the BET1 gene consisting of the nucleotide sequence of SEQ ID NO: 80 include variants encoding proteins registered in NCBI RefSeq as NM_001317739.2 and NP_001304668.1.

[0072] Specific examples of BET1L include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 93; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 93, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (d) a polypeptide consisting of 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 in intracellular vesicles (e.g., Golgi apparatus or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (d) above, has a transmembrane domain, and can be localized in 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 registered in NCBI RefSeq as NM_001098787.2 or NM_016526.5, and encoding proteins registered as NP_001092257.1 or NP_057610.2, respectively.

[0073] Specific examples of USE1 include the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 94; (b) a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 94, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (c) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 94, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides (a) to (c) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes.

[0074] The polynucleotides encoding SNARE proteins used in the present invention include polynucleotides that encode SNARE proteins and polynucleotides that encode polypeptides functionally equivalent to SNARE proteins. Specific examples of polynucleotides that encode polypeptides functionally equivalent to SNARE proteins include the following polynucleotides (g) to (k) and (m) to (o), or the following polynucleotides (g), (h), (j), (k), and (m) to (o):

[0075] Specific examples of polynucleotides encoding VAMP7 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 1, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 1, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 10, which has a transmembrane domain, and which can be localized in 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 is capable of localizing in intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes.

[0076] Specific examples of polynucleotides encoding GOSR2 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 2, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 2, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 11, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 11, which has a transmembrane domain, and which can be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus 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 be localized in intracellular vesicles (e.g., the endoplasmic reticulum-Golgi apparatus intermediate section or the Golgi apparatus) or exosomes.

[0077] Specific examples of polynucleotides encoding STX10 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to 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; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 3, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 12, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 12, and which has a transmembrane domain and can be localized in 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 be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0078] Specific examples of polynucleotides encoding STX18 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 4, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 4, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 13, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 13, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes.

[0079] Specific examples of polynucleotides encoding BNIP1 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 5, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 5, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 14, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes.

[0080] Specific examples of polynucleotides encoding STX7 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 6, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 6, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 15, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 15, which has a transmembrane domain, and which can be localized in 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 is capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0081] Specific examples of polynucleotides encoding VTI1A include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 7, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 7, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 16, and which has a transmembrane domain and can be localized in 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 be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0082] Specific examples of polynucleotides encoding STX16 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 8, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 8, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 17, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 17, and which has a transmembrane domain and can be localized in 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 be localized in intracellular vesicles (e.g., the trans-Golgi network) or exosomes.

[0083] Specific examples of polynucleotides encoding STX5 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 9, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 9, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (l) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18; (m) a polynucleotide encoding a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 18, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 18, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0084] Specific examples of polynucleotides encoding GOSR1 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 71; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to 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 the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 71, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 71, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 71, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 83, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 83, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 is capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes.

[0085] Specific examples of polynucleotides encoding STX8 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 72, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 72, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 84, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 84, which has a transmembrane domain, and which can be localized in 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 is capable of localizing in intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0086] Specific examples of polynucleotides encoding STX12 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 73; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 73, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 73, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 73, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ 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 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 85, which has a transmembrane domain, and which can be localized in 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 is capable of localizing to intracellular vesicles (e.g., Golgi apparatus, early endosomes, or recycling endosomes) or exosomes.

[0087] Specific examples of polynucleotides encoding VAMP8 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 74; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 74, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 74, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 74, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 86, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 86, which has a transmembrane domain, and which can be localized in 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 is capable of localizing in intracellular vesicles (e.g., lysosomes, early endosomes, or late endosomes) or exosomes.

[0088] Specific examples of polynucleotides encoding SEC22B include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 75; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 75, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 75, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 75, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 87, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 87, and having a transmembrane domain and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi apparatus 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 of the polypeptides (l) to (n) above, has a transmembrane domain, and is capable of localizing to intracellular vesicles (e.g., the endoplasmic reticulum, the endoplasmic reticulum-Golgi apparatus intermediate section, the Golgi apparatus, the cis-Golgi network, or the trans-Golgi network) or exosomes.

[0089] Specific examples of polynucleotides encoding STX6 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 76, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 76, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 88, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 88, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0090] Specific examples of polynucleotides encoding VAMP3 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 77; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 77, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 77, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 77, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 89, and having a transmembrane domain and capable of localizing in intracellular vesicles (e.g., Golgi apparatus) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can be localized in intracellular vesicles (e.g., Golgi apparatus) or exosomes.

[0091] Specific examples of polynucleotides encoding VAMP4 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 78; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to 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 the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 78, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 78, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 78, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 90, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 90, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 is capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes.

[0092] Specific examples of polynucleotides encoding VTI1B include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 79; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 79, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 79, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 79, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 91, which has a transmembrane domain, and which can be localized in 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 is capable of localizing in intracellular vesicles (e.g., early endosomes, late endosomes, lysosomes, or recycling endosomes) or exosomes.

[0093] Specific examples of polynucleotides encoding BET1 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 80; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 80, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 80, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 80, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 80, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 92, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 92, and which has a transmembrane domain and can be localized in 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 is capable of localizing in intracellular vesicles (e.g., the endoplasmic reticulum, the Golgi apparatus, or the cis-Golgi network) or exosomes.

[0094] Specific examples of polynucleotides encoding BET1L include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 81; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to 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 the trans-Golgi network) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 81, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (i) a polynucleotide encoding a polypeptide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 81, has a transmembrane domain, and is capable of localizing in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 81, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., the Golgi apparatus or the 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 93, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 is capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes.

[0095] Specific examples of polynucleotides encoding USE1 include the following: (f) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 82; (g) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 82, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (h) a polynucleotide encoding a polypeptide consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 82, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (j) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 82, has a transmembrane domain, and encodes a polypeptide that can be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (k) a polynucleotide that is a fragment of any of the polynucleotides (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can be localized in 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 consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 94, having a transmembrane domain, and capable of localizing in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes; (n) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 94, and which has a transmembrane domain and can be localized in intracellular vesicles (e.g., 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 be localized in intracellular vesicles (e.g., endoplasmic reticulum) or exosomes.

[0096] The method for obtaining polynucleotides encoding the SNARE proteins of the present invention is not particularly limited, and they can be obtained by conventional chemical synthesis or genetic engineering techniques. For example, polynucleotides encoding SNARE proteins can be artificially synthesized based on the nucleotide sequences of any of SEQ ID NOS: 1 to 9 and 71 to 82. For artificial synthesis, commercially available DNA synthesis services provided by, for example, GenScript, Inc. can be used. Alternatively, any of the nucleotide sequences of SEQ ID NOS: 1 to 9 and 71 to 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] Polynucleotides encoding the SNARE proteins of the present invention can also be produced by introducing mutations into DNA consisting of any of the nucleotide sequences of SEQ ID NOS: 1 to 9 and 71 to 82. Examples of methods for introducing mutations include ultraviolet irradiation and site-directed mutagenesis. Examples of site-directed mutagenesis methods include a method using splicing overlap extension (SOE) PCR (Horton et al., Gene, 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, the 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. Polynucleotides encoding the SNARE proteins of the present invention can be obtained by selecting from the mutated DNA those that have a transmembrane domain and are capable of localizing to intracellular vesicles. Whether or not a polypeptide encoded by a mutated DNA has a transmembrane domain can be determined, for example, based on the amino acid sequence of the polypeptide encoded by the DNA using prediction tools such as SOSUI (Hirokawa et al., Bioinformatics, 14(4):378-379, 1998) and TMHMM (Krogh et al., J. Mol. Biol. 305(3):567-580, 2001).

[0098] Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into a nucleotide sequence are described, for example, in Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, 581-621, 1995).

