WO2020085533A1 - Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed - Google Patents

Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed Download PDF

Info

Publication number
WO2020085533A1
WO2020085533A1 PCT/KR2018/012712 KR2018012712W WO2020085533A1 WO 2020085533 A1 WO2020085533 A1 WO 2020085533A1 KR 2018012712 W KR2018012712 W KR 2018012712W WO 2020085533 A1 WO2020085533 A1 WO 2020085533A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
atopic dermatitis
extracellular vesicles
derived
preventing
Prior art date
Application number
PCT/KR2018/012712
Other languages
French (fr)
Korean (ko)
Inventor
김형식
김태윤
Original Assignee
가톨릭대학교 산학협력단
부산대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 가톨릭대학교 산학협력단, 부산대학교 산학협력단 filed Critical 가톨릭대학교 산학협력단
Priority to KR1020217013938A priority Critical patent/KR20210066005A/en
Priority to PCT/KR2018/012712 priority patent/WO2020085533A1/en
Publication of WO2020085533A1 publication Critical patent/WO2020085533A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
  • S0D3 super oxide di smutase 3
  • Inflammatory disease refers to a condition in which edema, redness, and pain appear, and histological changes occur as well as infiltration of inflammatory immune cells in various organs or tissues of the human body due to various external stimuli or internal factors. This inflammation is triggered by a variety of chemical mediators produced from damaged tissues and moving cells (dragon 061 1), which are known to vary depending on the type of inflammatory process. In the normal case, the living body neutralizes or eliminates the pathogen through an inflammatory reaction and regenerates the upper tissue to restore normal structure and function. Otherwise, it progresses to a disease state such as chronic inflammation.
  • the most common inflammatory disease prevention or treatment for treating these inflammatory diseases is largely divided into steroidal and nonsteroidal inflammatory disease prevention or treatment, and most synthetic inflammatory disease prevention or treatment solvents have various side effects in addition to their main function. Since it is often accompanied, it has an excellent effect, and there is an urgent need for the development or treatment of inflammatory diseases with few side effects.
  • S0D super oxide di smutase
  • S0DCS0D 1 Mn with manganese atoms
  • -S0D (S0D 2) and EC-SOD extracel lular superoxide dismutase
  • the EC-S0D protein S0D3 (superoxide di smutase 3, extracel lular) is secreted outside the cell
  • the amino acid sequence of human normal (wi ld-type, WT) S0D3 protein is accession such as NP_003093.2 of NCBI Genbank database. It is known as number.
  • the base sequence of mRNA encoding human S0D3 protein is known as an accession number such as ⁇ _003102.2.
  • the present inventors studied a therapeutic agent having an excellent therapeutic effect in an inflammatory disease with a significantly low side effect, and immunomodulation and immunoregulation when administering extracellular vesicles derived from stem cells ( ⁇ 3 overexpressed) to individuals with atopy.
  • the present invention was completed by discovering that atopy is treated by remarkably improving functions such as antioxidant effect.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating atopic dermatitis consisting of extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
  • Another object of the present invention is to provide a cosmetic composition for preventing or improving atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a cosmetic composition for preventing or improving atopic dermatitis consisting of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
  • Another object of the present invention is a step of transfecting the stem cells with a recombinant expression vector containing a polynucleotide encoding the example; And extracting the extracellular vesicles by centrifuging the transfected stem cells; a method for manufacturing a pharmaceutical composition for preventing or treating atopy. Is to provide.
  • Another object of the present invention is to provide the use of superoxide dismutase 3 (super oxide dismutase 3, S0D3) overexpressing stem cell-derived extracellular vesicles for the preparation of atopic therapeutic agents.
  • superoxide dismutase 3 super oxide dismutase 3, S0D3
  • Another object of the present invention is to administer an effective amount of a composition containing an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient to an individual in need thereof It provides a method of treating atopic disease characterized by.
  • S0D3 super oxide di smutase 3
  • the present invention is a pharmaceutical composition for preventing or treating atopic dermatitis comprising extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3) as an active ingredient. to provide.
  • superoxide dismutase 3 superoxide dismutase 3 (super oxide di smutase 3,
  • composition for preventing or treating atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing S0D3).
  • present invention superoxide dismutase 3 (super oxide di smutase 3,
  • compositions for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing S0D3).
  • superoxide di smutase 3 (super oxide di smutase 3, S0D3) overexpressed stem cell-derived extracellular vesicles as an active ingredient to prevent or improve atopic dermatitis cosmetic composition for providing do.
  • the present invention provides a cosmetic composition for preventing or improving atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
  • the present invention provides a cosmetic composition for preventing or improving atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing superoxide oxidase 3 (S0D3).
  • the present invention comprises the steps of transfecting the stem cells with a recombinant expression vector containing a polynucleotide encoding ⁇ ⁇ ; It provides a method for manufacturing a pharmaceutical composition for preventing or treating atopy, characterized in that it comprises; extracting the extracellular vesicles by centrifuging the transfected stem cells.
  • the present invention provides the use of stem cell-derived extracellular vesicles overexpressing super oxide di smutase 3 (S0D3) for preparing an agent for the treatment of atopy. do.
  • S0D3 super oxide di smutase 3
  • the present invention requires an effective amount of a composition comprising extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3) as an active ingredient It provides a method of treating atopic disease characterized in that it is administered to an individual.
  • S0D3 superoxide di smutase 3
  • the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
  • the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3).
  • the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (313 ⁇ 4) 610 6 51111 336 3, example).
  • the inventors of the present invention have previously confirmed the effect of treating inflammatory diseases of stem cells overexpressing S0D3 (see Korean Patent Publication No. 10-2017-0032872).
  • stem cell-based therapeutics have many difficulties in commercialization of products as well as clinical trials due to high price, long-term side effects, and difficulty in managing raw materials. Therefore, the present inventors were studying the therapeutic agent that has the advantages of the therapeutic agent using stem cells, and confirmed that the extracellular vesicles derived from S0D3 overexpressing cells (extracel lular vesicle, EV) retain the advantages of the S0D3 overexpressing stem cells.
  • extracellular vesicle means that cells release various vesicles of various membrane types into the extracellular environment. Extracellular vesicles are also called cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes, etc. In some cases, they are used separately from exosomes. The present invention is not limited to this.
  • the extracellular vesicles of the present invention are derived from stem cells overexpressing S0D3, the S0D3 can be expressed as a protein, and the S0D3 protein catalyzes the dismutation reaction of anions, and is secreted outside the cell to extracellular matrix (extracel lular matrix), and exhibits anti-angiogenic, anti-chemotactic, and anti-cancer activities.
  • S0D3 refers to a mammal derived from humans and mice, preferably a protein derived from humans, and most preferably includes the amino acid sequence of a human normal S0D3 protein represented by SEQ ID NO: 1 or SEQ ID NO: 2. It may be. Or it may be to include the amino acid sequence of the recombinant S0D3 protein (209E-SOD3) represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  • the human normal S0D3 is a signal peptide up to the 18th alanine, including the starting methionine amino acid at the N-terminal site, and activated S0D3 is composed of 222 amino acids from which the signal peptide is removed.
  • the C-terminal region of S0D3 (amino acid residues 210 to 215) has a heparin binding domain ' .
  • the amino acid sequence of the full length human S0D3 consisting of 240 amino acids including the signal peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • S0D3 is a 209 amino acid in which 13 amino acids (amino acid residues 210 to 222) are removed, which contain a signal peptide at the N-terminal region and a heparin binding domain at the C-terminal region of human S0D3 of full length. It may be made of.
  • the signal peptide and the S0D3 protein from which the heparin binding domain is removed may be referred to as 209E- S0D3 or 209E in the present invention, and preferably may have an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  • the 209E-S0D3 can also bind to the anti-S0D3 antibody, and has been confirmed to have the same enzyme activity and R0S removal activity as the normal S0D3 (Korean Patent Publication No. 10-2008-0108876).
  • S0D3 includes all functional equivalents (funct ional equivalent), functional derivatives (funct ional der ivat ive), and fragments of the S0D3 protein, which have substantially equivalent physiological activity to the normal S0D3 or 209E-S0D3 protein.
  • Substantially equivalent physiological activity means having the same level of enzyme activity and / or extracellular secretion properties and intracellular permeability as normal S0D3, and overexpression in stem cells, preferably mesenchymal stem cells, due to the enzyme activity equivalent to S0D3 When it is done, it improves the immune and inflammation control ability of stem cells.
  • the immune and inflammatory control ability of the stem cells is specifically, suppression of invasion of immune cells due to inflammation, proliferation and differentiation of pro-inflammatory T cells, and suppression of expression of pro-inflammatory mediator / cytokine, Treg cells having inflammatory control ability Means proliferation and differentiation and increased expression of Treg-related cytokines, inhibition of phosphorylation of the NFkB signaling system, and the like as described in the characteristics of stem cells overexpressing S0D3 of the present invention described herein.
  • the functional equivalent of VIII is preferably SEQ ID NO: 1 to SEQ ID NO: 4 It may be a polyfeldtide having a sequence homology with at least 70%, preferably 80% or more, and more preferably 90% or more of the amino acid sequence displayed.
  • the functional equivalent may be generated as a result of addition, substitution, or deletion of some of the amino acid sequences of S0D3 of the present invention.
  • the substitution of amino acids in the above is preferably a conservative substitution.
  • amino acids examples include aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (lie, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp , Glu), basic amino acids (His, Lys, Arg, Gin, Asn) and sulfur-containing amino acids (Cys, Met).
  • the functional equivalent includes a variant in which a part of amino acids is deleted on the amino acid sequence of S0D3 of the present invention. The deletion or substitution of the amino acid is preferably located in a region not directly related to the physiological activity of the S0D3 protein.
  • S0D3 may be a peptide in which a portion of the entire length of S0D3 is removed without affecting the enzyme activity of S0D3, such as 209E-S0D3, and S0D3 such as SNP (smal 1 nucleotide polymorphism) having substantially equivalent physiological activity to S0D3 protein. It may be a polymorphism protein.
  • polypeptide derivatives in which some chemical structures of proteins have been modified while maintaining the basic framework and physiological activity of the S0D3 protein of the present invention.
  • structural modifications to change the stability, intracellular permeability, storage, volatile or solubility of the S0D3 protein of the present invention are included therein.
  • the extracellular vesicle included as an active ingredient in the pharmaceutical composition of the present invention is derived from stem cells overexpressing ⁇ , and the stem cells may preferably be mesenchymal stem cells.
  • the "mesenchymal stem cell (MSC)" of the present invention includes bones, cartilage, fat tissue, tendons (nerve tissue), fibroblasts ( Refers to a multipotent progenitor prior to differentiation into cells of specific organs such as fibroblasts and muscle cells.
  • Mesenchymal stem cells are included in the composition in an undifferentiated state, that is, a stem cell state.
  • the mesenchymal stem cells of the present invention may be derived from mammals, and preferably may be derived from humans.
  • the mesenchymal stem cells of the present invention may be derived from tissue selected from the group consisting of umbilical cord, umbilical cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, and amniotic membrane.
  • the mesenchymal stem cells of the present invention may be umbilical cord blood or placenta-derived mesenchymal stem cells, most preferably umbilical cord blood-derived mesenchymal stem cells.
  • Umbilical cord blood or placenta-derived mesenchymal stem cells have the characteristics of superior differentiation and proliferation ability than bone marrow-derived mesenchymal stem cells.
  • umbilical cord blood used in the present invention refers to blood collected from umbilical vein connecting the placenta and fetus of a mammal.
  • the umbilical cord blood can be easily collected from the donor's umbilical vein during childbirth. More specifically, in the case of normal vaginal delivery, the placenta still remains in the womb after childbirth, and can be collected from the umbilical vein exposed outside. Alternatively, in the case of a cesarean section, the placenta after birth is also taken from the umbilical vein in the state of being exposed outside the womb.
  • stem cells collected from the placenta may be described as a term "placental stem cells,” which are stem cells or precursors derived from mammalian placenta regardless of shape, cell surface label, or number of passages after primary culture.
  • a cell it refers to the attachment to a tissue culture substrate, such as tissue culture plastic or fibronectin coated tissue culture plate, but the term “placental stem cell” used herein refers to a trophoblast.
  • a cell is a stem cell if it has at least one of the characteristics of a stem cell, for example, the ability to differentiate into at least one cell type.
  • Mesenchymal stem cells can be isolated from placental or umbilical cord blood according to methods known in the art.
  • the mesenchymal stem cells can be separated by any conventionally known separation method. For example, a separation method using a density difference (density gradient fract ionat ion, immunoselection, and differential adhesion separat ion.
  • the method of separating and culturing mesenchymal stem cells from umbilical cord blood can be used for all methods that have been used previously.
  • Cultivation of the mesenchymal stem cells isolated from the above can be performed in a cell culture medium known in the art, for example, but not limited to, DMEM medium, McCoys 5A medium, Eagle's basal medium, CMRL Badges, Glasgow minimum required medium, Ham's F-12 medium, Iscove's modi f ied Dulbecco's medium, Liebovi tz 'L-15 medium, RPMI 1640 medium KSB-3 basal media and more.
  • one or more auxiliary components may be added to the cell culture medium, if necessary, including serum of fetal bovine serum, horse or human, and antibiotics and antifungal agents for preventing contamination of microorganisms. .
  • the isolated or cultured stem cells can be stored by methods known in the art until use. In general, stem cells can be stored frozen after treatment with cryoprotected roots !!).
  • the freeze protection treatment is known in the art ⁇ , glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dext tube, sucrose, ethylene glycol, ⁇ erythritol, I)-ribitol, I) -mannitol, I) -Can be performed using a cryoprotectant such as sorbitol, inositol, I) -lactose or choline chloride.
  • Stem cells over-expressing ⁇ ⁇ may be obtained by transducing a recombinant 'combination expression vector containing a polynucleotide encoding ⁇ into stem cells.
  • n expression refers to the production of a protein or nucleic acid in a cell
  • overexpression means that the level of expression of a particular gene is excessively increased than the normal or normal state.
  • stem cells over-expressing S0D3 are specifically stem cells having an increased expression level of S0D3 protein, thereby increasing the activity of S0D3 protein.
  • polynucleotide (1) 01 1111 (: 1601; 1 (16)" or nucleic acid refers to deoxyribonucleotide acids in single- or double-stranded form) or ribonucleotides (poetry. Otherwise, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.
  • the most common way to overexpress S0D3 in stem cells is to artificially or experimentally introduce polynucleotides containing the S0D3 gene into stem cells to increase the copy number of the S0D3 gene.
  • Injecting an exogenous polynucleotide that is not possessed by the original cell into the cell is called transfection, and a phenomenon in which the genetic trait of the cell is changed is called transformation.
  • the process in which the exogenous polynucleotide is injected into a cell through a virus or a virus-derived vector is called transduction ion.
  • transduction ion the process in which the exogenous polynucleotide is injected into a cell through a virus or a virus-derived vector.
  • Transfection and ‘transfection’ are used in a similar sense as referring to the process or introduction of an exogenous polynucleotide into a cell to have a genetic trait different from the normal type.
  • the polynucleotide encoding S0D3 may be a S0D3 gene derived from a mammal. Preferably, it may include a nucleotide sequence encoding human normal S0D3 represented by SEQ ID NO: 5 or SEQ ID NO: 6. Or it may include a nucleotide sequence encoding 209E-S0D3 represented by SEQ ID NO: 7 or SEQ ID NO: 8.
  • ⁇ D3 consisting of the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence shown in SEQ ID NO: 5, S0D3 consisting of the amino acid sequence of SEQ ID NO: 2 to the nucleotide sequence shown in SEQ ID NO: 6 Can be coded by
  • S0D3 consisting of the amino acid sequence of SEQ ID NO: 3 may be encoded by the nucleotide sequence represented by SEQ ID NO: 7
  • S0D3 consisting of the amino acid sequence of SEQ ID NO: 4 may be encoded by the nucleotide sequence represented by SEQ ID NO: 8 .
  • polynucleotide encoding ⁇ also includes a sequence showing substantial identity to the nucleotide sequence of human ⁇ ⁇ , preferably the nucleotide sequence represented by SEQ ID NO: 5 to SEQ ID NO: 8. Substantive identity encodes the human ⁇ 2020/085533 1 »(: 1 ⁇ 1 ⁇ 2018/012712
  • the protein encoded by the base sequence substantially identical to the base sequence of the polynucleotide encoding 3 ⁇ 3 is 8003 protein, preferably a functional equivalent of a protein comprising an amino acid sequence represented by SEQ ID NOs: 1 to 4 Can be.
  • the functional equivalents of the 5003 protein are as previously described herein.
  • ⁇ recombinant expression vector '' refers to a gene composition comprising an essential regulatory element operably linked to express a target protein or target nucleic acid (poetry-expressing vector, a polynucleotide (gene) insert) in a suitable host cell.
  • Sacrifice which is operatively connected ""
  • nucleic acid expression control sequence and the desired protein or nucleic acid sequence encoding a show are functionally linked (for £ 1111 101131 1 ⁇ ⁇ 6) to perform a general function. It means that the nucleic acid sequence is linked in such a way that gene expression is possible by an expression control sequence.
  • a promoter and a nucleic acid sequence encoding a protein or a shock can be operably linked to affect the expression of the encoding nucleic acid sequence.
  • Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art, and site-specific cleavage and linkage uses enzymes, etc., generally known in the art.
  • the recombinant expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the cloning field, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors.
  • vectors derived from viruses can be used.
  • the above plasmid is E. coli-derived plasmid 081 ⁇ 322 ,? 8 Mo 325, 1) 1018 and 1) 1019 ,? £ 1-225 (+)), plasmids derived from Bacillus subtilis (1) ⁇ 110 and? 5) and plasmids derived from yeast bamboo seedlings 1) 13, ⁇ 2 ⁇ and ⁇ 0? 50), etc.
