Extracellular vesicles linked with pro-inflammatory cytokines, and mesenchymal stem cells having improved Anti-inflammatory activity treated with extracellular vesicles

Abstract

The present invention relates to: extracellular vesicles linked with pro-inflammatory cytokines; and mesenchymal stem cells treated with the extracellular vesicles and having improved anti-inflammatory activity. Treating mesenchymal stem cells with extracellular vesicles according to an aspect during the culturing thereof can improve the tissue regeneration and inflammation regulatory factor secretion ability of the mesenchymal stem cells, and thus the mesenchymal stem cells can be effectively used for alleviating nervous system and musculoskeletal inflammatory diseases, and pain associated therewith.

Description

Extracellular vesicles linked to pro-inflammatory cytokines, and mesenchymal stem cells with enhanced anti-inflammatory activity treated with said extracellular vesicles

[0001] The present invention relates to extracellular vesicles and mesenchymal stem cells treated with the same that have enhanced anti-inflammatory activity.

[0002] Extracellular vesicles (EVs) are nano-sized membrane structures that have lower toxicity and reduced immunoactivity compared to other nanoparticles or small molecules, and their potential as drug carriers is being actively studied. Furthermore, through surface engineering that alters the activity of surface proteins, it is possible to improve target delivery efficiency and interaction with specific cell tissues.

[0003] Surface engineering of extracellular vesicles is broadly classified into genetic and chemical techniques. Genetic techniques require repetitive genetic manipulation of cells, which is time-consuming and can reduce engineering efficiency; furthermore, the range of molecules that can be engineered on the surface is limited to protein molecules. Among chemical techniques, non-covalent mediated chemistry may result in surface-engineered molecules failing to remain stable in specific environments or for extended periods due to the transient and unstable nature of the bonds themselves. In contrast, covalent mediated chemistry directly binds the molecule to be engineered to residues of surface protein molecules, thereby inducing changes in the activity of the surface protein. However, this technique is also highly challenging because it requires striking a balance between diversity and the need to minimize steric hindrance, and because the surface of the extracellular vesicle must be modified without compromising its integrity.

[0004] The researchers have made research efforts to develop novel surface engineering technology and have developed a chimeric adaptor protein (CAP) with a novel structure capable of being conjugated with various functional molecules. In particular, the researchers discovered that it is possible to develop mesenchymal stem cells with a potent anti-inflammatory effect by treating extracellular vesicles conjugated with pro-inflammatory cytokines using the chimeric adaptor protein during mesenchymal stem cell culture, thereby completing the present invention.

[0005] One aspect provides an extracellular vesicle comprising a chimeric adaptor protein comprising a pro-inflammatory cytokine; and an O6-benzyl guanine binding protein; a transmembrane domain; and a signaling domain.

[0006] Another aspect provides (a) a construct comprising (i) a gene encoding a pro-inflammatory cytokine; and (ii) a gene encoding a chimeric adapter protein comprising an O6-benzylguanine binding protein; a transmembrane domain; and a signaling domain; or (b) an extracellular vesicle derived from a recombinant cell comprising a vector comprising said construct.

[0007] Another aspect provides a method for producing mesenchymal stem cells with enhanced anti-inflammatory activity, comprising the step of culturing mesenchymal stem cells in the presence of the extracellular vesicles.

[0008] Another aspect is to provide mesenchymal stem cells treated with the above-mentioned extracellular vesicles.

[0009] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the above-mentioned mesenchymal stem cells as an active ingredient.

[0010] Another aspect provides a method for preventing or treating an inflammatory disease, comprising the step of administering an effective amount of the above-mentioned mesenchymal stem cells to an individual in need thereof.

[0011] Another aspect is to provide the use of the above mesenchymal stem cells for the prevention or treatment of inflammatory diseases.

[0012] Another aspect is to provide the use of the above mesenchymal stem cells for the manufacture of pharmaceutical preparations for the prevention or treatment of inflammatory diseases.

[0013] One aspect provides an extracellular vesicle comprising a chimeric adaptor protein comprising a pro-inflammatory cytokine; and an O6-benzyl guanine binding protein; a transmembrane domain; and a signaling domain.

[0014] The above "extracellular vesicle (EV)" refers to a nano-sized membrane-bound structure and refers to all types of membrane-bound vesicles generated outside the cell, which are produced in the endosome compartment of most eukaryotic cells. Terms such as "extracellular vesicle," "extracellular vesicle," and "vesicle or vesicle released outside the cell" are all used interchangeably, and extracellular vesicles may include a number of different species, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Small extracellular vesicles (sEVs) generally refer to extracellular vesicles with a diameter of 50 to 200 nm, and the extracellular vesicles of the present invention may be small extracellular vesicles, but are not limited thereto.

[0015] The above extracellular vesicles may have a diameter of 0.1 to 1000 nm. Specifically, they may have a diameter of 50 to 200 nm.