[0099] Alternatively, a polynucleotide encoding a SNARE protein of the present invention can be obtained, for example, by subjecting DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1 to 9 and 71 to 82 to genome editing using artificial DNA cleaving enzymes (artificial DNA nucleases or programmable nucleases).

[0100] In the present invention, any allergen can be used. In the present invention, an allergen refers to a protein or a peptide derived therefrom that enters the body from the outside by inhalation, puncture, ingestion, or contact and induces a hypersensitivity reaction or an allergic reaction. Examples of allergens include, but are not limited to, grass plant pollen (reed, timothy grass, foxtail, orchard grass, silvergrass, wheat, rice bran, paspalum, corn, longgrass, Japanese silvergrass, broad-leaved fescue, and ryegrass); weed pollen (goldenrod, nettle, giant ragweed, Japanese knotweed, dandelion, artemisia absinth, rhubarb, ragweed, false ragweed, oxeye daisy, and oak leaf pollen); Plantain, mugwort, etc.); tree pollen (acacia, olive, maple, walnut, mulberry, oak, birch, cedar 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, Helminthosporium, Ma Racetia, Mucor, etc.); Animal epidermis (duck feathers, cat dander, dog dander, cowhide dander, horse dander, rabbit epithelium, hamster epithelium, guinea pig epithelium, sheep epithelium, pig epithelium, goat epithelium, chicken feathers, goose feathers, budgie feathers, budgie droppings, mice, rats, etc.); Insects (paper wasps, moths, cockroaches, hornets, honeybees, Aedes mosquitoes, midges (adult), etc.); Parasites (Anisakis, roundworms, etc.); Mites (Acarina mites) , Tyrophagus putrescentiae, house dust mites, Dermoptera, and Pterygospermatidae; foods (eggs, milk, wheat, buckwheat, peanuts, shrimp, crab, almonds, abalone, squid, salmon roe, oranges, cashew nuts, kiwi fruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, etc.); proteins contained in human insulin, etc., or peptides derived therefrom. Of these, proteins contained in foods or peptides derived therefrom are preferred as allergens, and proteins contained in eggs or peptides derived therefrom are more preferred.

[0101] The method for obtaining polynucleotides encoding the allergens of the present invention is not particularly limited, and they can be obtained by conventional chemical synthesis or genetic engineering techniques, as in the case of obtaining polynucleotides encoding SNARE proteins described above. The polynucleotides encoding allergens may be polynucleotides encoding the full-length allergens, or polynucleotides encoding partial polypeptides of the allergens as long as they function as allergens. Furthermore, they may be polynucleotides in which multiple polynucleotides encoding one allergen are linked in an expressible manner, or polynucleotides in which polynucleotides encoding two or more allergens are linked in an expressible manner. The allergens used in the nucleic acid construct of the present invention are at least one type, and preferably five or fewer types, more preferably three or fewer types, and even more preferably two or fewer types. The allergens used in the nucleic acid construct of the present invention may be five, four, three, two, 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 operably linked downstream of a polynucleotide encoding a SNARE protein, more preferably a nucleic acid construct in which a polynucleotide encoding an allergen is operably 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 a nucleic acid construct in which a polynucleotide encoding an allergen is operably 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 contains a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence, the order of linking them 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. Alternatively, a sequence in which the proprotein convertase recognition sequence is repeated about 1 to 5 times may be linked, or a polynucleotide encoding a proprotein convertase recognition sequence may be linked so as to be sandwiched between multiple polynucleotides encoding linkers. Any polynucleotide other than the polynucleotide encoding a linker and the polynucleotide encoding a proprotein convertase recognition sequence may be contained between the polynucleotide encoding a SNARE protein and the polynucleotide encoding an antigen, as long as the expression of both is not impaired.

[0103] In the present invention, the term "linker" refers to a peptide linker that links two polypeptides. The linker is not particularly limited, as long as it allows the SNARE protein and allergen to function normally. The length of the linker is preferably 3 or more amino acid residues, more preferably 4 or more amino acid residues, and even more preferably 5 or more amino acid residues, and is preferably 30 or less amino acid residues, more preferably 25 or less amino acid residues, and even more preferably 20 or less amino acid residues. 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 containing G, GS, GGS, GGGS (SEQ ID NO: 96), GGGGS (SEQ ID NO: 97), EAAAK (SEQ ID NO: 98), or XP as a component. Here, X represents any amino acid residue. Specific examples include linkers consisting of a sequence in which each component 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). When the sequence component is GGGGS, the amino acid residue S may be added before the repeat sequence, or when it is EAAAK, the amino acid residue A may be added before and after the repeat sequence.

[0104] In the present invention, the term "proprotein convertase recognition sequence" refers to an amino acid sequence recognized by proprotein convertase, a serine protease that converts proproteins into physiologically active proteins or peptides in the Golgi apparatus and the like. Specifically, the sequence is an amino acid sequence consisting of X-Arg-X-(Arg / Lys)-Arg (e.g., SEQ ID NO: 22), where 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 may be Arg-X-(Arg / Lys)-Arg, where X represents any amino acid residue. The proprotein convertase recognizes the above amino acid sequence and cleaves the C-terminal side of the C-terminal arginine residue. In the present invention, the proprotein convertase is preferably furin, and the furin recognition sequence is the same as the above proprotein convertase recognition sequence.

[0105] The method for obtaining a polynucleotide encoding a linker or a polynucleotide encoding a proprotein convertase recognition sequence is not particularly limited, and they can be obtained by ordinary chemical synthesis or genetic engineering techniques.

[0106] The polynucleotide encoding a SNARE protein, the polynucleotide encoding an allergen, the polynucleotide encoding a linker, or the polynucleotide encoding a proprotein convertase recognition sequence contained in the nucleic acid construct of the present invention may be codon-optimized, if necessary, to suit the species to which the nucleic acid construct is to be administered. 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 and comprises a control 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 a SNARE protein and the polynucleotide encoding an allergen are operably linked to the control region. Examples of the control region include a promoter, a terminator, an enhancer, etc. Preferably, the expression cassette comprises a promoter linked upstream of the polynucleotide encoding a SNARE protein and the polynucleotide encoding an allergen.

[0108] The nucleic acid construct of the present invention can have a restriction enzyme recognition site at one or both ends. The restriction enzyme recognition site can be used to introduce the nucleic acid construct of the present invention into a vector. For example, the vector can be cleaved with a restriction enzyme and the nucleic acid construct of the present invention having a restriction enzyme recognition site at its end added thereto, thereby introducing the nucleic acid construct into the vector.

[0109] The type of vector is not particularly limited and may be any vector, such as a plasmid vector, a phage, a phagemid, a cosmid, or a viral vector. In one example, the vector into which the nucleic acid construct of the present invention is to be incorporated may be an expression vector; however, 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 containing a regulatory region, and is incorporated into any vector to construct an expression vector. In another example, the nucleic acid construct of the present invention is incorporated into an expression vector containing a regulatory region, thereby constructing an expression cassette on the expression vector.

[0110] In a preferred example, the nucleic acid construct of the present invention is a plasmid vector. Examples of plasmid vectors include, but are not limited to, plasmid vectors such as pVAX1. Generally, a drug resistance gene is incorporated into the sequence of a plasmid vector for the purpose of selectively maintaining the plasmid vector during bacterial culture. However, due to the risk of the gene being transmitted to bacterial flora in vivo or being activated and expressed via a mammalian promoter, a plasmid vector from which the gene sequence has been removed is more preferred.

[0111] In a preferred embodiment, the nucleic acid construct of the present invention is a viral vector, including, but not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, Sendai viral vectors, and herpes viral vectors.