  • Expression vectors comprising nucleic acids according to the present invention are methods known in the art, for example, but not limited to, transient transfection (transient transfect ion), micro injection, transduction (transduct ion), cell fusion, calcium Phosphate precipitation method, liposome-mediated transfection, DEAE dextran-mediated transfect ion, polybrene-mediated transfect ion, It can be introduced into stem cells by electroporat ion, gene gun, and known methods for introducing nucleic acids into cells.
  • Stem cells over-expressing 003 according to the present invention are preferably expressed in a mesenchymal stem cell using a recombinant expression vector containing a polynucleotide encoding 3 (X ) 3, preferably using an electroporation method or a virus-mediated transduction method. It may be transformed by injection.
  • a recombinant expression vector containing a polynucleotide encoding 3 (X ) 3 preferably using an electroporation method or a virus-mediated transduction method. It may be transformed by injection.
  • the stem cells of the present invention can be prepared by using a recombinant virus vector through the following steps: (a) Shuttle vector, DNA construct encoding a S0D3 nucleic acid and / or protein transduction domain operably linked thereto. Preparing a recombinant viral vector comprising; (b) transfecting the recombinant virus vector into a virus-producing cell line to produce a S0D3 expressing recombinant virus; And (c) infecting mesenchymal stem cells with the S0D3 expressing recombinant virus.
  • the viral vector of the present invention is characterized in that it is selected from the group consisting of lentiviral vector, retroviral vector, adenovirus vector, adeno-associated virus ( ⁇ 0 vector, vaccinia virus vector, herpesvirus vector and abipoxvirus vector).
  • lentiviral vector retroviral vector
  • adenovirus vector adeno-associated virus
  • ⁇ 0 vector vaccinia virus vector
  • herpesvirus vector adeno-associated virus vector
  • abipoxvirus vector adeno-associated virus
  • the present invention comprises the steps of (transfecting stem cells with a recombinant expression vector containing a polynucleotide encoding ⁇ 3; and extracting extracellular vesicles by centrifuging the transfected stem cells).
  • the pharmaceutical composition according to the present invention may contain 8003 overexpressing stem cell-derived extracellular vesicles alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may additionally contain excipients or diluents.
  • 'pharmaceutically acceptable' refers to a non-toxic composition that does not cause allergic reactions or similar reactions, such as gastrointestinal disorders, dizziness, etc., when physiologically acceptable and administered to humans.
  • a carrier for oral administration or a carrier for parenteral administration may be further included.
  • Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to the peptide preparation.
  • the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl guaraben and chlorobutanol.
  • the pharmaceutical composition of the present invention may include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a prestigious agent, etc. in addition to the above components.
  • Other pharmaceutically acceptable carriers and formulations can be referenced as described in the following literature (Remington's Pharmaceut ical Sciences, 19th ed., Mack Publ i shing Company, Easton, PA, 1995).
  • composition of the present invention can be administered in any way to mammals, including humans.
  • it can be administered orally or parenterally.
  • the parenteral administration method is not limited to this, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or intrarectal administration.
  • the pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.
  • the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. Can be.
  • tablets or dragees can be obtained by mixing the active ingredient with a solid excipient and then grinding it and adding a suitable adjuvant to the granule mixture.
  • excipients examples include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starch including wheat starch, rice starch and potato starch, cellulose, etc.
  • Fillers such as celluloses, gelatin, polyvinylpyrrolidone, etc. may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose.
  • crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant.
  • the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.
  • the total effective amount of the composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractional ionized treatment protocol that is administered for a long time in multiple doses. have.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease.
  • the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 // g to 10,000 mg per 1 kg of patient body weight per day, and most preferably 0.1 / to 500 mg per day.
  • the dose of the pharmaceutical composition is determined by considering various factors such as the formulation method, the administration route and the number of treatments, as well as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, etc.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effects of the present invention.
  • S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention were treated in an animal model inducing atopic dermatitis and visually evaluated for changes in lesions, S0D3 overexpressing umbilical cord blood stem cell-derived extracellular cells When the vesicles were treated, it was confirmed that the most improved symptoms (Example 2-1, see Fig. 3).
  • the vesicles of 5003 overexpressing stem cell-derived cells according to the present invention are treated in an animal model inducing atopic dermatitis, and the severity of the lesion is evaluated through tissue staining, and the skin is coated. It was confirmed that the thickness and the degree of lymphocyte infiltration were reduced. In addition, it was confirmed that the invasion of mast cells, which play a key role in the chronicization of atopic dermatitis, was also suppressed (see Examples 2-2 and 4 to 7).
  • 3, IL-6 As a result, it was confirmed that the proliferation of peripheral blood mononuclear cells (PBMC), which are immune cells, was inhibited by the treatment of the extracellular vesicles according to the present invention (see Examples 3, 8 and 9). .
  • PBMC peripheral blood mononuclear cells
  • S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention are used for differentiation of regulatory T cells (regulatory T cells) that play a key role in suppressing the overall immune response and excessive activation of other immune cells.
  • regulatory T cells regulatory T cells
  • the extracellular vesicles of the present invention have excellent Treg-inducing ability (see Example 4-2, FIG. 12).
  • the extracellular vesicles show the effect of treating atopic dermatitis through suppressing proliferation of immune cells secreting pro-inflammatory cytokines and inducing Treg.
  • the S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention inhibit the invasion of mast cell lesions and suppress the Thl differentiation in the differentiation of helper T cells, but show significant results that do not affect the differentiation of Th2. .
  • Atopic dermatitis is most often caused by immune mechanisms associated with seedlings, and there are many reports that immune responses by T cell abnormalities are involved, and the role of helper T cells is known to be important.
  • Helper T cells include helper T1 type (Thl) cells and helper T2 type (Th2) cells.
  • Cytokines secreted by Thl cells are involved in cell-mediated immune responses, and in the case of atopic dermatitis, secretion of Thl cytokines such as interferon-Y (IFN-Y) secreted by Thl cells is known to decrease. It is known to be initiated by the Th2 immune response and gradually converted into a chronic Thl immune response (see Am J Cl in Dermatol 5; 281-294, 2004 and Nature 413: 531-534, 2001).
  • IFN-Y interferon-Y
  • the above cell test and animal test results show that the 8003 overexpressing stem cell-derived extracellular vesicles according to the present invention show excellent atopy treatment effect, and can be used as a safe and effective pharmaceutical candidate substance having a treatment effect of chronic atopic dermatitis. Suggests
  • the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, which comprises extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient.
  • a cosmetic composition for preventing or improving atopic dermatitis which comprises extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient.
  • S0D3 super oxide di smutase 3
  • superoxide dismutase 3 superoxide dismutase 3
  • a cosmetic composition for preventing or improving atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing S0D3).
  • the present invention superoxide dismutase 3 (super oxide di smutase 3,
  • the cosmetic composition of the present invention contains S0D3 overexpressing stem cell-derived extracellular vesicles as an active ingredient and is a basic cosmetic composition (face wash, cream, essence, cleansing foam and cleansing water-like face wash, pack, body) Oil), hue cosmetic composition (foundation, lipstick, mascara, makeup base), hair product composition (shampoo, conditioner, hair conditioner, hair gel) and soap.
  • the excipient is not limited to this, for example, may include skin emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners and solvents.
  • fragrances, pigments, disinfectants, antioxidants, preservatives and moisturizers may be further included, and thickeners, inorganic salts, and synthetic polymer materials may be included for the purpose of improving physical properties.
  • the S0D3 overexpressing stem cell-derived extracellular vesicles can be easily added to the normal face wash and soap base.
  • a cream In the case of manufacturing a cream, it can be prepared by adding S0D3 overexpressing stem cell-derived extracellular vesicles to a general oil-in-water type (0 / W) cream base.
  • synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving physical properties such as fragrances, chelating agents, pigments, antioxidants, and preservatives can be added .
  • the content of S0D3 overexpressing stem cell-derived extracellular vesicles contained in the cosmetic composition of the present invention is not limited to this, but is preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight relative to the total weight of the total composition. If the content is less than 0.001% by weight, the desired effect of treating atopic dermatitis cannot be expected, and if it is more than 10% by weight, there may be difficulties in safety or preparation of the formulation.
  • the present invention provides the use of extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (S0D3) for preparing an atopy treatment preparation.
  • S0D3 superoxide dismutase 3
  • the present invention is characterized by administering an effective amount of a composition containing extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient to an individual in need thereof. How to treat disease to provide.
  • S0D3 super oxide di smutase 3
  • the 'effective amount' of the present invention when administered to an individual, refers to the amount of improving, treating, preventing, detecting, diagnosing or suppressing or reducing the effects of atopic dermatitis disease when administered to an individual, the 'individual' being an animal, preferably For example, it may be a mammal, particularly an animal including humans, or may be cells, tissues, organs, etc. derived from animals. The individual may be a patient who needs the effect).
  • the 'treatment' of the present invention collectively refers to improving the symptoms of atopic dermatitis disease or atopic dermatitis disease, which may include healing, substantially preventing, or improving the condition of atopic dermatitis disease, It includes, but is not limited to, alleviating, healing or preventing one symptom or most symptoms resulting from atopic dermatitis disease.
  • the term 'compr i sing' is used the same as 'containing' or 'characterizing' and does not exclude additional component elements or method steps not mentioned in the composition or method. .
  • the term (consi st ing of) ” means excluding additional elements, steps or components, which are not described separately.
  • the term 'essentially composed (essent i al ly consi st ing of)' includes a component element or step described in the scope of the composition or method, as well as a component element or step that does not substantially affect its basic properties. It means to do.
  • the present invention provides a composition for preventing or treating atopic dermatitis comprising extracellular vesicles derived from C3 overexpressing stem cells as an active ingredient and a method for treating atopic dermatitis using the same.
  • the composition according to the present invention suppresses the proliferation of immune cells that secrete pro-inflammatory cytokines, shows the effect of treating atopic dermatitis through the induction effect of ⁇ 6, inhibits the invasion of lesions of mast cells and inhibits 3 ⁇ 41 differentiation, chronicating atopic dermatitis It can be useful in the prevention or treatment of chronic atopic dermatitis.
  • (k) is the date of manufacture of the atopic dermatitis animal model, and the date of administration of the extracellular vesicles derived from 8003 overexpressing umbilical cord blood stem cells is set, and (indicates the change in lesions of atopic dermatitis by administration of the extracellular vesicles) It is the result of confirmation.
  • FIG. 6 is a graph showing the numerical results of tissue staining after administration of 3 ⁇ 3 overexpressing cord blood stem cell-derived extracellular vesicles to an atopy animal model, and (3) in FIG. 6 is a result of digitizing the result of confirming the thickness of the envelope. (Is the result of quantifying lymphocyte infiltration.
  • FIG. 7 is a result of confirming the degree of infiltration of lesions of mast cells that play a key role in chronic atopic dermatitis after administration of S0D3 overexpressing umbilical cord blood stem cell-derived extracellular vesicles through atopic animal model through toluidine blue staining to be.
  • 8 and 9 is a result of confirming the effect on the proliferation of urine (:) after treatment of the extracellular vesicles derived from the overexpressed umbilical cord blood stem cells from? 83 ⁇ 48 separated from normal blood.
  • FIG. 12 is a result of confirming the effect on ⁇ differentiation after treatment of 8003 overexpressing cord blood stem cell-derived extracellular vesicles to uncontacted I cells.
  • MSC Mesenchymal stem cells derived from human umbilical cord blood were collected from blood samples of human umbilical cord with the consent of the donor. Umbilical cord blood samples were collected and stored in blood collection packs containing citrate phosphate glucose, which acts as an anticoagulant. Treatment for the experiment was carried out within 24 hours. The mononuclear cell fraction was isolated by centrifugation under a F i co 1 -Paque PLUS concentration gradient (Amersham Biosciences).
  • Antibiotic / antifungal agents include 100 U / ml penicillin, 100 ug / ml streptomycin, and 25 yg / ml amphotericin table. 7 days later The non-adherent cells were removed, and the attached cells were cultured by changing two cultures every week. Cells were cultured in 5% C02, 37 ° C wet conditions.
  • hUCB-MSC The immunophenotype of hUCB-MSC was analyzed to investigate the presence of antigen-positive markers associated with MSC and the absence of hematopoietic lineage markers by flow cytometry (Epi cs XL, Beckman Coul ter). Positive markers include CD90 (Thy-l), CD10 endogl in) and SH3 (CD73), and hematopoietic lineage markers include endothelial cell markers such as CD34, CD45 and CD31. Cells were positive for HLA cl ass I, but negative for HLA-m.
  • Each fluorescently labeled monoclonal antibody was purchased from Beet on Dickinson (results not shown). Through this, three cell lines of P7 U192, P7 U197, and P7 U218 were obtained (P7: cells cultured over seven cell passages, U192 / 197/218: different cell numbers for donors).
  • S0D3 Transformation S0D3 was transformed by electroporation on hUCB-MSC prepared through the above.
  • the viral expression vector and transduction conditions for S0D3 transduction were that lentivirus was used as a viral expression vector, as well as for the pharmaceutical composition for the prevention or treatment of inflammatory diseases including stem cells overexpressing S0D3, which is a prior art, as an active ingredient. It was performed in the same way as the patent (refer to the experimental method of Korean Patent Publication No. 10-2017-0032872).
  • Extracellular vesicles derived from S0D3 overexpressing umbilical cord blood stem cells The extracellular vesicles were obtained by ultracentrifugation in hUCB-MSC overexpressing S0D3. At this time, extracellular vesicles were extracted from hUCB-MSC where S0D3 was not overexpressed and used as a control.
  • 3X106 S0D3 overexpressing hUCB-MSC and control hUCB-MSC are placed in a 150X25mm culture dish and fetal bovine serum (Thermo Fi sher Scient if ic, Inc. tham, MA, USA) was cultured with 25 ml of a culture solution containing 2%.
  • the culture was collected in a tube, and ultracentrifugation was performed in the following order using an OPTIMA MAX-XP / MLA-55 rotor (Beckman) equipment.
  • the supernatant was collected and centrifuged for 45 minutes at a rate of 3,000 8 .
  • the supernatant was collected and centrifuged for 30 minutes at a rate of 10,00 kPa, and the supernatant was collected and centrifuged at a rate of 100,00 for 70 minutes.
  • the pellet was suspended on the surface and centrifuged at a rate of 100,00 for 70 minutes. Then pellet Melted to obtain extracellular vesicles.
  • anti_S0D3 (Abeam, Cambridge, United Kingdom)
  • Atopy animal model preparation and extracellular vesicle administration Atopy animal model was prepared by a known method in the art.
  • the mice used in the experiment were C57BL / 6 mice at week 8, were raised with standard mouse feed and water in the absence of specific pathogens, and were treated according to the guidelines of the Catholic University clergy Committee according to the guidelines of the Ministry of Health and Welfare.
  • 1% 2,4- ⁇ 11 ⁇ ; 1 0 (: 11101 0561 6116 (0 8) was applied for 3 days]! 11).
  • 0.2% ⁇ ⁇ was repeatedly applied to the skin of the animal model of the 6th day to prepare (0131 ⁇ ⁇ 6).
  • mice Histological evaluation of mouse model and overexpression of fluorescent tissue staining
  • the back skin tissue was obtained from the mice to which the derived extracellular vesicles were administered, fixed in 4% paraformaldehyde, and then embedded in paraffin.
  • Tissue sections were prepared. Subsequently, it was degreased using xylene) and dehydrated with a concentration gradient alcohol. The pretreated tissue sections were stained with hematoxylin and eosindae staining). In addition, the degree of invasion of mast cells It was confirmed by staining. Sections of paraffin sections of mouse skin tissues were soaked in 0.
  • 1% toluidine blue staining solution 1% toluidine blue solution prepared in 1% (prepared by diluting 10 times in 1 solution) for 2-3 minutes), and then washed 3 times with distilled water. And then dehydrated by dipping in 95% and 100% alcohol one after another to complete the staining by drying with xylten and staining. The stained tissue was observed and counted by granules of mast cells stained with toluidine blue through a microscope.
  • CFSE Carboxyf luoresce in succ inimi dyl ester
  • hUCB-MSC-derived extracellular vesicles with factors that promote T lymphocyte proliferation (day 0), how the proliferation pattern changes compared to the other group. Compared.
  • naive T cel l (ThO) was isolated from normal PBMC, followed by treatment with ant i_IL4 + IFNr or ant i_IFNr + IL4 for 5 days to induce differentiation into Thl and Th2, respectively.
  • Example 3 Confirmation of the effect of suppressing the proliferation of immune cells
  • Immune cells that release pro-inflammatory cytokines such as -1 and -6 from the derived extracellular vesicles The proliferation inhibitory effect was confirmed.
  • Figs. 8 and 9 it was confirmed that the proliferation of epidermis was significantly suppressed when the extracellular vesicles derived from 11'8-3 ⁇ 41 'overexpressing 03 were administered.
  • Example 4 I cell differentiation inhibitory effect confirmation 4-1. Checking the effect on helper 1 cell differentiation Most atopic dermatitis It is caused by an associated immune mechanism, and there are many reports that immune responses by I cell abnormalities are involved, and the role of helper cells (3 ⁇ 4061 1) is known to be important, so that overexpression of ⁇ ⁇ according to the present invention The influence of the derived extracellular vesicles on I cell differentiation was confirmed. As a result, as shown in FIG. 10, it was confirmed that the administration of 111 «3 ⁇ 4-1 ⁇ 1 ⁇ -derived extracellular vesicles overexpressing ⁇ ⁇ reduced! 3 ⁇ 41 differentiation. On the other hand, as shown in Figure 11, it was found that it did not significantly affect the differentiation into 3 ⁇ 42 cells. 2020/085533 1 »(: 1 ⁇ 1 ⁇ 2018/012712
  • composition according to the present invention and the method using the same show the effect of treating atopic dermatitis through suppression of proliferation of immune cells secreting pro-inflammatory cytokines and induction of agitation, and lesion of mast cells It can be used for the prevention of chronic atopic dermatitis or for the treatment of chronic atopic dermatitis by showing the effect of inhibiting site infiltration and inhibiting 3 ⁇ 41 differentiation.