[0016] The above "Chimeric adaptor protein (CAP)" refers to a synthetic complex protein designed to perform a new or enhanced function by combining domains of different proteins. A chimeric adaptor protein may include a specific protein binding domain, a functional domain that influences the activity of a pathway, and a linker region connecting each domain. The chimeric adaptor protein of the present invention may be bound to the extracellular endoplasmic reticulum and expressed on the surface of the extracellular endoplasmic reticulum. That is, the chimeric adaptor protein of the present invention may include a specific protein binding domain, a transmembrane domain, and a signal transduction domain. The chimeric adaptor protein of the present invention may bind to the extracellular endoplasmic reticulum and be expressed on the surface of the extracellular endoplasmic reticulum, thereby inducing signal transduction within the extracellular endoplasmic reticulum and altering the activity of the extracellular endoplasmic reticulum by binding to a specific protein.

[0017] The above "O6-benzylguanine" is a compound in which a benzyl group is attached to guanine, and is used as a substrate specifically recognized by self-labeling enzymes such as SNAP-tags. SNAP-tags can bind to benzylguanine derivatives to label molecules. The above O6-benzylguanine is used interchangeably with 6-benzylguanine, O6-benzylguanine, 6-(benzyloxy)-7H-purin-2-amine, O6-Benzyl, NSC 637037, 6-O-benzylguanine, 6-benzyloxyguanine, 6-oxo-benzylguanine, or 4-6-Benzyloxyguanine, etc.

[0018] The aforementioned "self-labeling enzyme" or "self-labeling protein" refers to a protein capable of self-attaching specific molecular tags or markers to a target protein. Self-labeling proteins are highly useful tools in the fields of cell biology, molecular biology, and biochemistry because they can attach specific molecular tags or markers to target proteins without complex chemical reactions or modification procedures. The term "self-labeling protein" may be used interchangeably with "self-labeling protein" or "self-labeling protein." Unless otherwise specified, the term "self-labeling protein" used without modification is used to include, without limitation, any protein capable of attaching molecular tags or markers to the extracellular endoplasmic reticulum.

[0019] In one embodiment, the O6-benzylguanine binding protein may include a self-labeling protein.

[0020] In one embodiment, the O6-benzylguanine binding protein may include a SNAP-tag. The SNAP-tag has a high affinity for O6-benzylguanine.

[0021] In one embodiment, the O6-benzylguanine binding protein may be expressed on the surface of an extracellular endoplasmic reticulum.

[0022] In one embodiment, the O6-benzylguanine binding protein may comprise the amino acid sequence of SEQ ID NO. 2, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0023] The chimeric adapter protein of the present invention can perform site-specific binding with various functional molecules through the O6-benzylguanine binding protein. For example, it can form a covalent bond with O6-benzylguanine through the O6-benzylguanine binding protein and bind to various functional molecules through O6-benzylguanine.

[0024] The above "transmembrane domain" refers to a domain located on the cell membrane that connects the extracellular domain and the intracellular domain. The transmembrane domain of the present invention can serve to immobilize, display, or bind O6-benzylguanine binding protein to the cell membrane of an extracellular vesicle.

[0025] In one embodiment, the transmembrane domain is one or more transmembrane domains selected from the group consisting of PDGFR (platelent-derived growth factor receptor), EGFR (epidermal growth factor receptor), FGFR (fibroblast growth factor receptor), VEGFR (vascular endothelial growth factor receptor), HGFR (hepatocyte growth factor receptor), Trk (tropomyosin receptor kinase), IR (insulin receptor), LTK (leukocyte receptor tyrosine kinase), angiopoietin receptor, CCK (cholecystokinin) receptor, Neurotrophic factor (NGF) receptor, ROR (receptor tyrosine kinase-like orphan receptors), DDR (discoidin domain receptor), RETR (rearranged during transfection receptor), PTK (tyrosine-protein kinase-like), RYK (related to receptor tyrosine kinase), MuSK (muscle-specific kinase), CD63, CD9, and CD81. It may include a domain. Preferably, the transmembrane domain may include a transmembrane domain of platelet-derived growth factor receptor (PDGFR).

[0026] In one embodiment, the transmembrane domain may comprise the amino acid sequence of SEQ ID NO. 4, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0027] The aforementioned "signaling domain" refers to a region located inside the cell membrane, specifically within the extracellular vesicle, which transmits signals through the binding of the extracellular domain and ligands.

[0028] In one embodiment, the signaling domain may include one or more signaling domains selected from the group consisting of CD9, CD63, the C-terminus of CD63, CD81, LAMP-1, LAMP-2, Syntaxin-3, Syntenin-1, Syntenin-2, Syndecan-1, Syndecan-4, and Prostaglandin F2 Receptor Negative Regulator. Preferably, the signaling domain may include an N-terminal sorting domain (NTD) of Syntenin. An N-terminal sorting domain is a signal sequence required for a protein to move to a specific organelle or location within a cell, and is involved in the localization and transport of the protein.

[0029] In the chimeric adapter protein of the present invention, the signaling domain can play a role in enriching the chimeric adapter protein on the surface during the biosynthesis of extracellular vesicles.

[0030] In one embodiment, the signal transduction domain may comprise the amino acid sequence of SEQ ID NO. 6, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0031] In one embodiment, the O6-benzylguanine binding protein and the transmembrane domain may be connected directly or via a linker. That is, the linker domain may be located between the O6-benzylguanine binding protein and the transmembrane domain.