[0112] In a preferred embodiment, the nucleic acid construct of the present invention is an mRNA construct. For example, a DNA containing a polynucleotide encoding a SNARE protein and a polynucleotide encoding an allergen is used as a template to generate an mRNA using a HiScribe T7 ARCA mRNA Kit (New England Biolabs), T7 mScript TM The nucleic acid construct of the present invention can be obtained as mRNA by using commercially available in vitro transcription kits such as Standard mRNA Production System (CELLSCRIPT) and HighYield T7 ARCA mRNA Synthesis Kit (Jena Bioscience).

[0113] As shown in the Examples below, a nucleic acid construct comprising a polynucleotide encoding a SNARE protein of the present invention and a polynucleotide encoding an allergen has 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 comprising only an allergen-encoding polynucleotide (hereinafter referred to as a control). This is presumably because, in the antigen presentation process in which a nucleic acid construct taken up into a cell is translated or transcribed and translated to express an antigen protein, which is then transported to intracellular vesicles, where a portion of the antigen binds to major histocompatibility complex (MHC) molecules and is transported to the cell surface, in the nucleic acid construct of the present invention, the allergen protein is expressed as a fusion polypeptide of a SNARE protein and an allergen, which targets the allergen protein to intracellular vesicles and increases the probability of association with MHC molecules, thereby 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 expressing allergen proteins as fusion polypeptides of SNARE proteins and allergens may target the allergen proteins to exosomes, thereby improving their immunogenicity. Furthermore, as shown in the Examples below, in an allergy model, the nucleic acid construct of the present invention can improve the drop in body temperature associated with anaphylaxis and reduce the blood concentration of allergen-specific IgE antibodies compared to controls. These results demonstrate that the nucleic acid construct of the present invention has an excellent allergy-suppressing effect. Furthermore, in conventional allergen immunotherapy, it is known that a portion of allergens ingested from outside the body is released into the blood and captured by allergen-specific IgE antibodies, causing side 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 blood after administration. These results indicate that the nucleic acid construct of the present invention may be able to reduce the risk of anaphylaxis.

[0114] The production levels of cytokines such as IFNγ and IL-4, which can serve as indicators for determining whether an immune response is Th1 or Th2 type, or whether it is a cellular or humoral immune response, can be measured by conventionally known methods. Examples of such methods include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT) assay, immunohistochemical staining, in situ hybridization, RT-PCR, microarray, and flow cytometry. Commercially available reagents and kits for measuring cytokine production, such as the ELISPOT assay kit (CTL) used in the Examples below, may also be used for measurement. Furthermore, the production amount of each IgG subclass, which can be used as another indicator for determining whether an 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 as ELISA and immunoturbidimetry. Th1-type immune responses and cellular immune responses can be evaluated using the amount of cytokines produced and / or the amount of IgG subclasses produced as indicators. For example, an increase in the amount of IFNγ produced or the ratio of IFNγ to IL-4 produced (IFNγ / IL-4) can be evaluated as indicating that a Th1-type immune response and a cellular immune response have been induced or enhanced, whereas a decrease in the amount of IFNγ produced or IFNγ / IL-4 can be evaluated as indicating that a Th1-type immune response and a cellular immune response have been weakened. Alternatively, an increase in the amount of IgG2a produced and / or the ratio of IgG2a produced to IgG1 produced (IgG2a / IgG1) can be evaluated as indicating that a Th1-type immune response and a cellular immune response have been induced or enhanced, whereas a decrease in the amount of IgG2a produced and / or IgG2a / IgG1 can be evaluated as indicating that a Th1-type immune response and a cellular immune response have been weakened.

[0115] Therefore, the nucleic acid construct of the present invention can be an allergen-specific IFNγ production enhancer, an IgG2a production enhancer, a Th1-type immune response inducer or enhancer, or a cellular immune response inducer or enhancer (hereinafter referred to as a cellular immune response inducer or enhancer, etc.), and the nucleic acid construct can be used to manufacture a cellular immune response inducer or enhancer, 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 a Th1-type immune response, or induce or enhance a cellular immune response. Such uses may involve administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic. The term "non-therapeutic" does not include medical procedures, i.e., methods of surgery, therapy, or diagnosis of humans, and more specifically, does not include methods of surgery, therapy, or diagnosis performed on humans by physicians or those acting under the direction of a physician.

[0116] The cellular immune response inducer or enhancer of the present invention can be used by itself as a pharmaceutical or quasi-drug for enhancing allergen-specific IFNγ production, enhancing IgG2a production, inducing or enhancing Th1-type immune responses, or inducing or enhancing cellular immune responses, or can be used as a material or preparation to be incorporated into such pharmaceuticals or quasi-drugs.

[0117] When the cellular immune response inducer or enhancer of the present invention is used as a pharmaceutical (including quasi-drugs), the pharmaceutical may be administered in any dosage form, such as 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., with parenteral administration being preferred, and parenteral administration in the form of injections being more preferred. Such pharmaceutical formulations in various dosage forms can be prepared by appropriately combining the nucleic acid construct of the present invention with other pharmaceutically acceptable excipients, binders, fillers, disintegrants, diluents, thickeners, emulsifiers, lubricants, dispersants, coating agents, surfactants, coating agents, osmotic pressure adjusters, buffers, pH adjusters, preservatives, stabilizers, antioxidants, colorants, flavorings, odorants, 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 subject to be administered, and the route of administration, and is therefore not particularly limited and can be appropriately selected within a wide range. For example, the nucleic acid construct may be contained in an amount of 0.00001 to 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, body weight, sex, age, condition, or other factors of the subject. The dosage, route, and interval of administration can be determined appropriately by those skilled in the art. For example, the dosage is determined as the amount of the nucleic acid construct of the present invention, between 1 ng and 10 mg per adult (body weight 60 kg) per day. When the nucleic acid construct is a viral vector, the dosage is, for example, between 10 and 1 × 10 per adult (body weight 60 kg) per day. 15 It may be a viral particle.

[0120] The cellular immune response inducer or enhancer of the present invention can be administered to both humans and non-human animals. Non-human animals include non-human mammals, such as apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, 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 patients or humans at risk of allergies.

[0121] Furthermore, the nucleic acid construct of the present invention can be a nucleic acid vaccine, and can be used to produce a nucleic acid vaccine. Furthermore, the nucleic acid constructs of the present invention can be used to induce or enhance an allergen-specific Th1-type immune response and / or to induce or enhance an allergen-specific cellular immune response, which may be administered to humans or non-human animals or in specimens derived therefrom, and which may be therapeutic or non-therapeutic.

[0122] The nucleic acid vaccine of the present invention can be used as a pharmaceutical product by itself for inducing or enhancing an allergen-specific Th1-type immune response and / or for inducing or enhancing an allergen-specific cellular immune response, and can also be used as a material or formulation to be incorporated into such pharmaceutical products.

[0123] In one embodiment, the nucleic acid vaccine is a DNA vaccine. The DNA vaccine comprises the nucleic acid construct of the present invention, which is a plasmid vector. The plasmid vector is not particularly limited, but an example thereof is the pVAX1 vector. Among these, a plasmid vector that does not contain a drug resistance gene is 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. 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 with some base modifications (e.g., uridine replaced with pseudouridine or 1-methylpseudouridine). Alternatively, the nucleic acid construct may be self-amplifying RNA containing the nucleic acid construct of the present invention and the sequence of a virus-derived RNA-dependent RNA polymerase (RdRP) complex and its replication origin (5'CSE, 3'CSE), or trans-amplifying RNA, which is a mixture of the nucleic acid construct of the present invention, RNA containing the replication origin of the RdRP complex, and mRNA containing the sequence of the RdRP complex. The mRNA vaccine preferably further comprises a drug delivery construct, such as a liposome or lipid nanoparticle composed of a lipid, or a polymer nanoparticle such as PLGA nanoparticle composed of a polymer, as a carrier for stabilizing and delivering the mRNA. 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. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, Sendai viral vectors, and herpes viral vectors.