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising, as an active ingredient, extracellular vesicles derived from stem cells in which superoxide dismutase 3 (SOD3) is overexpressed. A composition according to the present invention exhibits the effects of treating atopic dermatitis by inhibiting the proliferation of immune cells which secrete pro-inflammatory cytokines and a Treg-inducing effect, and exhibits the effects of inhibiting the infiltration of lesions of mast cells and inhibiting Th1 differentiation, and thus, can be effectively used for preventing the chronicity of atopic dermatitis or treating chronic atopic dermatitis.

Description

【명세세 【발명의 명칭】 엤此를 과발현시킨 줄기세포 유래 세포외 소낭의 신규한 용도  [Specifications [Name of invention] New use of extracellular vesicles derived from stem cells overexpressing 엤 此
【기술분야】 . 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학 조성물에 관한 것이다. 【Technical Field】. The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
【배경기술】 염증성 질환이란 여러 외부 자극 혹은 내부 요인으로 인해 인체 여러 장기나 조직에 염증성 면역세포의 침윤과 더불어 부종, 발적 및 동통이 나타나고 조직학적 변화를 보이는 상태를 말한다. 이러한 염증은 손상조직과 이동하는 세포 ( 용 061 1 )로부터 생산되는 다양한 화학매개인자에 의하여 촉발되며, 이들 화학매개인자들은 염증 과정의 형태에 따라 다양한 것으로 알려져 있다. 정상적인 경우에 생체는 염증 반응을 통하여 발병 요인을 중화시키거나 제거하고 상한 조직을 재생시켜서 정상적인 구조와 기능을 회복시키지만， 그렇지 못한 경우에는 만성 염증과 같은 질병 상태로 진행되기도 한다. [Background Technology] Inflammatory disease refers to a condition in which edema, redness, and pain appear, and histological changes occur as well as infiltration of inflammatory immune cells in various organs or tissues of the human body due to various external stimuli or internal factors. This inflammation is triggered by a variety of chemical mediators produced from damaged tissues and moving cells (dragon 061 1), which are known to vary depending on the type of inflammatory process. In the normal case, the living body neutralizes or eliminates the pathogen through an inflammatory reaction and regenerates the upper tissue to restore normal structure and function. Otherwise, it progresses to a disease state such as chronic inflammation.
이러한 염증 질환을 치료하기 위한 가장 일반적인 염증성 질환 예방 또는 치료제는 크게 스테로이드성 및 비스테로이드성 염증성 질환 예방 또는 치료용제로 구분되며， 이중 대부분의 합성 염증성 질환 예방 또는 치료용제는 주작용 이외에 여러 가지 부작용을 수반하는 경우가 많으므로 효과가 탁월하며 부작용이 적은 염증성 질환 예방 또는 치료제의 개발이 절실히 요구되고 있는 실정이다. The most common inflammatory disease prevention or treatment for treating these inflammatory diseases is largely divided into steroidal and nonsteroidal inflammatory disease prevention or treatment, and most synthetic inflammatory disease prevention or treatment solvents have various side effects in addition to their main function. Since it is often accompanied, it has an excellent effect, and there is an urgent need for the development or treatment of inflammatory diseases with few side effects.
활성산소를 제거할 뿐만 아니라 다른 항산화 효소가 작용할 수 있도록 하여 세포를 보호하는 기능을 가지는 S0D( super oxide di smutase)는 구리와 아연 원자를 가지고 있는 Cu/Zn S0DCS0D 1)， 망간 원자를 가지고 있는 Mn-S0D(S0D 2) 및 세포 표면 또는 세포 외액에 존재하는 EC-SOD(extracel lular superoxide dismutase)로 구분된다. 그 중 EC-S0D 단백질인 S0D3(superoxide di smutase 3， extracel lular)는 세포 외부로 분비되며 인간의 정상형 (wi ld-type , WT) S0D3 단백질의 아미노산 서열은 NCBI Genbank database의 NP_003093.2 등의 accession number로 공지되어 있다. 또한 인간의 S0D3 단백질을 암호화하는 mRNA의 염기서열은 ■_003102.2 등의 accession number로 공지되어 있다. S0D (super oxide di smutase), which has the function of protecting cells by removing free radicals and allowing other antioxidant enzymes to act, is Cu / Zn with copper and zinc atoms. S0DCS0D 1), Mn with manganese atoms. -S0D (S0D 2) and EC-SOD (extracel lular superoxide dismutase) present on the cell surface or extracellular fluid Are distinguished. Among them, the EC-S0D protein S0D3 (superoxide di smutase 3, extracel lular) is secreted outside the cell, and the amino acid sequence of human normal (wi ld-type, WT) S0D3 protein is accession such as NP_003093.2 of NCBI Genbank database. It is known as number. In addition, the base sequence of mRNA encoding human S0D3 protein is known as an accession number such as ■ _003102.2.
종전 요(©3를 과발현하는 줄기세포를 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물에 대해서 공지된바 있다 (한국공개특허 10-2017- 0032872 참고) . 하지만 줄기세포를 이용한 치료제는 생명윤리 문제뿐만 아니라 분화조절의 어려움， 변이발생， 암세포화 등 부작용 발생의 위험성에 대한 이론적 우려가 꾸준히 제기되고 있어 임상시험 적용 및 제품 상용화에 많은 어려움을 가지고 있다. 이러한 부작용은 투여 즉시 발생하기보다는 수개월 내지 수년에 걸쳐 발생할 가능성이 있으므로， 장기간에 걸쳐 부작용 발생 여부를 확인해야 한다. 더군다나 줄기세포들을 채취한 후 많은 조작과정을 수행하게 되는데, 그 과정에서 세포의 특성이 변할 수도 있고， 박테리아나 바이러스 등 질병을 유발하는 병원체에 감염될 수도 있다. 특히 줄기세포의 경우, 살아있는 유기체이기 때문에 관리는 더욱더 어렵다. Previously, a pharmaceutical composition for the prevention or treatment of inflammatory diseases including stem cells overexpressing © 3 as an active ingredient has been known (see Korean Patent Publication No. 10-2017-0032872). In addition to the bioethics problem, there is a lot of difficulty in applying clinical trials and commercializing products, as theoretical concerns about the risk of side effects such as difficulty in differentiation control, mutation, and cancer cellization have been raised. Since there is a possibility of occurrence over several months to several years, it is necessary to check whether side effects occur over a long period of time.In addition, after the stem cells are collected, many manipulations are performed. In the process, the characteristics of the cells may change, and bacteria or viruses You may also be infected with pathogens that cause disease, such as In the case of gas cells, management is even more difficult because they are living organisms.
실제 임상 또는 제품화를 위해서는 효능이 우선시 되어야 하고， 효능이 동등한 경우에는 시술의 용이성， 가격 등이 경쟁력이 있어야 한다. 하지만 줄기세포를 이용한 치료제는 개인별 맞춤 시술이어야 하므로 고비용일 수밖에 없다. 또한 상기와 같은 장기간에 걸친 부작용 발생 가능성 추적과 원료 관리의 어려움은 제품 가격 상승의 요인이 된다. 따라서 줄기세포를 이용한 치료제의 장점을 그대로 보유할 뿐만 아니라， 부작용의 위험이 적으며 쉬운 보관을 통해 가격이 저감된 치료제의 개발이 필요하다. For actual clinical or commercialization, efficacy should be given priority, and if efficacy is equal, ease of treatment and price must be competitive. However, the treatment using stem cells must be expensive because it must be customized for each individual. In addition, the difficulty of tracking the likelihood of side effects and managing raw materials over a long period of time is a factor in raising product prices. Therefore, it is necessary to develop a therapeutic agent that not only retains the advantages of a therapeutic agent using stem cells, but also has a low risk of side effects and has a reduced cost through easy storage.
【발명의 상세한 설명】 【기술적 과제】 이에， 본 발명자들은 부작용은 현저히 낮으면서 염증성 질환에 있어 뛰어난 치료 효과를 갖는 치료제에 대하여 연구하던 중, 요(©3가 과발현된 줄기세포 유래 세포외 소낭을 아토피를 가진 개체에 투여시 면역조절과 항산화 효과 등의 기능이 현저하게 향상되어 아토피가치료되는 것을 발견하여 본 발명을완성하였다. 【Detailed description of invention】 【Technical problem】 Accordingly, the present inventors studied a therapeutic agent having an excellent therapeutic effect in an inflammatory disease with a significantly low side effect, and immunomodulation and immunoregulation when administering extracellular vesicles derived from stem cells (© 3 overexpressed) to individuals with atopy. The present invention was completed by discovering that atopy is treated by remarkably improving functions such as antioxidant effect.
따라서 본 발명의 목적은 슈퍼옥사이드 디스무타아제 3(superoxide di smutase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방또는 치료용 약학조성물을 제공하는 것이다. 또한 본 발명의 목적은 슈퍼옥사이드 디스무타아제 3(superoxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방또는 치료용 약학조성물을 제공하는 것이다. 또한 본 발명의 목적은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 치료용 약학조성물을 제공하는 것이다. Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating atopic dermatitis consisting of extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3).
본 발명의 또 다른 목적은 슈퍼옥사이드 디스무타아제 3( super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방또는 개선용 화장료조성물을 제공하는 것이다. 또한 본 발명의 목적은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방또는 개선용 화장료조성물을 제공하는 것이다. 또한 본 발명의 목적은 슈퍼옥사이드 디스무타아제 3( super oxi'de di smutase 3 , S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 개선용화장료조성물을 제공하는 것이다. Another object of the present invention is to provide a cosmetic composition for preventing or improving atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing superoxide di smutase 3 (S0D3). It is also an object of the present invention to provide a cosmetic composition for preventing or improving atopic dermatitis consisting of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3). It is also an object of the present invention to provide a cosmetic composition for preventing or improving atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (super oxi'de di smutase 3, S0D3).
본 발명의 또 다른 목적은 엤예를 암호화하는 폴리뉴클레오티드를 포함하는 재조합발현 벡터로줄기세포를 형질감염 시키는 단계; 및 상기 형질감염된 줄기세포를 원심분리하여 세포외 소낭을 추출하는 단계;를 포함하는 것을 특징으로 하는, 아토피 예방또는 치료용 약학조성물의 제조 방법을 제공하는 것이다. Another object of the present invention is a step of transfecting the stem cells with a recombinant expression vector containing a polynucleotide encoding the example; And extracting the extracellular vesicles by centrifuging the transfected stem cells; a method for manufacturing a pharmaceutical composition for preventing or treating atopy. Is to provide.
본 발명의 또 다른 목적은 아토피 치료용 제제를 제조하기 위한 슈퍼옥사이드 디스무타아제 3 (super oxide dismutase 3, S0D3)를 과발현시킨 줄기세포유래 세포외 소낭의 용도를 제공하는 것이다. Another object of the present invention is to provide the use of superoxide dismutase 3 (super oxide dismutase 3, S0D3) overexpressing stem cell-derived extracellular vesicles for the preparation of atopic therapeutic agents.
본 발명의 또 다른 목적은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 아토피 질환의 치료방법을 제공하는 것이다. Another object of the present invention is to administer an effective amount of a composition containing an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient to an individual in need thereof It provides a method of treating atopic disease characterized by.
【기술적 해결방법】 【Technical Solution】
상기와 같은 목적을 달성하기 위하여, 본 발명은 슈퍼옥사이드 디스무타아제 3(superoxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로포함하는 아토피 피부염 예방또는치료용 약학조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 , In order to achieve the above object, the present invention is a pharmaceutical composition for preventing or treating atopic dermatitis comprising extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3) as an active ingredient. to provide. In addition, the present invention superoxide dismutase 3 (super oxide di smutase 3,
S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방 또는 치료용 약학조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 ,It provides a pharmaceutical composition for preventing or treating atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing S0D3). In addition, the present invention superoxide dismutase 3 (super oxide di smutase 3,
S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 치료용 약학조성물을 제공한다. It provides a pharmaceutical composition for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing S0D3).
본 발명의 다른 목적을 달성하기 위하여， 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로포함하는 아토피 피부염 예방또는 개선용 화장료조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방 또는 개선용화장료조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxi de di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 개선용화장료조성물을 제공한다. In order to achieve another object of the present invention, superoxide di smutase 3 (super oxide di smutase 3, S0D3) overexpressed stem cell-derived extracellular vesicles as an active ingredient to prevent or improve atopic dermatitis cosmetic composition for providing do. In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3). In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing superoxide oxidase 3 (S0D3).
본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 엤此를 암호화하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터로 줄기세포를 형질감염 시키는 단계; 미 상기 형질감염된 줄기세포를 원심분리하여 세포외 소낭을 추출하는 단계;를 포함하는 것을 특징으로 하는， 아토피 예방또는 치료용 약학조성물의 제조 방법을 제공한다. In order to achieve another object of the present invention, the present invention comprises the steps of transfecting the stem cells with a recombinant expression vector containing a polynucleotide encoding 엤 此; It provides a method for manufacturing a pharmaceutical composition for preventing or treating atopy, characterized in that it comprises; extracting the extracellular vesicles by centrifuging the transfected stem cells.
본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 아토피 치료용 제제를 제조하기 위한 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 , S0D3)를 과발현시킨 줄기세포유래 세포외 소낭의 용도를 제공한다. In order to achieve another object of the present invention, the present invention provides the use of stem cell-derived extracellular vesicles overexpressing super oxide di smutase 3 (S0D3) for preparing an agent for the treatment of atopy. do.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을특징으로 하는 아토피 질환의 치료 방법을 제공한다. In order to achieve another object of the present invention, the present invention requires an effective amount of a composition comprising extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3) as an active ingredient It provides a method of treating atopic disease characterized in that it is administered to an individual.
이하본 발명을상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 , S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방또는 치료용 약학조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3(superoxide di smutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방 또는 치료용 약학조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3(31¾)610 6 51111 336 3, 엤예)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 치료용 약학조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient an extracellular vesicle derived from stem cells overexpressing super oxide di smutase 3 (S0D3). In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing superoxide di smutase 3 (S0D3). In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (31¾) 610 6 51111 336 3, example).
본 발명의 발명자는 종전 S0D3를 과발현하는 줄기세포의 염증성 질환 치료 효과를 확인한 바 있다 (한국공개특허 10-2017-0032872 참고). 그러나 줄기세포 기반의 치료제는 상기에 기재한 바와 같이 고가의 가격， 장기간의 부작용 발생 가능성 추적과 원료 관리의 어려움으로 인해 임상 시험뿐만 아니라 제품의 상용화에 많은 어려움이 있다. 따라서 본 발명자는 줄기세포를 이용한 치료제의 장점을 그대로 보유한 치료제에 대해 연구하던 중， S0D3 과발현 세포 유래 세포외 소낭 (extracel lular vesicle, EV)가 S0D3 과발현 줄기세포의 장점을 그대로 보유한 것을 확인하였다. 본 발명에서 사용된 “세포외 소낭” 은 세포가 세포외 환경에 다양한 막 (membrane) 유형의 소포체를 방줄하는데, 통상 이러한 방줄 소포체들을 의미한다. 세포외 소낭은 세포막 유래 소포체, 엑토좀 (ectosomes) , 쉐딩 소포체 (shedding vesicles) , 마이크로파티클 (microparticles) , 엑소좀 등으로 불려지기도 하며, 경우에 따라서는 엑소좀과는 구별되어 사용되기도 한다. 본 발명에서는 이에 제한되지 않는다. The inventors of the present invention have previously confirmed the effect of treating inflammatory diseases of stem cells overexpressing S0D3 (see Korean Patent Publication No. 10-2017-0032872). However, as described above, stem cell-based therapeutics have many difficulties in commercialization of products as well as clinical trials due to high price, long-term side effects, and difficulty in managing raw materials. Therefore, the present inventors were studying the therapeutic agent that has the advantages of the therapeutic agent using stem cells, and confirmed that the extracellular vesicles derived from S0D3 overexpressing cells (extracel lular vesicle, EV) retain the advantages of the S0D3 overexpressing stem cells. The term “extracellular vesicle” as used in the present invention means that cells release various vesicles of various membrane types into the extracellular environment. Extracellular vesicles are also called cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes, etc. In some cases, they are used separately from exosomes. The present invention is not limited to this.
본 발명의 세포외 소낭은 S0D3를 과발현하는 줄기세포 유래로， 상기 S0D3는 단백질로 발현될 수 있고， 상기 S0D3 단백질은 음이온의 부동 변화 (dismutation) 반응을 촉매하고， 세포 외부로 분비되어 세포외 기질 (extracel lular matrix)에 존재하며， 항혈관신생， 항화학주성, 항암활성 등의 효과를 나타낸다. The extracellular vesicles of the present invention are derived from stem cells overexpressing S0D3, the S0D3 can be expressed as a protein, and the S0D3 protein catalyzes the dismutation reaction of anions, and is secreted outside the cell to extracellular matrix (extracel lular matrix), and exhibits anti-angiogenic, anti-chemotactic, and anti-cancer activities.
본 발명에서 S0D3는 인간 및 마우스를 포함하는 포유동물， 바람직하게는 인간으로부터 유래한 단백질을 의미하며, 가장 바람직하게는 서열번호 1 또는 서열번호 2로 표시되는 인간의 정상형 S0D3 단백질의 아미노산 서열을 포함하는 것일 수 있다. 또는 서열번호 3 또는 서열번호 4로 표시되는 재조합 S0D3 단백질 (209E-SOD3)의 아미노산서열을포함하는 것일 수 있다. 상기 인간의 정상형 S0D3는 N-말단 부위의 개시 메치오닌 아미노산을 포함하여 18번째의 알라닌까지는 시그날 펩타이드로 되어 있으며, 활성화된 S0D3는 상기 시그날 펩타이드가 제거된 222개의 아미노산으로 구성된다. S0D3의 C-말단 영역 (아미노산 잔기 210번〜 215번)에는 헤파린 결합 도메인 (hepar in binding domain)을 가지고 있다'. 시그널 펩타이드를 포함하는 240개의 아미노산으로 이루어진 전체 길이의 인간 S0D3의 아미노산 서열은 서열번호 1 또는 서열번호 2에 표시된 바와같다. In the present invention, S0D3 refers to a mammal derived from humans and mice, preferably a protein derived from humans, and most preferably includes the amino acid sequence of a human normal S0D3 protein represented by SEQ ID NO: 1 or SEQ ID NO: 2. It may be. Or it may be to include the amino acid sequence of the recombinant S0D3 protein (209E-SOD3) represented by SEQ ID NO: 3 or SEQ ID NO: 4. The human normal S0D3 is a signal peptide up to the 18th alanine, including the starting methionine amino acid at the N-terminal site, and activated S0D3 is composed of 222 amino acids from which the signal peptide is removed. The C-terminal region of S0D3 (amino acid residues 210 to 215) has a heparin binding domain ' . The amino acid sequence of the full length human S0D3 consisting of 240 amino acids including the signal peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
