Composition for reducing the size or volume of target tissue, or kit comprising same

Abstract

The present invention provides a pharmaceutical composition for treating obesity, the composition comprising: one or more viruses selected from the group consisting of yellow fever virus, herpes zoster virus and rubella virus; or genetic materials encoding proteins derived from these viruses. Preferably, the pharmaceutical composition is a vaccine composition. The composition provides an effect of reducing a target tissue, preferably a tissue containing adipocytes, or causing adipocyte death.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application No. 10-2022-0050367 filed on April 22, 2022, and all the contents disclosed in the specification and drawings of the corresponding application are incorporated into this application. The present invention relates to a composition for reducing the size or volume of a target tissue, a medicine box comprising the composition, its use, or a method for treating or improving obesity using the composition. More particularly, it relates to a composition for reducing the size or volume of a target tissue by killing mast cells, inhibiting mast cell accumulation or reducing mast cells. In addition, it includes a medicine box containing the composition, its use, or a method for treating or improving obesity using the composition. Background Art

[0002] Obesity refers to "a condition in which excess body fat accumulates in the body" rather than simply being overweight, and is considered a risk factor for increasing the incidence of various chronic diseases, such as metabolic diseases, including diabetes, cardiovascular diseases, cancer, etc. Obesity, defined as excessive fat accumulation, is accompanied by a loss of metabolic, endocrine, and immune functions of adipose tissue, and this pathological remodeling of adipose tissue is attracting attention as a pathophysiological factor of major metabolic diseases.

[0003] As a drug treatment for obesity, the lipolytic enzyme inhibitor "Orlistat" is used, which plays a role in excreting some fat from the body, and side effects include diarrhea and steatorrhea, etc. In addition, in the case of drugs for treating obesity, they not only have many side effects, but also break down fat in undesirable parts of the female body. Currently, there are no appetite suppressants that can be used safely, and in the case of severely obese patients with complications, gastrointestinal bariatric surgery can be helpful.

[0004] Meanwhile, although technologies have been developed to prevent fat formation, the field of reducing already formed fat cells and reducing the size of adipose tissue still requires much research. Summary of the invention

[0005] Technical issues

[0006] In order to solve the above problems, the present invention provides a novel method or pharmaceutical composition which can reduce the size or volume of a target tissue by being administered into the target tissue.

[0007] One problem to be solved by the present invention is to provide a new composition for reducing target cells, which helps the immune system to attack target cells expressing antigenic proteins, leading to apoptosis of target cells, while not affecting other cells in undesirable areas.

[0008] In addition, the present invention seeks to provide novel uses of the compositions, which can reduce, decrease or induce apoptosis in desired cells or tissues by administering foreign antigens.

[0009] Technical Solution

[0010] One embodiment of the present invention provides a composition, obesity treatment vaccine, treatment method or use for treating obesity using a virus or its fragment. One embodiment of the present invention provides a new pharmaceutical composition (preferably a vaccine composition) for reducing tissue size or volume and a pharmaceutical composition (preferably a vaccine composition) for improving, suppressing or treating obesity, which applies a virus or a genetic material encoding a virus to a target tissue, and when an antigen gene is expressed in a cell constituting the target tissue by the genetic material applied, a pre-existing immunity acts thereon to kill cells. More specifically, the present invention is intended to provide a pharmaceutical composition containing a virus or a genetic material encoding a virus for preventing or treating obesity, a composition for removing subcutaneous fat cells, a pharmaceutical composition (preferably a vaccine composition) for reducing the size or volume of a tissue and a pharmaceutical composition for improving, suppressing or treating obesity. Preferably, the composition may include a vaccine formulation.

[0011] The virus of the present invention includes the virus itself, the protein of virus origin and the genetic material of the antigen of coding virus origin, and may include its fragment, variant or derivative. Preferably, the genetic material may be understood as the genetic material including the antigen of coding virus origin. In particular, the virus may include one or more selected from yellow fever virus, varicella-zoster virus and rubella virus. In addition, the virus belonging to the higher taxonomic group or higher level including these viruses may also be included in the present invention, as long as they do not hinder the purpose of the present invention.

[0012] The inventors of the present invention have determined that the tissue composed of cells can be reduced by killing the cells constituting the target site using an immune response, thereby completing the present invention. In particular, it has been determined through experiments that the present invention can achieve effective removal and / or reduction of fat cells, and through this, can show body shape correction and obesity treatment effects.

[0013] One embodiment of the present invention provides a new use of a pharmaceutical composition comprising genetic material, wherein a virus or a composition comprising the virus is administered to a target tissue, and the antigen gene is expressed in cells constituting the target tissue through the administered genetic material, thereby reducing the size of fat, especially reducing subcutaneous fat cells. The use provides a use in which, after administration of the composition, a pre-existing immune response plays a role in killing corresponding cells, thereby reducing the size or volume of the tissue, and provides a use in improving, inhibiting or treating obesity.

[0014] The inventors of the present invention determined that the cells constituting the target area can be killed by using an immune response via a virus, thereby reducing the tissue, and thus completed the present invention. In particular, it was determined through experiments that the present invention can effectively remove and / or reduce target cells, thereby showing body shape correction and obesity treatment effects. In particular, when the virus is yellow fever virus, herpes zoster virus and / or rubella virus, it is beneficial to achieve the effects of the present invention.

[0015] One embodiment of the present invention is a pharmaceutical composition for use in reducing tissue by administering to a target tissue, wherein the composition may include genetic material encoding a virus, and the virus is preferably at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus. One embodiment of the present invention provides the use of genetic material encoding a virus for killing target cells and reducing target tissue, and more specifically, provides the use of the genetic material for reducing local adipose tissue. Preferably, the genetic material encoding the virus comprises at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus; or its epitope. One embodiment may provide the use of a virus or its epitope for killing target cells or reducing target tissue. Tissue may be understood as tissue comprising fat cells. The immune effect of the present invention may reduce fat cells or reduce the size of fat tissue. For example, fat cells or fat tissue may more preferably be subcutaneous fat tissue. The composition may induce a reduction, reduction or death of fat cells, particularly fat cells distributed in subcutaneous fat cells.

[0016] The inventors of the present invention provide compositions or kits that have fewer side effects than existing fat removal techniques, drugs, etc., and that can anticipate body shape correction effects by targeting only local target areas (e.g., adipose tissue in a specific area). The present invention provides pharmaceutical compositions that act locally on a target area and use immunotherapy to reduce the size or volume of a target tissue. The present invention may preferably provide pharmaceutical compositions, vaccine compositions, or therapeutic vaccine compositions that induce death of adipocytes, particularly subcutaneous adipocytes. The present invention may provide pharmaceutical compositions, vaccine compositions, or therapeutic vaccine compositions that act locally on a target area and use immunotherapy to improve, inhibit, or treat obesity.

[0017] The term "target tissue or target site" used in this specification means a cell group with a structure and function of reducing size or volume. "Body correction or body shape correction" means to reduce the size or volume of tissue by killing or reducing the cells of the constituent tissue (e.g., adipose tissue) accumulated in undesirable parts and having an external slimming and balancing effect. "Local effect" used in this specification means, based on the injection site, causing cell death or reducing the size or volume of tissue within a radius of 10cm, 9cm, 8cm, 7cm, 6cm, 5cm, 4cm, 3cm, 2cm, 1cm, and preferably, it can cause the effect within a range of 3cm.

[0018] When the object is exposed to the antigen of virus origin, immune cells functionally perform the function of phagocytosis and antigen presentation. In one embodiment, the pharmaceutical composition of the present invention can be applied after producing immune environment by intentionally or unintentionally infiltrating foreign substances (such as protein antigens or their epitopes, etc.) to induce immune response. By another example, the pharmaceutical composition can be repeatedly applied in vivo. In another example, the genetic material contained in the pharmaceutical composition of the present invention is applied to induce pre-existing immunity, and then in the case of repeated application of the composition, the fat cells presenting antigens are killed by the material (such as monocytes, neutrophils, natural killer cells, a kind of lymphocytes, cytotoxic T cell immune cells, physiologically active substances, antibodies, complement, etc.) secreted by immunity in vivo, and therefore, the size or volume of adipose tissue can be reduced. The composition can be used as the purpose of body correction or body shape correction, and it can be used as the purpose of body shape correction in local parts. In addition, the composition can be applied to muscle tissue, and is used as the effect (such as botulinum toxin) of improving skin wrinkles, the purpose of body shape correction by muscle contraction, etc. Additionally, it can be used to remove benign or malignant tumors, or to remove warts on the skin.