[0126] The nucleic acid vaccine of the present invention contains, in addition to the above-mentioned nucleic acid construct, a pharmaceutically acceptable carrier as appropriate, and can be formulated in a predetermined form. Here, examples of the carrier include carriers commonly used in the production of vaccines, and specific examples include buffers, emulsifiers, preservatives (e.g., thimerosal), isotonicity agents, pH adjusters, thickening agents, adjuvants, or immunostimulants. An adjuvant is a substance that enhances the immune response to an antigen when administered together with the antigen, but the nucleic acid vaccine of the present invention can itself function as an adjuvant, so the addition of an adjuvant is not necessarily required, and the composition may be adjuvant-free.

[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, with intramuscular, intradermal, or subcutaneous administration being preferred. Injectable formulations include, for example, solutions, emulsions, water-soluble or hydrophobic suspensions, and dry powder formulations that are dissolved or suspended in 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 subject to be administered, and the administration route, and is therefore not particularly limited and can be appropriately selected from a wide range. For example, the nucleic acid construct may be contained in an amount of 0.00001 to 100% by mass of the entire 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 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 the nucleic acid construct of the present invention between 1 ng and 10 mg per dosage unit. When the nucleic acid construct is a viral vector, the dosage is, for example, between 10 and 1 × 10 per dosage unit. 15 It may be a viral particle.

[0130] The nucleic acid vaccine of the present invention can be administered to both humans and non-human animals. Examples of non-human animals include 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 patients or humans at risk of allergy.

[0131] The number of administrations of the nucleic acid vaccine of the present invention may be appropriately determined depending on the application, and is at least once, but may be two or more times from the viewpoint of efficacy. Further administration is sometimes called booster immunization, and this can achieve more effective infection prevention or therapeutic effects. The interval between booster immunizations is recommended to be at least one week, and intervals of one to four weeks are preferable.

[0132] Nucleic acid vaccines can be used to treat a wide variety of allergens by modifying the nucleic acid that encodes the allergen, and because they are nucleic acids, they can be produced quickly and at low cost.

[0133] In a preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for preventing or treating allergies, preferably a vaccine for allergy immunotherapy. Specifically, the allergy preventive or therapeutic vaccine comprises a nucleic acid construct comprising a polynucleotide encoding a SNARE protein of the present invention and a polynucleotide encoding an allergen. The allergy preventive or therapeutic vaccine can be administered directly to a living body. It is believed that such an allergy preventive or therapeutic vaccine can exert an inhibitory effect against allergic responses caused by excessive activation of Th2-type immune responses by inducing or enhancing allergen-specific Th1-type immune responses and cellular immune responses.

[0134] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed herein, but 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 described in [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 (wherein X represents any amino acid residue), preferably an amino acid sequence consisting of X-Arg-X-(Arg / Lys)-Arg (wherein X represents any amino acid residue), and more preferably an amino acid sequence set forth in SEQ ID NO: 22. [5] The nucleic acid construct according to any one of [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, 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 any polynucleotide selected from the group consisting of polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1 to 9 and 71 to 82 and polynucleotides having equivalent functions thereto, and more preferably a polynucleotide consisting of any of the nucleotide sequences of SEQ ID NOs: 1 to 9 and 71 to 82. [8] The SNARE protein is preferably any one 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 any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B. The nucleic acid construct according to any one of [1] to [7], wherein the nucleic acid construct is any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, and GOSR1, more preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, and GOSR1, even more preferably any one selected from the group consisting of VAMP7, GOSR2, STX10, and GOSR1, even more preferably VAMP7.

[0136] [9] A pharmaceutical composition comprising the nucleic acid construct according to any one of [1] to [8].

[10] A nucleic acid vaccine containing the nucleic acid construct according to any one of [1] to [8] as an active ingredient.

[11] The nucleic acid vaccine described in

[10] , which induces or enhances an allergen-specific Th1-type immune response.

[12] The nucleic acid vaccine described in

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

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

[10] to

[12] , which is a vaccine for preventing or treating allergies.

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

[10] to

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

[0137]

[15] Use of the nucleic acid construct according to any one of [1] to [8] for producing a nucleic acid vaccine.

[16] The use described in

[15] , wherein the nucleic acid vaccine is a vaccine for preventing or treating allergies.

[17] The use according to

[15] or

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

[0138]

[18] Use of the nucleic acid construct according to any one of [1] to [8] for inducing or enhancing an allergen-specific Th1-type immune response and / or inducing or enhancing an allergen-specific cellular immune response.

[19] The nucleic acid construct according to any one of [1] to [8], for use in inducing or enhancing an allergen-specific Th1-type immune response and / or inducing or enhancing an allergen-specific cellular immune response.

[20] A method for inducing or enhancing an allergen-specific Th1-type immune response and / or an allergen-specific cellular immune response, comprising administering an effective amount of the nucleic acid construct described in any one of [1] to [8] to a subject in need thereof.

[0139]

[21] Use of the nucleic acid vaccine according to any one of

[10] to

[14] for inducing or enhancing an allergen-specific Th1-type immune response and / or inducing or enhancing an allergen-specific cellular immune response.

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

[10] to

[14] , for use in inducing or enhancing an allergen-specific Th1-type immune response and / or inducing or enhancing an allergen-specific cellular immune response.

[23] A method for inducing or enhancing an allergen-specific Th1-type immune response and / or an allergen-specific cellular immune response, comprising administering an effective amount of the nucleic acid vaccine described in any one of

[10] to

[14] to a subject in need thereof.

[24] A method for preventing or treating allergies, comprising administering an effective amount of the nucleic acid vaccine according to

[13] to a subject in need thereof.

[25] The dose of the nucleic acid vaccine is preferably 1 ng to 10 mg per dosage unit in terms of the amount of the nucleic acid construct when the nucleic acid vaccine is a DNA vaccine or an mRNA vaccine, and is preferably 10 to 1 × 10 per dosage unit in terms of the amount of the nucleic acid construct when the nucleic acid vaccine is a viral vector vaccine. 15 The method according to

[23] or

[24] , wherein the particle is a virus particle.

[0140]

[26] An allergen-specific IFNγ production enhancer comprising the nucleic acid construct according to any one of [1] to [8] as an active ingredient.

[27] An allergen-specific IgG2a production enhancer comprising the nucleic acid construct according to any one of [1] to [8] as an active ingredient.

[28] An agent for inducing or enhancing an allergen-specific Th1 type immune response, comprising the nucleic acid construct according to any one of [1] to [8] as an active ingredient.

[29] An agent for inducing or enhancing an allergen-specific cellular immune response, comprising the nucleic acid construct according to any one of [1] to [8] as an active ingredient.

[30] The agent according to any one of

[26] to

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

[0141]

[31] Use of the nucleic acid construct according to any one of [1] to [8] for producing an allergen-specific IFNγ production enhancer.

[32] Use of the nucleic acid construct according to any one of [1] to [8] for producing an allergen-specific IgG2a production enhancer.

[33] Use of the nucleic acid construct according to any one of [1] to [8] for producing an agent for inducing or enhancing an allergen-specific Th1-type immune response.

[34] Use of the nucleic acid construct according to any one of [1] to [8] for producing an agent for inducing or enhancing an allergen-specific cellular immune response.

[35] The use according to any one of

[31] to

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

[0142]

[36] Use of the nucleic acid construct according to any one of [1] to [8] for enhancing allergen-specific IFNγ production.

[37] Use of the nucleic acid construct according to any one of [1] to [8] for enhancing allergen-specific IgG2a production.

[38] Use of the nucleic acid construct according to any one of [1] to [8] for inducing or enhancing an allergen-specific Th1-type immune response.

[39] Use of the nucleic acid construct according to any one of [1] to [8] for inducing or enhancing an allergen-specific cellular immune response.