[0032] Each domain of the chimeric adapter protein of the present invention may optionally be connected by a short oligopeptide or polypeptide linker. The linker may be a rigid linker or a flexible linker, and may be applied without limitation by any linker known in the art, without being particularly limited in its length or type.

[0033] In the present invention, the linker may preferably include a rigid linker. More specifically, the linker may be (EAAAK)n, and the number of copies "n" may be adjusted considering the optimization of the linker. For example, the number of copies n may be an integer from 5 to 10, and preferably n may be 8.

[0034] In one embodiment, the linker may comprise the amino acid sequence of SEQ ID NO. 3, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0035] In the chimeric adapter protein of the present invention, the linker can improve the conjugation efficiency of the O6-benzylguanine binding protein and O6-benzylguanine.

[0036] In one embodiment, the chimeric adapter protein may additionally include a reporter.

[0037] The above "reporter" is used to monitor whether the self-labeled protein of the present invention is introduced or the expression efficiency, and can be used without limitation as long as it is a protein that can be monitored without damage.

[0038] In one embodiment, the reporter may be luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), or enhanced indigo fluorescent protein (ECFP).

[0039] In one embodiment, the reporter may be located between the membrane transversal domain and the signal transduction domain.

[0040] In one embodiment, the reporter may comprise the amino acid sequence of SEQ ID NO. 5, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0041] As described above, the chimeric adapter protein of the present invention is abundantly expressed on the surface of extracellular vesicles and exhibits excellent binding efficiency with O6-benzylguanine. Furthermore, it is in a form that minimizes interference, such as by not interacting with other components of the cell membrane, including neighboring proteins. The chimeric adapter protein of the present invention strategically integrates a long, rigid linker with syntenin NTDs to minimize steric interference of the local environment on the surface of extracellular vesicles. This enables efficient binding with target molecules and allows for enhanced functionality. Additionally, the extracellular vesicle into which the chimeric adapter protein of the present invention has been introduced maintains its integrity as a separate entity. In other words, the chimeric adapter protein of the present invention can impart modularity without disturbing the surrounding environment of the extracellular vesicle surface.

[0042] In one embodiment, the chimeric adapter protein may comprise the amino acid sequence of SEQ ID NO. 1, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0043] "Homology" and "identity" refer to the degree of association between two given sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences have homology or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology or identity of sequences can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0044] Each domain of the chimeric adapter protein of the present invention may include the domains themselves as well as modified forms of each of the domains. In this case, the modification may be performed by substituting, deleting, or adding one or more amino acids to the amino acid sequence of the wild-type domain without altering the function of the domains. Typically, the substitution may be performed by a conservative amino acid substitution that does not affect the charge, polarity, or hydrophobicity of the entire protein.

[0045] The extracellular vesicle engineered with the chimeric adapter protein of the present invention can be used as a powerful vehicle for targeted drug delivery based on the excellent bioavailability and pharmacokinetic properties of the extracellular vesicle itself.

[0046] The extracellular vesicle of the present invention comprises a chimeric adapter protein and may be linked to a pro-inflammatory cytokine through the chimeric adapter protein.

[0047] Specifically, the extracellular vesicle of the present invention may externally express a chimeric adapter protein and be linked to a pro-inflammatory cytokine via O6-benzylguanine.

[0048] In one embodiment, the extracellular endoplasmic reticulum may comprise a pro-inflammatory cytokine; O6-benzylguanine; and the chimeric adapter protein.

[0049] In one embodiment, the pro-inflammatory cytokine may be one or more selected from the group consisting of TNF-α, IFN-γ, IL-1α, IL-1β, IL-6, IL-8, IL-12, IL-17, IL-18, and IL-33.

[0050]

[0051] Another aspect provides (a) a construct comprising (i) a gene encoding a pro-inflammatory cytokine; and (ii) a gene encoding the chimeric adapter protein; or (b) an extracellular vesicle derived from a recombinant cell comprising a vector comprising the construct.

[0052] The above "construct" refers to a macromolecule or molecular complex containing a polynucleotide that is delivered to a host cell in vitro, in vivo, or ex vivo. The construct may additionally include a promoter sequence.

[0053] The term “coding” above refers to the inherent properties of a specific sequence of nucleotides in polynucleotides, such as genes, cDNA, or mRNA, which act as a template for the synthesis of other polymers and macromolecules in biological processes having either a limited sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a limited sequence of amino acids, and the biological properties arising therefrom. Thus, a gene encodes a protein when the transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produce a protein. Both the coding strand, which is a nucleotide sequence identical to the mRNA sequence and usually provided in sequence lists, and the non-coding strand used as a template for the transcription of the gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.

[0054] The above "vector" refers to any nucleic acid construct capable of delivering or directing the transfer of foreign genetic material to a target cell where the polynucleotide may be replicated and / or expressed. The vector comprises the construct to be delivered. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0055] In one embodiment, the vector may be any one selected from the group consisting of plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons, but is not limited thereto; it is obvious that any vector among those commonly available that is suitable for the purpose of the present invention may be used.

[0056] It is obvious that if a gene sequence having a deletion, modification, substitution, or addition of a portion of the sequence from the construct or vector of the present invention has the same or corresponding function as the gene of the present invention, it may also be used in the present application.