[0019] The pharmaceutical composition comprises genetic material encoding one or more antigens of viral origin, and the genetic material comprises not only DNA and / or RNA, but also viral vectors. In addition, it comprises antisense nucleotides, mRNA, ssRNA (single-stranded RNA), cDNA, etc. associated with the genetic material. The antigen may also comprise an epitope of an antigen that induces an immune response.

[0020] The inventors of the present invention have determined that when a virus-derived antigen, preferably a virus-derived antigen whose association with obesity is unknown, more preferably the genetic material of a virus-derived antigen, and even more preferably the DNA or RNA of a virus-derived antigen, is administered, an excellent effect of reducing the number of fat cells, reducing the size of adipose tissue, or inhibiting fat accumulation is exhibited. In particular, it has been determined that among viruses, yellow fever virus, herpes zoster virus, and / or rubella virus (which were previously unknown to have any association with obesity) have an excellent effect of reducing the size of fat cells.

[0021] Genetic material of viral origin can be expressed as a viral peptide or complete protein or a portion thereof. For example, some non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantaan virus, Bunga virus, and phlebovirus and Nairovirus); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Herpesviridae (herpes simplex virus, her ... simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes simplex virus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus).Preferably, it may comprise the Rubivirus genus, more preferably Rubella virus (also referred to as rubella virus); preferably the Flavivirus genus, more preferably the Yellow Fever virus; preferably the Varicellovirus genus, more preferably the Varicella-Zoster virus. When the virus is administered, the effect of reducing tissue size is excellent. In addition, when the above virus is administered, there are few side effects and it is safe. The virus is easy to formulate. Even if a small amount of the virus is administered, pre-existing immunity can be stimulated, thereby rapidly producing a lipolytic effect.

[0022] Preferably, the genetic material encoding the virus may comprise a nucleic acid molecule having a nucleic acid sequence consisting of a polynucleotide selected from SEQ ID NOs: 1 to 6. Alternatively, the genetic material may comprise a nucleic acid molecule encoding any one or more peptides selected from SEQ ID NOs: 12 to 14.

[0023] One embodiment of the present invention may provide a pharmaceutical composition, a vaccine composition or a therapeutic vaccine composition comprising a nucleic acid molecule comprising a polynucleotide selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 may include a polynucleotide sequence encoding at least one virus selected from yellow fever virus, varicella-zoster virus and rubella virus (or a protein derived from a virus), followed by a capping sequence, a 5'-UTR sequence, and a Kozak sequence. Unless otherwise specified, a 3'-UTR sequence, a poly A tail and a primer may be connected after the polynucleotide sequence encoding the virus (or a protein derived from a virus), and these are connected in a 5' to 3' direction. See Figures 16 to 18 .

[0024] In addition, the genetic material may include a nucleic acid sequence consisting of: a polynucleotide selected from SEQ ID NO: 1 to 6; or a polynucleotide having a nucleic acid sequence having at least 85% sequence homology with the nucleic acid sequence and capable of reducing the size or volume of the target tissue, decomposing fat, or treating, improving or preventing obesity (i.e., the present invention can achieve this purpose). The sequence homology may include 85% or more, 90% or more, 95% or more, or 99% or more sequence homology. Alternatively, the genetic material may include a polynucleotide having a nucleic acid sequence having at least 85% sequence homology with a nucleic acid molecule encoding any one or more peptides selected from SEQ ID NO: 12 to 14 and capable of reducing the size or volume of the target tissue (i.e., the present invention can achieve this purpose). The sequence homology may include 85% or more, 90% or more, 95% or more, 99% or more, or 100% sequence homology.

[0025] The composition may also include genetic material encoding at least one cytokine selected from IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β, and it preferably includes IL-12 to further maximize the immunostimulatory effect of the antigen expressed by the genetic material. For example, IL-12 may include a DNA sequence consisting of SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0026] In a preferred embodiment, the viral genetic material can be included in a plasmid (eg, a gWiz™ vector) such that the plasmid is capable of expressing genes corresponding to viral structural proteins in animal cells.

[0027] In a preferred embodiment, the virus may include yellow fever virus, for example, but not limited to, the yellow fever virus may include the 17D strain. The plasmid may include a nucleic acid molecule encoding a virus or a fragment, variant, or analog thereof. For example, it may include SEQ ID NO: 1 or SEQ ID NO: 2; or a fragment, variant, or analog thereof.

[0028] The nucleic acid molecule encoding the virus may include a nucleic acid molecule encoding a structural protein. For example, it may include a nucleic acid molecule encoding the protein of SEQ ID NO: 12 or a fragment, variant or analog of the protein of SEQ ID NO: 12.

[0029] In a preferred embodiment, the viral genetic material may be included in a plasmid (e.g., a gWiz™ vector) such that the plasmid is capable of expressing proteins corresponding to genes for Rubella virus structural proteins in animal cells. Rubella virus may be used without limitation, and for example, Rubella virus may include, but is not limited to, the RA27 / 3 strain. The gene encoding the virus may include a gene encoding a spike glycoprotein. The plasmid may include a nucleic acid molecule encoding the virus or a fragment, variant, or analog thereof. For example, it may include SEQ ID NO:5 or SEQ ID NO:6; or a fragment, variant, or analog thereof.

[0030] Some examples of spike glycoproteins may include E2-E1 hybrids. For example, nucleic acid molecules encoding the protein of SEQ ID NO: 14 or encoding a fragment, variant or analog of the protein of SEQ ID NO: 14 may be included.

[0031] In a preferred embodiment, the composition may include genes corresponding to structural proteins of Rubella virus genes in a plasmid, such that the plasmid is capable of expressing the protein in animal cells (e.g., gWiZ TM Vector). Rubella virus can be used without limitation, and for example, Rubella virus can include RA27 / 3 strain, but is not limited thereto. The gene encoding the virus can include a gene encoding a spike glycoprotein. Examples of spike glycoproteins can include E2-E1 hybrids. For example, it can include a nucleic acid molecule encoding a protein of SEQ ID NO: 5 or a fragment, variant or analog of a protein of SEQ ID NO: 5.

[0032] In a preferred embodiment, the composition may include genes corresponding to structural proteins of varicella-zoster virus genes in a plasmid, such that the plasmid is capable of expressing the protein in animal cells (e.g., gWiZ TM Vector). Varicella-zoster virus can be used without limitation, and for example, varicella-zoster virus can include Oka strain, but is not limited thereto. The above plasmid can include a nucleic acid molecule encoding a virus or a fragment, variant or analog thereof. For example, it can include SEQ ID NO:3 or SEQ ID NO:4; or a fragment, variant or analog thereof.

[0033] The nucleic acid molecule encoding the virus may include a nucleic acid virus encoding the surface protein gE protein. For example, it may include a nucleic acid molecule encoding the protein of SEQ ID NO: 13 or a fragment, variant or analog of the protein of SEQ ID NO: 13.

[0034] Here, the term "fragment, variant or analog thereof" can be understood to mean a portion of a protein, peptide, nucleic acid, nucleic acid molecule or gene, which achieves the intended purpose of the protein, peptide, nucleic acid, nucleic acid molecule or gene of the present invention, but may have a sequence homology of 85% or more, 90% or more, 95% or more or 99% or more. For example, the meaning of comprising a fragment, variant or analog can be understood to mean that if it comprises a nucleic acid molecule consisting of any one or more polynucleotides selected from SEQ ID NO: 1 to 6, it is not excluded from the scope of the present invention.

[0035] The virus can have an excellent effect of killing target tissue (preferably fat cells) and reducing tissue size. In addition, this effect can be quickly manifested and has little side effects. In addition, even if it is stored for a long time as an injection, there is almost no risk of deterioration.

[0036] In another embodiment, the composition can also enhance the effect of adipocyte death or reduce target tissue by including an immunopotentiator that can enhance cellular immune response. The composition can also include genetic material encoding an immunopotentiator that can improve the immunopotentiating effect, such as auxiliary genetic material encoding interleukin 12 (IL-12). The auxiliary genetic material can be provided by, for example, inserting the coding region into the pSF-CMV-CMV-Sbf1 vector.

