Therapeutic agent composition for treatment of immunopathies through mitochondrial transplantation

Abstract

The present invention relates to an immunomodulatory therapeutic composition for immunomodulation through the transplantation of induced pluripotent stem cell (iPSC)-derived mitochondria. The iPSC-derived mitochondria according to the present invention overexpress UCP2, and the expression of UCP2 was found to be associated with an immunomodulatory effect. In addition, the administration of iPSC-derived mitochondria was found to enhance mitochondrial function and thereby increase ATP synthesis and mitochondrial membrane potential. In addition, the present invention was found to regulate the expression of autoimmune disease-related immune cells and to increase the synergistic effect of immune cell regulation when used in combination with metformin. In addition, the present invention was found to reduce the activity of autoimmune disease symptoms and inhibit cell expression related to autoimmunity. In addition, treating peripheral blood mononuclear cells with iPSC-derived mitochondria was found to inhibit Th17 expression and increase Treg expression, as well as improve immunomodulation and ameliorate the pathological activity of various immune diseases.

Description

Composition for the treatment of immunopathology through mitochondrial transplantation

[0001] The present invention relates to a composition for treating immunopathology through mitochondrial transplantation.

[0002] Immune disorders are conditions in which components of the immune system cause, mediate, or otherwise contribute to pathology. Inflammatory disorders, in particular, represent one of the most significant global health problems. Inflammation generally refers to the localized protective response of body tissues against host invasion by foreign substances or harmful stimuli. Causes of inflammation include infectious agents such as bacteria, viruses, and parasites; physical agents such as burns or radiation; chemical agents such as toxins, drugs, or industrial agents; immune responses such as allergies and autoimmune reactions; and conditions associated with oxidative stress.

[0003] Autoimmune diseases, also known as autoimmune diseases, are characterized by the immune system attacking its own organs, triggering an unprovoked response. These responses stem from the recognition of autoantigens by T lymphocytes, triggering humoral (autoantigen production) and cellular (increased lymphocyte and macrophage cytotoxic activity) immune responses. Autoimmune diseases include rheumatoid arthritis, psoriasis, systemic dermatomyositis, multiple sclerosis, and lupus erythematosus, as well as conditions characterized by antigen-induced exacerbations of immune responses, such as asthma, drug or food allergies, and inflammatory bowel disease. These diseases are all chronic and limited in scope, sometimes fatal, and currently, no effective treatment exists. Therefore, any drug, medication, or medium that can alleviate or alleviate the disease during its progression would be a valuable tool for the patient's health.

[0004] Meanwhile, mitochondria are eukaryotic organelles involved in the synthesis and regulation of adenosine triphosphate (ATP), the cellular energy source. Mitochondria are involved in various metabolic pathways within the body, including cell signaling, cell differentiation, apoptosis, as well as the control of the cell cycle and cell growth. Mitochondria possess their own genome and are central organelles in cellular energy metabolism. Mitochondria produce energy through electron transport and oxidative phosphorylation, and play a crucial role in apoptotic signaling pathways.

[0005] Reduced energy production due to mitochondrial dysfunction has been reported to cause various diseases. Mutations in the mitochondrial genome and proteome, which impair the electron transport chain, can lead to decreased ATP production, excessive reactive oxygen species production, and impaired calcium regulation. These changes can alter mitochondrial membrane permeability, leading to abnormal cell death and the development of cancer and other intractable diseases.

[0006] Human diseases caused by mitochondrial dysfunction have been reported to include mitochondrial-related genetic disorders, diabetes, heart disease, dementias such as Parkinson's and Alzheimer's, and the development and metastasis of various cancers. Furthermore, common characteristics found in over 200 different cancer types include impaired apoptosis, increased inflammatory responses, and increased abnormal metabolic activity. Furthermore, research is currently underway into the relationship between mitochondria and immune diseases.