[0143]

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

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

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

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

[0144]

[44] A method for enhancing allergen-specific IFNγ production, comprising administering an effective amount of the nucleic acid construct according to any one of [1] to [8] to a subject in need thereof.

[45] A method for enhancing allergen-specific IgG2a production, comprising administering an effective amount of the nucleic acid construct according to any one of [1] to [8] to a subject in need thereof.

[46] A method for inducing or enhancing an allergen-specific Th1-type immune response, comprising administering an effective amount of the nucleic acid construct according to any one of [1] to [8] to a subject in need thereof.

[47] A method for inducing or enhancing an allergen-specific cellular immune response, comprising administering an effective amount of the nucleic acid construct according to any one of [1] to [8] to a subject in need thereof.

[48] ​​The dosage 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, the dosage is preferably 10 to 1 × 10 per day. 15 The method according to any one of

[44] to

[47] , wherein the viral particles are administered at a dose of 60 kg body weight.

[0145]

[49] In

[10] to

[17] and

[21] to

[25] , the nucleic acid vaccine is administered orally or parenterally, preferably parenterally.

[50] In

[20] ,

[23] to

[25] and

[44] to

[48] , the subject is an allergy sufferer or a person at risk of allergy. [Example]

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

[0147] Example 1: Examination of SNARE proteins We constructed mRNAs encoding the amino acid sequences of fusion polypeptides (SEQ ID NOS: 25-45) or the amino acid sequence of OVA alone, in which the allergen ovalbumin (OVA, amino acid sequence: SEQ ID NOS: 24) was linked to the C-terminus of each of 21 human SNARE proteins via a linker (nucleotide sequence: SEQ ID NOS: 19, amino acid sequence: SEQ ID NOS: 20). The nucleotide sequences encoding the antigens or fusion polypeptides were codon-optimized and then incorporated into plasmid DNA (pVAX1 vector, Thermo Fisher Scientific). The nucleotide sequence of OVA incorporated into the plasmid DNA is shown in SEQ ID NOS: 95, and the nucleotide sequences of the fusion polypeptides are shown in SEQ ID NOS: 48-68. The nucleotide sequence of OVA before codon optimization is shown in SEQ ID NOS: 23. mRNAs were prepared using the HiScribe T7 ARCA mRNA Kit (New England Biolabs). Specifically, the plasmid DNAs incorporating each sequence were linearized using restriction enzymes, and then in vitro mRNA synthesis was performed using the DNA as a template with T7 RNA polymerase and 5'-end capping using ARCA. Next, DNase was added to degrade the DNA used as a template, and poly(A) tails were added using Poly(A) Polymerase. The resulting mRNA was purified using an RNeasy kit (QIAGEN) and used for the subsequent procedures. The purified mRNA was transfected into mature human CD14 using TransIT-mRNA Transfection Reagent (Mirus Bio) according to the attached protocol. + Dendritic cells (Lonza) were transfected. As a control, only transfection reagent without mRNA was added to the dendritic cells. From the day after transfection, human CD4 + T cells (Lonza) 1.0×10 6 2.5 × 10 transfected dendritic cells 5 The cells were co-cultured for a total of 7 days and presented with mRNA-derived OVA as an antigen. + T cells 1.0×106 Human CD14 cells from the same donor + 1.0 x 10 dendritic cells 5 The cells were co-cultured again with 200 μg / mL of OVA protein for antigen stimulation. After 24 hours, an ELISPOT assay was performed using an ELISPOT assay kit (CTL) according to the attached protocol to detect IFNγ-producing CD4 + T cells were detected.

[0148] The results are shown in Figure 1. Expression of fusion proteins of OVA with specific SNARE proteins (VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, or SEC22B) in dendritic cells induced OVA-specific IFNγ-producing CD4 + The number of T cells was significantly elevated. Generally, CD4 + Upon antigen presentation, T cells differentiate into various cell types, such as Th1, Th2, Th17, and Treg, and release specific cytokines. IFNγ is a cytokine characteristically secreted by Th1 cells. These findings demonstrate that the introduction of a specific SNARE-OVA fusion protein according to the present invention into antigen-presenting cells, such as dendritic cells, can induce or enhance Th1 immune responses. This experiment was performed with n=3 per group. A control group (Empty) that underwent restimulation with OVA protein was used as a control. Groups with a p<0.05 result in Dunnett's test were considered to have significant differences. Furthermore, a Student's t-test using Empty as a control also indicated a significant difference. The Empty (NT) group was a control group (Empty) that did not undergo restimulation with OVA protein.

[0149] Example 2 IgG, IgG1, and IgG2a antibody titers VAMP7 was selected from the SNARE proteins that increased IFNγ production in Example 1. A fusion polypeptide (V7-OVA) was constructed by linking OVA to the C-terminus of VAMP7 via a linker (SEQ ID NO: 25), or a plasmid vector (pVAX1 vector, Thermo Fisher Scientific) encoding only the amino acid sequence of OVA (SEQ ID NO: 24) was constructed. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each of the constructed vectors was intramuscularly administered to the thigh muscles of female BALB / c mice, followed by application of voltage using an electroporator (NEPA21, Nepa Gene Co., Ltd.) to perform gene transfer. The above treatments were performed on days 0, 7, and 14 after the start of the experiment. On day 35, the mice were euthanized and blood was collected. Serum was separated from the collected blood and antibody titers were measured. For antibody titer measurements, 10 μg / mL OVA (Merck) in DPBS was added to a 96-well ELISA plate (IWAKI) at 100 μL / well to immobilize the OVA. The next day, the plate was washed and blocked with 100 μL / well of 1% BSA in DPBS for 1 hour. After washing the plate, 100 μL / well of the isolated serum was added, serially diluted 2-fold. After 2 hours, the serum was removed by washing, and 100 μL / well of 1 μg / mL anti-mouse IgG, IgG1, or IgG2a antibody (Abcam) was added and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (Abcam) was added to initiate the color reaction. After 10 minutes, 50 μL / well of Stop solution (Abcam) was added to stop the color reaction, and the absorbance was measured at 450 nm. The maximum dilution factor at which the absorbance was measured was two times or more higher than the absorbance of the control in which DPBS was added instead of serum, and this value was defined as the antibody titer of each sample.

[0150] The results are shown in Figure 2. Compared with OVA alone, VAMP7-OVA (V7-OVA) significantly reduced the OVA-specific IgG1 antibody titer. On the other hand, a significant increase in OVA-specific IgG2a antibody titer was observed with VAMP7-OVA alone compared with the control group (Empty). There was no significant difference in the overall OVA-specific IgG antibody titer between OVA alone and VAMP7-OVA. Generally, Th1-type immune responses are known to produce IgG2a antibodies from B cells, whereas Th2-type immune responses are known to produce IgG1 antibodies from B cells. Therefore, measuring the titers of allergen-specific IgG1 and IgG2a antibodies can estimate the Th1 / Th2 balance of immune responses in vivo. These results demonstrate that VAMP7-OVA can induce or enhance Th1-type immune responses in vivo, as the OVA-specific IgG1 antibody titer was reduced and the IgG2a antibody titer was increased with VAMP7-OVA compared with OVA alone. This experiment was carried out with n=10 for each group, and when a Tukey test was carried out for each group and p<0.05 was determined, it was determined that there was a significant difference.