[0037] The composition of the present invention can be used not only with cytokines, genes encoding the cytokines or other immunoadjuvants, but also with pharmaceutical carriers or excipients widely used in the art. The composition can be formulated for human or veterinary use and applied in a variety of ways. As a route of administration, oral, transdermal, intramuscular, peritoneal, intravenous, subcutaneous or intranasal routes can be used, but are not limited thereto. More preferably, transdermal, intramuscular, peritoneal or subcutaneous routes can be used. In addition, the composition can be applied by any device or approach in which the active substance can be moved to the target cell, and the method of application is not particularly limited, as long as the composition of the present invention or the genetic material contained in the composition can be delivered to the target tissue or the cell constituting the tissue. For example, it can be formulated into an injection, a microneedle patch, etc. and provided as an injection, a microneedle patch, etc. For example, when used as an injection, the following can be used to prepare the injection within the range that does not hinder the action of the genetic material: aqueous solvents (e.g., physiological saline solution, Ringer solution, etc.), non-aqueous solvents (e.g., vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerol, etc.)), etc., and may contain drug carriers, such as stabilizers for preventing deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for adjusting pH, preservatives for preventing microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.), etc. When used as a microneedle patch, the microneedles may include both insoluble microneedles and soluble microneedles.

[0038] In addition, the genetic material and / or auxiliary genetic material contained in the composition can be delivered to the body according to the gene delivery method commonly used in the art. As a non-limiting example, gene delivery methods such as physical delivery methods such as electroporation or gene guns and chemical delivery methods such as lipid-gene complexes (lipid-DNA complexes: Lipoplex), polymer-gene complexes (polymer-DNA complexes: Polyplex), liposomes, dendrimers, nanoparticles or other suitable transfer vectors can be used.

[0039] The composition of the present invention is administered in a pharmaceutically effective dose. The term "pharmaceutically effective dose" refers to an amount sufficient to make the composition show a therapeutic, ameliorative or preventive effect or to show a vaccine effect, and in addition, it means an amount sufficient not to cause side effects or a serious or excessive immune response. Due to the characteristics of the purpose of inducing an immune response and inducing cell death, the composition of the present invention can be administered together with genetic material, and preferably administered 4 to 8 times, preferably 5 times, at intervals of 2 to 3 days. When administered as described above, the effect of reduction or reduction of tissue can be obtained by cell death at the target site. When the composition is administered in large quantities or repeatedly, the composition may have differences in cell death effects, and can be used by adjusting the appropriate dose for each site.

[0040] Another embodiment of the present invention provides a method for reducing or decreasing the volume size of tissue, which comprises applying genetic material encoding an antigen, preferably a virus (e.g., yellow fever virus, herpes zoster virus, or rubella virus), a vector comprising the genetic material, or a composition comprising the same to a target tissue of a subject. The present method can effectively kill cells constituting subcutaneous fat cell tissue, reduce fat weight, or reduce cell size. i) Method; or ii) Genetic material encoding a virus or a protein derived from a virus, a vector comprising the genetic material, or a composition comprising the same, or a kit comprising the same can be used for improving or treating obesity in a subject, for improving wrinkles, for reducing muscle, for removing or reducing benign or malignant tumors, for removing warts, etc., and can be used for removing fat bags under the eyes for cosmetic purposes, and can be used for thereby reducing dark circles.

[0041] The method may also include constructing an immune environment in vivo before administering the genetic material, a vector comprising the genetic material, or a composition comprising the genetic material to the target tissue. Constructing an immune environment in vivo may include administering a foreign substance capable of inducing an immune response. The foreign substance may include any substance as long as it uses a protein as an antigen without limitation.

[0042] One embodiment of the present invention may be provided as a use for treating, ameliorating or preventing a variety of indications, and may also be provided for cosmetic purposes for improving appearance.

[0043] One embodiment of the present invention may be provided as a vaccine for treating obesity. A therapeutic vaccine may be defined as a vaccine that utilizes an immune response in the body, and may be defined as a therapeutic vaccine that affects already produced fat, fat cells, or fat tissue to reduce their size or kill cells. Therefore, a vaccine for treating obesity may be understood as a different concept from a preventive vaccine that simply inhibits the formation of fat cells or inhibits fat accumulation.

[0044] Another example of the present invention provides a kit comprising (a) a vaccine composition for reducing the size or volume of a target tissue, the vaccine composition comprising at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus; a protein derived from the virus; or a genetic material encoding the virus or protein, preferably comprising at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus or a genetic material encoding the virus or protein, and (b) an administration guide for administering the vaccine. The kit may also comprise (c) a composition containing a protein antigen or an epitope thereof for constituting an immune environment prior to administering the composition (a). Herein, (c) may be interpreted as at least one pre-vaccine or composition for inducing an immune response.

[0045] The "prevaccine" mentioned herein may be understood as a vaccine composition that is administered to obtain pre-existing immunity before administering the "pharmaceutical composition for improving, inhibiting or treating obesity" or "composition for reducing the size or volume of a target tissue" of the present invention. The prevaccine may contain the same antigenic substance as the antigenic substance contained in the "pharmaceutical composition for improving, inhibiting or treating obesity" or "composition for reducing the size or volume of a target tissue" of the present invention, but is not limited thereto. For example, (a) the "pharmaceutical composition for improving, inhibiting or treating obesity" or "composition for reducing the size or volume of a target tissue" containing genetic material encoding a virus, preferably yellow fever virus, herpes zoster virus and / or rubella virus may contain at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus, and (c) may also contain at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus. As another example, (a) a “pharmaceutical composition for improving, inhibiting or treating obesity” or a “composition for reducing the size or volume of a target tissue” comprising genetic material encoding a virus, preferably at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus may comprise at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus, and (c) may comprise a vaccine administered to infants to induce antibody retention in the body.

[0046] The above composition (a) may also include genetic material encoding cytokines, and may include, but is not limited to, materials that can be used to improve the immunological effect of the above composition (a). The genetic material encoding cytokines may be replaced by proteins expressed by the genetic material. The cytokine may include at least one selected from the following: IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β, and in addition to the above cytokines, may include, but is not limited to, cytokines known in the art.

[0047] The medicine box of the present invention may include at least one other medicament as defined in this specification sheet about pharmaceutical composition, antimicrobial, DNA enzyme inhibitor, RNA enzyme inhibitor, solubilizing agent, etc. The medicine box may, for example, be composed of at least one medicine box part (e.g., container). For example, each container may be a syringe, a pre-filled syringe, a vial, a bottle, a jar, a sealing sleeve, an envelope or a pouch, a tube or a blister pack or any other suitable form wherein the container is configured to prevent premature mixing of elements. Each of the different elements may be provided separately, or some of the different elements may be provided together (i.e., in the same container). The medicine box may also include an application guide having information about the application, method of application and dosage of any component.

[0048] Another embodiment of the present invention provides a syringe filled with a composition for improving, inhibiting or treating obesity, preferably a vaccine composition, the syringe comprising genetic material encoding a virus or viral protein. Another embodiment of the present invention provides a syringe filled with a pharmaceutical composition for reducing the size or volume of a target tissue, preferably a vaccine composition, the syringe comprising genetic material encoding a virus or viral protein. Preferably, the syringe is filled with a composition for improving, inhibiting or treating obesity, preferably a vaccine composition, the syringe comprising at least one virus selected from yellow fever virus, varicella-zoster virus and rubella virus; or genetic material encoding a protein of viral origin; or a vaccine composition for reducing the size or volume of a target tissue. The syringe may also contain genetic material encoding a cytokine. The cytokine may include at least one selected from the following: IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β.

[0049] One embodiment of the present invention is to provide a pharmaceutical composition for improving, inhibiting or treating obesity, the pharmaceutical composition comprising genetic material encoding at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus; or a protein derived from the virus.

[0050] The pharmaceutical composition can reduce the size or volume of a target tissue.

[0051] The genetic material may be a polynucleotide encoding at least one protein selected from the group consisting of a structural protein of yellow fever virus, glycoprotein E of herpes zoster virus, and E2 and E1 proteins of rubella virus.

[0052] The polynucleotide may be DNA or RNA, and preferably may be DNA or mRNA.

[0053] The genetic material may comprise a nucleic acid molecule consisting of one or more polynucleotides selected from SEQ ID NOs: 1 to 6.