[0007] Mitochondria contain signaling mechanisms closely linked to cell death, and if we can interpret and control these signals, we can apply them to disease treatment. Mitochondrial dysfunction is not a single symptom or disease, but can manifest as a variety of diseases in various organs within the body. Therefore, the possibility of lesion- and disease-specific treatments related to diseased mitochondria has been raised. However, the development of precise targets and appropriate therapeutic technologies remains urgent. In particular, increased oxidative stress due to increased intracellular reactive oxygen species has recently been proposed as a fundamental cellular and molecular biological mechanism in cardiovascular, metabolic, and neurodegenerative diseases. However, the efficacy of metabolic or antioxidant therapies currently used in clinical and preclinical trials remains controversial. Therefore, gene therapy, which involves transplanting intact mitochondria or modulating the expression of membrane and matrix proteins that directly regulate mitochondrial function, may be promising strategies for permanent or semi-permanent treatment.

[0008] The purpose of the present invention is to provide mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs).

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0010] Another object of the present invention is to provide a food composition for preventing or improving autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0011] Another object of the present invention is to provide an immunomodulatory pharmaceutical composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0012] Another object of the present invention is to provide an immune-modulating food composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0013] Another object of the present invention is to provide a method for treating an autoimmune disease, comprising the step of administering a pharmaceutically effective amount of the above mitochondria to a subject.

[0014] To achieve the above purpose, the present invention provides mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs).

[0015] In addition, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0016] In addition, the present invention provides a food composition for preventing or improving autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0017] In addition, the present invention provides an immunomodulatory pharmaceutical composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0018] In addition, the present invention provides an immune-modulating food composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0019] In addition, the present invention provides a method for treating an autoimmune disease, comprising the step of administering a pharmaceutically effective amount of the above mitochondria to a subject.

[0020] The iPSC-derived mitochondria of the present invention overexpress UCP2, and it was confirmed that UCP2 expression is related to an immune-modulatory effect. In addition, it was confirmed that the administration of iPSC-derived mitochondria enhances mitochondrial function, increasing ATP synthesis and mitochondrial membrane potential. In addition, it was confirmed that it regulates the expression of immune cells related to autoimmune diseases, and when used in combination with metformin, a synergistic effect of immune cell regulation was confirmed. In addition, it was confirmed that it reduces the disease activity of autoimmune diseases and suppresses the expression of autoimmune-related cells. In addition, it was confirmed that treatment of peripheral blood mononuclear cells with iPSC-derived mitochondria suppresses the expression of Th17 and increases the expression of Treg, improving immune regulation and disease activity in various immune diseases, and thus can be usefully utilized in the field of immunotherapy.

[0021] Figure 1 is a diagram showing the quantitative analysis of UCP2 protein expression in mitochondria according to cell origin using Western blot.

[0022] Figure 2 shows a Western blot analysis of UCP2 protein expression in mitochondria derived from iPSCs treated with UCP2 siRNA (A: Western blot results, B: quantification of analysis results).

[0023] Figure 3 is a diagram analyzing immune cell activity according to treatment of mitochondria derived from iPSCs treated with UCP2 siRNA (A: flow cytometry results, B: quantification of Th17 expression, C: quantification of IL-17).

[0024] Figure 4 is a diagram confirming an increase in mitochondrial function in mouse spleen cells following treatment with iPSC-derived mitochondria of the present invention (A: confirmation of intracellular ATP content, B: confirmation of ATP synthesis ability, C: confirmation of membrane potential).

[0025] Figure 5 is a diagram confirming the expression of immune cells in mouse spleen cells according to treatment with iPSC-derived mitochondria of the present invention (A: confirmation of IFN-γ positive cells, B: confirmation of IL-17 positive cells, C: confirmation of Foxp3 positive cells, D: confirmation of Th17 / Treg ratio).

[0026] Figure 6 is a diagram showing the amounts of IFN-γ and IL-17 in a mouse spleen cell culture solution confirmed by ELISA according to treatment with iPSC-derived mitochondria of the present invention (A: IFN-γ quantification, B: IL-17 quantification).