[0057] In one embodiment, the above-mentioned composition may comprise the gene sequence of SEQ ID NO. 7, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0058] In one embodiment, the above-mentioned composition may comprise one or more gene sequences of SEQ ID NOs 8 to 12, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0059]

[0060] Another aspect provides a method for producing mesenchymal stem cells with enhanced anti-inflammatory activity, comprising the step of culturing mesenchymal stem cells in the presence of the extracellular vesicles.

[0061] Another aspect is to provide mesenchymal stem cells treated with the above-mentioned extracellular vesicles.

[0062] The above "mesenchymal stem cells" are capable of self-renewal and possess pluripotency, and CD73 + , CD90 + , CD105 +, CD14 - , CD33 - , CD45 - It refers to cells that exhibit a human cell phenotype.

[0063] In one embodiment, the mesenchymal stem cells may be derived from a tissue selected from the group consisting of the umbilical cord, umbilical cord blood, synovium, bone marrow, fat, muscle, nerve, skin, hair, amniotic membrane, and placenta, and preferably may be bone marrow-derived mesenchymal stem cells, but are not limited thereto.

[0064] The culture of the above mesenchymal stem cells may involve growing a population of mesenchymal stem cells in a culture medium.

[0065] In one embodiment, the mesenchymal stem cells may have increased expression of inflammation-suppressing genes and / or increased expression of growth factors by being cultured in the presence of the extracellular vesicles. The mesenchymal stem cells may possess excellent tissue regeneration ability and the ability to secrete inflammation-regulating factors due to having increased levels of inflammation-suppressing gene expression and / or increased levels of growth factor expression, and may be very effectively utilized for the prevention, improvement, or treatment of inflammatory diseases by controlling inflammatory responses.

[0066] The above mesenchymal stem cells may have increased expression of inflammation-suppressing genes. Specifically, the above mesenchymal stem cells may have increased expression of one or more inflammatory genes selected from the group consisting of COX2, A20, and TSG6.

[0067] The aforementioned "COX2" is an inducible enzyme that delivers inflammation-related substances and plays a role in inflammation, cell proliferation, and angiogenesis through the production of prostaglandin E2 (PGE2).

[0068] The above "A20" is a protein that inhibits inflammatory responses and exerts an anti-inflammatory effect by inhibiting NF-κB signaling.

[0069] The aforementioned "TSG6" is an anti-inflammatory protein induced by TNF-α that plays a role in regulating inflammatory responses through hyaluronic acid matrix reorganization.

[0070] The above mesenchymal stem cells may have increased expression of growth factors. Specifically, the above mesenchymal stem cells may have increased expression of hepatocyte growth factor (HGF) and / or platelet-derived growth factor-AA (PDGF-AA).

[0071] The aforementioned "HGF" can regulate cell growth, cell motility, and morphogenesis, and can induce somatic cell division and stimulate the growth of hematopoietic progenitor cells. Accordingly, it can play an important role in embryonic organ development, muscle development, organ regeneration and wound healing in adults, angiogenesis, and tissue regeneration.

[0072] The above "PDGF-AA" is a form of platelet-derived growth factor composed of two A subunits and can play an important role in regulating the growth and survival of specific cell types during embryonic development and in tissue repair in adults.

[0073]

[0074] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the above-mentioned mesenchymal stem cells as an active ingredient.

[0075] In this specification, the term "prevention" refers to any act of suppressing or delaying a disease by administering the composition of the present invention to an individual. For preventive benefits, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet appeared.

[0076] In this specification, the term "treat" refers to any act of administering the composition of the present invention to an individual to improve or benefit from the symptoms of a disease. As used herein, "treat," "alleviate," or "improvement" may be used interchangeably. A therapeutic benefit means any therapeutically significant improvement of one or more diseases, conditions, or symptoms under treatment, or an effect thereon.

[0077] In one embodiment, the inflammatory disease may be an inflammatory disease of the nervous system or musculoskeletal system.

[0078] In one embodiment, the inflammatory disease may be one or more selected from the group consisting of arthritis, myositis, tendinitis, vasculitis, respiratory inflammation, rhinitis, neuroinflammation, vestibular neuritis, peripheral neuritis, multiple sclerosis, encephalitis, and meningitis.

[0079] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" means introducing a specific substance to a patient by any appropriate method, and the route of administration of said pharmaceutical composition may be administered through any general route as long as the drug can reach the target tissue. Administration may be administered by methods known in the art, such as, for example, ocular local administration (e.g., periocular (e.g., subtenon's), subconjunctival, intraocular, intravitreal, anterior chamber, subretinal, supracorbital, and retroocular administration), intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto. Additionally, it may be administered by any device capable of delivering the active ingredient to target cells, and it is preferable that the route of administration be determined according to the type of disease to which it is applied.

[0080] The above administration is 0.00001 mg to 1,000 mg of the composition according to one embodiment per individual per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or It may be administered 10 mg to 25 mg.

[0081] However, the dosage may vary depending on factors such as the formulation method, method of administration, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art may appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or two or more times within the range of clinically acceptable side effects, and the administration site may be one or two or more sites, and the total duration of administration may be from 1 to 30 days per treatment, administered daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after the appropriate time. For animals other than humans, the dosage may be the same as that for humans per kg, or an amount calculated by converting the above dosage based on, for example, the ratio of organ (e.g., heart) volume between the target animal and humans (e.g., average value).