[0151] Example 3 ELISPOT Assay A plasmid vector (pVAX1 vector, Thermo Fisher Scientific) was constructed encoding either the amino acid sequence of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 via a linker (V7-OVA), or the amino acid sequence of OVA alone. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each of the constructed vectors was intramuscularly administered to the thigh muscles of female BALB / c mice, followed by application of voltage using an electroporator (NEPA21) for gene transfer. The above treatment was performed on days 0, 7, and 14 after the start of the experiment. On day 35, the mice were euthanized and their spleens were collected. The collected spleens were ground on a 40 μm cell strainer with the addition of 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the resulting solution was then lysed using Pharm Lyse. TMThe suspension was then centrifuged again at 200 × g for 5 minutes, the supernatant was removed, and the cells were washed twice with DPBS. TM A splenocyte suspension was obtained by adding medium (CTL). 7 Splenocytes at 100 μL / well were seeded onto an ELISPOT plate and mixed with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck) for antigen stimulation at a final concentration of 50 μg / mL. A negative control group (NT) was prepared without OVA protein. After 24 hours, ELISPOT assays were performed using an ELISPOT assay kit (CTL) according to the attached protocol to detect cells producing IFNγ and IL-4 specifically 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 allergen-specific immune responses.

[0152] The results are shown in Figure 3. When VAMP7-OVA (V7-OVA) was used, the number of OVA-specific IL-4-secreting T cells remained unchanged compared to OVA alone, while the number of OVA-specific IFNγ-secreting T cells significantly increased. Generally, IL-4 is secreted by Th2 cells, and IFNγ is secreted by Th1 cells or cytotoxic T cells (CD8 + Since VAMP7 is known to be secreted by T cells, these results indicate that fusing VAMP7 with an allergen can selectively induce or enhance allergen-specific Th1-type immune responses and cellular immunity without inducing allergen-specific Th2-type immune responses. This experiment was performed with n = 10 per group, and a Tukey test was performed for each group under non-stimulated conditions (NT) or OVA-stimulated conditions (OVA), and a p < 0.05 result was considered to indicate a significant difference.

[0153] Example 4 IgG, IgG1, and IgG2a antibody titers Plasmid vectors (pVAX1 vector, Thermo Fisher Scientific) encoding the amino acid sequences (SEQ ID NOs: 25, 27, 28, 41) of fusion polypeptides in which OVA was linked to the C-terminus of VAMP7, STX10, STX18, or GOSR1 via a linker (V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA) or the OVA amino acid sequence (SEQ ID NO: 24) alone were constructed. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each vector was intramuscularly administered to female BALB / c mice in the thigh muscle, followed by application of voltage using an electroporator (NEPA21) for gene transfer. The above treatments were performed on days 0, 7, and 14 after the start of the experiment. On day 35, the mice were euthanized and blood was collected. Serum was isolated from the collected blood and antibody titers were measured. For antibody titer measurements, 10 μg / mL OVA (Merck) in DPBS was added to a 96-well ELISA plate (IWAKI) at 100 μL / well to immobilize the OVA. The next day, the plate was washed and blocked for 1 hour with 100 μL / well of 1% BSA in DPBS. Subsequently, the plate was washed and 100 μL / well of the isolated serum was added, serially diluted 2-fold. After 2 hours, the serum was removed by washing, and 100 μL / well of 1 μg / mL anti-mouse IgG, IgG1, or IgG2a antibody (Abcam) was added and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (Abcam) was added to initiate the color reaction. After 10 minutes, 50 μL / well of Stop solution (Abcam) was added to stop the color reaction, and the absorbance was measured at 450 nm. The maximum dilution factor at which the absorbance was measured was two times or more higher than the absorbance of the control in which DPBS was added instead of serum, and this value was defined as the antibody titer of each sample.

[0154] The results are shown in Figure 4. Five weeks after vector administration, a significant increase in OVA-specific IgG antibody titers was observed in all groups administered with OVA alone and each SNARE sequence linked to OVA compared to the empty vector group. VAMP7-OVA (V7-OVA) and GOSR1-OVA showed no change in OVA-specific IgG antibody titers compared to OVA alone, whereas STX10-OVA and STX18-OVA showed a significant decrease in OVA-specific IgG1 antibody titers compared to OVA alone. A significant decrease in OVA-specific IgG2a antibody titers was observed only with V7-OVA compared to the empty vector group. The ratio of OVA-specific IgG1 and IgG2a antibody titers was calculated for each individual, i.e., IgG2a / IgG1, but was increased in all groups administered with each SNARE sequence linked to OVA compared to OVA alone, although no significant difference was observed. In the IgG2a / IgG1 graph, the average value is shown above the dot plot for each group. These results show that OVA-specific IgG1 antibody titers and IgG2a / IgG1 ratios are also increased for SNARE family members other than VAMP7, such as STX10, STX18, and GOSR1. These results suggest that linking these SNARE sequences to allergens can induce or enhance allergen-specific Th1-type immune responses in vivo. This experiment was performed with n = 5 per group, and a Dunnett's test determined a p < 0.05 to indicate a significant difference. Significant differences are indicated by * when compared with the empty group, and by † when compared with the OVA group.

[0155] Example 5 ELISPOT Assay Plasmid vectors (pVAX1 vector, Thermo Fisher Scientific) encoding the amino acid sequences (SEQ ID NOs: 25, 27, 28, 41) of fusion polypeptides in which OVA was linked to the C-terminus of VAMP7, STX10, STX18, or GOSR1 via a linker (V7-OVA, STX10-OVA, STX18-OVA, GOSR1-OVA) or the OVA amino acid sequence (SEQ ID NO: 24) alone were constructed. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each vector was intramuscularly administered to female BALB / c mice in the thigh muscle, followed by application of voltage using an electroporator (NEPA21) for gene transfer. The above treatments were performed on days 0, 7, and 14 after the start of the experiment. On day 35, the mice were euthanized and their spleens were collected. The collected spleens were mashed on a 40 μm cell strainer in the presence of 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, the supernatant was removed, and then Pharm Lyse solution (BD Biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells contained in the suspension. The suspension was centrifuged again at 200 × g for 5 minutes, the supernatant was removed, and the cells were washed twice with DPBS. CTL-Test Medium (CTL) was added to obtain a splenocyte suspension. 1.0 × 10 7 Splenocytes from the cells were seeded at 100 μL / well on an ELISPOT plate and mixed with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck) for antigen stimulation at a final concentration of 50 μg / mL. A group without OVA protein (NT) was prepared as a negative control. After 24 hours, ELISPOT assays were performed using an ELISPOT assay kit (CTL) according to the attached protocol to detect cells producing IFNγ and IL-4 specifically 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 allergen-specific immune responses.

[0156] The results are shown in Figure 5. Compared with OVA alone, the number of OVA-specific IFNγ-secreting T cells was significantly increased in all groups in which each SNARE sequence was linked to OVA. On the other hand, although significant differences were observed between groups in the number of OVA-specific IL-4-secreting T cells under both unstimulated (NT) and OVA-stimulated (OVA) conditions, the IL-4 ratio, calculated as the ratio of individual T cells under each NT and OVA condition, was unchanged in all groups. These results suggest that SNARE family members other than VAMP7, such as STX10, STX18, and GOSR1, can selectively induce or enhance allergen-specific Th1-type immune responses and cellular immunity without inducing allergen-specific Th2-type immune responses by linking them to allergens. Each group consisted of five individuals. A Dunnett's test (p<0.05) was used to determine whether a significant difference existed between groups under unstimulated or OVA-stimulated conditions. Significant differences are indicated by * when compared with the Empty group, and by † when compared with the OVA group.