[0054] The pharmaceutical composition may also contain genetic material encoding a cytokine.

[0055] The cytokine may be one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α, and TNF-β. For example, the genetic material may comprise polynucleotide DNA sequences represented by SEQ ID NOs: 7 and 8, respectively.

[0056] Genetic material can induce an intracellular immune response.

[0057] The composition acts locally and can be administered to a subject after first administering to the subject at least one antigen for generating an immune environment.

[0058] Antigens may include viruses, proteins or epitopes thereof introduced from outside the human body. Preferably, the genetic material may be administered orally, injected transdermally, intramuscularly, intraperitoneally, intravenously, subcutaneously or intranasally, or administered to the target tissue by electroporation, gene guns, liposomes, dendrimers, nanoparticles or transfer vectors.

[0059] The dosage of the genetic material contained in the composition may be 0.1 to 1000 ug / site per one-time inoculation.

[0060] The composition is administered to an individual for the purpose of reducing or shrinking the size of or killing adipose tissue, and for the purpose of reducing or shrinking the size of or killing adipose tissue, the composition can be applied to the site at least once after administration of a vaccine for preventing any one or more viruses selected from yellow fever virus, herpes zoster virus and rubella virus, after determining that antibodies against at least one virus have been produced.

[0061] The composition can be provided as a vaccine (eg, a therapeutic agent, a therapeutic vaccine, or a prophylactic vaccine).

[0062] Another embodiment may provide a kit for providing the composition. In addition to the composition, the kit may also include instructions for administering the composition.

[0063] The kit may also include an additional composition containing an antigen for generating an immune environment in vivo prior to administration of the composition. The kit may also include genetic material encoding a cytokine.

[0064] Another embodiment may provide a pharmaceutical composition for reducing the size or volume of a target tissue, the pharmaceutical composition comprising at least one virus selected from yellow fever virus, varicella-zoster virus, and rubella virus; or genetic material encoding a protein derived from a virus.

[0065] The pharmaceutical composition may reduce the number of adipocytes or reduce the size of adipocyte tissue, or induce cell or tissue death.

[0066] According to one embodiment, the present invention provides a method for reducing the size or volume of a target tissue by administering a composition to a subject in need thereof. Another embodiment provides a method for inhibiting, improving or treating obesity by administering a composition to a subject in need thereof. The subject may include, but is not limited to, mammals, such as dogs, cattle, pigs, horses, humans, etc., and preferably humans.

[0067] In one embodiment, for the purpose of reducing, shrinking or killing adipose tissue, the method can provide a method for improving, inhibiting or treating obesity in a subject, or a method for reducing, shrinking or killing adipose tissue, the method comprising the following steps: i) administering a vaccine for preventing at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus, ii) determining antibodies produced by the vaccine, and iii) administering a pharmaceutical composition comprising: at least one virus selected from yellow fever virus, herpes zoster virus and rubella virus; a protein derived from a virus; or genetic material encoding a virus or a protein. The above method can repeat step iii) 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 times or more, preferably 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 times or more, more preferably 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 times or more. Preferably, step iii) can be repeated at intervals of 1 to 10 days, at intervals of 2 to 8 days, or at intervals of 3 to 7 days.

[0068] The composition used in step iii) above may also contain one or more cytokines, preferably, the cytokines may contain one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β. As another alternative, step iii) above may include step iv) administering a composition comprising one or more cytokines, preferably, the cytokines may contain one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β. That is, the cytokines may be administered together or sequentially with a composition containing the genetic material of the virus or virus-derived protein of the present invention.

[0069] One embodiment of the present invention provides a method of improving or therapeutically improving a viral portion selected from yellow fever virus, herpes zoster virus, and rubella virus; a viral protein; or a material encoding a virus. Another example provides the use of a composition comprising the following for reducing the size or volume of a target tissue: at least one virus selected from yellow fever virus, varicella-zoster virus, and rubella virus; a protein derived from the virus; or genetic material encoding the virus or protein.

[0070] The above uses may be used for the purpose of reducing the size or volume of a target tissue. Preferably, the target tissue may include a tissue containing fat cells.

[0071] The genetic material may be a polynucleotide encoding at least one protein selected from the group consisting of a structural protein of yellow fever virus, glycoprotein E of varicella-zoster virus, and E2 and E1 proteins of rubella virus.

[0072] The polygon may comprise DNA or RNA, and the RNA may alternatively comprise mRNA.

[0073] The genetic material may comprise a nucleic acid molecule consisting of one or more polynucleotides selected from SEQ ID NOs: 1 to 6.

[0074] The genetic material can be used to induce an immune response within a cell.

[0075] The composition may be administered to act locally, and may be administered to a subject after first administering to the subject at least one antigen for generating an immune environment.

[0076] In one embodiment, the composition is administered to a subject to reduce the size, decrease the size, or kill adipose tissue, and for the purpose of reducing the size, decreasing the size, or killing adipose tissue, the composition can be applied to the area at least once after administering a vaccine for preventing any one or more viruses selected from yellow fever virus, herpes zoster virus, and rubella virus and determining that antibodies against any one or more viruses are produced.

[0077] Beneficial Effects

[0078] The present invention provides an effect of reducing the size or volume of tissue in a specific target area. The present invention provides a novel composition or therapeutic vaccine that can improve or treat obesity. The composition has the ability to destroy cells in a specific target tissue, resulting in a reduction in the size or volume of the tissue. By administering the composition, a body shaping effect or body improvement effect can be obtained.

[0079] The present invention can provide a novel obesity treatment which can resolve local obesity and achieve smooth body lines.

[0080] The composition for improving, inhibiting or treating obesity of the present invention is effective for selective local site reduction, allowing selective reduction of tissue only in desired areas without affecting tissues in undesirable areas or cells constituting them.

[0081] Additionally, the compositions of the present invention may be used for cosmetic purposes to improve appearance. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 A vector map of the gWiz™ vector used to insert the genetic material of the present invention is shown. The gene is inserted into the restriction enzyme sites SalI and NotI.

[0083] Figure 2 The vector map of the pSF-CMV-CMV-Sbf1 vector is shown, which is used to insert nucleic acid sequences corresponding to the coding regions of IL-12p35 (GenBank: M86672.1) and p40 (GenBank: M86671.1) (p35; 127 to 774 bp, p40; 35 to 1042 bp). IL-12p35 is inserted into the restriction enzyme sites EcoRI and XhoI in MCS site I, while IL-12p40 is inserted into the restriction enzyme sites SalI and SpeI in MCS site II.

[0084] Figure 3 Vectors used for in vitro transcription (IVT) are shown.

[0085] Figure 4Results of inducing pre-existing immunity by administering YFV vaccine strain (live attenuated virus) to mice are shown.After three administrations of the vaccine strain at two-week intervals, the levels of YFV-specific IgG antibodies in the blood were measured two weeks after the last administration.

[0086] NC (negative control) indicates normal mice receiving medium only, YFV-ND indicates YFV-immunized normal mice fed a normal diet, and YFV-HFD indicates YFV-immunized obese mice fed a high-fat diet.

[0087] Figure 5 Also shown is the result of inducing pre-existing immunity by administering YFV vaccine strain (live attenuated virus) to mice.After administering the vaccine strain twice at intervals of two days, the YFV-specific IgG antibody level in the blood was measured three weeks later.Before vaccination refers to some individuals before administering YFV, and after vaccination refers to obese mice of YFV immunization fed with a high-fat diet.

[0088] Figure 6 and Figure 7 Results of inducing pre-existing immunity by administering a VZV vaccine strain (live attenuated virus) to mice are shown. After two administrations of the vaccine strain at four-week intervals, VZV gE protein-specific IgG antibody levels in the blood were measured three weeks later. Before vaccination refers to individuals before administration of VZV, while after vaccination refers to VZV-immunized obese mice fed a high-fat diet.

[0089] Figure 8 and Fig. 9 Shown are the results following administration of a DNA vaccine against YFV. Figure 8 Based on the body weight before the YFV genetic material (DNA) was to be administered, the body weight change before and after the administration was measured. Fig. 9 The weight loss effect of inguinal adipose tissue (Ing AT) and interscapular adipose tissue (Ins AT) due to treatment containing YFV genetic material (DNA) is shown.