[0082] The pharmaceutical composition of the present invention may be used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, topical preparations, suppositories, sterile injectable solutions, and implantable preparations, which are formulated according to conventional methods. In addition to the active ingredient, the pharmaceutical composition may further include pharmaceutically acceptable excipients that can be used for formulation.

[0083] The pharmaceutical composition of the present invention being formulated in the form of an oral administration formulation may be, for example, a tablet, a pill, a hard or soft capsule, a liquid, a suspension, an emulsifier, a syrup, a granule, an elixir, etc. Depending on the conventional composition of each formulation, such oral administration formulations may include, in addition to the active ingredient, a pharmaceutically acceptable carrier such as a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, or a lubricant such as silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol.

[0084] The fact that the pharmaceutical composition of the present invention is formulated in the form of a parenteral administration formulation may mean that it is administered by a method of administration such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the pharmaceutical composition into the parenteral administration formulation, the active ingredient is mixed in water with a stabilizer or a buffer to prepare a solution or suspension, and such a solution or suspension may be prepared in a unit dosage form in an ampoule or vial.

[0085] The content of stem cells in the pharmaceutical composition of the present invention can be appropriately adjusted according to the purpose of use of the pharmaceutical composition, the form of the formulation, etc., and, for example, may be 0.001 to 99 weight %, 0.001 to 90 weight %, 0.001 to 50 weight %, 0.01 to 50 weight %, 0.1 to 50 weight %, or 1 to 50 weight % based on the total weight of the pharmaceutical composition.

[0086] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. "Pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of disease of the patient, the severity of the disease, the type of active ingredient administered, the type of formulation, the age, gender, weight, health condition, diet, sensitivity, the time and method of administration of the drug, the combination of the composition or drugs used concurrently, and other factors well known in the medical field.

[0087] Another aspect provides a method for preventing or treating an inflammatory disease, comprising the step of administering an effective amount of the above-mentioned mesenchymal stem cells to an individual in need thereof.

[0088] Another aspect provides the use of the above mesenchymal stem cells for the prevention or treatment of inflammatory diseases.

[0089] Another aspect provides the use of the above mesenchymal stem cells for the manufacture of pharmaceutical preparations for the prevention or treatment of inflammatory diseases.

[0090]

[0091] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0092] When extracellular vesicles of a specific type are treated during mesenchymal stem cell culture, the tissue regeneration and secretion of inflammation-regulating factors of the mesenchymal stem cells can be enhanced, making them useful for the treatment of inflammatory diseases of the nervous and musculoskeletal systems and for the alleviation of associated pain.

[0093] Figure 1 is a graph confirming the cell viability of mesenchymal stem cells treated with extracellular vesicles according to one embodiment.

[0094] Figure 2 is a graph confirming the retention of cell surface markers in mesenchymal stem cells treated with extracellular vesicles according to one embodiment.

[0095] Figure 3 is a graph showing the expression levels of COX2, A20, and TSG6 in mesenchymal stem cells treated with extracellular vesicles according to one embodiment.

[0096] Figure 4 is a graph showing the expression levels of HGF, PDGF-AA, and IGFBP-3 in mesenchymal stem cells treated with extracellular vesicles according to one embodiment.

[0097] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since the embodiments are subject to various modifications, they are not limited to the embodiments disclosed below but can be implemented in various forms.

[0098]

[0099] Example 1. Manufacture of sEV-CAP

[0100] 1.1 Introduction of CAP

[0101] An extracellular vesicle (sEV-CAP) expressing a chimeric adapter protein (CAP) according to one embodiment was prepared as follows.

[0102] A vector encoding CAP was constructed by synthesizing the CAP gene and introducing it into a pLV2 vector. HEK293FT cells were transfected with the vector encoding CAP using Lipofectamine 2000, and then the cells were cultured for 21 days in a culture medium supplemented with the antibiotic Hygromycin (300 μg ml-1).

[0103] The structure of CAP is as follows, and information on the gene and amino acid sequence is shown in Table 1.

[0104] CAP = SNAP-tag + rigid linker + PDGFR β TM + EGFP + Syntenin NTD

[0105]

[0106] Structural Amino Acid Sequence Gene Sequence CAP Full Length Sequence No. 1 Sequence No. 7 SNAP-tag Sequence No. 2 Sequence No. 8 rigid linker Sequence No. 3 Sequence No. 9 PDGFR β TM Sequence No. 4 Sequence No. 10 EGFP Sequence No. 5 Sequence No. 11 Syntenin NTD Sequence No. 6 Sequence No. 12

[0107] 1.2 Separation of sEV-CAP

[0108] sEVs were isolated from the culture supernatant of cells stably expressing the chimeric adapter protein (CAP) as in Example 1.