[0027] Figure 7 is a diagram confirming the immune cell expression of mouse spleen cells following combined treatment with iPSC-derived mitochondria and metformin of the present invention (A: confirmation of IFN-γ positive cells, B: confirmation of IL-17 positive cells).

[0028] Figure 8 is a diagram confirming the disease activity according to the treatment of iPSC-derived mitochondria in an animal model of inflammatory bowel disease (A: confirmation of weight change, B: quantification of intestinal length).

[0029] Figure 9 is a diagram showing intestinal tissue damage confirmed by H&E staining following treatment with iPSC-derived mitochondria in an inflammatory bowel disease animal model (A: staining results, B: quantification of intestinal damage index).

[0030] Figure 10 is a diagram confirming the expression of immune cells in spleen cells according to treatment with iPSC-derived mitochondria in an animal model of inflammatory bowel disease (A: Th1 quantification, B: Th2 quantification, C: Th17 quantification).

[0031] Figure 11 is a diagram showing the expression of Th17 and Treg cells in spleen cells confirmed by confocal analysis according to treatment with iPSC-derived mitochondria in an animal model of inflammatory bowel disease (A: Th17 analysis results, B: Th17 quantification, C: Treg analysis results, D: Treg quantification).

[0032] Figure 12 is a diagram showing the expression of Th17 in peripheral blood mononuclear cells confirmed by flow cytometry according to treatment with iPSC-derived mitochondria of the present invention (A: flow cytometry results, B: Th17 quantification).

[0033] Figure 13 is a diagram showing the expression of Th17 in peripheral blood mononuclear cells according to the iPSC-derived mitochondria and MTDR Treg conditions of the present invention, confirmed by flow cytometry (A: flow cytometry results, B: Th17 quantification).

[0034] Figure 14 is a diagram showing the amount of IL-17 in a cell culture solution confirmed by ELISA according to the treatment of iPSC-derived mitochondria of the present invention.

[0035] Figure 15 is a diagram showing the expression of Treg in peripheral blood mononuclear cells confirmed by flow cytometry according to the treatment of iPSC-derived mitochondria of the present invention (A: flow cytometry results, B: Treg quantification).

[0036] Figure 16 is a diagram showing the expression of Tregs in peripheral blood mononuclear cells according to the iPSC-derived mitochondria and MTDR Tregs conditions of the present invention, confirmed by flow cytometry (A: flow cytometry results, B: Treg quantification).

[0037] Figure 17 is a diagram showing the amount of IL-10 in a cell culture solution confirmed by ELISA according to the treatment of iPSC-derived mitochondria of the present invention.

[0038] Figure 18 is a diagram confirming the immune modulatory effect in wild-type mouse spleen cells according to treatment with iPSC-derived mitochondria of the present invention (A: quantification of immune cell expression, B: quantification of autoimmune antibodies).

[0039] Figure 19 is a diagram showing the expression of pathogenic B cells in mouse B lymphocytes according to treatment with iPSC-derived mitochondria of the present invention, analyzed by flow cytometry (A: flow cytometry analysis results, B: quantification of analysis results).

[0040] Figure 20 is a diagram showing the expression of immune regulatory B cells in mouse B lymphocytes according to treatment with iPSC-derived mitochondria of the present invention, analyzed by flow cytometry (A: flow cytometry results, B: quantification of analysis results).

[0041] Figure 21 is a diagram showing the improvement effect of lupus nephritis in an animal model of lupus with metabolic disorder administered with iPSC-derived mitochondria of the present invention, confirmed by H&E staining.

[0042] A: Confirmation of increased nephritis due to metabolic abnormalities

[0043] B: Confirmation of improvement in renal function following iPSC mitochondria treatment

[0044] C: Confirmation of a decrease in the urinary nephritis index

[0045] Figure 22 is a diagram showing the B cell regulatory effect in a lupus animal model with metabolic disorder to which the iPSC-derived mitochondria of the present invention were administered, analyzed by flow cytometry (A: flow cytometry results, B: quantification of analysis results).