또한본 발명에서의 S0D3는 전체 길이의 인간 S0D3 중 N-말단부위의 시그날 펩티드 및 C-말단 부위의 헤파린 결합 도메인이 포함된 13개의 아미노산 (아미노산 잔기 210번〜 222번)이 제거된 209개의 아미노산으로 이루어진 것일 수도 있다. 상기 시그널 펩타이드와 헤파린 결합 도메인이 제거된 S0D3 단백질은 본 발명에서 209E- S0D3 또는 209E로 지칭할 수 있으며， 바람직하게는 서열번호 3 또는 서열번호 4로 표시되는 아미노산 서열을 가질 수 있다. 상기 209E-S0D3는 항- S0D3 항체와도 결합할 수 있으며， 정상형 S0D3와 동일한 효소 활성 및 R0S 제거 활성을 갖는 것으로 확인된 바 있다 (대한민국공개특허 제 10-2008-0108876호) . In addition, in the present invention, S0D3 is a 209 amino acid in which 13 amino acids (amino acid residues 210 to 222) are removed, which contain a signal peptide at the N-terminal region and a heparin binding domain at the C-terminal region of human S0D3 of full length. It may be made of. The signal peptide and the S0D3 protein from which the heparin binding domain is removed may be referred to as 209E- S0D3 or 209E in the present invention, and preferably may have an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. The 209E-S0D3 can also bind to the anti-S0D3 antibody, and has been confirmed to have the same enzyme activity and R0S removal activity as the normal S0D3 (Korean Patent Publication No. 10-2008-0108876).
본 발명에서의 S0D3는 정상형 S0D3 또는 209E-S0D3 단백질과 실질적으로 동등한 생리 활성을 갖는 기능적 동등물 ( funct ional equivalent ) , 기능적 유도체 (funct ional der ivat ive) 및 상기 S0D3 단백질의 단편이 모두 포함된다. 실질적으로 동등한 생리 활성이란 정상형 S0D3와 동등한 수준의 효소 활성 및/또는 세포외 분비 특성과 세포내 투과성을 갖는 것을 의미하며, S0D3와 동등한 효소 활성으로 인하여 줄기세포， 바람직하게는 중간엽 줄기세포에서 과발현되었을 때 줄기세포의 면역 및 염증 조절능력을 향상시키게 된다. 상기 줄기세포의 면역 및 염증조절능력이란구체적으로는 염증으로 인한 면역 세포의 침윤 억제， 전염증성 T 세포의 증식과 분화 및 전염증성 매개체/사이토카인의 발현의 억제， 염증조절능을 갖는 Treg 세포의 증식과 분화 및 Treg 관련 사이토카인의 발현 증가, NFkB 신호전달계의 인산화 억제 등을 의미하며， 본 명세서에서 서술한 본 발명의 S0D3를 과발현하는줄기세포가갖는특성에서 기재한바와같다. In the present invention, S0D3 includes all functional equivalents (funct ional equivalent), functional derivatives (funct ional der ivat ive), and fragments of the S0D3 protein, which have substantially equivalent physiological activity to the normal S0D3 or 209E-S0D3 protein. Substantially equivalent physiological activity means having the same level of enzyme activity and / or extracellular secretion properties and intracellular permeability as normal S0D3, and overexpression in stem cells, preferably mesenchymal stem cells, due to the enzyme activity equivalent to S0D3 When it is done, it improves the immune and inflammation control ability of stem cells. The immune and inflammatory control ability of the stem cells is specifically, suppression of invasion of immune cells due to inflammation, proliferation and differentiation of pro-inflammatory T cells, and suppression of expression of pro-inflammatory mediator / cytokine, Treg cells having inflammatory control ability Means proliferation and differentiation and increased expression of Treg-related cytokines, inhibition of phosphorylation of the NFkB signaling system, and the like as described in the characteristics of stem cells overexpressing S0D3 of the present invention described herein.
상기 엤此의 기능적 동등물은 바람직하게는 서열번호 1 내지 서열번호 4로 표시되는 아미노산 서열과 적어도 70% 이상， 바람직하게는 80% 이상， 보다 바람직하게는 90% 이상의 서열 상동성 (homology)을 갖는 폴리펠티드일 수 있다. 또한 상기 기능적 동등물은 본 발명의 S0D3의 아미노산 서열 중 일부가 부가, 치환 또는 결실의 결과 생성될 것일 수 있다. 상기에서 아미노산의 치환은 바람직하게는 보존적 치환이다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다: 지방족 아미노산 (Gly, Ala, Pro) , 소수성 아미노산 (lie, Leu, Val) , 방향족 아미노산 (Phe, Tyr , Trp) , 산성 아미노산 (Asp, Glu) , 염기성 아미노산 (His, Lys, Arg, Gin, Asn) 및 황 함유 아미노산 (Cys， Met) . 또한 상기 기능적 동등물에는 본 발명의 S0D3의 아미노산 서열상에서 아미노산의 일부가 결실된 변형체도 포함된다. 상기 아미노산의 결실 또는 치환은 바람직하게는 S0D3 단백질의 생리활성에 직접적으로 관련되지 않은 영역에 위치해 있다. 아울러 상기 S0D3의 아미노산 서열의 양 말단또는서열 내에 몇몇의 아미노산이 부가된 변형체도 포함된다. 예를 들어 209E-S0D3과 같이 S0D3의 효소 활성에는 영향을 주지 않으면서 S0D3 전체 길이의 일부분이 제거된 펩티드일 수 있으며， S0D3 단백질과 실질적으로 동등한 생리 활성을 갖는 SNP(smal 1 nucleotide polymorphism) 등 S0D3의 다형성 (polymorphism) 단백질일 수도 있다. The functional equivalent of VIII is preferably SEQ ID NO: 1 to SEQ ID NO: 4 It may be a polyfeldtide having a sequence homology with at least 70%, preferably 80% or more, and more preferably 90% or more of the amino acid sequence displayed. In addition, the functional equivalent may be generated as a result of addition, substitution, or deletion of some of the amino acid sequences of S0D3 of the present invention. The substitution of amino acids in the above is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (lie, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp , Glu), basic amino acids (His, Lys, Arg, Gin, Asn) and sulfur-containing amino acids (Cys, Met). In addition, the functional equivalent includes a variant in which a part of amino acids is deleted on the amino acid sequence of S0D3 of the present invention. The deletion or substitution of the amino acid is preferably located in a region not directly related to the physiological activity of the S0D3 protein. In addition, a variant in which several amino acids are added to both ends or sequences of the amino acid sequence of S0D3 is also included. For example, S0D3 may be a peptide in which a portion of the entire length of S0D3 is removed without affecting the enzyme activity of S0D3, such as 209E-S0D3, and S0D3 such as SNP (smal 1 nucleotide polymorphism) having substantially equivalent physiological activity to S0D3 protein. It may be a polymorphism protein.
본 발명의 기능적 동등물의 범위에는 본 발명의 S0D3 단백질의 기본 골격 및 이의 생리 활성을 유지하면서 단백질의 일부 화학 구조가 변형된 폴리펩티드 유도체도 포함된다. 예를 들어, 여기 한정되는 것은 아니지만， 본 발명의 S0D3 단백질의 안정성， 세포내 투과성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한구조 변경이 이에 포함된다. The scope of the functional equivalents of the present invention includes polypeptide derivatives in which some chemical structures of proteins have been modified while maintaining the basic framework and physiological activity of the S0D3 protein of the present invention. For example, but not limited to this, structural modifications to change the stability, intracellular permeability, storage, volatile or solubility of the S0D3 protein of the present invention are included therein.
본 발명의 약학적 조성물에 유효성분으로 포함되는 세포외 소낭은 此를 과발현하는 줄기세포 유래로, 상기 줄기세포는 바람직하게는 중간엽 줄기세포일 수 있다. The extracellular vesicle included as an active ingredient in the pharmaceutical composition of the present invention is derived from stem cells overexpressing 此, and the stem cells may preferably be mesenchymal stem cells.
본 발명의 "중간엽 줄기세포 (mesenchymal stem cell, MSC)”는 뼈 (bone), 연골 (carti lage) , 지방조직 (fat tissue) , 힘줄 (건)， 신경조직 (nerve tissue) , 섬유아세포 (fibroblast) 및 근육세포 (muscle cell) 등 구체적인 장기의 세포로 분화되기 전의 다분화 전구 세포 (multipotent progenitor)를 말한다. 본 발명에서 중간엽 줄기세포는 분화되지 않은상태, 즉줄기세포 상태로 조성물 중에 포함된다. 본 발명의 중간엽 줄기세포는 포유류에서 유래한 것일 수 있으며 바람직하게는 인간에서 유래한 것일 수 있다. The "mesenchymal stem cell (MSC)" of the present invention includes bones, cartilage, fat tissue, tendons (nerve tissue), fibroblasts ( Refers to a multipotent progenitor prior to differentiation into cells of specific organs such as fibroblasts and muscle cells. Mesenchymal stem cells are included in the composition in an undifferentiated state, that is, a stem cell state. The mesenchymal stem cells of the present invention may be derived from mammals, and preferably may be derived from humans.
본 발명의 중간엽 줄기세포는 제대, 제대혈， 태반， 골수， 지방조직, 근육， 양수 및 양막으로 이루어진 군으로부터 선택된 조직에서 유래한 것일 수 있다. 바람직하게는 본 발명의 중간엽 줄기세포는 제대혈 또는 태반 유래 중간엽 줄기세포일 수 있으며， 가장 바람직하게는 제대혈 유래 중간엽 줄기세포일 수 있다. 제대혈 또는 태반 유래 중간엽 줄기세포는 골수 유래 중간엽 줄기세포보다 분화 능력 및 증식 능력이 뛰어난특징을지닌다. The mesenchymal stem cells of the present invention may be derived from tissue selected from the group consisting of umbilical cord, umbilical cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, and amniotic membrane. Preferably, the mesenchymal stem cells of the present invention may be umbilical cord blood or placenta-derived mesenchymal stem cells, most preferably umbilical cord blood-derived mesenchymal stem cells. Umbilical cord blood or placenta-derived mesenchymal stem cells have the characteristics of superior differentiation and proliferation ability than bone marrow-derived mesenchymal stem cells.
본 발명에서 사용된 용어 제대혈은 포유동물의 태반과 태아를 연결하는 제대정맥으로부터 채취된 혈액을 말한다. 상기 제대혈은 출산시 공여자의 제대정맥으로부터 용이하게 채취할 수 있다. 보다 구체적으로， 정상질식분만의 경우에는 출산 후 자궁 내에 아직 태반이 남아있는 상태에서 밖으로 만출된 제대정맥으로부터 채취할 수 있다. 또는 제왕절개의 경우에는 출산 후 태반 역시 자궁밖으로 만출된 상태에서 제대정맥으로부터 채취한다. The term umbilical cord blood used in the present invention refers to blood collected from umbilical vein connecting the placenta and fetus of a mammal. The umbilical cord blood can be easily collected from the donor's umbilical vein during childbirth. More specifically, in the case of normal vaginal delivery, the placenta still remains in the womb after childbirth, and can be collected from the umbilical vein exposed outside. Alternatively, in the case of a cesarean section, the placenta after birth is also taken from the umbilical vein in the state of being exposed outside the womb.
본 명세서에서, 태반에서 채취된 줄기세포는 "태반 줄기세포'’라는 용어로 기재될 수 있고, 이는 형상, 세포 표면 표지 또는 1차 배양 후 계대 수에 무관하게 포유류 태반에서 유래한 줄기세포 또는 전구세포로서 조직 배양 기질 (t issue culture substrate) , 예를 들어 조직 배양 플라스틱 또는 피브로넥틴 피복 조직 배양판에 부착하는 것을 말한다. 그러나본 명세서에서 "태반줄기세포"라는 용어는 영양막 세포 (trophoblast )를 가리키는 것은 아니다. 세포는 줄기세포의 특징 중 적어도 하나, 예를 들어 적어도 하나 이상의 세포 유형으로 분화할 수 있는 능력 등을 갖추고 있으면 줄기세포이다. In the present specification, stem cells collected from the placenta may be described as a term "placental stem cells," which are stem cells or precursors derived from mammalian placenta regardless of shape, cell surface label, or number of passages after primary culture. As a cell, it refers to the attachment to a tissue culture substrate, such as tissue culture plastic or fibronectin coated tissue culture plate, but the term "placental stem cell" used herein refers to a trophoblast. No. A cell is a stem cell if it has at least one of the characteristics of a stem cell, for example, the ability to differentiate into at least one cell type.
태반 또는 제대혈로부터 당업계에 공지된 방법에 따라 중간엽 줄기세포를 분리할 수 있다. 상기 중간엽 줄기세포의 분리는 종래 알려진 어떤 분리방법에 의해서도 분리될 수 있다. 예를 들면, 밀도차를 이용한 분리법 (density gradient fract ionat ion) , 면역선택 ( immunose lect ion) 및 감별 부착 분리법 (di f ferent ial adhesion separat ion) 등이 있다. 제대혈으로부터 중간엽 줄기세포를 분리, 배양하는 방법은 기존에 사용되어 온 방법은 모두 사용할수 있다. Mesenchymal stem cells can be isolated from placental or umbilical cord blood according to methods known in the art. The mesenchymal stem cells can be separated by any conventionally known separation method. For example, a separation method using a density difference (density gradient fract ionat ion, immunoselection, and differential adhesion separat ion. The method of separating and culturing mesenchymal stem cells from umbilical cord blood can be used for all methods that have been used previously.
상기에서 분리된 중간엽 줄기세포의 배양은 당업계에 공지된 세포 배양용 배지에서 수행할 수 있으며, 예를 들면， 이에 한정되지는 않으나， DMEM 배지, McCoys 5A 배지, Eagle ' s basal 배지, CMRL 배지, Glasgow 최소 필수 배지， Ham' s F-12 배지， Iscove’s modi f ied Dulbecco’s 배지, Liebovi tz ' L-15 배지, RPMI 1640 배지 KSB-3 basal media등이 있다. 또한, 본 발명에서 세포 배양 배지에는 필요에 따라 한 가지 이상의 보조 성분을 첨가할 수 있는데, 우태아혈청, 말 또는 인간 등의 혈청을 비롯하여, 미생물의 오염을 막기 위한 항생제 및 항진균제 등을 사용할 수 있다. Cultivation of the mesenchymal stem cells isolated from the above can be performed in a cell culture medium known in the art, for example, but not limited to, DMEM medium, McCoys 5A medium, Eagle's basal medium, CMRL Badges, Glasgow minimum required medium, Ham's F-12 medium, Iscove's modi f ied Dulbecco's medium, Liebovi tz 'L-15 medium, RPMI 1640 medium KSB-3 basal media and more. In addition, in the present invention, one or more auxiliary components may be added to the cell culture medium, if necessary, including serum of fetal bovine serum, horse or human, and antibiotics and antifungal agents for preventing contamination of microorganisms. .
분리 또는 배양된 줄기세포는 사용 전까지 당업계에 공지된 방법에 의해 보관될 수 있다. 일반적으로 줄기세포는 동결보호 아근 )!!) 처리한 후 냉동 보관할 수 있다. 상기 동결보호 처리는 당업계에 공지된 엤, 글리세롤， 폴리비닐피롤리돈， 폴리에틸렌 글리콜， 알부민, 덱스트관， 수크로스， 에틸렌 글리콜， 卜에리스리톨, I) -리비톨, I)-만니톨 , I) -솔비톨， 卜이노시톨, I)-락토스 또는 콜린 클로라이드와 같은 동결보호제를 이용하여 수행할 수 있다. The isolated or cultured stem cells can be stored by methods known in the art until use. In general, stem cells can be stored frozen after treatment with cryoprotected roots !!). The freeze protection treatment is known in the art 엤, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dext tube, sucrose, ethylene glycol, 卜 erythritol, I)-ribitol, I) -mannitol, I) -Can be performed using a cryoprotectant such as sorbitol, inositol, I) -lactose or choline chloride.
본 발명에 따른 엤此를 과발현하는 줄기세포는 此를 암호화하는 폴리뉴클레오티드를 포함하는 재조’합 발현 벡터를 줄기세포에 형질도입시켜 수득한 것일 수 있다. Stem cells over-expressing 엤 此 according to the present invention may be obtained by transducing a recombinant 'combination expression vector containing a polynucleotide encoding 此 into stem cells.
본 명세서에서 용어 n발현 (expression) "은 세포에서 단백질 또는 핵산의 생성을 의미하며, “과발현 (overexpression) "은 정상적인 상태 또는 일반적인 상태보다 특정 유전자의 발현의 수준이 과도하게 증가한 것을 의미한다. 본 발명에서 S0D3를 과발현하는 줄기세포는 구체적으로는 S0D3 단백질의 발현 수준이 증가하여 S0D3 단백질의 활성이 증가되어 있는 줄기세포이다. 본 명세서에서 "폴리뉴클레오티드 (1)01 1111(：1601；1(16) " 또는 핵산은 단일-또는 이중-가닥의 형태로 된 데옥시리보뉴클레오티드山 ) 또는 리보뉴클레오티드 ( 시를 말한다. 다른 제한이 없는 한， 자연적으로 생성되는 뉴클레오티드와 비슷한 방법으로 핵산에 혼성화되는자연적 뉴클레오티드의 공지된 아날로그도포함된다. As used herein, the term n expression (expression) refers to the production of a protein or nucleic acid in a cell, and "overexpression (overexpression)" means that the level of expression of a particular gene is excessively increased than the normal or normal state. In the present invention, stem cells over-expressing S0D3 are specifically stem cells having an increased expression level of S0D3 protein, thereby increasing the activity of S0D3 protein. As used herein, "polynucleotide (1) 01 1111 (： 1601; 1 (16)" or nucleic acid refers to deoxyribonucleotide acids in single- or double-stranded form) or ribonucleotides (poetry. Otherwise, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.
줄기세포에서 S0D3를 과발현시키는 가장 일반적인 방법은 S0D3 유전자를 포함하는 폴리뉴클레오티드를 줄기세포에 인위적으로 또는 실험적으로 도입하여 S0D3 유전자의 복제개수 (copy number)를 증가시키는 것이다. 원래의 세포가 가지고 있던 것이 아닌 외인성 (exogenous) 폴리뉴클레오티드를 세포 내부로 주입하는 것을 형질감염 (transfect ion) , 그리고 이로 인하여 세포의 유전적 형질이 변화되는 현상을 형질전환 (transformat ion)이라고 한다. 상기 외인성 폴리뉴클레오티드가 바이러스 또는 바이러스 유래 벡터를 통하여 세포 내부로 주입되는 과정을 형질도입 (transduct ion)이라고 부른다. 본 명세서에서는 용어 ‘형질전환’ ,The most common way to overexpress S0D3 in stem cells is to artificially or experimentally introduce polynucleotides containing the S0D3 gene into stem cells to increase the copy number of the S0D3 gene. Injecting an exogenous polynucleotide that is not possessed by the original cell into the cell is called transfection, and a phenomenon in which the genetic trait of the cell is changed is called transformation. The process in which the exogenous polynucleotide is injected into a cell through a virus or a virus-derived vector is called transduction ion. In this specification, the term 'transformation',
‘형질감염’ 그리고 ‘형질도입’ 이 외인성 폴리뉴클레오티드를 세포에 도입되어 정상형과 다른 유전적 형질을 갖게 된 것 또는 그러한 과정을 일컫는 것으로서 유사한의미로사용된다. ‘Transfection’ and ‘transfection’ are used in a similar sense as referring to the process or introduction of an exogenous polynucleotide into a cell to have a genetic trait different from the normal type.
본 발명에서 S0D3를 암호화하는 폴리뉴클레오티드는 포유류에서 유래한 S0D3 유전자일 수 있다. 바람직하게는 서열번호 5 또는 서열번호 6으로 표시되는 인간의 정상형 S0D3를 암호화하는 염기서열을 포함하는 것일 수 있다. 또는 서열번호 7 또는 서열번호 8로 표시되는 209E-S0D3를 암호화하는 염기서열을 포함하는 것일 수 있다. 보다 상세하게는 서열번호 1의 아미노산 서열로 이루어지는 就) D3는 서열번호 5로 표시되는 염기서열에 의하여 코딩될 수 있고, 서열번호 2의 아미노산 서열로 이루어지는 S0D3는 서열번호 6으로 표시되는 염기서열에 의하여 코딩될 수 있다. 또한 서열번호 3의 아미노산 서열로 이루어지는 S0D3는 서열번호 7로 표시되는 염기서열에 의하여 코딩될 수 있고, 서열번호 4의 아미노산 서열로 이루어지는 S0D3는서열번호 8로 표시되는 염기서열에 의하여 코딩될 수 있다. In the present invention, the polynucleotide encoding S0D3 may be a S0D3 gene derived from a mammal. Preferably, it may include a nucleotide sequence encoding human normal S0D3 represented by SEQ ID NO: 5 or SEQ ID NO: 6. Or it may include a nucleotide sequence encoding 209E-S0D3 represented by SEQ ID NO: 7 or SEQ ID NO: 8. More specifically, 就) D3 consisting of the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence shown in SEQ ID NO: 5, S0D3 consisting of the amino acid sequence of SEQ ID NO: 2 to the nucleotide sequence shown in SEQ ID NO: 6 Can be coded by In addition, S0D3 consisting of the amino acid sequence of SEQ ID NO: 3 may be encoded by the nucleotide sequence represented by SEQ ID NO: 7, S0D3 consisting of the amino acid sequence of SEQ ID NO: 4 may be encoded by the nucleotide sequence represented by SEQ ID NO: 8 .
또한 상기 此를 암호화하는 폴리뉴클레오티드는 인간의 엤此의 염기서열, 바람직하게는 서열번호 5 내지 서열번호 8로 표시되는 염기서열과 실질적인 동일성을 나타내는 서열도 포함한다. 실질적인 동일성은 인간 엤 를 암호화하는 2020/085533 1»（：1^1{2018/012712 In addition, the polynucleotide encoding 此 also includes a sequence showing substantial identity to the nucleotide sequence of human 엤 此, preferably the nucleotide sequence represented by SEQ ID NO: 5 to SEQ ID NO: 8. Substantive identity encodes the human 엤 2020/085533 1 »（： 1 ^ 1 {2018/012712