[0109] First, the cells were stabilized for 16 hours after inoculation. Then, the cells were washed three times with PBS and cultured for 24 hours in serum-free DMEM supplemented with 1% A / A, insulin (1 mg ml-1), transferrin (550 μg ml-1), and sodium selenium (670 ng ml-1). The culture supernatant was collected, centrifuged serially at 300 g for 5 minutes and at 2,500 g for 20 minutes, and then filtered through a 0.22 μm filter. sEVs were purified via a tangential flow filtration (TFF) system using a hollow fiber filter (polysulfone MidiKros TFF device with a 0.05 μm pore size, Spectrum Laboratories). The filtered supernatant was continuously circulated while maintaining a pressure of 20 psi or less, monitored by an in-line analog pressure gauge. The sEVs were then concentrated after dialyzed filtration using PBS.

[0110]

[0111] Example 2. Surface engineering of sEV-CAP and pro-inflammatory cytokines

[0112] Surface engineering with various functional molecules of extracellular vesicles (sEV-CAP) expressing a chimeric adapter protein prepared according to Example 1 was performed as follows.

[0113] Specifically, 20 μM TNF-α (#300-01A; PeproTech) and 20 μM IFN-γ (#300-02; PeproTech) were each pre-incubated with 100 μM BG-GLA-NHS (# S9151S; NEB) at room temperature for 30 minutes. Each mixture was buffer-exchanged with PBS using a 7K MWCO zeba spin desalting column (# 89862; Thermo Scientific). Subsequently, BG-linker-conjugated TNF-α and BG-linker-conjugated IFN-γ were incubated with sEV at a molar concentration of 1:100, respectively, at 4 °C for 16 hours. Unconjugated cytokines were removed using PBS via a TFF system.

[0114]

[0115] Example 3. Culture of Bone Marrow-Derived Mesenchymal Stem Cells (B-MSCs)

[0116] 3.1 Bone Marrow Harvesting

[0117] First, to collect a specimen from the ilium (pelvic bone), blood pressure, pulse, and body temperature were measured to ensure the donor's body was suitable for the procedure. Subsequently, the donor was laid down, and their lower body was covered with a sterile drape, leaving only the area around the collection site exposed. After cleaning the collection site with an antiseptic such as iodine, a general anesthetic was injected into the donor. Once sensation was lost at the injection site, a needle was inserted through the skin into the bone to collect bone marrow. After removing the needle, a sterile bandage was placed over the collection site and pressure was applied to stop bleeding at the procedure site.

[0118]

[0119] 3.2 Isolation of Bone Marrow Stem Cells from Bone Marrow Fluid

[0120] Bone marrow tissue was slowly divided into 50 mL leucosep tubes (Greiner LeucoSep-tube, #Z642843, MERCK) containing Ficoll (Ficoll-Paque™ PLUS, #17144002, Cytivalifesciences) and centrifuged at 2000 rpm for 15 minutes. The buffy coat remaining in the lower layer was collected without mixing with the red blood cell layer and transferred to a 50 mL tube (Corning® 50 mL centrifuge tubes, #CLS430828, MERCK). The tube was then filled to 50 mL with 1X PBS (PBS-1X, #LB001-02, WELGENE) and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were rehydrated with 10 mL of RBC lysis buffer (eBioscience™ 1X RBC Lysis Buffer, #00-4333-57, ThermoFisher), transferred to a 15 mL tube, incubated in a 37℃ incubator for 5 minutes, and then centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were washed with 10 mL of 1X PBS (PBS-1X, #LB004-01, WELGENE) and then centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, cells were dispersed in 15 mL of 10% FBS (Fetal Bovine Serum, #16000-044, Gibco) in Alpha MEM culture medium (Alpha MEM, #LM008-01, WELGENE), seeded in one T75 Flask (T175 Flask, #159910, ThermoFisher), and cultured in a 37℃ incubator at 5% CO2.

[0121]

[0122] 3.3 Culture of Bone Marrow Stem Cell Proliferation

[0123] To proliferate and culture bone marrow stem cells, the supernatant of the inoculated T75 flask was removed, and after washing twice with 5 mL of 1X PBS, the culture medium was changed to Alpha MEM (Alpha MEM, #LM008-01, WELGENE) with 15 mL of 10% FBS (Fetal Bovine Serum, #16000-044, Gibco) and 20 ng / mL bFGF (Recombinant Human FGF basic, #233-FB-500, R&D system), and then cultured in a 37℃ incubator at 5% CO2. After washing twice with 5 mL of 1X PBS every 2 days, the medium was changed to 15 mL of 10% FBS (Fetal Bovine Serum, #16000-044, Gibco) and 20 ng / mL bFGF (Recombinant Human FGF basic, #233-FB-500, R&D system) in Alpha MEM (Alpha MEM, #LM008-01, WELGENE), and cultured in a 37℃ incubator at 5% CO2. When the cell confluency of the T75 flask was 90% or higher, the cells were washed twice with 5 mL of 1X PBS, then 5 mL of 1X TrypLE (TrypLE™ Express Enzyme (1X) no phenol red, #12604021, ThermoFisher) was added and incubated in a 37℃ incubator for 5 minutes. Afterward, the cells were suspended in a 50 mL tube and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were washed with 10 mL of 1X PBS (PBS-1X, #LB004-01, WELGENE) and centrifuged at 1500 rpm for 5 minutes.After removing the supernatant, cells were suspended in 30 mL of 20 ng / mL bFGF (Recombinant Human FGF basic, #233-FB-500, R&D system) and 10% FBS (Fetal Bovine Serum, #16000-044, Gibco) in Alpha MEM (Alpha MEM, #LM008-01, WELGENE), then inoculated into a T175 Flask (T175 Flask, #159910, ThermoFisher) and cultured in a 37℃ incubator at 5% CO2.