[0046] Figure 23 is a diagram showing the analysis of the pathogenic T cell regulation effect in a lupus animal model with metabolic disorder administered with iPSC-derived mitochondria of the present invention using flow cytometry.

[0047] Figure 24 is a diagram showing the B cell regulatory effect of iPSC-derived mitochondria administration in naturally occurring lupus spleen cells analyzed by flow cytometry (A: flow cytometry results, B: quantification of analysis results).

[0048] Figure 25 is a flow cytometric analysis of the B cell regulatory effect of iPSC-derived mitochondria administration to serum-treated splenocytes with spontaneous lupus and metabolic disorders (A: flow cytometric analysis results, B: quantification of analysis results).

[0049] Figure 26 is a diagram confirming the effect of suppressing T cell proliferation in a homologous reaction according to the treatment of iPSC-derived mitochondria of the present invention.

[0050] Figure 27 is a diagram showing the specific increase in IFN-r and IL-10 in a homogeneous reaction following treatment with iPSC-derived mitochondria of the present invention, confirmed by ELISA.

[0051] Figure 28 shows the quantification of disease indices and body weight in a GVHD animal model according to treatment with iPSC-derived mitochondria of the present invention (A: disease indices over time, B: body weight over time).

[0052] Figure 29 is a diagram quantifying the regulation of pathogenic immune cells in the blood and spleen of a GVHD animal model according to treatment with iPSC-derived mitochondria of the present invention.

[0053] Figure 30 is a diagram showing the analysis of the inhibitory effect of the iPSC-derived mitochondria of the present invention on CD4 and CD8 lineage inflammatory IL-17 expressing T cells by flow cytometry (A: analysis results, B: quantification of Th17 and Tc17 cells).

[0054] Figure 31 is a diagram showing the analysis of subtypes of CD4 and CD8 lineage tissue-resident memory T cells by flow cytometry according to the treatment of iPSC-derived mitochondria of the present invention (A: flow cytometry results, B: CD4 and CD8 TRM quantification).

[0055] Figure 32 is a diagram confirming the disease activity of an acute psoriasis animal model according to the administration of iPSC-derived mitochondria of the present invention (A: Schematic diagram of psoriasis animal model and dosage schedule, B: Visual confirmation of psoriasis activity, C: Quantification of psoriasis index).

[0056] Figure 33 is a diagram showing the expression of inflammatory immune cells in an acute psoriasis animal model confirmed by flow cytometry following administration of iPSC-derived mitochondria of the present invention (A: CD4 lineage cell quantification, B: CD8 lineage cell quantification, C: Treg expression confirmation).

[0057] Figure 34 is a diagram showing the expression of tissue-resident memory cells in an acute psoriasis animal model analyzed by flow cytometry following administration of iPSC-derived mitochondria of the present invention (A: CD4 TRM quantification, B: CD8 TRM quantification).

[0058] Figure 35 is a diagram showing histological changes in an acute psoriasis animal model following administration of iPSC-derived mitochondria of the present invention, confirmed by H&E staining (A: staining results, B: quantification of staining results).

[0059] Figure 36 shows the quantification of immune cell expression in PBMCs derived from gastric cancer patients following treatment with iPSC-derived mitochondria of the present invention.

[0060] Figure 37 shows the quantification of TRM subtypes in PBMCs derived from gastric cancer patients according to treatment with iPSC-derived mitochondria of the present invention.

[0061] Figure 38 is a schematic diagram illustrating an experimental schedule for an avatar animal model of a gastric cancer patient following administration of iPSC-derived mitochondria of the present invention.

[0062] Figure 39 is a diagram confirming a reduction in the size of a gastric cancer tumor following administration of iPSC-derived mitochondria of the present invention.

[0063] Figure 40 is a diagram quantifying the reduction in gastric cancer tumor size following administration of iPSC-derived mitochondria of the present invention.