[0157] Example 6 Antibody titer over time Plasmid vectors encoding the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide in which OVA is linked to the C-terminus of VAMP7 via a linker (V7-OVA), the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide in which a proprotein convertase recognition sequence (nucleotide sequence: SEQ ID NO: 21, amino acid sequence: SEQ ID NO: 22) is added to the C-terminus of the linker in the fusion polypeptide (V7-pc-OVA), the amino acid sequence (SEQ ID NO: 47) of a fusion polypeptide in which the amino acid sequence of OVA is inserted into the lysosome-associated protein LAMP (Patent Document 1), or the amino acid sequence of OVA alone (SEQ ID NO: 24) were constructed. The nucleotide sequence of V7-pc-OVA incorporated into the 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 encoding no allergen (OVA) was used as a control. 50 μg of each of the constructed vectors was intramuscularly administered into the thigh muscle of female BALB / c mice, followed by voltage application using an electroporator (NEPA21) for gene transfer. The above treatments were performed on days 0, 7, and 14 after the start of the experiment. Blood samples were collected weekly from the tail vein throughout the experiment, and on day 35, the animals were euthanized and blood was collected. Serum was isolated from the collected blood and antibody titers were measured. For antibody titer measurements, 10 μg / mL OVA (Merck) in DPBS was added to a 96-well ELISA plate (IWAKI) at 100 μL / well to immobilize OVA. The following day, the plate was washed and blocked with 100 μL / well of 1% BSA in DPBS for 1 hour. After washing, the isolated serum was added at 100 μL / well in two-fold serial dilutions. After 2 hours, the serum was removed by washing, and 1 μg / mL anti-mouse IgG, IgG1, or IgG2a antibody (Abcam) was added at 100 μL / well and allowed to stand for 1 hour. Then, 50 μL / well of TMB solution (Abcam) was added for color development. After 10 minutes, 50 μL / well of Stop solution (Abcam) was added to stop the color reaction, and absorbance was measured (450 nm).The maximum dilution factor at which the absorbance was measured was two times or more higher than the absorbance of the control in which DPBS was added instead of serum, and this value was defined as the antibody titer of each sample.

[0158] The results are shown in Figure 6. As in Example 2, a significant decrease in OVA-specific IgG1 antibody titer was observed with 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 titer was observed with VAMP7-pc-OVA. Regarding overall OVA-specific IgG, the antibody titer tended to be higher than with OVA alone at weeks 3 and 4. However, at week 5, VAMP7-pc-OVA showed an increase in antibody titer comparable to that of OVA alone. Furthermore, almost no increase in OVA-specific antibody titer was observed with LAMP[OVA]. The ratio of OVA-specific IgG1 and IgG2a antibody titers in each individual at week 5, calculated as IgG2a / IgG1, was higher with VAMP7-OVA and VAMP7-pc-OVA compared to OVA alone and LAMP[OVA], although no significant difference was observed. These results indicate that the introduction of a proprotein convertase recognition sequence further promotes the production of IgG2a antibodies, inducing or enhancing allergen-specific Th1-type immune responses in vivo. Furthermore, compared to LAMP[OVA], VAMP7-OVA and VAMP7-pc-OVA are superior in allergen-specific IgG antibody production and enhance allergen-specific Th1-type immune responses. This experiment was performed with n=5 per group, and a Tukey test was performed for each group at each week. Significant differences were determined to be p<0.05. Significant differences compared with the Empty group are indicated by *, and compared with the OVA group by †.

[0159] Example 7 ELISPOT Assay Plasmid vectors encoding the amino acid sequence of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 via a linker (SEQ ID NO: 25), the amino acid sequence 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 (SEQ ID NO: 46), or the amino acid sequence of OVA alone (SEQ ID NO: 24) were constructed. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each vector was intramuscularly administered to female BALB / c mice in the thigh muscle, followed by application of voltage using an electroporator (NEPA21) for gene transfer. The above treatments were performed on days 0, 7, and 14 after the start of the experiment. On day 35, the mice were euthanized and their spleens were collected. The collected spleens were mashed on a 40 μm cell strainer in the presence of 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, the supernatant was removed, and then Pharm Lyse solution (BD Biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells contained in the suspension. The suspension was centrifuged again at 200 × g for 5 minutes, the supernatant was removed, and the cells were washed twice with DPBS. CTL-Test Medium (CTL) was added to obtain a splenocyte suspension. 1.0 × 10 7 Splenocytes from the cells were seeded at 100 μL / well on an ELISPOT plate and mixed with 100 μL of CTL-Tes Medium supplemented with OVA protein (Merck) for antigen stimulation at a final concentration of 50 μg / mL. A group without OVA protein (NT) was prepared as a negative control. After 24 hours, ELISPOT assays were performed using an ELISPOT assay kit (CTL) according to the attached protocol to detect cells producing IFNγ and IL-4 specifically 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 allergen-specific immune responses.

[0160] The results are shown in Figure 7. The introduction of a proprotein convertase recognition sequence further increased the number of OVA-specific IFNγ-secreting T cells. Meanwhile, although significant differences were observed between groups in the number of OVA-specific IL-4-secreting T cells under both unstimulated (NT) and OVA-stimulated (OVA) conditions, the IL-4 ratio, calculated as the ratio of individual T cells under each NT and OVA condition, remained unchanged in all groups. These results suggest that the introduction of a proprotein convertase recognition sequence can further enhance allergen-specific Th1 immune responses and cellular immunity. Proprotein convertase is a type of protease that cleaves intracellular precursor and immature proteins, converting them into mature and active forms. Because proprotein convertase is localized in the Golgi apparatus and endosomes, it is thought that by cleaving the VAMP7-pc-OVA fusion protein transported to intracellular vesicles in vivo, it releases OVA into the vesicles, allowing it to more efficiently associate with MHC molecules. In addition, it is possible that the release of OVA encapsulated in exosomes and secreted may have induced an enhanced immune response. This experiment was performed with n = 5 in each group, and a Tukey test was performed in each group under non-stimulated conditions (NT) or OVA-stimulated conditions, and a p < 0.05 result was considered to indicate a significant difference.

[0161] Example 8 ELISPOT Assay Plasmid vectors encoding the amino acid sequence of a fusion polypeptide (V7-OVA) in which OVA was linked to the C-terminus of VAMP7 via a linker (SEQ ID NO: 25), 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 (SEQ ID NO: 46), a fusion polypeptide in which the amino acid sequence of OVA was inserted into the lysosome-associated protein LAMP (Patent Document 1) (LAMP[OVA]) (SEQ ID NO: 47), or the amino acid sequence of OVA alone (SEQ ID NO: 24) were constructed. An empty vector encoding no allergen (OVA) was used as a control. 50 μg of each of the constructed vectors was intramuscularly administered into the thigh muscle of female BALB / c mice, followed by application of voltage using an electroporator (NEPA21) for gene transfer. Seven days after the above treatment, the mice were euthanized and their spleens were collected. The collected spleens were mashed on a 40 μm cell strainer in the presence of 2% FBS-containing DPBS. The resulting suspension was centrifuged at 200 × g for 5 minutes, the supernatant was removed, and then Pharm Lyse solution (BD Biosciences) was added and allowed to stand for 3 minutes to lyse the red blood cells contained in the suspension. The suspension was centrifuged again at 200 × g for 5 minutes, the supernatant was removed, and the cells were washed twice with DPBS. CTL-Test Medium (CTL) was added to obtain a splenocyte suspension. 1.0 × 10 7Splenocytes from the cells were seeded at 100 μL / well on an ELISPOT plate and mixed with 100 μL of CTL-Test Medium supplemented with OVA protein (Merck) for antigen stimulation at a final concentration of 50 μg / mL. A group without OVA protein (NT) was prepared as a negative control. After 24 hours, ELISPOT assays were performed using an ELISPOT assay kit (CTL) according to the attached protocol to detect cells producing IFNγ and IL-4 specifically 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 allergen-specific immune responses.