12 폴리뉴클레오티드의 염기서열과 비교 대상이 되는 임의의 다른 염기서열을 최대한 대응되도록 정렬하고， 당해 기술분야에서 통상적으로 이용되는 분석 방법과 알고리즘을 이용하여 서열을 비교 분석하는 경우， 최소 70% 이상의 상동성을 나타내는 서열을 의미한다. 상기 3孤3를 암호화하는 폴리뉴클레오티드의 염기서열과 실질적으로 동일한 염기서열에 의해 암호화되는 단백질은 8003 단백질， 바람직하게는 서열번호 1 내지 서열번호 4로 표시되는 아미노산 서열을 포함하는 단백질의 기능적 동등물일 수 있다. 5003 단백질의 기능적 동등물에 대하여서는 본 명세서에서 앞서 서술한 바와 같다.  12 Align the nucleotide sequence of the polynucleotide with any other nucleotide sequence to be compared as much as possible, and compare and analyze the sequence using analysis methods and algorithms commonly used in the art. It means the sequence showing the same sex. The protein encoded by the base sequence substantially identical to the base sequence of the polynucleotide encoding 3 孤 3 is 8003 protein, preferably a functional equivalent of a protein comprising an amino acid sequence represented by SEQ ID NOs: 1 to 4 Can be. The functional equivalents of the 5003 protein are as previously described herein.
본 명세서에서 ’’재조합 발현 벡터’’란 적합한 숙주세포에서 목적 단백질 또는 목적 핵산 ( 시을 발현할 수 있는 벡터로서， 폴리뉴클레오티드 (유전자) 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다. 작동가능하게 연결된 (애 크비
Figure imgf000014_0001
"이란 일반적 기능을 수행하도록 핵산 발현 조절 서열과 목적하는 단백질 또는 쇼를 코딩하는 핵산 서열이 기능적으로 연결 ( £1111 101131 1 止^용6)되어 있는 것을 말한다. 즉, 단백질 또는
Figure imgf000014_0002
핵산 서열이 발현 조절 서열에 의해 유전자 발현이 가능하게 되는 방식으로 연결된 것을 의미하는 것으로, 예를 들어 프로모터와 단백질 또는 例쇼를 코딩하는 핵산 서열이 작동가능하게 연결되어야 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을사용한다. In the present specification, `` recombinant expression vector '' refers to a gene composition comprising an essential regulatory element operably linked to express a target protein or target nucleic acid (poetry-expressing vector, a polynucleotide (gene) insert) in a suitable host cell. Sacrifice, which is operatively connected
Figure imgf000014_0001
"" Means that the nucleic acid expression control sequence and the desired protein or nucleic acid sequence encoding a show are functionally linked (for £ 1111 101131 1 止 ^ 6) to perform a general function.
Figure imgf000014_0002
It means that the nucleic acid sequence is linked in such a way that gene expression is possible by an expression control sequence. For example, a promoter and a nucleic acid sequence encoding a protein or a shock can be operably linked to affect the expression of the encoding nucleic acid sequence. Can be crazy. Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art, and site-specific cleavage and linkage uses enzymes, etc., generally known in the art.
본 발명의 재조합 발현 벡터는 클로닝 분야에서 통상적으로 사용되는 벡터라면 그 종류가 특별히 제한되지 않으며， 그 예로는 플라스미드 벡터, 코즈미드 벡터， 박테리오파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 바람직하게는 바이러스에서 유래한 벡터를 사용할 수 있다. 상기 플라스미드에는 대장균 유래 플라스미드 081^322 , ?8모325 , 1)1018 및 1)1019 , ?£1-225(+) ) , 바실러스 서브틸리스 유래 플라스미드 (1)仰110 및 ?5) 및 효모 유래 플라스미드竹묘1)13 , ^2^ 및 ¥0?50) 등이 있으며, 상기 바이러스는 레트로바이러스, 아데노바이러스 또는 백시니아 바이러스와 같은 동물 바이러스, 배큘로 바이러스와 같은 곤충 바이러스 등이 사용될 수 있으며， 이에 제한되지 않는다. 본 발명에 따른 핵산을 포함하는 발현 벡터는 당업계에 공지된 방법， 예를 들어 이에 한정되지는 않으나, 일시적 형질감염 (transient transfect ion) , 미세주사， 형질도입 (transduct ion) , 세포융합, 칼슘 포스페이트 침전법， 리포좀 매개된 형질감염 ( l iposome-mediated transfect ion) , DEAE 덱스트란-매개된 형질감염 (DEAE Dextran- mediated transfect ion) , 폴리브렌-매개된 형질감염 (polybrene-mediated transfect ion) , 전기천공법 (electroporat ion) , 유전자 종 (gene gun) 및 세포 내로 핵산을유입시키기 위한공지의 방법에 의해 줄기세포 내로도입할수 있다. The recombinant expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the cloning field, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Preferably, vectors derived from viruses can be used. The above plasmid is E. coli-derived plasmid 081 ^ 322 ,? 8 Mo 325, 1) 1018 and 1) 1019 ,? £ 1-225 (+)), plasmids derived from Bacillus subtilis (1) 仰 110 and? 5) and plasmids derived from yeast bamboo seedlings 1) 13, ^ 2 ^ and ¥ 0? 50), etc. Animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus, etc. may be used, but are not limited thereto. Expression vectors comprising nucleic acids according to the present invention are methods known in the art, for example, but not limited to, transient transfection (transient transfect ion), micro injection, transduction (transduct ion), cell fusion, calcium Phosphate precipitation method, liposome-mediated transfection, DEAE dextran-mediated transfect ion, polybrene-mediated transfect ion, It can be introduced into stem cells by electroporat ion, gene gun, and known methods for introducing nucleic acids into cells.
본 발명에 따른 003를 과발현하는 줄기세포는 3(X)3를 암호화하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터를 바람직하게는 전기천공법 또는 바이러스로 매개되는 형질도입 방법을 이용하여 중간엽 줄기세포에 주입하여 형질전환한 것일 수 있다. Stem cells over-expressing 003 according to the present invention are preferably expressed in a mesenchymal stem cell using a recombinant expression vector containing a polynucleotide encoding 3 (X ) 3, preferably using an electroporation method or a virus-mediated transduction method. It may be transformed by injection.
또한 본 발명의 줄기세포는 재조합 바이러스 벡터를 이용하여 다음의 단계를 거쳐 제조될 수 있다: (a) 셔틀벡터， S0D3를 암호화하는 핵산 및/또는 단백질 형질도입 도메인이 작동가능하게 연결된 DNA 컨스트럭트를 포함하는 재조합 바이러스 벡터를 제조하는 단계; (b) 상기 재조합 바이러스 벡터를 바이러스 생산 세포주에 형질감염시켜 S0D3 발현 재조합 바이러스를 제조하는 단계; 및 (c) 상기 S0D3발현 재조합바이러스로 중간엽 줄기세포를 감염시키는 단계. In addition, the stem cells of the present invention can be prepared by using a recombinant virus vector through the following steps: (a) Shuttle vector, DNA construct encoding a S0D3 nucleic acid and / or protein transduction domain operably linked thereto. Preparing a recombinant viral vector comprising; (b) transfecting the recombinant virus vector into a virus-producing cell line to produce a S0D3 expressing recombinant virus; And (c) infecting mesenchymal stem cells with the S0D3 expressing recombinant virus.
본 발명의 바이러스 벡터는 렌티바이러스 벡터, 레트로바이러스 벡터， 아데노바이러스 벡터， 아데노연관바이러스 ( \0 벡터, 벡시니아바이러스 벡터, 허피스바이러스 벡터 및 아비폭스바이러스 벡터로 이루어진 군에서 선택되는 것을 특징으로 한다. 바람직하게는 렌티바이러스 벡터일 수 있다. The viral vector of the present invention is characterized in that it is selected from the group consisting of lentiviral vector, retroviral vector, adenovirus vector, adeno-associated virus (\ 0 vector, vaccinia virus vector, herpesvirus vector and abipoxvirus vector). Preferably it can be a lentiviral vector.
따라서 본 발명은 (©3를 암호화하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터로줄기세포를 형질감염 시키는 단계; 및 상기 형질감염된 줄기세포를 원심분리하여 세포외 소낭을 추출하는 단계;를 포함하는 것을특징으로 하는， 아토피 예방또는 치료용 약학조성물의 제조 방법을 제공한다. 본 발명에 따른 약학적 조성물은 8003 과발현 줄기세포 유래 세포외 소낭을 단독으로 함유하거나 약학적으로 허용되는 담체와 함께 적합한 형태로 제형화 될 수 있으며， 부형제 또는 희석제를 추가로 함유할 수 있다. 상기에서 ’약학적으로 허용되는’이란 생리학적으로 허용되고 인간에게 투여될 때 , 통상적으로 위장 장애 , 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한다. Therefore, the present invention comprises the steps of (transfecting stem cells with a recombinant expression vector containing a polynucleotide encoding © 3; and extracting extracellular vesicles by centrifuging the transfected stem cells). To provide a method for manufacturing a pharmaceutical composition for preventing or treating atopy. The pharmaceutical composition according to the present invention may contain 8003 overexpressing stem cell-derived extracellular vesicles alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may additionally contain excipients or diluents. As used herein, 'pharmaceutically acceptable' refers to a non-toxic composition that does not cause allergic reactions or similar reactions, such as gastrointestinal disorders, dizziness, etc., when physiologically acceptable and administered to humans.
약학적으로 허용되는 담체로는 예컨대， 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분， 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 아울러， 펩티드 제제에 대한 경구투여용으로 사용되는 다양한 약물전달물질을 포함할 수 있다. 또한, 비경구 투여용 담체는 물， 적합한 오일， 식염수， 수성 글루코오스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨， 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다 . 적합한 보존제로는 벤즈알코늄 클로라이드， 메틸-또는 프로필 과라벤 및 클로로부탄올이 있다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제 , 습윤제 , 감미제 , 향미제 , 유화제， 현믿 i제 등을 주가로 포함할 수 있다 . 그 밖의 약학적으로 허용되는 담체 및 제제는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다 (Remington’ s Pharmaceut ical Sciences , 19th ed. , Mack Publ i shing Company, Easton, PA, 1995) . As a pharmaceutically acceptable carrier, for example, a carrier for oral administration or a carrier for parenteral administration may be further included. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to the peptide preparation. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl guaraben and chlorobutanol. The pharmaceutical composition of the present invention may include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a prestigious agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations can be referenced as described in the following literature (Remington's Pharmaceut ical Sciences, 19th ed., Mack Publ i shing Company, Easton, PA, 1995).
본 발명의 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나， 정맥내， 근육내， 동맥내， 골수내， 경막내， 심장내, 경피, 피하， 복강내， 비강내, 장관， 국소, 설하 또는 직장내 투여일 수 있다. 보다 바람직하게는 피하에 주입되는 것이나， 이에 제한되는 것은 아니다. The composition of the present invention can be administered in any way to mammals, including humans. For example, it can be administered orally or parenterally. The parenteral administration method is not limited to this, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or intrarectal administration. Can be More preferably, it is injected subcutaneously, but is not limited thereto.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다. - 경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제， 캡슐제， 액제， 겔제， 시럽제, 슬러리제， 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈， 덱스트로즈， 수크로즈， 솔비톨， 만니톨， 자일리톨， 에리스리톨 및 말티톨 등을 포함하는 당류와옥수수 전분, 밀 전분， 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈， 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 · 셀룰로즈류， 젤라틴， 폴리비닐피롤리돈등과 같은충전제가포함될 수 있다. 또한, 경우에 따라가교결합 폴리비닐피롤리돈, 한천， 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은항응집제， 윤활제 , 습윤제 , 향료, 유화제 및 방부제 등을추가로포함할수 있다. 비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제， 외용연고제， 오일제, 보습제, 겔제， 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌 (Remington’s Pharmaceut ical Science , 19th ed. , Mack Publ i shing Company, Easton, PA, 1995)에 기재되어 있다. The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above. - In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. Can be. For example, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient and then grinding it and adding a suitable adjuvant to the granule mixture. Examples of suitable excipients are sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starch including wheat starch, rice starch and potato starch, cellulose, etc. Fillers such as celluloses, gelatin, polyvinylpyrrolidone, etc. may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose. In addition, if desired, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative. In the case of preparations for parenteral administration, injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol, and nasal inhaler may be formulated by a method known in the art. These formulations are described in Remington's Pharmaceut ical Science, 19th ed., Mack Publ Shing Company, Easton, PA, 1995, a prescription generally known to all pharmaceutical chemistries.
본 발명의 조성물의 총 유효량은 단일 투여량 (single dose)으로 환자에게 투여될 수 있으며， 다중 투여량 (mul t iple dose)으로 장기간 투여되는 분할 치료 방법 (fract ionated treatment protocol )에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게 본 발명의 약학적 조성물의 바람직한 전체 용량은 1일당 환자 체중 lkg 당 약 0.01//g 내지 10,000mg， 가장 바람직하게는 0.1/ 내지 500mg일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법， 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중， 건강 상태， 성별， 질환의 중증도, 식이 및 배설율 등 다양한 요인들을 고려하여 환자에 대한유효투여량이 결정되는 것이므로， 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한그 제형， 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. 본 발명의 일실시예에 따르면， 본 발명에 따른 S0D3 과발현 줄기세포 유래 세포외 소낭을 아토피성 피부염을 유도한 동물 모델에 처리하고 병변의 변화를 육안으로 평가한 결과 S0D3과발현 제대혈 줄기세포 유래 세포외 소낭을 처리하였을 때 가장증상이 개선된 것을 확인하였다（실시예 2-1， 도 3 참조）. The total effective amount of the composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractional ionized treatment protocol that is administered for a long time in multiple doses. have. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 // g to 10,000 mg per 1 kg of patient body weight per day, and most preferably 0.1 / to 500 mg per day. However, the dose of the pharmaceutical composition is determined by considering various factors such as the formulation method, the administration route and the number of treatments, as well as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, etc. Considering this, those skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effects of the present invention. According to one embodiment of the present invention, S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention were treated in an animal model inducing atopic dermatitis and visually evaluated for changes in lesions, S0D3 overexpressing umbilical cord blood stem cell-derived extracellular cells When the vesicles were treated, it was confirmed that the most improved symptoms (Example 2-1, see Fig. 3).
본 발명의 또 다른 일실시예에서는 본 발명에 따른 5003 과발현 줄기세포 유래 세포의 소낭을 아토피성 피부염을 유도한 동물 모델에 처리하고 병변의 중증도를 조직염색을 통해 평가한 결과， 외피（印 하미 ） 두께와 림프구 침윤 정도가 감소된 것을 확인하였다. 또한 아토피성 피부염의 만성화에 핵심적인 역할을 하는 비만세포의 침윤 또한 억제하는 것을 확인할수 있었다（실시예 2-2, 도 4내지 도 7참조）. In another embodiment of the present invention, the vesicles of 5003 overexpressing stem cell-derived cells according to the present invention are treated in an animal model inducing atopic dermatitis, and the severity of the lesion is evaluated through tissue staining, and the skin is coated. It was confirmed that the thickness and the degree of lymphocyte infiltration were reduced. In addition, it was confirmed that the invasion of mast cells, which play a key role in the chronicization of atopic dermatitis, was also suppressed (see Examples 2-2 and 4 to 7).
본 발명의 또 다른 일실시예에서는 본 발명에 따른 S0D3 과발현 줄기세포 유래 세포의 소낭이 TNF-a, IL-1 |3 , IL-6과 같은 전염증성 사이토카인을 방출하는 면역세포에 미치는 영향을 확인한 결과， 본 발명에 따른 세포외 소낭의 처리에 의해 면역 세포인 말초혈액 단핵세포（peripheral blood mononuclear cell, PBMC）의 증식을 억제하는 것을 확인할수 있었다（실시예 3, 도 8 및 도 9참조）. In another embodiment of the present invention, the effect of the vesicles of S0D3 overexpressing stem cell-derived cells according to the present invention on immune cells releasing pro-inflammatory cytokines such as TNF-a, IL-1 | 3, IL-6 As a result, it was confirmed that the proliferation of peripheral blood mononuclear cells (PBMC), which are immune cells, was inhibited by the treatment of the extracellular vesicles according to the present invention (see Examples 3, 8 and 9). .
본 발명의 또 다른 일실시예에서는 본 발명에 따른 S0D3 과발현 줄기세포 유래 세포외 소낭이 헬퍼 T세포（helper T cell） 분화에 미치는 영향을 확인한 결과, Thl의 분화가 억제된 반면, Th2의 분화에는 영향을 미치지 않음을 확인하였다（실시예 4-1, 도 W및 도 11참조）. In another embodiment of the present invention, as a result of confirming the effect of S0D3 overexpressing stem cell-derived extracellular vesicles on helper T cell differentiation according to the present invention, Thl differentiation was inhibited, whereas Th2 differentiation It was confirmed that there was no effect (see Example 4-1, Fig. W and Fig. 11).
본 발명의 또 다른 일실시예에서는 본 발명에 따른 S0D3 과발현 줄기세포 유래 세포외 소낭이 전체적인 면역 반응과 타면역세포의 과도한활성화 억제에 핵심 역할을 하는 조절 T세포（regulatory T cell, Treg） 분화에 미치는 영향을 확인한 결과, 본 발명의 세포외 소낭은 뛰어난 Treg유도 능력이 있음을 확인하였다（실시예 4-2, 도 12참조）. In another embodiment of the present invention, S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention are used for differentiation of regulatory T cells (regulatory T cells) that play a key role in suppressing the overall immune response and excessive activation of other immune cells. As a result of confirming the effect, it was confirmed that the extracellular vesicles of the present invention have excellent Treg-inducing ability (see Example 4-2, FIG. 12).
상기 실시예의 결과를 통해, 본 발명에 따른 8003 과발현 줄기세포 유래 세포외 소낭은 전염증성 사이토카인을 분비하는 면역세포의 증식 억제, Treg 유도 효과를통해 아토피 피부염 치료효과를보이는 것을 확인하였다. 특히， 본 발명에 따른 S0D3 과발현 줄기세포 유래 세포외 소낭은 비만세포의 병변부위 침윤을 억제하며， 헬퍼 T 세포의 분화에 있어서 Thl의 분화는 억제하지만 Th2의 분화에는 영향을 미치지 않는유의미한결과를보였다. 아토피성 피부염은 대부분 묘와 연관된 면역기전에 의해 발병되는데， T 세포 이상에 의한 면역반응이 관여한다는 보고가 많으며， 헬퍼 T세포 (helper T cel l )의 역할이 중요하다고 알려져 있다. 헬퍼 T세포에는 헬퍼 T1 형 (Thl) 세포와 헬퍼 T2형 (Th2) 세포가 포함된다. Thl 세포가 분비하는 사이토카인은 세포매개형 면역반응에 관여하며， 아토피성 피부염의 경우 Thl 세포가 분비하는 인터페론- Y ( IFN- Y )과 같은 Thl 사이토카인의 분비가 감소하는 것으로 알려져 있다. 초기에 Th2 면역반응에 의하여 개시되어 점차 만성적인 Thl 면역반응으로 전환된다고 알려져 있다 (Am J Cl in Dermatol 5； 281-294, 2004 및 Nature 413:531-534, 2001 참고) . Through the results of the above example, 8003 overexpressing stem cells derived from the present invention It was confirmed that the extracellular vesicles show the effect of treating atopic dermatitis through suppressing proliferation of immune cells secreting pro-inflammatory cytokines and inducing Treg. In particular, the S0D3 overexpressing stem cell-derived extracellular vesicles according to the present invention inhibit the invasion of mast cell lesions and suppress the Thl differentiation in the differentiation of helper T cells, but show significant results that do not affect the differentiation of Th2. . Atopic dermatitis is most often caused by immune mechanisms associated with seedlings, and there are many reports that immune responses by T cell abnormalities are involved, and the role of helper T cells is known to be important. Helper T cells include helper T1 type (Thl) cells and helper T2 type (Th2) cells. Cytokines secreted by Thl cells are involved in cell-mediated immune responses, and in the case of atopic dermatitis, secretion of Thl cytokines such as interferon-Y (IFN-Y) secreted by Thl cells is known to decrease. It is known to be initiated by the Th2 immune response and gradually converted into a chronic Thl immune response (see Am J Cl in Dermatol 5; 281-294, 2004 and Nature 413: 531-534, 2001).