[0090] Fig.10 and Fig.11 Shown are the results following administration of a DNA vaccine for VZV. Fig.10 Based on the body weight before the VZV genetic material (DNA) was to be administered, the change in body weight before and after administration was measured. Fig.11 The weight-reducing effect on inguinal adipose tissue due to administration of a vaccine containing VZV genetic material (DNA) is shown.

[0091] Fig.12 and Fig.13Shown are the results after administration of an mRNA vaccine against YFV. Fig.12 Based on the body weight before the YFV genetic material (mRNA) was to be administered, the body weight change before and after the administration was measured. Fig.13 The weight-reducing effect on inguinal adipose tissue due to treatment containing YFV genetic material (mRNA) is shown.

[0092] Fig.14 and Fig.15 Shown are the results following administration of an mRNA vaccine against VZV. Fig.14 Based on the body weight before the VZV genetic material (mRNA) was to be administered, the change in body weight before and after administration was measured. Fig.15 The weight-reducing effect on inguinal adipose tissue due to administration of a vaccine containing VZV genetic material (mRNA) is shown.

[0093] Figures 16 to 18 The mRNA sequences of SEQ ID NOs. 2, 4 and 6 are shown respectively. Here, 1 refers to the capping sequence, 2 refers to the 5'-UTR sequence, 3 refers to the Kozak sequence, 5 refers to the 3'-UTR sequence, 6 refers to the poly A sequence, and 7 refers to the primer sequence. The unmarked segment (4) represents the GOI (Gene Of Interest) sequence. Fig.16 Includes genes encoding structural proteins of yellow fever virus, Fig.17 including the gene for the gE protein of the varicella-zoster virus, and Fig.18 Includes genes encoding the E2-E1 proteins of RuV. DETAILED DESCRIPTION

[0094] Hereinafter, in order to help understand the present invention, it will be described in detail by examples etc. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be interpreted as being limited to the following examples. The embodiments of the present invention are provided to more fully illustrate the present invention to those skilled in the art. Unless otherwise stated, all sequences are described in a 5' to 3' direction.

[0095] 1. Preparation of vaccines (treatment) for degrading fat cells

[0096] For this experiment, genetic material to be administered to individuals with pre-existing immunity and an immunological adjuvant that can be administered with the genetic material were prepared as follows:

[0097] (1) Preparation of foreign antigens

[0098] 1) Preparation of genetic material of yellow fever virus (Yellow fever virus (strain: 17D); YFV)

[0099] First, genetic materials (SEQ ID NOs. 1 and 2) encoding structural proteins (capsid protein, prM protein, envelope protein, positions 119 to 2452 (2,334 bp)) were prepared from the YFV genome (GenBank: X03700.1).

[0100] 2) Preparation of genetic material of varicella-zoster virus (Varicella zoster virus (strain: Oka); VZV)

[0101] The genetic material (SEQ ID NOs. 3 and 4) encoding the gE protein (1,872 bp) was prepared from the VZV genome.

[0102] 3) Preparation of genetic material of Rubella virus (Rubella virus (strain: RA27 / 3); RuV)

[0103] The genetic material encoding the E2-E1 protein (2,358 bp) was prepared from the RuV genome (SEQ ID NOs. 5 and 6).

[0104] 4) Preparation of genetic material encoding firefly luciferase (FLuc) (SEQ ID NO. 15).

[0105] (2) Preparation of vectors expressed in animal cells (DNA vaccine therapy)

[0106] The genetic material of YFV was inserted into the gWiz that allows the protein to be expressed in animal cells. TM The Kozak sequence (GCCACC) was added to the front of the gene, and a stop codon was added to the end of the gene. TM The RuV gene is inserted into the vector and a Kozak sequence is added at the front end of the gene. TM The vector was modified to add a Kozak sequence (GCCACC) and a start codon (ATG) to the front end of the gene.

[0107] The vector map is Figure 1 The gene was inserted into the restriction enzyme sites of SalI and NotI.

[0108] The cloned YFV antigen plasmid (vector backbone: gWiZ TM)、VZV antigen plasmid (vector backbone: gWiz TM ) and RuV antigen plasmid (vector backbone: gWiz TM ) was transformed into DH5α competent cells. The cells were then cultured in large quantities and the plasmid was recovered using a plasmid preparation kit.

[0109] (3) Preparation of auxiliary genetic material

[0110] Next, prepare an immune adjuvant as auxiliary genetic material. The gene sequences (SEQ ID NOs. 7 and 8) of the coding regions of IL-12 p35 (GenBank: M86672.1) and p40 (GenBank: M86671.1) (p35; 127 to 774 (648 bp), p40; 35 to 1042 (1,008 bp)) were inserted into the pSF-CMV-CMV-Sbfl vector together with the kozak sequence. P35 was inserted into the EcoRI and XhoI sites, and p40 was inserted into the SalI and SpeI sites. The vector map is in Figure 2 Shown in.

[0111] The cloned IL-12 plasmid (vector backbone: pSF-CMV-CMV-Sbfl) was transformed into DH5a competent cells, the cells were cultured in large quantities, and the plasmid was recovered using a plasmid preparation kit.

[0112] (4) Preparation of vectors for in vitro transcription (IVT)

[0113] The genetic material of YFV was inserted into pcDNA by cloning TM 3.1(+) between the T7 promoter sequence and the bGH poly(A) signal sequence. The 5' untranslated region sequence and Kozak sequence of the Homo sapiens PPARG-related coactivator 1 (PPRC1) gene were added to the front of the gene, and the termination codon (TGA), the 3' untranslated region sequence of the Homo sapiens α-1-globin gene, the poly A sequence, and the Esp3i restriction site sequence were added to the end of the gene. The vector map is in Figure 3 Shown in.

[0114] The genetic material of VZV was inserted into pcDNA3.1 by cloning TM The 5' untranslated region sequence and Kozak sequence of the Homo sapiens PPARG-related coactivator 1 (PPRC1) gene were added to the front of the gene, and the 3' untranslated region sequence, poly A sequence and Esp3i restriction site sequence of the Homo sapiens α-1-globin gene were added to the end of the gene.

[0115] The RuV genetic material was inserted into pcDNA3.1 by cloning TM The 5' untranslated region sequence, Kozak sequence and start codon of the Homo sapiens PPARG-related coactivator 1 (PPRC1) gene were added to the front of the gene, and the 3' untranslated region sequence, poly A sequence and Esp3i restriction site sequence of the Homo sapiens α-1-globin gene were added to the end of the gene.

[0116] The genetic material encoding FLuc was inserted into the pcDNA by cloning TM 3.1(+) between the T7 promoter sequence and the bGH poly(A) signal sequence. The 5' untranslated region sequence and Kozak sequence of the Homo sapiens PPARG-related coactivator 1 (PPRC1) gene were added to the front end of the gene, and the stop codon, the 3' untranslated region sequence of the Homo sapiens α-1-globin gene, the poly A sequence and the Esp3i restriction site sequence were added to the end of the gene.

[0117] The cloned YFV antigen plasmid (vector backbone: pcDNA TM 3.1(+)), VZV antigen plasmid (vector backbone: pcDNA TM 3.1(+)), RuV antigen plasmid (vector backbone: pcDNA TM 3.1(+)) and FLuc plasmid (vector backbone: pcDNA TM 3.1(+)) was transformed into DH5α competent cells. The cells were cultured in large quantities and the plasmid was recovered using a plasmid preparation kit.

[0118] (5) Genetic material expressed in animal cells prepared by IVT (mRNA vaccine therapeutics)

[0119] YFV antigen plasmid (vector backbone: pcDNA3.1 TM (+))、VZV antigen plasmid (vector backbone: pcDNA TM 3.1(+)) and RuV antigen plasmid (vector backbone: pcDNA TM 3.1(+)) was used as a template for PCR to prepare linearized DNA (SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11). The primer information is shown in Table 1 below.

[0120] Table 1.

[0121]

[0122] Use FLuc plasmid (vector backbone: pcDNA TM 3.1(+)) as a template, PCR was performed to induce a frameshift of 12 bp downstream of the start codon, and a linearized DNA with a stop codon was prepared (SEQ ID NO. 15). The primer information is shown in Table 2 below.

[0123] Table 2.

[0124]

[0125] IVT was performed using linearized DNA as a template. Subsequently, DNase treatment was performed and the mRNA was precipitated and purified using LiCl. The prepared mRNA was transfected into the Vero E6 cell line to determine antigen expression. It was determined that the FLuc mRNA containing a stop codon did not result in protein expression before it was used.