[0124]

[0125] 3.4 Recovery of Bone Marrow Stem Cells

[0126] To recover proliferated bone marrow stem cells, when the cell confluency of the T175 flask was 90% or higher, the cells were washed twice with 5 mL of 1X PBS, followed by the addition of 15 mL of 1X TrypLE (TrypLE™ Express Enzyme (1X) no phenol red, #12604021, ThermoFisher). The mixture was then incubated in a 37°C incubator for 5 minutes, after which the cells were suspended in a 50 mL tube and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were washed with 10 mL of 1X PBS (PBS-1X, #LB004-01, WELGENE) and centrifuged at 1500 rpm for 5 minutes. The cells were recovered after removing the supernatant and used in the experiment.

[0127]

[0128] General B-MSCs were prepared and used according to the methods of Examples 3.1 to 3.4 above, and the B-MSCs of the present invention were used by treating cultured B-MSCs with Con-sEV and / or TNFα-sEV and culturing them together.

[0129]

[0130] Experimental Example 1. Confirmation of maintenance of cell viability

[0131] The cell viability of the mesenchymal stem cells of Example 3 was confirmed.

[0132] Specifically, 5 × 10⁶ B-MSCs in a 96-well plate 3 Inoculated at cell / well density, and treated with various substances the next day (Con-sEV / IFNγ-sEV+TNFα-sEV: 6.76 × 10⁻⁶ 10 particles / ml, TNFα-sEV: 3.38 × 10 10 particles / ml) and cultured for 24 hours.

[0133] At the end of the culture, MTS reagent (# G3582; Promega) was added to each well and incubated at 37°C for 2 hours. Afterward, the MTS reagent containing the supernatant of each well was removed, and the absorbance was measured at 490 nm using a microplate reader.

[0134] As a result, as shown in Figure 1, when mesenchymal stem cells were cultured with TNFα-sEV or Con-sEV, no significant changes in cell viability were observed in comparisons between the controls. In summary, TNFα-engineered EVs were found not to inhibit the growth of mesenchymal stem cells.

[0135]

[0136] Experimental Example 2. Confirmation of Cell Surface Marker Retention

[0137] The cell surface markers of the mesenchymal stem cells of Example 3 were identified.

[0138] Specifically, 1 × 10⁶ in a 6-well plate of B-MSCs 6 Inoculated at cell / well density, and treated with various substances the next day (Con-sEV / IFNγ-sEV+TNFα-sEV: 6.76 × 10⁻⁶ 10 particles / ml, IFNγ-sEV / TNFα-sEV: 3.38 × 10 10particles / ml, rIFNγ+rTNFα: rIFNγ (10 ng / ml), rTNFα (15 ng / ml), rIFNγ+rTNFα (1 / 10): rIFNγ (1 ng / ml), rTNFα (1.5 ng / ml), and were incubated for 24 hours. Subsequently, flow cytometry was performed on CD14, CD34, CD45, CD73, CD90, and CD105. A CytoFLEX Flow Cytometer (Beckman Coulter) was used for flow cytometry, and FlowJo software (Flowjo, Ashland, OR, USA) was used for data analysis.

[0139] The primary antibodies used in this experiment are as follows: PE-conjugated anti-CD14 antibody, PE-conjugated anti-CD34 antibody, PE-conjugated anti-CD45 antibody, PE-conjugated anti-CD73 antibody, PE-conjugated anti-CD90, PE-conjugated anti-CD105 antibody.

[0140] As a result, as shown in Figure 2, no significant change in the cell surface morphology of mesenchymal stem cells was observed when cultured with TNFα-sEV or Con-sEV. In summary, culturing mesenchymal stem cells with TNFα-engineered EVs did not affect changes in cell-specific surface morphology.

[0141]

[0142] Experimental Example 3. Confirmation of Inflammation Marker Expression

[0143] The degree of expression of inflammation-suppressing genes in the mesenchymal stem cells of Example 3 was confirmed. COX2 is an enzyme that delivers inflammation-related substances, A20 is an inflammation-suppressing factor, and TSG6 is an anti-inflammatory protein induced by TNF-α.

[0144] 2 x 10 B-MSCs in a 24-well plate 5 Inoculated at cell / well density, and treated with various substances the next day (Con-sEV / IFNγ-sEV+TNFα-sEV: 6.76 × 10⁻⁶ 10 particles / ml, TNFα-sEV: 3.38 × 10 10 particles / ml) and cultured for 4 hours. After culture, mRNA (MiniBEST Universal RNA Extraction Kit, # 9767; TaKaRa) was extracted from the cells, and the concentration of mRNA was measured using a nanodrop (DS-11 Series Spectrophotometer; DeNovix). Subsequently, cDNA (PrimeScript™ 1st strand cDNA Synthesis Kit, # 6110A; TaKaRa) was synthesized using 100 ng of mRNA, and gene expression was detected using the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (# RR420A; TaKaRa).