따라서 이상의 세포 실험과 동물 실험 결과는 본 발명에 따른 8003 과발현 줄기세포 유래 세포외 소낭이 뛰어난 아토피 치료 효과를 보일뿐만 아니라, 만성적인 아토피 피부염의 치료 효과를 갖는 안전하고 효과적인 의약 후보 물질로 사용될 수 있음을 시사한다. Therefore, the above cell test and animal test results show that the 8003 overexpressing stem cell-derived extracellular vesicles according to the present invention show excellent atopy treatment effect, and can be used as a safe and effective pharmaceutical candidate substance having a treatment effect of chronic atopic dermatitis. Suggests
본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 , S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방또는 개선용 화장료조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 ,The present invention provides a cosmetic composition for preventing or improving atopic dermatitis, which comprises extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient. In addition, the present invention superoxide dismutase 3 (super oxide di smutase 3,
S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 구성되는 아토피 피부염 예방 또는 개선용 화장료조성물을 제공한다. 또한 본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3 ,Provides a cosmetic composition for preventing or improving atopic dermatitis composed of extracellular vesicles derived from stem cells overexpressing S0D3). In addition, the present invention superoxide dismutase 3 (super oxide di smutase 3,
S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭으로 필수적으로 구성되는 아토피 피부염 예방또는 개선용화장료조성물을 제공한다. 상기 화장료 조성물을 제조하기 위한 S0D3 과발현 줄기세포 유래 세포외 소낭에 대하여서는 전술한 바와 같다. 본 발명의 화장료 조성물은 S0D3 과발현 줄기세포 유래 세포외 소낭을 유효성분으로 함유하며 피부학적으로 허용 가능한 부형제와 함께 기초 화장품 조성물 (화장수, 크림， 에센스, 클렌징 폼 및 클렌징 워터와 같은 세안제, 팩, 보디오일) , 색조 화장품 조성물 (화운데이션， 립스틱， 마스카라， 메이크업 베이스)， 두발 제품 조성물 (샴푸, 린스， 헤어컨디셔너， 헤어젤) 및 비누등의 형태로 제조될 수 있다. Provides a cosmetic composition for preventing or improving atopic dermatitis consisting essentially of extracellular vesicles derived from stem cells overexpressing S0D3). The S0D3 overexpressing stem cell-derived extracellular vesicles for preparing the cosmetic composition are as described above. The cosmetic composition of the present invention contains S0D3 overexpressing stem cell-derived extracellular vesicles as an active ingredient and is a basic cosmetic composition (face wash, cream, essence, cleansing foam and cleansing water-like face wash, pack, body) Oil), hue cosmetic composition (foundation, lipstick, mascara, makeup base), hair product composition (shampoo, conditioner, hair conditioner, hair gel) and soap.
상기 부형제로는 이에 한정되지는 않으나 예를 들어, 피부연화제， 피부 침투 증강제， 착색제， 방향제, 유화제, 농화제 및 용매를 포함할 수 있다. 또한， 향료， 색소, 살균제, 산화방지제， 방부제 및 보습제 등을 추가로 포함할 수 있으며， 물성개선을 목적으로 점증제， 무기염류， 합성 고분자 물질 등을 포함할 수 있다. 예를 들면， 본 발명의 화장료조성물로 세안제 및 비누를 제조하는 경우에는통상의 세안제 및 비누 베이스에 상기 S0D3 과발현 줄기세포 유래 세포외 소낭을 첨가하여 용이하게 제조할 수 있다. 크림을 제조하는 경우에는 일반적인 수중유적형 (0/W)의 크림베이스에 S0D3 과발현 줄기세포 유래 세포외 소낭을 첨가하여 제조할 수 있다. 여기에 향료， 킬레이트제, 색소， 산화방지제, 방부제 등과 물성개선을 목적으로 한 단백질, 미네랄， 비타민 등 합성 또는 천연소재를 추가로 첨가할 수 .있다. 본 발명의 화장료조성물에 함유되는 S0D3 과발현 줄기세포유래 세포외 소낭의 함량은 이에 한정되지 않지만 전체 조성물 총중량에 대하여 0.001 내지 10 중량%인 것이 바람직하고, 0.01 내지 5 중량%인 것이 더욱 바람직하다. 상기 함량이 0.001 중량% 미만에서는 목적하는 아토피 피부염 치료 효과를 기대할 수 없고， 10 중량% 초과에서는 안전성 또는 제형상의 제조에 어려움이 있을수 있다. The excipient is not limited to this, for example, may include skin emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners and solvents. In addition, fragrances, pigments, disinfectants, antioxidants, preservatives and moisturizers may be further included, and thickeners, inorganic salts, and synthetic polymer materials may be included for the purpose of improving physical properties. For example, in the case of preparing the face wash and soap with the cosmetic composition of the present invention, the S0D3 overexpressing stem cell-derived extracellular vesicles can be easily added to the normal face wash and soap base. In the case of manufacturing a cream, it can be prepared by adding S0D3 overexpressing stem cell-derived extracellular vesicles to a general oil-in-water type (0 / W) cream base. To this, synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving physical properties such as fragrances, chelating agents, pigments, antioxidants, and preservatives can be added . have. The content of S0D3 overexpressing stem cell-derived extracellular vesicles contained in the cosmetic composition of the present invention is not limited to this, but is preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight relative to the total weight of the total composition. If the content is less than 0.001% by weight, the desired effect of treating atopic dermatitis cannot be expected, and if it is more than 10% by weight, there may be difficulties in safety or preparation of the formulation.
본 발명은 아토피 치료용 제제를 제조하기 위한 슈퍼옥사이드 디스무타아제 3(superoxide dismutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭의 용도를 제공한다. The present invention provides the use of extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (S0D3) for preparing an atopy treatment preparation.
본 발명은 슈퍼옥사이드 디스무타아제 3 (super oxide di smutase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 아토피 질환의 치료 방법을 제공한다. The present invention is characterized by administering an effective amount of a composition containing extracellular vesicles derived from stem cells overexpressing super oxide di smutase 3 (S0D3) as an active ingredient to an individual in need thereof. How to treat disease to provide.
본 발명의 상기 ‘유효량’ 이란 개체에게 투여하였을 때， 아토피 피부염 질환의 개선， 치료， 예방， 검출， 진단 또는 아토피 피부염 질환의 억제 또는 감소 효과를 나타내는 양을 말하며， 상기 ‘개체’ 란 동물, 바람직하게는 포유동물, 특히 인간을 포함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직， 기관 등일 수도 있다. 상기 개체는상기 효과가필요한환자 ) 일 수 있다. 본 발명의 상기 ‘치료’ 는 아토피 피부염 질환 또는 아토피 피부염 질환의 증상을 개선시키는 것을 포괄적으로 지칭하고， 이는 아토피 피부염 질환을 치유하거나, 실질적으로 예방하거나， 또는 상태를 개선시키는 것을 포함할 수 있으며， 아토피 피부염 질환으로부터 비롯된 한 가지 증상 또는 대부분의 증상을 완화시키거나， 치유하거나 예방하는 것을포함하나, 이에 제한되는 것은 아니다. The 'effective amount' of the present invention, when administered to an individual, refers to the amount of improving, treating, preventing, detecting, diagnosing or suppressing or reducing the effects of atopic dermatitis disease when administered to an individual, the 'individual' being an animal, preferably For example, it may be a mammal, particularly an animal including humans, or may be cells, tissues, organs, etc. derived from animals. The individual may be a patient who needs the effect). The 'treatment' of the present invention collectively refers to improving the symptoms of atopic dermatitis disease or atopic dermatitis disease, which may include healing, substantially preventing, or improving the condition of atopic dermatitis disease, It includes, but is not limited to, alleviating, healing or preventing one symptom or most symptoms resulting from atopic dermatitis disease.
본 발명의 용어 을 포함하는 (compr i sing)’ 이란 ‘함유하는’ 또는 ‘특징으로 하는’ 과동일하게 사용되며， 조성물 또는 방법에 있어서, 언급되지 않은 추가적인 성분 요소 또는 방법 단계 등을 배제하지 않는다. 용어 로 구성되는 (consi st ing of )’ 이란 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외하는 것을 의미한다. 용어 ‘필수적으로 구성되는 (essent i al ly consi st ing of )’ 이란 조성물 또는 방법의 범위에 있어서， 기재된 성분 요소 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 성분 요소 또는 단계 등을포함하는 것을 의미한다. The term 'compr i sing' is used the same as 'containing' or 'characterizing' and does not exclude additional component elements or method steps not mentioned in the composition or method. . The term (consi st ing of) ”means excluding additional elements, steps or components, which are not described separately. The term 'essentially composed (essent i al ly consi st ing of)' includes a component element or step described in the scope of the composition or method, as well as a component element or step that does not substantially affect its basic properties. It means to do.
【발명의 효과】 따라서, 본 발명은 으孤3 과발현 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 조성물 및 이를 이용한 아토피 피부염 치료 방법을 제공한다. 본 발명에 따른 조성물은 전염증성 사이토카인을 분비하는 면역세포의 증식 억제， 竹6용유도 효과를 통해 아토피 피부염 치료 효과를 보이며， 비만 세포의 병변부위 침윤 억제 및 ¾1 분화 억제 효과를 보여 아토피 피부염의 만성화 예방또는 만성화된 아토피 피부염 치료에 유용하게 이용될 수 있다. 【도면의 간단한설명】 도 1은 엤的를 과발현시킨 제대혈 줄기세포의 3003 단백질 발현량을 확인한 결과이다 7 : 세포 계대를 7번 넘겨 배양한 세포, 11192/197/218 : 공여자에 따른 세포 번호) .
Figure imgf000022_0001
과발현시킨 제대혈 줄기세포 유래 세포외 소낭의 엤況 단백질 발현량을 확인한 결과이다. [Effects of the Invention] Accordingly, the present invention provides a composition for preventing or treating atopic dermatitis comprising extracellular vesicles derived from C3 overexpressing stem cells as an active ingredient and a method for treating atopic dermatitis using the same. The composition according to the present invention suppresses the proliferation of immune cells that secrete pro-inflammatory cytokines, shows the effect of treating atopic dermatitis through the induction effect of 竹 6, inhibits the invasion of lesions of mast cells and inhibits ¾1 differentiation, chronicating atopic dermatitis It can be useful in the prevention or treatment of chronic atopic dermatitis. [Brief Description of Drawings] FIG. 1 is a result of confirming 3003 protein expression level of umbilical cord blood stem cells overexpressing 7: 7: cells cultured over 7 cell passages, 11192/197/218: cell number according to donor) .
Figure imgf000022_0001
This is the result of confirming the expression level of the protein in the extracellular vesicles derived from the overexpressed cord blood stem cells.
도 3 중 (크)는 아토피 피부염 동물 모델을 제조 순서에서, 8003 과발현 제대혈 줄기세포 유래 세포외 소낭의 투여 날짜를 설정한 것이고， ( 는 상기 세포외 소낭의 투여에 의한 아토피성 피부염의 병변 변화를 확인한 결과이다. In FIG. 3, (k) is the date of manufacture of the atopic dermatitis animal model, and the date of administration of the extracellular vesicles derived from 8003 overexpressing umbilical cord blood stem cells is set, and (indicates the change in lesions of atopic dermatitis by administration of the extracellular vesicles) It is the result of confirmation.
도 4는 S0D3 과발현 제대혈 줄기세포 유래 세포외 소낭을 아토피 동물 모델에 투여한 후 병변의 중증도를 조직 염색을 통해 확인한 결과로, 외피 (epidermi s)의 두께를 확인한 결과이다. 4 is a result of confirming the severity of the lesion through tissue staining after administering S0D3 overexpressing umbilical cord blood stem cell-derived extracellular vesicles to an atopy animal model, and confirming the thickness of the epidermis (epidermi s).
도 5는 과발현 제대혈 줄기세포 유래 세포외 소낭을 아토피 동물 모델에 투여한 후 병변의 중증도를 조직 염색을 통해 확인한 결과로, 림프구의
Figure imgf000022_0002
확인한 결과이다. 5 is a result of confirming the severity of the lesion through tissue staining after administering an overexpressed umbilical cord blood stem cell-derived extracellular vesicle to an atopy animal model.
Figure imgf000022_0002
It is the result of confirmation.
도 6은 3抑3 과발현 제대혈 줄기세포 유래 세포외 소낭을 아토피 동물 모델에 투여한 후 조직 염색 결과를 수치화한 그래프로， 도 6 중 (3)는 외피의 두께를 확인한 결과를 수치화한 결과이고, ( 는 림프구의 침윤을 수치화한 결과이다. FIG. 6 is a graph showing the numerical results of tissue staining after administration of 3 抑 3 overexpressing cord blood stem cell-derived extracellular vesicles to an atopy animal model, and (3) in FIG. 6 is a result of digitizing the result of confirming the thickness of the envelope. (Is the result of quantifying lymphocyte infiltration.
도 7은 S0D3 과발현 제대혈 줄기세포 유래 세포외 소낭을 아토피 동물 모델에 투여한 후 아토피성 피부염의 만성화에 핵심적인 역할을 하는 비만세포의 병변부위 침윤 정도를 톨루이딘 블루 (Toluidine blue) 염색을 통해 확인한 결과이다. 도 8 및 도 9는 엤예 과발현 제대혈 줄기세포 유래 세포외 소낭을 정상 혈액에서 분리한 ?8¾犯에 처리한후, 요 (:의 증식에 미치는 영향을 확인한 결과이다. 7 is a result of confirming the degree of infiltration of lesions of mast cells that play a key role in chronic atopic dermatitis after administration of S0D3 overexpressing umbilical cord blood stem cell-derived extracellular vesicles through atopic animal model through toluidine blue staining to be. 8 and 9 is a result of confirming the effect on the proliferation of urine (:) after treatment of the extracellular vesicles derived from the overexpressed umbilical cord blood stem cells from? 8¾8 separated from normal blood.
도 10 및 도 11은 S0D3 과발현 제대혈 줄기세포 유래 세포외 소낭을 PBMC에 서 분리한 미접촉 T 세포 (naive T cel l )에 처리한 후， 헬퍼 T세포의 분화에 미치는 영향을 확인한 결과이다. . 10 and 11 are the results confirming the effect on the differentiation of helper T cells after treatment with S0D3 overexpressing umbilical cord blood stem cell-derived extracellular vesicles isolated from PBMC (naive T cel l). .
도 12는 8003 과발현 제대혈 줄기세포 유래 세포외 소낭을 미접촉 I 세포에 처리한후, ^분화에 미치는 영향을 확인한 결과이다. FIG. 12 is a result of confirming the effect on ^ differentiation after treatment of 8003 overexpressing cord blood stem cell-derived extracellular vesicles to uncontacted I cells.
【발명의 실시를 위한 형태】 [Mode for the Invention]
이하 본 발명을 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
실험방법 Experiment method
MSC의 배양 및 동정 인간의 제대혈에서 유래한 중간엽 줄기세포 (hUCB-MSC)는 제공자의 동의하에 인간 탯줄의 혈액 샘플로부터 수집하였다. 탯줄 혈액 샘플은 채취 후 항응고제로 작용하는 구연산 인산 포도당을 포함하는 혈액수집팩에 보관하였다. 실험을 위한 처리는 24시간 이내에 실시하였다. F i co 1 1 -Paque PLUS 농도 구배 (Amersham Biosciences)하에 원심분리하여 단핵 세포 분획을 분리하였다. HBSSCei l Biotech Services)로 세척하고， 저혈당 Dulbecco의 변형된 Eagle 배양액 (DMEM, Invitrogen Corp) , 20%우태아 혈청 (Gibco-BRL) , 2mM L_글루타민, ImM 피루브산 나트륨 및 1%의 항생제/항진균제 (Li fe Technologies)로 재현탁하였다. 항생제/항진균제는 100U/ml 페니실린, 100 u g/ml 스트렙토마이신, 25 y g/ml의 암포테리신표를 포함한다. 7일 후， 부착되지 않은 세포는 제거하고， 부착된 세포를 두 가지 배양액을 일주일마다 바꿔가며 배양하였다. 세포는 5% C02 , 37°C의 습윤 조건에서 배양하였다. 융합성 세포의 약 60%를 0.1%트림신- EDTA로 분리하였고, 배양플레이트에 옮겨 담았다. hUCB-MSC의 면역표현형을 분석하여 MSC와 관련된 항원 양성 마커의 존재와 조혈 계통 마커의 부재를 유동세포 분석법 (Epi cs XL, Beckman Coul ter)으로 조사하였다. 양성 마커로는 CD90(Thy-l) , CD10 endogl in) 및 SH3(CD73)가 있으며， 조혈계통 마커로는 CD34, CD45 및 CD31과 같은 내피 세포 마커가 있다. 세포는 HLA cl ass I에는 양성이었지만, HLA-m에는 음성이었다. 각각의 형광표지 단일클론 항체는 Beet on Dickinson에서 구입하였다 (결과도시하지 않음) . 이를 통해 P7 U192, P7 U197, P7 U218의 3 가지 세포주를 수득하였다 (P7: 세포 계대를 7번 넘겨 배양한세포， U192/197/218： 공여자에 다른 세포 번호) . Culture and identification of MSC Mesenchymal stem cells (hUCB-MSC) derived from human umbilical cord blood were collected from blood samples of human umbilical cord with the consent of the donor. Umbilical cord blood samples were collected and stored in blood collection packs containing citrate phosphate glucose, which acts as an anticoagulant. Treatment for the experiment was carried out within 24 hours. The mononuclear cell fraction was isolated by centrifugation under a F i co 1 -Paque PLUS concentration gradient (Amersham Biosciences). HBSSCei l Biotech Services), hypoglycemic Dulbecco's modified Eagle culture (DMEM, Invitrogen Corp), 20% fetal calf serum (Gibco-BRL), 2 mM L_glutamine, ImM sodium pyruvate and 1% antibiotic / antifungal ( Li fe Technologies). Antibiotic / antifungal agents include 100 U / ml penicillin, 100 ug / ml streptomycin, and 25 yg / ml amphotericin table. 7 days later The non-adherent cells were removed, and the attached cells were cultured by changing two cultures every week. Cells were cultured in 5% C02, 37 ° C wet conditions. About 60% of the fused cells were separated with 0.1% trimcine-EDTA and transferred to a culture plate. The immunophenotype of hUCB-MSC was analyzed to investigate the presence of antigen-positive markers associated with MSC and the absence of hematopoietic lineage markers by flow cytometry (Epi cs XL, Beckman Coul ter). Positive markers include CD90 (Thy-l), CD10 endogl in) and SH3 (CD73), and hematopoietic lineage markers include endothelial cell markers such as CD34, CD45 and CD31. Cells were positive for HLA cl ass I, but negative for HLA-m. Each fluorescently labeled monoclonal antibody was purchased from Beet on Dickinson (results not shown). Through this, three cell lines of P7 U192, P7 U197, and P7 U218 were obtained (P7: cells cultured over seven cell passages, U192 / 197/218: different cell numbers for donors).
S0D3형질전환 상기를 통해 준비한 hUCB-MSC에 전기천공법을 이용하여 S0D3를 형질전환시켰다. S0D3 형질도입을 위한 바이러스 발현 벡터 및 형질도입 조건은 렌티바이러스를 바이러스 발현 벡터로 사용하였다는 점 외에 종전 기술인 S0D3를 과발현하는 줄기세포를 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물에 대한 특허와 동일하게 수행하였다 (한국공개특허 10-2017-0032872의 실험방법 참고) . S0D3 Transformation S0D3 was transformed by electroporation on hUCB-MSC prepared through the above. The viral expression vector and transduction conditions for S0D3 transduction were that lentivirus was used as a viral expression vector, as well as for the pharmaceutical composition for the prevention or treatment of inflammatory diseases including stem cells overexpressing S0D3, which is a prior art, as an active ingredient. It was performed in the same way as the patent (refer to the experimental method of Korean Patent Publication No. 10-2017-0032872).
S0D3과발현 제대혈 줄기세포유래 세포외 소낭수득 상기 S0D3 과발현 hUCB-MSC에서 초원심분리법을 통해 세포외 소낭을 수득하였다. 이때 S0D3가 과발현되지 않은 hUCB-MSC에서 세포외 소낭을 추출하여 대조군으로사용하였다. 세포 배양액으로부터 세포외 소낭의 분리를 위해 각각 3X106 개의 S0D3 과발현 hUCB-MSC와 대조군 hUCB-MSC를 150X25mm 배양 접시에 깔고 세포외 소낭이 포함되지 않은 fetal bovine serum (Thermo Fi sher Scient i f ic , Inc . Wal tham, MA, USA)를 2% 함유한 배양액 25ml과 함께 배양하였다. 3일 후 배양액을 튜브에 모으고 OPTIMA MAX-XP/MLA-55 rotor (Beckman) 장비를 사용하여 다음 순서를 따라 초원심 분리를 진행하였다. 먼저 30½의 속도로 5분동안 원심분리한 후， 상층액을 수거하여 3,0008의 속도로 45분동안 원심분리하였다. 그 후 상층액을 수거하여 10,00始의 속도로 30분동안 원심분리하였으며 이의 상층액을 수거하여 100,00 의 속도로 70분동안 원심분리하였다. 마지막으로 상층액을 제거후 펠렛을 표요에 부유시킨 후 100,00 의 속도로 70분 동안 원심분리하였다. 그 후 펠렛을