[0126] 2. Determination of Adipocyte Death

[0127] (1) Experimental methods

[0128] 1) Diet-induced obese (DIO) mice

[0129] C57BL / 6J, 3w, female mice were fed a normal diet and a high-fat diet containing 60% kcal from fat, respectively. Body weight was measured at weekly intervals, and the animals were housed for 10 to 15 weeks to examine body weight gain before being used in experiments.

[0130] C57BL / 6J, 4w, male mice were fed a high fat diet containing 60% kcal from fat. Body weight was measured at weekly intervals, and the animals were housed for 10 to 15 weeks to examine body weight gain before being used in experiments.

[0131] 2) Induction of pre-existing immunity and measurement of antibody titers

[0132] Attenuated live viruses were used as vaccine strains to induce pre-existing immune responses. YFV (strain: 17D, GenBank: X03700.1) and VZV (strain: Oka, Sky Zoster strain (SK Bioscience)) were used as vaccine strains.

[0133] (i) YFV immunization: YFV vaccine strain (attenuated live virus) was injected into the muscle of the left hind limb of mice at a concentration of 2E5 pfu / time, 3 times at intervals of 2 weeks. Blood was collected 2 weeks after the last injection, and serum was separated.

[0134] Determination of antibody titer: In the separated serum, antibodies specific to YFV were detected by ELISA analysis. By ELISA analysis, YFV for injection was coated on the plate at a concentration of 5E4pfu / well, and the analysis sample (serum) was diluted and reacted for 2 hours. After washing, the anti-mouse IgG-HRP secondary antibody was diluted and processed at 1 / 4000, and then reacted for 2 hours. After washing, a color formation solution was added and reacted for 10 minutes to measure the OD value.

[0135] Results: Normal immune induction (antibody response) occurred by administration of the YFV vaccine.

[0136] The results are Figure 4 As shown in FIG. , an increase in YFV-specific antibodies was confirmed in the YFV-ND group and the YFV-HFD group administered with YFV, compared with the negative control (NC) group administered with the medium.

[0137] The induction of YFV-specific pre-existing immunity was determined for therapeutic administration.

[0138] (ii) YFV immunization (2): YFV (live attenuated virus) was administered twice to the left hind limb muscle of mice at 2-day intervals 3 weeks before the start of treatment administration (intramuscular injection, 1st injection: 2E4 pfu / injection, 2nd injection: 4E4 pfu / injection). An additional YFV immunization was administered 2 to 3 days before treatment administration, i.e. 3 weeks after the 1st injection (intramuscular injection, 3rd injection: 1E5 pfu / injection).

[0139] Determination of antibody titer: Blood samples were collected and serum was separated before and 3 weeks after the first injection of YFV. For ELISA analysis, YFV for injection was coated on the plate at a concentration of 1E4 pfu / well, blocked with 2% skim milk, and then the diluted serum was reacted with the antigen for 1 hour. After washing, the anti-mouse IgG-HRP secondary antibody was diluted 1 / 1000 and reacted for 1 hour. After washing, the color development solution was added and reacted for 10 minutes, and the OD value was measured.

[0140] Results: Normal immune induction (antibody response) occurred by administration of the YFV vaccine strain. Figure 5 The increase in YFV-specific antibodies was determined in sera collected after two YFV immunizations (post-vaccination) compared to sera before YFV immunization (pre-vaccination). The induction of YFV-specific pre-existing immunity was determined for therapeutic administration.

[0141] (iii) VZV immunization: Mice were immunized twice with VZV (live attenuated virus) at 4-week intervals. (Subcutaneous injection, 1st injection: 9E3 pfu / injection, 2nd injection: 9E3 pfu / injection). An additional VZV immunization was performed one week before treatment administration. (Subcutaneous injection, 3rd injection: 9E3 pfu / injection)

[0142] Determination of antibody titer: Blood was collected before administration of VZV and 3 weeks after administration of the second injection and serum was separated. The separated serum was analyzed by ELISA to detect VZVgE protein-specific antibodies. For ELISA analysis, recombinant VZVgE protein was coated on the plate at a concentration of 100 ng / well, blocked with 2% skim milk, and then the diluted serum was reacted with the antigen for 1 hour. After washing, the anti-mouse IgG-HRP secondary antibody was diluted 1 / 1000 and reacted for 1 hour. After washing, the color development solution was added and reacted for 10 minutes to measure the OD value.

[0143] Results: Normal immunity (antibody response) occurred with the administration of the VZV vaccine. Figure 6 and Figure 7 An increase in VZV gE protein-specific antibodies was determined in sera collected after two VZV immunizations (post-vaccination) compared to sera collected before VZV immunization (pre-vaccination). The induction of VZV-specific pre-existing immunity was determined for therapeutic administration.

[0144] (iv) RuV immunization: Experimental animals were immunized 2 to 3 times with MMR vaccine (a vaccine containing live attenuated RuV virus) at 4-week intervals (subcutaneous injection)

[0145] Determination of antibody titer: Blood samples were collected before MMR vaccination and 3 weeks after the second vaccination and serum was separated. The RuV E1 protein-specific antibodies in the separated serum were detected by ELISA analysis. For ELISA analysis, recombinant RuV E1 protein was coated on the plate at a concentration of 100 ng / well, blocked with 2% skim milk, and the diluted serum was reacted with the antigen for 1 hour. After washing, the anti-mouse IgG-HRP secondary antibody was diluted 1 / 1000 and reacted for 1 hour. After washing, the color development solution was added and reacted for 10 minutes, and the OD value was measured.

[0146] Results: Similar to administration of YFV and VZV, induction of RuV-specific pre-existing immunity was determined for therapeutic administration.

[0147] 3) Therapeutic vaccine administration and determination of adipose tissue reduction effect

[0148] (i) DNA vaccine administration (1) (YFV vaccination group)

[0149] Methods: Normal mice or mice induced to become obese were divided into a YFV vaccine group and a non-YFV vaccine group, and used for experiments. Starting 3 days before the administration of the therapeutic vaccine, all mice were provided with a normal diet (ND). The DNA vaccine (therapeutic agent) was administered to the inguinal (scapular) adipose tissue and inguinal (inguinal) (groin) adipose tissue of the mice. The treatment was administered 5 times at intervals of 2 to 3 days at 2 sites on the left or right side of each tissue. The treatment was administered at a concentration of 100 μg / tissue of DNA encoding YFV antigen and 50 μg / tissue of DNA encoding IL-12, totaling 150 μg / time, and the mock group was administered with the same amount of empty vector as the total amount of DNA vaccine administered.

[0150] Body weight was measured before and after administration of the therapeutic vaccine. Mice were subjected to necropsy 7 days after the last administration of the therapeutic vaccine. Left / right inguinal adipose tissue and interscapular adipose tissue were separated and their respective weights were measured.

[0151] Table 3.

[0152]

[0153] (DIO: diet-induced obesity, ND: normal diet, HFD: high-fat diet; YFV-antigen DNA: SEQ ID NO: 1, IL-12 DNA: combination of SEQ ID NO: 5 and 6)

[0154] Results: The average weight change rate and adipose tissue reduction effect after treatment administration were determined for each group in Table 3 above and Figure 8 and Fig. 9 Shown in.

[0155] Reference Figure 8 , body weight is measured before and after treatment is applied, and weight change is measured based on the body weight just before application.In the case of the group (control 1) that is not processed completely, body weight gradually increases, but simulation group, processing 1 group and processing 2 groups all show weight loss effect, and compared with simulation group, processing 1 group and processing 2 groups show greater reduction.Processing 1 group also shows excellent weight loss effect, and processing 2 groups (which are used together with IL-12) show maximum weight loss.These results show that only processing 1 just shows excellent weight loss effect.

[0156] Fig. 9The fat weight reducing effect by treatment administration is shown. Treatment was administered only to the left or right side, and on day 7 after 5 administrations of treatment, the inguinal AT (adipose tissue) and interscapular AT on the left and right sides of the mice were isolated and the AT weight was measured. Fig. 9 The results are shown by group, and the effect of reducing fat weight is calculated by comparing the weight of the left and right AT in the same individual. When comparing the control group (no treatment) and the mock group (empty vehicle), it can be seen that the adipose tissue weight is reduced by the administration of treatment 1 and treatment 2.