[0162] The results are shown in Figure 8. Even just one week after vector administration, VAMP7-pc-OVA significantly increased the number of OVA-specific IFNγ-secreting T cells compared with VAMP7-OVA and LAMP[OVA]. Meanwhile, significant differences were observed between groups in the number of OVA-specific IL-4-secreting T cells under both non-stimulated (NT) and OVA-stimulated (OVA) conditions. However, the IL-4 ratio, calculated for each individual under NT and OVA conditions, remained unchanged in all groups. Furthermore, the ratio of IFNγ to IL-4-producing T cells (IFNγ / IL-4) was calculated for each individual, and the IFNγ / IL-4 ratio was significantly increased in V7-pc-OVA compared with V7-OVA and LAMP[OVA] after OVA stimulation. These results indicate that VAMP7-pc-OVA can selectively induce allergen-specific Th1 immunity and cellular immunity without inducing allergen-specific Th2 immune responses from as early as the first week, and that it has a superior immune induction effect compared to conventional techniques. This experiment was performed with n=5 per group, and a Tukey test was performed for each group under non-stimulated conditions (NT) or OVA-stimulated conditions, and a p<0.05 result was considered to indicate a significant difference.

[0163] Example 9: OVA food allergy model efficacy A sensitization mixture was prepared by mixing 100 μg of OVA and 1 mg of Alum adjuvant (Thermo Fisher Scientific) in 200 μL of DPBS and mixing by inversion for 30 minutes. The resulting sensitization mixture was intraperitoneally administered to female BALB / c mice on days 0 and 14. Plasmid vectors encoding the OVA amino acid sequence (SEQ ID NO: 24) alone, the amino acid sequence of a fusion polypeptide in which OVA was linked to the C-terminus of VAMP7 via a linker and a proprotein convertase recognition sequence (V7-pc-OVA) (SEQ ID NO: 46), or the amino acid sequence of a fusion polypeptide in which the OVA amino acid sequence was inserted into LAMP (LAMP[OVA]) (SEQ ID NO: 47) were also prepared. An empty vector (Empty) encoding no allergen (OVA) was also prepared as a control. On days 21, 28, and 35, BALB / c mice were intramuscularly injected with 50 μg of each vector into the thigh muscle, followed by application of voltage using an electroporator (NEPA21) for gene transfer. On days 42, 43, 44, 45, and 46, a challenge mixture (50 mg of OVA in 200 μL of DPBS) was orally administered once daily to induce food allergy symptoms. On day 46, rectal temperatures were measured using a thermometer probe (Natsume Seisakusho Co., Ltd.) before and 15, 30, 45, and 60 minutes after challenge mixture administration. Mice were then euthanized and blood samples were collected. Serum was isolated from the collected blood and OVA-specific IgE concentrations were measured using the LBIS Mouse Anti-OVA-IgE ELISA Kit (Fujifilm Wako Shibayagi Co., Ltd.). 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 plate was left to react at room temperature for 1 hour. After washing three times with washing solution, 100 μL of peroxidase-avidin conjugate was added and the plate was left to react at room temperature for 30 minutes. After washing three times, 100 μL of TMB coloring 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 (main wavelength 450 nm, secondary wavelength 620 nm). The OVA-specific IgE concentration in each serum sample was calculated using a calibration curve obtained from the absorbance values ​​of the standard solutions.

[0164] The results are shown in Figure 9. Compared with the non-OVA-sensitized Empty group (Empty(-)), the OVA-sensitized Empty group (Empty(+)), the OVA-only group (OVA(+)), and the LAMP[OVA] group (LAMP[OVA](+)) showed a significant 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(+)) showed no decrease in body temperature compared with the Empty(-) group, and the decrease in body temperature was significantly suppressed compared with the Empty(+), OVA(+), and LAMP[OVA](+) groups. Furthermore, OVA-specific IgE antibody concentrations were significantly elevated in the OVA(+) group compared with the Empty(-) group, whereas OVA-specific IgE antibody concentrations were significantly reduced in the V7-pc-OVA(+) and LAMP[OVA] groups compared with the OVA(+) group. Considering that the drop in body temperature associated with anaphylaxis is a common phenotype in animal models of allergy, and that blood allergen-specific IgE levels are a common marker of allergic disease progression, these results indicate that VAMP7-pc-OVA has a superior allergy-suppressing effect compared with OVA alone and conventional techniques. Rectal temperature measurements were performed on n = 8 subjects per group. A Dunnett's test was performed at each measurement time, and a significant difference was determined if p < 0.05. Significant differences are indicated by * for comparison with the Empty(-) group and † for comparison with the V7-pc-OVA(+) group. OVA-specific IgE levels were measured on n = 7 subjects in the Empty(+) group and n = 8 subjects in the other groups. A Tukey test was performed on each group, and a significant difference was determined if p < 0.05. For the V7-pc-OVA and LAMP[OVA] groups, the mean values ​​are shown on the dot plots.

[0165] Example 10 OVA blood concentration Plasmid vectors encoding the amino acid sequence (SEQ ID NO: 25) of a fusion polypeptide in which VAMP7 and OVA are linked via a linker (V7-OVA), the amino acid sequence (SEQ ID NO: 46) of a fusion polypeptide in which a proprotein convertase recognition sequence has been added to the end of the linker in the fusion polypeptide (V7-pc-OVA), or the amino acid sequence of OVA alone (SEQ ID NO: 24) were prepared. An empty vector (Empty) encoding no allergen (OVA) was prepared as a control. 50 μg of each vector was intramuscularly administered to female BALB / c mice in the thigh muscle, followed by application of voltage using an electroporator (NEPA21) for gene transfer. Seven days after the treatment, the mice were euthanized and blood was collected. Serum was separated from the collected blood and OVA concentration was measured. OVA concentration was measured using an ITEA ovalbumin (OVA) ELISA kit (ITEA Corporation). 100 μL / well of serum or standard solution was added to a 96-well plate coated with anti-OVA antibody and allowed to react at room temperature for 1 hour. After washing three times with washing solution, 100 μL / well of enzyme-labeled anti-OVA antibody was added and allowed to react at room temperature for 1 hour. After washing three times, 100 μL / well of TMB chromogenic substrate solution was added, and the color reaction was allowed to proceed at room temperature. After 15 minutes, 100 μL / well of reaction stop solution was added to stop the color reaction, and absorbance measurements were performed (main 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 with the group administered with the empty vector (Empty), OVA protein was detected in the blood of mice administered with OVA alone one week after vector administration. On the other hand, compared with OVA alone, the blood OVA protein concentrations of VAMP7-OVA (V7-OVA) and VAMP7-pc-OVA (V7-pc-OVA) were reduced to almost the same level as those of Empty. This result suggests that when OVA alone is administered in vivo, some of the OVA protein expressed in vivo is released into the blood. On the other hand, the blood OVA protein concentrations of V7-OVA and V7-pc-OVA were reduced to the same level as those of Empty, which does not express OVA protein, suggesting that almost no OVA protein was present in the blood at one week. In conventional allergen immunotherapy, it is known that a portion of ingested allergens is released into the blood and captured by allergen-specific IgE antibodies, causing side effects classified as type I allergies, such as anaphylaxis. As shown in these results, if VAMP7, or VAMP7 linked to a proprotein convertase recognition sequence, is used instead of the allergen alone, it is possible that the release of the allergen into the bloodstream can be suppressed, potentially reducing the side effects associated with the above-mentioned treatment, which are classified as type I allergies. This experiment was performed with n=5 per group, and a Tukey test with p<0.05 was considered to indicate a significant difference.

Claims

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 claim 1, wherein the polynucleotide encoding the allergen is linked downstream of the 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. The nucleic acid construct according to claim 1, wherein the SNARE protein is any one selected from the group consisting of VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, and SEC22B.

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

7. An inducer or enhancer of allergen-specific cellular immune response, comprising the nucleic acid construct according to any one of claims 1 to 5 as an active ingredient.

8. A nucleic acid vaccine, comprising the nucleic acid construct according to any one of claims 1 to 5 as an active ingredient.

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

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

11. The nucleic acid vaccine according to claim 8, which is a vaccine for preventing or treating allergy.

12. The nucleic acid vaccine according to claim 8, which is administered parenterally.

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