[0145] The primers used in this experiment are as shown in Table 1:

[0146]

[0147] Primer typeBase sequenceSEQ ID numberHuman COX2 specific sense primer5'-CGG TGA AAC TCT GGC TAG ACA G-3'(22bp)13Human COX2 specific anti-sense primer5'- GCA AAC CGT AGA TGC TCA GGG A-3'(22bp)14Human A20 specific sense primer5'- CTC AAC TGG TGT CGA GAA GTC C-3'(22bp)15Human A20 specific anti-sense primer5'- TTC CTT GAG CGT GCT GAA CAG C-3'(22bp)16Human TSG6 specific sense primer5'- TCA CCT ACG CAG AAG CTA AGG C-3'(22bp)17Human TSG6 specific anti-sense primer5'- TCC AAC TCT GCC CTT AGC CAT C-3' (22bp)18Human GAPDH specific sense primer5'- TGC ACC ACC AAC TGC TTA GC -3'(20bp)19Human GAPDH specific anti-sense primer5'- GGC ATG GAC TGT GGT CAT GAG -3'(21bp)20

[0148] Real-time polymerase chain reaction was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The relative mRNA levels of the samples were calculated by Ct (comparative threshold cycle) analysis after normalization with respect to the amount of GAPDH of the same samples, and expressed as 2-△△Ct values ​​modified from the initial Ct values.

[0149] As a result, as shown in Figure 3, it was confirmed that the TNFα-sEV group showed an increase in COX2 gene expression of approximately 41.67%, an increase in A20 gene expression of approximately 300%, and an increase in TSG6 gene expression of approximately 242% compared to the control group.

[0150]

[0151] Experimental Example 4. Cytokine Analysis

[0152] Multiplex analysis was performed to quantify cytokines and the analytes of interest, hepatocyte growth factor (HGF) and platelet-derived growth factor-AA (PDGF-AA). The analysis was conducted using a Bio-Plex 200 Reader (Bio-Rad) and the associated Bio-Plex Manager MP / Bio-Plex Manager 6.1 software (Bio-Rad). Multiplex analysis allows for the simultaneous quantification of multiple targets, providing more information while requiring smaller sample volumes and shorter processing times compared to conventional immunoassays; this is achieved by utilizing differentially detectable bead sets and fluorescence-based detection. The concentration of each analyte was determined based on individual calibration curves generated using standards provided by the manufacturer. Data were expressed in pg / ml and reported as mean ± standard error of mean (SEM) after normalization to an internal standard control. Statistical analysis was performed using one-way analysis of variance (ANOVA), and a p-value < 0.05 was considered statistically significant. The data were shown in Figure 4, expressed as mean ± standard deviation (SD) or SEM along with the distribution where appropriate.

[0153] As shown in Figure 4, it was confirmed that the TNFα-sEV group showed an increase in HGF secretion of approximately 14.81% and an increase in PDGF-AA secretion of approximately 58.88% compared to the control group.

[0154]

[0155] In summary, mesenchymal stem cells treated with cytokine-linked sEV-CAP exhibit excellent tissue regeneration and the ability to secrete inflammation-regulating factors, and have potential for the treatment of inflammatory diseases of the nervous and musculoskeletal systems.

[0156]

[0157] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Pro-inflammatory cytokines; and A chimeric adaptor protein comprising an O6-benzyl guanine binding protein; a transmembrane domain; and a signaling domain Extracellular vesicles containing 2. The extracellular vesicle of Claim 1, wherein the O6-benzylguanine binding protein comprises a SNAP-tag.

3. The extracellular endoplasmic reticulum of claim 1, wherein the chimeric adapter protein comprises an amino acid sequence represented by SEQ ID NO.

1.

4. The extracellular vesicle of Claim 1, wherein the pro-inflammatory cytokine is one or more selected from the group consisting of TNF-α, IFN-γ, IL-1α, IL-1β, IL-6, IL-8, IL-12, IL-17, IL-18, and IL-33.

5. (a) (i) a gene encoding a pro-inflammatory cytokine; and (ii) a construct comprising a gene encoding a chimeric adapter protein comprising an O6-benzylguanine binding protein; a transmembrane domain; and a signaling domain; or (b) Vector containing the above-mentioned work Extracellular vesicles derived from recombinant cells containing 6. A method for producing mesenchymal stem cells with enhanced anti-inflammatory activity, comprising the step of culturing mesenchymal stem cells in the presence of extracellular vesicles according to any one of claims 1 to 5.

7. Mesenchymal stem cells treated with the extracellular vesicles of any one of claims 1 to 5.

8. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the mesenchymal stem cells of Claim 7 as an active ingredient.

9. A pharmaceutical composition according to claim 8, wherein the inflammatory disease is an inflammatory disease of the nervous system or musculoskeletal system.

10. A pharmaceutical composition according to claim 8, wherein the inflammatory disease is one or more selected from the group consisting of arthritis, myositis, tendinitis, vasculitis, respiratory inflammation, rhinitis, neuroinflammation, vestibular neuritis, peripheral neuritis, multiple sclerosis, encephalitis, and meningitis.

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