[0157] These results show that the treatment of the present invention is not only effective in reducing body weight, but also has the effect of reducing adipose tissue weight. Compared with the simulation group, the mean value of adipose tissue weight is significantly reduced. It can be seen that the change of adipose tissue weight in the treatment 2 application group used together with IL-12 is much greater.

[0158] (ii) DNA vaccine administration (2) (VZV vaccination group)

[0159] Methods: Among mice induced to become obese by a high-fat diet (HFD), mice with pre-existing VZV-specific immunity were selected for DNA vaccine (therapeutic agent) administration. The mice were fed HFD until the administration of the therapeutic agent, and starting from the day of administration, the mice were fed alternately with normal diet (ND) and HFD every three days.

[0160] The treatment was administered to the inguinal fat tissue of the mice. The agent was injected at four sites on the left or right side of each tissue, with a total of five administrations at two-day intervals. The treatment was administered at a concentration of 100 μg / tissue of DNA encoding VZV antigen, totaling 100 μg / administration, and an equal amount of empty vector was administered to the mock group.

[0161] Body weight was measured before and after administration of the therapeutic vaccine. Mice were necropsy 12 to 14 days after the last administration of the therapeutic vaccine. Left and right inguinal adipose tissue was isolated and its weight was measured.

[0162] Table 4.

[0163]

[0164] (DIO: diet-induced obesity, ND: normal diet, HFD: high-fat diet; VZV-antigen DNA: SEQ ID NO. 3)

[0165] Results: The average weight change rate and adipose tissue reduction effect after treatment administration were determined for each group in Table 4 above, and Fig.10 and 11 Shown in.

[0166] Reference Fig.10 , body weight was measured before and after treatment administration, and the rate of body weight change was calculated based on the body weight immediately before administration. Weight loss was observed in all groups fed alternately with ND and HFD for 3 days starting from the day of treatment administration. In particular, greater weight loss was determined in the mock group and the treatment group administered with DNA compared to the control group (no treatment). In the mock group and the treatment group, the rate of weight loss before administration (day 0) and immediately before autopsy was similar.

[0167] Fig.11 The fat weight reducing effect by treatment administration is shown. Treatment was administered only to the left or right side, and the weight of the left and right inguinal fat tissue of the mice was measured on days 12 to 14 after administration of treatment 5 times. Fig.11 The results in the are shown in groups, and the adipose tissue reduction rate is calculated by comparing the weight of the left and right adipose tissues separated from the same individual. Compared with the control group (no treatment), it is determined that adipose tissue is reduced in the simulation group and the treatment group receiving DNA. Especially, compared with the simulation group, a higher reduction rate is determined in the treatment group.

[0168] Table 5

[0169] Comparison simulation deal with 1 4.6 -31.9 -44.3 2 12.3 -28.9 -41.3 3 8.6 -20.2 -32.9 4 -12.4 -26.9 -34.3 5 -6.1 -21.4 -23.0 6 0.2 -47.5 -37.2 7 -10.5 -33.2 -44.9 8 -9.7 -30.1 -34.9 9 -12.2 -39.6 -49.8 10 - -37.1 - (average value) -2.80 -31.69 -38.05 (SD) 9.47 8.26 8.02

[0170] As shown in Table 5 and Fig.11 As can be seen, the mean value of the reduction of adipose tissue in the treatment group of the present invention is significantly reduced compared to the control group. In addition, when compared to the simulation group, the degree of adipose tissue reduction is even more impressive. Considering the weight and size of adipose tissue, the realization of such a reduction effect is a significant outcome.

[0171] (iii) mRNA vaccine administration (3) (YFV vaccination group)

[0172] Methods: In mice induced to be obese by a high-fat diet (HFD), mice with established YFV-specific pre-existing immunity were selected for mRNA vaccine (treatment) administration. The mice were fed HFD until treatment was administered, and starting on the day of treatment, the mice were fed a normal diet (ND) and HFD alternately every three days. Treatment was administered to the inguinal adipose tissue of the mice. Treatment was given at four spots on the left or right side of each tissue, at intervals of two days, for a total of five administrations. Treatment was administered at a concentration of 10 μg / tissue of mRNA encoding YFV antigen per session. The mock group received the same total amount of mRNA as the treated group, consisting of an mRNA encoding FLu that did not express protein due to the presence of a stop codon. cmRNA composition of . Body weight was measured before and after administration of the therapeutic vaccine. Twelve to fourteen days after the last administration of the therapeutic vaccine, the mice were euthanized, and the left and right inguinal adipose tissues were isolated and weighed.

[0173] Table 6

[0174]

[0175] (DIO: diet-induced obesity, ND: normal diet, HFD: high-fat diet; treatment: SEQ ID No. 2)

[0176] Results: The average weight change rate and fat tissue reduction effect of each group after treatment administration are shown in Table 6. Fig.12 and 13 See Fig.12 , body weight was measured before and after treatment, and the rate of change in body weight was calculated based on the body weight before administration. Starting from the day of treatment administration, weight loss was observed in all groups alternating between a normal diet (ND) and a high-fat diet (HFD) every three days. It is worth noting that greater weight loss was determined in the simulation group and the treatment group receiving mRNA (therapeutic) compared to the control group (untreated). In particular, the weight loss rate before euthanasia after treatment in the treatment group was higher than that in the simulation group.

[0177] Table 7

[0178]

[0179] Fig.13 The effect of treatment administration on fat weight reduction is shown. Treatment was administered to one side only (left or right), and on days 12 to 14 after 5 administrations of treatment, the left and right inguinal (subcutaneous) adipose tissue of the mice was isolated and weighed. Fig.13 The result in shows the adipose tissue reduction rate calculated by comparing the weight of the left and right adipose tissue separated from the same individual, and is shown in group.Compared with the control group (untreated), in the simulation group and the processing (therapeutic) group accepting mRNA, it is observed that adipose tissue reduces.It should be noted that, compared with the simulation group, a higher reduction rate is determined in the treatment group.The result is shown in Table 8.

[0180] Table 8

[0181] Comparison simulation deal with 1 4.6 6.6 -35.4 2 12.3 -7.0 -28.7 3 8.6 -9.9 -17.6 4 -12.4 3.4 -24.1 5 -6.1 -31.9 -28.3 6 0.2 -14.3 -35.4 7 -10.5 -24.1 -41.9 8 -9.7 -20.8 -43.5 9 -12.2 -25.4 -32.6 10 - - -37.3 (average value) .2.80 -13.7 -32.5 (SD) 9.47 13.2 8.0

[0182] (iv) mRNA vaccine administration (4) (VZV vaccination group)

[0183] Methods: In mice induced to become obese by a high-fat diet (HFD), mice with pre-existing VZV-specific immunity were selected for mRNA vaccine (therapeutic agent) administration. The mice were fed HFD until the administration of the therapeutic agent, and starting from the day of administration, the mice were fed a normal diet (ND) and HFD alternately every three days.

[0184] The treatment was administered to the inguinal fat tissue of the mice. The agent was injected at four sites on the left or right side of each tissue, with a total of five administrations at two-day intervals. The treatment was administered at a concentration of 10 μg / tissue of mRNA encoding VZV antigen per administration. c The mRNA of was administered to the mock group in an amount equal to the total amount of mRNA in the administered therapeutic agent.

[0185] Body weight was measured before and after administration of the therapeutic vaccine. Mice were euthanized 12 to 14 days after the last administration of the therapeutic vaccine. Left and right inguinal adipose tissues were isolated and their weights were measured.

[0186] Table 9

[0187]

[0188] (DIO: diet-induced obesity, ND: normal diet, HFD: high-fat diet; VZV antigen mRNA: SEQ ID No. 4)

[0189] Results: The mean weight change rate and adipose tissue reduction effect of each group after administration of the treatment agent were determined as shown in Table 9 and Fig.14 and 15 Shown in.

[0190] Reference Fig.14 , body weight was measured before and after the administration of the therapeutic agent, and the body weight change rate was calculated based on the body weight immediately before administration. From the day of administration, weight loss was observed in all groups fed with a normal diet (ND) and a high-fat diet (HFD) alternately every three days. It is worth noting that, compared with the control group (untreated), a greater weight loss was determined in the simulation group and the treatment group (therapeutic agent) receiving mRNA. Before administration (day 0) and before euthanasia after administration, the weight loss rate of the simulation group and the treatment group was similar. These values ​​are shown in Table 10.