Figure imgf000025_0001
녹여 세포외 소낭 수득하였다. Extracellular vesicles derived from S0D3 overexpressing umbilical cord blood stem cells The extracellular vesicles were obtained by ultracentrifugation in hUCB-MSC overexpressing S0D3. At this time, extracellular vesicles were extracted from hUCB-MSC where S0D3 was not overexpressed and used as a control. For separation of extracellular vesicles from cell culture, 3X106 S0D3 overexpressing hUCB-MSC and control hUCB-MSC are placed in a 150X25mm culture dish and fetal bovine serum (Thermo Fi sher Scient if ic, Inc. tham, MA, USA) was cultured with 25 ml of a culture solution containing 2%. After 3 days, the culture was collected in a tube, and ultracentrifugation was performed in the following order using an OPTIMA MAX-XP / MLA-55 rotor (Beckman) equipment. First, after centrifugation for 5 minutes at a rate of 30½, the supernatant was collected and centrifuged for 45 minutes at a rate of 3,000 8 . Thereafter, the supernatant was collected and centrifuged for 30 minutes at a rate of 10,00 kPa, and the supernatant was collected and centrifuged at a rate of 100,00 for 70 minutes. Finally, after removing the supernatant, the pellet was suspended on the surface and centrifuged at a rate of 100,00 for 70 minutes. Then pellet
Figure imgf000025_0001
Melted to obtain extracellular vesicles.
S0D3과발현 유무 확인 S0D3 overexpression check
S0D3 유전자로 형질전환된 hUCB-MSC및 이로부터 수득한 세포외 소낭의 S0D3 과발현 유무를 S0D3 단백질 발현을 분석하여 확인하였다. hUCB-MSC 및 세포외 소낭으로부터 단백질을 추출하였다. 그 다음 12% SDS-PAGE 젤에서 분리한 후, nitrocellulose membrane (Bio-Rad Laboratories Inc.)으로 이동시키고， 상기 membrane을 5% 탈지 분유로 blocking한 후, 1:1000으로 희석된 1차 항체 및 2차 항체와 각각 반응시켰다. 항체와 반응시킨 membranes를 PBST 용액으로 씻은 후, Enhancecd Chemi -Luminescence 시약 (Thermo Fisher Scientific, Inc . Waltham, MA, USA)에 반응시키고， X-ray film에 노출시켜 단백질 밴드를분석하였다. 상기 방법에서 사용한항체는 다음과 같다. The presence of S0D3 overexpression of hUCB-MSC transformed with S0D3 gene and the extracellular vesicles obtained therefrom was confirmed by analyzing S0D3 protein expression. Protein was extracted from hUCB-MSC and extracellular vesicles. Then, after separation from a 12% SDS-PAGE gel, it was transferred to a nitrocellulose membrane (Bio-Rad Laboratories Inc.), the membrane was blocked with 5% skim milk powder, and then the primary antibody diluted 1: 1000 and 2 Each was reacted with a secondary antibody. The membranes reacted with the antibody were washed with PBST solution, reacted with Enhancecd Chemi-Luminescence reagent (Thermo Fisher Scientific, Inc. Waltham, MA, USA), and exposed to X-ray film to analyze protein bands. The antibody used in the above method is as follows.
1차항체: anti_S0D3 (Abeam, Cambridge, United Kingdom) Primary antibody: anti_S0D3 (Abeam, Cambridge, United Kingdom)
2차 항체: ant i -rabbit IgG (Thermo Fisher Scientific, Inc. Waltham, MA,Secondary antibody: ant i-rabbit IgG (Thermo Fisher Scientific, Inc. Waltham, MA,
USA) USA)
S0D3 과발현 hUCB-MSC 유래 세포외 소낭의 경우 1차 항체로 엑소좀 (exosome)에 대한 항체인 ant i -CD9 , ant i -CD63 , ant i~CD81, anti- HSP70( System Bioscience, Palo Alto, CA, USA)를추가하였다. For S0D3 overexpressed hUCB-MSC-derived extracellular vesicles, ant i -CD9, ant i -CD63, ant i ~ CD81, anti- HSP70 (System Bioscience, Palo Alto, CA) , USA).
아토피 동물모델 제조 및 세포외 소낭투여 아토피 동물모델은 본 분야의 공지된 방법으로 제조하였다. 실험에 사용한 마우스는 8주차의 C57BL/6 마우스 (mouse)로， 특정 병원균이 없는 상태에서 표준 마우스사료와물로사육하였으며， 보건복지부의 지침에 따른 가톨릭 대학교 가톨릭 윤리위원회의 규정에 따라다루었다. 먼저 마우스 동물 모델의 피부의 털을 제거한 후， 3일간 1% 2,4 - ^11^；1 0(：11101 0561 6116(0 8)를 도포하였다 ］! )11) . 그 후 6일차 마우스 동물 모델의 피부에 0 .2% 例期를 반복 도포하여 제조하였다 (0131比明6) . 상기 0^8 도포 과정 중 (사육 7일차) 상기에서 제조한, 4 X
Figure imgf000026_0001
유래의 세포외 소낭을 병변 부위 피하로 단회 주사하였고， 일주일 후 아토피 피부염의 병변을 육안으로 평가하였다. 그 결과 抑3를 과발현시킨 11況841엤 유래의 세포외 소낭을 투여하였을 때 가장 증상이 개선된 것을 확인하였다. Atopy animal model preparation and extracellular vesicle administration Atopy animal model was prepared by a known method in the art. The mice used in the experiment were C57BL / 6 mice at week 8, were raised with standard mouse feed and water in the absence of specific pathogens, and were treated according to the guidelines of the Catholic University Catholic Ethics Committee according to the guidelines of the Ministry of Health and Welfare. First, after removing the hairs of the skin of the mouse animal model, 1% 2,4-^ 11 ^; 1 0 (： 11101 0561 6116 (0 8) was applied for 3 days]!) 11). After that, 0.2% 例 期 was repeatedly applied to the skin of the animal model of the 6th day to prepare (0131 比 明 6). During the 0 ^ 8 application process (day 7 of breeding), 4 X prepared above
Figure imgf000026_0001
The derived extracellular vesicles were injected once subcutaneously to the lesion site, and a week later the lesions of atopic dermatitis were visually evaluated. As a result, when the extracellular vesicles derived from 11 況 841 엤 overexpressing 抑 3 were administered, it was confirmed that the symptoms were most improved.
마우스 모델의 조직학적 평가와 형광조직염색 此를 과발현시킨
Figure imgf000026_0002
유래의 세포외 소낭을 투여한 마우스로부터 등 피부 조직을 수득하고, 4% 파라포름알데하이드어시에 고정한 후, 파라핀에 내장하였다 . 피부 샘플은 회전식 마이크로톰此 03)을 이용하여
Figure imgf000026_0003
조직 절편을 준비하였다. 이후 자일렌 )을 이용하여 탈지하고， 농도구배 알코올로 탈수하였다. 상기 전처리한 조직 절편은 헤마톡실린과 에오신대湖 염색)으로 염색하였다. 더불어 비만세포의 침윤 정도를 톨루이
Figure imgf000026_0004
염색을 통해 확인하였다. 마우스 피부 조직의 파라핀 섹션 절편을 0. 1% 톨루이딘 블루 염색 용액 ( 1%의 톨루이딘 블루 용액을 1% (：1 용액에 10배 희석하여 준비)에 2-3분간 담궜다. 이후 증류수로 3회 세척하고 95%와 100% 알코올에 차례대로 담궈서 탈수시킨 뒤 자일텐으로 탈지하고 말려서 염색을 완료하였다. 염색된 조직은 현미경을 통해 톨루이딘 블루로 염색된 비만세포의 과립상을 관찰하고 계수하였다. Histological evaluation of mouse model and overexpression of fluorescent tissue staining
Figure imgf000026_0002
The back skin tissue was obtained from the mice to which the derived extracellular vesicles were administered, fixed in 4% paraformaldehyde, and then embedded in paraffin. For skin samples, use a rotating microtome 03)
Figure imgf000026_0003
Tissue sections were prepared. Subsequently, it was degreased using xylene) and dehydrated with a concentration gradient alcohol. The pretreated tissue sections were stained with hematoxylin and eosindae staining). In addition, the degree of invasion of mast cells
Figure imgf000026_0004
It was confirmed by staining. Sections of paraffin sections of mouse skin tissues were soaked in 0. 1% toluidine blue staining solution (1% toluidine blue solution prepared in 1% (prepared by diluting 10 times in 1 solution) for 2-3 minutes), and then washed 3 times with distilled water. And then dehydrated by dipping in 95% and 100% alcohol one after another to complete the staining by drying with xylten and staining.The stained tissue was observed and counted by granules of mast cells stained with toluidine blue through a microscope.
유세포 분석 (Flow cytometry) Flow cytometry
S0D3를 과발현시킨 hUCB-MSC 유래의 세포외 소낭이 면역세포에 미치는 영향을 평가하기 위해 유세포 분석을 수행하였다. 먼저 정상 혈액에서 분리한 말초혈액 단핵세포 (PBMC)을 CFSE(Carboxyf luoresce in succ inimi dyl ester )로 표지하였다. 그 후 증식을 촉진하는 미토겐 (mi togen)인 콘카나발린 ACConcanaval in A) (G， n=2) 또는 T 림프구 증식을 특징적으로 촉진할 수 있는 CD3/28 (H， n=4)을 처리 (day 0)하여 5일 후 (day 5) 유세포 분석기로 증식 정도를 측정하였다. 이때 hUCB-MSC유래 세포외 소낭을 T 림프구 증식을 촉진하는 인자들과 함께 처치함으로써 (day 0) 그렇지 않은 그룹에 비해 증식 양상이 어떻게 변화하는지 비교하였다. 더불어 유세포 분석을 통해 S0D3를 과발현시킨 hUCB-MSC 유래의 세포외 소낭이 헬퍼 T세포의 분화에 미치는 영향을 확인하였다. 이를 위해 정상 PBMC에서 naive T cel l (ThO)을 분리한 뒤 5일간의 ant i_IL4+IFNr 혹은 ant i_IFNr+IL4를 처리하여 각각 Thl과 Th2로의 분화를 유도하였다. 이때 일부 그룹을 정해 S0D3를 과발현시킨 hUCB-MSC 유래의 세포외 소낭을 분화를 유도하는 시점에 함께 처치하여 분화된 세포 비율을 분석하였다. 분화된 헬퍼 T 세포의 마커로 Thl 세포는 Interferon-r 항체 (e-bioscience, Wal tham, MA, USA)를 사용하였고 Th2 세포는 Inter leukin-4항체 (e-bioscience, Waltham, MA, USA)를사용하였다. 또한 S0D3를 과발현시킨 hUCB-MSC 유래의 세포외 소낭이 용의 분화에 미치는 영향을 확인하였다. 이를 위해 Naive T cel l에 hUCB-MSC 유래의 세포외 소낭을 처치하고 5일 후 Treg 마커인 FoxP3 (e-bioscience , Waltham, MA, USA) 발현을 확인하였다. Flow cytometry was performed to evaluate the effect of extracellular vesicles derived from hUCB-MSC overexpressing S0D3 on immune cells. First, peripheral blood mononuclear cells (PBMC) isolated from normal blood were labeled with CFSE (Carboxyf luoresce in succ inimi dyl ester). After that, treatment with contonavalin ACConcanaval in A) (G, n = 2) or CD3 / 28 (H, n = 4) that can characteristically promote T lymphocyte proliferation. (day 0) and 5 days later (day 5) The degree of proliferation was measured by a flow cytometer. At this time, by treating hUCB-MSC-derived extracellular vesicles with factors that promote T lymphocyte proliferation (day 0), how the proliferation pattern changes compared to the other group. Compared. In addition, through flow cytometry analysis, the effect of extracellular vesicles derived from hUCB-MSC overexpressing S0D3 on the differentiation of helper T cells was confirmed. To this end, naive T cel l (ThO) was isolated from normal PBMC, followed by treatment with ant i_IL4 + IFNr or ant i_IFNr + IL4 for 5 days to induce differentiation into Thl and Th2, respectively. At this time, some groups were selected to treat extracellular vesicles derived from hUCB-MSC overexpressing S0D3 at the time of inducing differentiation, and the percentage of differentiated cells was analyzed. As a marker of differentiated helper T cells, Thl cells used Interferon-r antibody (e-bioscience, Wal tham, MA, USA) and Th2 cells used Inter leukin-4 antibody (e-bioscience, Waltham, MA, USA). Used. In addition, it was confirmed that the effect of extracellular vesicles derived from hUCB-MSC overexpressing S0D3 on the differentiation of the dragon. To this end, treatment of hUCB-MSC-derived extracellular vesicles was performed on Naive T cel l, and expression of the Treg marker FoxP3 (e-bioscience, Waltham, MA, USA) was confirmed after 5 days.
실시예 1： S0D3과발현 여부확인 Example 1: Confirmation of overexpression of S0D3
S0D3를 과발현시킨 hUCB-MSC의 S0D3 단백질 발현을 확인한 결과도 1과 같이 , S0D3 발현이 크게 증가된 것을 확인할 수 있었다. 더불어 도 2와 같이 S0D3를 과발현시킨 hUCB-MSC 유래 세포외 소낭의 경우， S0D3가 과발현되기 전에는 엑소좀 마커의 발현이 높았으나， S0D3 형질도입 후 S0D3 단백질이 다량포함되어 있는 것을 확인할수 있었다. As a result of confirming S0D3 protein expression of hUCB-MSC overexpressing S0D3, it was confirmed that S0D3 expression was significantly increased. In addition, in the case of hUCB-MSC-derived extracellular vesicles overexpressing S0D3, as shown in FIG. 2, the expression of the exosome marker was high before S0D3 was overexpressed, but it was confirmed that a large amount of S0D3 protein was contained after S0D3 transduction.
실시예 2： 아토피 치료 효과확인 Example 2: Confirmation of atopy treatment effect
2-1. 8(形3를 과발현시킨
Figure imgf000027_0001
유래 세포외 소낭의 아토피 치료 효과 확인 도 3 중 (幻와 같이 아토피 동물 모델을 확립하고 此를 과발현시킨 111£ 엤 유래 세포외 소낭을 투여한 후 아토피 치료 효과를 확인하였다. 그 결과 도 3 중 )와 같이 엤此를 과발현시킨 1x1X3-10:( 1925 묘 , 19花 표재)를 투여하였을 때 증상이 개선되는 것을 확인하였다. 2020/085533 1»（：1^1{2018/012712 2-1. 8 (Overexpressed form 3
Figure imgf000027_0001
Confirmation of atopic treatment effect of derived extracellular vesicles In FIG. 3 (established atopic animal model as shown in FIG. 3, and after the administration of 111 £ 엤 derived extracellular vesicles overexpressing 此, the effect of atopic treatment was confirmed. It was confirmed that the symptoms were improved when 1x1X3-10: (1925 seedlings, 19 花 surface) overexpressed 엤 此 as shown. 2020/085533 1 »（： 1 ^ 1 {2018/012712
26  26
2-2. 8(形3를 과발현시킨 유래 세포외 소낭 투여에 따른 병변의 중증도 변화 확인 아토피 동물 모델에 확립하고 엤此를 과발현시킨 11況84!엤 유래 세포외 소낭을 투여한 후 병변의 중증도를 조직염색으로 평가하였다. 그 결과 도 4 및 도 6 중 (3)와 같이， 외피의 두께가 엤此를 과발현시킨
Figure imgf000028_0001
유래 세포외 소낭 투여에 의해 감소하는 경향을 보임을 확인하였다. 더불어 도 5 및 도 6 중 )도 마찬가지로 림프구의 침윤 정도도 孤3를 과발현시킨 11況8-1\1 유래 세포외 소낭 투여에 의해 감소하는 것을 확인하였다. 특히 아토피 피부염의 만성화에 핵심적인 역할을 하는 비만세포의 침윤 정도를 평가한 도 7도 마찬가지로 세포외 소낭 투여에 의해 감소된 것을 확인하였다. 따라서 본 발명에 따른 此를 과발현시킨 느況요 엤 유래 세포외 소낭은 아토피 피부염의 만성화 억제에도 뛰어난 효과를 보임을 알수 있었다. 2-2. 8 (Confirmation of the severity change of the lesion following the administration of the extracellular vesicles overexpressing form 3 Established in the atopy animal model and administered the extracellular vesicles derived from 11 況 84! 엤 overexpressing 엤 此, the severity of the lesion as tissue staining As a result, as shown in Fig. 4 and Fig. 6, (3), the thickness of the skin overexpressed 엤 此.
Figure imgf000028_0001
It was confirmed that the tendency to decrease by administration of the derived extracellular vesicles. In addition, in FIGS. 5 and 6), the degree of lymphocyte infiltration was also decreased by administration of extracellular vesicles derived from 11 況 8-1 \ 1 overexpressing 孤 3. In particular, FIG. 7, which assessed the degree of invasion of mast cells, which plays a key role in chronicization of atopic dermatitis, was also confirmed to be reduced by administration of extracellular vesicles. Therefore, it was found that the extracellular vesicles derived from Nyoyo 엤 overexpressing 此 according to the present invention show excellent effects in suppressing chronicity of atopic dermatitis.
실시예 3： 면역세포 증식 억제 효과 확인 더불어 본 발명에 따른 쌨예를 과발현시킨
Figure imgf000028_0002
유래 세포외 소낭의 -1 ， -6과 같은 전염증성 사이토카인을 방출하는 면역세포인
Figure imgf000028_0003
증식 억제 효과를 확인하였다. 그 결과 도 8 및 도 9에 나타낸 바와 같이， 03를 과발현시킨 11狀8-¾1洗 유래 세포외 소낭을 투여하였을 때 표祀의 증식이 현저히 억제되는 것을 확인할 수 있었다. Example 3: Confirmation of the effect of suppressing the proliferation of immune cells In addition to overexpressing the example according to the present invention
Figure imgf000028_0002
Immune cells that release pro-inflammatory cytokines such as -1 and -6 from the derived extracellular vesicles
Figure imgf000028_0003
The proliferation inhibitory effect was confirmed. As a result, as shown in Figs. 8 and 9, it was confirmed that the proliferation of epidermis was significantly suppressed when the extracellular vesicles derived from 11'8-¾1 'overexpressing 03 were administered.
실시예 4： I세포 분화 억제 효과 확인 4-1. 헬퍼 1세포분화에 미치는 영향 확인 아토피성 피부염은 대부분
Figure imgf000028_0004
연관된 면역기전에 의해 발병되는데, I 세포 이상에 의한 면역반응이 관여한다는 보고가 많으며， 헬퍼 세포 (¾ 061 1 )의 역할이 중요하다고 알려져 있으므로， 본 발명에 따른 엤此를 과발현시킨
Figure imgf000028_0005
유래 세포외 소낭의 I세포 분화에 미치는 영향을 확인하였다. 그 결과 도 10에 나타낸 바와 같이， 엤此를 과발현시킨 111«¾-1\1就 유래 세포외 소낭의 투여는 !¾1의 분화를 감소시킨 것을 확인하였다. 반면 도 11과 같이， ¾2 세포로의 분화에는 유의적인 영향을 끼치지 않은 것을 알수 있었다. 2020/085533 1»（：1^1{2018/012712 Example 4: I cell differentiation inhibitory effect confirmation 4-1. Checking the effect on helper 1 cell differentiation Most atopic dermatitis
Figure imgf000028_0004
It is caused by an associated immune mechanism, and there are many reports that immune responses by I cell abnormalities are involved, and the role of helper cells (¾061 1) is known to be important, so that overexpression of 엤 此 according to the present invention
Figure imgf000028_0005
The influence of the derived extracellular vesicles on I cell differentiation was confirmed. As a result, as shown in FIG. 10, it was confirmed that the administration of 111 «¾-1 \ 1 就 -derived extracellular vesicles overexpressing 엤 此 reduced! ¾1 differentiation. On the other hand, as shown in Figure 11, it was found that it did not significantly affect the differentiation into ¾2 cells. 2020/085533 1 »（： 1 ^ 1 {2018/012712
27 따라서 본 발명에 따른 此를 과발현시킨
Figure imgf000029_0001
유래 세포외 소낭은 아토피 피부염의 만성화 예방 또는 만성화된 아토피 피부염의 치료 효과를 갖는 것을 확인하였다. 27 Therefore, overexpressing 此 according to the present invention
Figure imgf000029_0001
It was confirmed that the derived extracellular vesicles had the effect of preventing chronic atopic dermatitis or treating chronic atopic dermatitis.
4-2. 조절 I세포 분화에 미치는 영향 확인 더불어 전체적인 면역 반응과 타면역세포의 과도한 활성화 억제에 중요한 역할을 하는 조절 I세포奸 요）의 분화에 요（犯3를 과발현시킨 !遇-!\1엤 유래 세포외 소낭이 미치는 영향을 확인하였다. 그 결과 도 12에 나타낸 바와 같이， 脚 3를 과발현시킨 느況표생앴 유래 세포외 소낭은 뛰어난 요 유도 효과를 가지는 것을 확인하였다. 4-2. Influencing the effect on the differentiation of regulatory I cells In addition, the cells derived from! 요-! \ 1 엤 overexpressing urine (犯 3) in the differentiation of regulatory I cells, which play an important role in suppressing the overall immune response and excessive activation of other immune cells The influence of the vesicles was confirmed. As a result, as shown in Fig. 12, it was confirmed that the extracellular vesicles derived from N. epoxidosa 과 3 overexpressed have an excellent urinary induction effect.
이 연구는 한국 정부가 후원하는 의 바이오 & 의료 기술 개발 프로그램인 ¥311）（ 則'-2016 3쇼986903020）의 지원을 받았다. This study was supported by the government's sponsored bio & medical technology development program ¥ 311） (則' -2016 3Show986903020).
【산업상 이용가능성】 이상 살펴본 바와 같이 본 발명에 따른 조성물 및 이를 활용한 방법은 전염증성 사이토카인을 분비하는 면역세포의 증식 억제， 근요 유도 효과를 통해 아토피 피부염 치료 효과를 보이며， 비만 세포의 병변부위 침윤 억제 및 ¾1 분화 억제 효과를 보여 아토피 피부염의 만성화 예방 또는 만성화된 아토피 피부염을 치료에 유용하게 이용될 수 있다. [Industrial applicability] As described above, the composition according to the present invention and the method using the same show the effect of treating atopic dermatitis through suppression of proliferation of immune cells secreting pro-inflammatory cytokines and induction of agitation, and lesion of mast cells It can be used for the prevention of chronic atopic dermatitis or for the treatment of chronic atopic dermatitis by showing the effect of inhibiting site infiltration and inhibiting ¾1 differentiation.