[0191] Table 10

[0192] (％) Day 0 Day 2 Day 4 Day 6 Day 8 Day 11 Day 13 Day 15 Day 20 Comparison 0 -10.17 -7.90 -8.13 -11.91 -5.20 -10.68 -5.75 -7.40 simulation 0 -5.63 -1.61 -6.18 -10.24 -4.00 -9.74 -6.15 -10.75 deal with 0 -6.44 -1.65 -7.14 -10.77 -3.09 -9.36 -4.89 -10.09

[0193] Fig.15The effect of weight loss in adipose tissue due to administration of the therapeutic agent is shown. The treatment was administered to the left or right side only, and on days 12 to 14 after five administrations of the therapeutic agent, the left and right inguinal (groin) adipose tissue of the mice was isolated and weighed. Fig.15 The result in shows the adipose tissue reduction rate calculated by comparing the weight of the left and right adipose tissues separated from the same individual. Compared with the control group (untreated), in the simulation group and the treatment group (therapeutic agent) receiving mRNA, the reduction of adipose tissue was determined. It should be noted that, compared with the simulation group, a higher reduction rate was observed in the treatment group.

[0194] (v) DNA vaccine administration (5) (MMR vaccination group)

[0195] Methods: In mice induced to become obese by a high-fat diet (HFD), mice with pre-existing RuV-specific immunity were selected for mRNA vaccine (therapeutic agent) administration. The mice were fed HFD until the administration of the therapeutic agent, and starting from the day of administration, the mice were fed alternately with normal diet (ND) and HFD every three days.

[0196] Treatment was administered to the inguinal adipose tissue of mice. The agent was injected at four sites on the left or right side of each tissue, with a total of five administrations at two-day intervals. Treatment was administered at a concentration of 100 μg / tissue of DNA encoding RuV antigen, totaling 100 μg / application, and an equal amount of empty vector was administered to the mock group.

[0197] Body weight was measured before and after administration of the therapeutic vaccine. Mice were euthanized 12 to 14 days after the last administration of the therapeutic vaccine. Left and right inguinal adipose tissues were isolated and their weights were measured.

[0198] Results: Similar to the YFV and VZV treated groups, body weight loss and adipose tissue reduction effects were determined in the treatment administration groups.

[0199] (vi) mRNA vaccine administration (6) (MMR vaccination group)

[0200] Methods: In mice induced to become obese by a high-fat diet (HFD), mice with pre-existing RuV-specific immunity were selected for mRNA vaccine (therapeutic agent) administration. The mice were fed HFD until the administration of the therapeutic agent, and starting from the day of administration, the mice were fed alternately with normal diet (ND) and HFD every three days.

[0201] The treatment was administered to the inguinal adipose tissue of the mice. The agent was injected at four sites on the left or right side of each tissue, with a total of five administrations at two-day intervals. The treatment was administered at a concentration of 10 μg / tissue of mRNA encoding RuV antigen per administration. An mRNA encoding FLuc containing a stop codon that does not express protein was administered to the mock group in an amount equal to the total amount of mRNA in the administered therapeutic agent.

[0202] Body weight was measured before and after administration of the therapeutic vaccine. Mice were euthanized 12 to 14 days after the last administration of the therapeutic vaccine. Left and right inguinal adipose tissues were isolated and their weights were measured.

[0203] Results: Similar to the YFV and VZV treatment experiments, body weight loss and adipose tissue reduction effects were determined in the treatment administration groups.

[0204] Table 11. Sequence information

[0205]

[0206]

[0207]

[0208] Industrial Applicability

[0209] The present invention can prevent and / or treat obesity. The present invention provides a method for preventing and / or treating obesity by injection into a local area.

Claims

1. A pharmaceutical composition for improving, inhibiting or treating obesity or reducing the size or volume of a target tissue, the pharmaceutical composition comprising at least one virus selected from yellow fever virus, varicella-zoster virus and rubella virus; a protein derived from said virus; or The genetic material encoding the virus or protein. 2 . The pharmaceutical composition according to claim 1 , which is a pharmaceutical composition for reducing the size or volume of a target tissue.

3. The pharmaceutical composition according to claim 1, wherein the genetic material is a polynucleotide encoding at least one protein selected from the group consisting of a structural protein of yellow fever virus, glycoprotein E of varicella-zoster virus, and E2 and E1 proteins of rubella virus.

4. The pharmaceutical composition according to claim 3, wherein the polynucleotide is DNA or RNA.

5. The pharmaceutical composition according to claim 1, wherein the genetic material comprises a nucleic acid sequence consisting of a polynucleotide selected from SEQ ID NO: 1 to 6.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises genetic material encoding cytokines.

7. The pharmaceutical composition according to claim 6, wherein the cytokine is one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β.

8. The pharmaceutical composition of claim 1, wherein the genetic material induces an intracellular immune response.

9. The pharmaceutical composition according to claim 1, wherein the composition acts locally, and The pharmaceutical composition is administered to the subject after first administering at least one antigen for generating an immune environment.

10. The pharmaceutical composition according to claim 9, wherein the antigen comprises a virus, a protein or an epitope thereof introduced from outside the human body.

11. The pharmaceutical composition according to claim 1, wherein the genetic material is orally administered to the target tissue, Administration to the target tissue by transdermal, intramuscular, peritoneal, intravenous, subcutaneous or nasal injection, or Administration to target tissues is via electroporation, gene guns, liposomes, dendrimers, nanoparticles or transfer vectors.

12. The pharmaceutical composition according to claim 1, wherein the dosage of the genetic material in the composition is 0.1 to 1000 ug / site per one-time inoculation.

13. The pharmaceutical composition according to claim 1, wherein the composition is administered to a subject for reducing or shrinking the size of adipose tissue, or killing adipose tissue, and Wherein, after determining that antibodies against at least one of the viruses are produced by administering a vaccine for preventing any one or more viruses selected from yellow fever virus, varicella-zoster virus and rubella virus, the composition is applied to the area at least once for reducing or shrinking the size of adipose tissue or killing adipose tissue.

14. The pharmaceutical composition of claim 1, wherein the composition is a vaccine.

15. A kit comprising (a) a pharmaceutical composition according to any one of claims 1 to 14; and (b) administration instructions for administering the pharmaceutical composition.

16. The kit according to claim 15, The kit further comprises (c) a composition containing an antigen for generating an immune environment before administering the composition (a).

17. The kit according to claim 15, Wherein (a) the pharmaceutical composition further comprises genetic material encoding cytokines.

18. A syringe filled with a vaccine composition for reducing the size or volume of a target tissue, the syringe comprising at least one virus selected from the group consisting of yellow fever virus, varicella-zoster virus and rubella virus; or genetic material encoding a protein derived therefrom.

19. The syringe of claim 18, wherein the syringe further comprises genetic material encoding a cytokine.

20. A method for reducing the size or volume of a target tissue in a subject, comprising administering to the target tissue in the subject genetic material encoding at least one virus selected from the group consisting of yellow fever virus, varicella-zoster virus, and rubella virus.

21. The method of claim 20, wherein the method induces a decrease in the number of adipocytes or death of adipocytes or a decrease in the size of adipose tissue in the subject.

22. The method of claim 20, wherein the method comprises: i) administering to a subject a vaccine for the prevention of at least one virus selected from the group consisting of yellow fever virus, varicella-zoster virus and rubella virus for reducing or shrinking the size of adipose tissue or killing adipose tissue, ii) determining the production of antibodies achieved by the vaccine, and iii) administering a pharmaceutical composition comprising: at least one virus selected from the group consisting of yellow fever virus, varicella-zoster virus and rubella virus; a protein derived from said virus; or genetic material encoding said virus or protein.

23. The method according to claim 21 or 22, wherein the method further comprises: iv) administering simultaneously or sequentially with the pharmaceutical composition at least one cytokine selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α and TNF-β.

24. Use of a composition comprising: at least one virus selected from yellow fever virus, varicella-zoster virus and rubella virus; a protein derived from the virus; or genetic material encoding the virus or protein for reducing the size or volume of a target tissue or improving, inhibiting or treating obesity.

25. The use according to claim 24, wherein the genetic material comprises a nucleic acid sequence consisting of a polynucleotide selected from SEQ ID NO: 1 to 6.

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