Claims

【청구의 범위】 【Scope of request】
【청구항 1】 슈퍼옥사이드 디스무타아제 3(superoxide dismutase 3, S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학조성물.  [Claim 1] A pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (S0D3) as an active ingredient.
【청구항 2] 제 1항에 있어서, 상기 조성물은 피하에 주입되는 것을 특징으로 하는 아토피 피부염 예방 또는 치료용 약학조성물. [Claim 2] The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the composition is injected subcutaneously.
【청구항 3] 제 1항에 있어서, 상기 줄기세포는 중간엽줄기세포인 것을 특징으로 하는 아토피 피부염 예방 또는 치료용 약학조성물. [Claim 3] The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the stem cells are mesenchymal stem cells.
【청구항 4] [Claim 4]
제3항에 있어서， 상기 중간엽 줄기세포는 제대， 제대혈, 태반, 골수, 지방조직, 근육， 양수 및 양막으로 이루어진 군으로부터 선택된 조직에서 유래한 것을 특징으로 하는 아토피 피부염 예방또는 치료용 약학조성물.  The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 3, wherein the mesenchymal stem cells are derived from tissue selected from the group consisting of umbilical cord, cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, and amniotic membrane.
【청구항 5】 제 1항에 있어서, [Claim 5] According to claim 1,
상기 此는 서열번호 1 내지 서열번호 4로 표시되는 아미노산 서열 중 선택된 어느 하나를 포함하는 것을 특징으로 하는 아토피 피부염 예방 또는 치료용 약학조성물. The 此 is a pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that it comprises any one selected from the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 4.
【청구항 6] 제 1항에 있어서， 상기 아토피는 만성화된 아토피인 것을 특징으로 하는 아토피 피부염 예방 또는 치료용 약학 조성물. [Claim 6] The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the atopy is chronicized atopy.
【청구항 7】 제 6항에 있어서， 상기 만성화된. 아토피는 ¾1 매개 면역반응에 의한 것을 특징으로 하는 아토피 피부염 예방 또는 치료용 약학 조성물. [Claim 7] The method of claim 6, wherein the chronicized . Atopy is a pharmaceutical composition for preventing or treating atopic dermatitis, characterized by a ¾-mediated immune response.
【청구항 8】 【Claim 8】
슈퍼옥사이드 디스무타아제 3(superoxide dismutase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물.  A cosmetic composition for preventing or improving atopic dermatitis, comprising extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (S0D3) as an active ingredient.
【청구항 9] 엤03를 암호화하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터로 줄기세포를 형질감염 시키는 단계; 및 상기 형질감염된 줄기세포를 원심분리하여 세포외 소낭을 추출하는 단계;를 포함하는 것을 특징으로 하는, 아토피 예방 또는 치료용 약학 조성물의 제조 방법 . [Claim 9] Transfecting the stem cells with a recombinant expression vector containing a polynucleotide encoding # 03; And Extracting the extracellular vesicles by centrifuging the transfected stem cells; characterized in that it comprises, a method for producing a pharmaceutical composition for preventing or treating atopy.
【청구항 10】 제 9항에 있어서, 상기 재조합 발현 벡터는 렌티바이러스 벡터， 레트로바이러스 벡터, 아데노바이러스 벡터， 아데노연관바이러스 ( \0 벡터， 벡시니아바이러스 벡터， 허피스바이러스 벡터 및 아비폭스바이러스 벡터로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는， 아토피 예방 또는 치료용 약학 조성물의 제조 방법 [Claim 10] The recombinant expression vector is composed of a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus (\ 0 vector, vaccinia virus vector, herpesvirus vector and abipoxvirus vector). Which is selected from the county Method of manufacturing a pharmaceutical composition for preventing or treating atopy, characterized in that it is one
【청구항 11】 제 9항에 있어서， 상기 를 암호화하는 폴리뉴클레오티드는 서열번호 5 내지 서열번호 8로 표시되는 염기서열 중 선택된 어느 하나를 포함하는 것을 특징으로 하는， 아토피 예방 또는 치료용 약학 조성물의 제조 방법 . [Claim 11] The preparation of a pharmaceutical composition for preventing or treating atopy according to claim 9, wherein the polynucleotide encoding the above comprises any one selected from nucleotide sequences represented by SEQ ID NOs: 5 to 8 Way .
【청구항 12】 아토피 치료용 제제를 제조하기 위한 슈퍼옥사이드 디스무타아제[Claim 12] Superoxide dismutase for manufacturing atopy treatment preparation
3(31 61'0 (16 3011^336 3， 3(¾3)를 과발현시킨 줄기세포 유래 세포외 소낭의 용도 . 3 (31 61'0 (16 3011 ^ 336 3, 3 (¾3) Overexpression of stem cell-derived extracellular vesicles).
【청구항 13】 슈퍼옥사이드 디스무타아제 3 (super oxide di srautase 3， S0D3)를 과발현시킨 줄기세포 유래 세포외 소낭을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 아토피 질환의 치료 방법. [Claim 13] An effective amount of a composition comprising extracellular vesicles derived from stem cells overexpressing super oxide di srautase 3 (S0D3) as an active ingredient is administered to an individual in need thereof Methods of treatment of atopic diseases.
PCT/KR2018/012712 2018-10-25 2018-10-25 Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed WO2020085533A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020217013938A KR20210066005A (en) 2018-10-25 2018-10-25 Novel use of stem cell-derived extracellular vesicles overexpressing SOD3
PCT/KR2018/012712 WO2020085533A1 (en) 2018-10-25 2018-10-25 Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2018/012712 WO2020085533A1 (en) 2018-10-25 2018-10-25 Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed

Publications (1)

Publication Number Publication Date
WO2020085533A1 true WO2020085533A1 (en) 2020-04-30

Family

ID=70331064

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/012712 WO2020085533A1 (en) 2018-10-25 2018-10-25 Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed

Country Status (2)

Country Link
KR (1) KR20210066005A (en)
WO (1) WO2020085533A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6894652B1 (en) * 2020-09-08 2021-06-30 デクソンファーマシューティカルズ株式会社 Tissue repair agent, how to use tissue repair agent and screening method
CN115381880A (en) * 2022-08-31 2022-11-25 中山大学 Fusion nano vesicle and preparation method and application thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023195570A1 (en) * 2022-04-08 2023-10-12 이왕재바이오연구소 주식회사 Anticancer compositoin comprising superoxide dismutase

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170032872A (en) * 2015-09-15 2017-03-23 주식회사 강스템바이오텍 Composition for preventing or treating inflammatory diseases comprising stem cells overexpressing SOD3

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170032872A (en) * 2015-09-15 2017-03-23 주식회사 강스템바이오텍 Composition for preventing or treating inflammatory diseases comprising stem cells overexpressing SOD3

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHO, BYONG SEUNG ET AL.: "Exosomes derived from human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis", STEM CELL RES . THER. ELECTRONIC, vol. 9, no. 187, 11 July 2018 (2018-07-11), pages 1 - 5, XP055709528 *
DATABASE Protein 23 March 2015 (2015-03-23), "superoxide dismutase 3, extracellular [Homo sapiens", XP055709545, retrieved from NCBI Database accession no. EAW92823.1. *
LO SICCO, CLAUDIA ET AL.: "Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization", STEM CELLS TRANSL. MED, vol. 6, 2017, pages 1018 - 1028, XP055709542 *
SAH, S. K. ET AL.: "Enhanced therapeutic effects of human mesenchymal stem cells transducec with superoxide dismutase 3 in a murine atopic dermatitis-like skin inflammation model", ALLERGY. ELECTRONIC, vol. 73, no. 12, 11 October 2018 (2018-10-11), pages 2364 - 2376, XP055709532 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6894652B1 (en) * 2020-09-08 2021-06-30 デクソンファーマシューティカルズ株式会社 Tissue repair agent, how to use tissue repair agent and screening method
CN115381880A (en) * 2022-08-31 2022-11-25 中山大学 Fusion nano vesicle and preparation method and application thereof
CN115381880B (en) * 2022-08-31 2023-05-12 中山大学 Fused nano vesicle and preparation method and application thereof

Also Published As

Publication number Publication date
KR20210066005A (en) 2021-06-04

Similar Documents

Publication Publication Date Title
EP3479831B1 (en) Composition comprising thrombin-treated stem cell-derived exosome for use in treating skin wound
WO2017048097A1 (en) Composition for preventing or treating inflammatory diseases, containing, as active ingredient, stem cells overexpressing sod3
WO2020085533A1 (en) Novel use of extracellular vesicles derived from stem cells in which sod3 is overexpressed
KR101775262B1 (en) A composition for preventing or treating of skin inflammation disease comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof
JP5856117B2 (en) Method for promoting wound healing and muscle regeneration using cell signaling protein NELL1
JP7452798B2 (en) Disease therapeutics based on mesenchymal stem cell mobilization
KR101987354B1 (en) Composition comprising peptide derived from thioredoxin-interacting protein or polynucleotide encoding the peptide for rejuvenation of stem cell
JP2021505611A (en) Combination of RIPK1 and IKK inhibitors for the prevention or treatment of immune disorders
EP3656850A1 (en) Mesenchymal-stem-cell induction agent
KR20170072291A (en) A method of treatment
KR20130060670A (en) Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8
Lucas-Ruiz et al. Mesenchymal stromal cell therapy for damaged retinal ganglion cells, is gold all that glitters?
CN113785068A (en) Hybrid promoters and their use in therapy, in particular in the treatment of type II collagen diseases
KR101796826B1 (en) Mesenchymal Stem Cell overexpressing Oct4 and Sox2 and its use
KR101721228B1 (en) Composition for Promoting Hair Growth Comprising Culture Medium of Adipose-Derived Stem Cells
CN112402592B (en) Application of SPINK7 protein in preparation of medicine for promoting healing of excessive inflammatory wound surface
MXPA02002765A (en) Opaminergic neuronal survival promoting factors and uses thereof.
CN112143705A (en) Double-gene modified stem cell and application thereof
EP3967706A1 (en) Aav mutant that efficiently infects supporting cells
WO2020085534A1 (en) Composition comprising sod3-overexpressed, cord blood-derived stem cell as active ingredient for prevention or treatment of atopic dermatitis
US10722563B2 (en) Prostate-specific tumor antigens and uses thereof
EP2275437A1 (en) Polypeptide and pharmaceutical composition containing the polypeptide
CN114432427B (en) Application of anti-aging active peptide and bone marrow mesenchymal stem cells in preparation of anti-aging drugs
CN114712393B (en) Application of Hnf-1 alpha gene modified mesenchymal stem cells in preventing and treating liver cancer
CN114569702B (en) Pharmaceutical composition containing anti-aging active peptide and stem cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18938034

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20217013938

Country of ref document: KR

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 18938034

Country of ref document: EP

Kind code of ref document: A1