[0064] Figure 41 is a diagram analyzing immune cell subtypes in an avatar animal model of a gastric cancer patient following administration of iPSC-derived mitochondria of the present invention.

[0065] Figure 42 is a schematic diagram illustrating an experimental schedule for an avatar animal model of liver cancer patients administered with iPSC-derived mitochondria of the present invention.

[0066] Figure 43 is a diagram confirming a reduction in liver cancer tumor size following administration of iPSC-derived mitochondria of the present invention.

[0067] Figure 44 is a diagram showing the purification quality of iPSC-derived mitochondria of the present invention and the delivery efficiency into chondrocytes (A: Mitochondrial quality confirmation, B: Delivery efficiency and quantification).

[0068] Figure 45 is a diagram confirming the effect of regulating the expression of cartilage metabolic factors of iPSC-derived mitochondria of the present invention (A: quantification of q-PCR results, B: quantification of ELISA results).

[0069] Figure 46 is a diagram showing the expression of inflammatory apoptotic factors in mitochondria derived from iPSCs of the present invention analyzed by Western blot (A: Western blot results, B: quantification of analysis results).

[0070] Figure 47 is a diagram confirming the osteoarthritis improvement effect of iPSC-derived mitochondria of the present invention through pain analysis (A: Schematic representation of the animal model production process, B: Quantification of pain analysis results).

[0071] Figure 48 is a histological analysis of the cartilage damage protective effect of iPSC-derived mitochondria of the present invention (A: Safranin O staining and quantification, B: Immunohistochemical staining and quantification).

[0072] Figure 49 is a diagram showing the analysis of immune cell subtypes in an animal model of osteoarthritis following administration of iPSC-derived mitochondria of the present invention using flow cytometry.

[0073] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0074] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0075] The present invention provides mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs).

[0076] The "induced pluripotent stem cells (iPSCs)" of the present invention are pluripotent stem cells that can be produced directly from adult cells. iPSCs are created by reprogramming cells by expressing specific transcription factor genes in adult somatic cells, thereby transforming them into induced pluripotent stem cells. The key to producing iPSCs lies in the use of reprogramming factors. Representative factors include the "Yamanaka factors" used in the method announced by Yamanaka's research team, as well as transcription factors Oct4, Sox2, cMyc, and Klf4. Continued follow-up research on reprogramming factors is uncovering not only transcription factors but also new regulatory factors such as miRNAs. The method for producing iPSCs involves (1) isolating and culturing necessary cells from adults, and (2) introducing genes that can induce stem cells into the isolated cells using a viral vector. (3) Afterwards, cells expressing the introduced gene are labeled with the embryonic stem cell marker Fbx15 and isolated using antibiotics. The isolated cells are then cultured on a support cell layer according to stem cell culture methods. (4) Among the cultured cells, a portion of the transformed cells are reprogrammed into iPSCs, and colonies resembling embryonic stem cells are selected to create iPSCs.

[0077] The "mitochondria" of the present invention is one of the cellular organelles that participate in cellular respiration, and the more actively respiring cells are, the more mitochondria they contain. Mitochondria are surrounded by a double membrane, and the inside is made up of a winding inner membrane called cristae. DNA and RNA exist inside, and they are 0.2-3 μm in size and participate in cellular respiration. The most important function of mitochondria is to synthesize ATP, an energy source, from food taken into the body. There is a protein called 'ATP synthase' in the inner membrane of the mitochondria, and this is responsible for producing ATP. Hydrogen ions created between the inner and outer membranes of the mitochondria through food flow into the inner mitochondrial membrane, and ATP synthase combines phosphate and ADP (a form in which two phosphates are combined with adenosine) to produce ATP (a form in which three phosphates are combined with adenosine). Mitochondria also play a role in killing cells that have lost their function, which is called apoptosis, and they absorb cells with damaged DNA or fragment the DNA, leading to cell death. They also prevent cells that have already lost their function from transforming into cancer cells or other cells. Mitochondria are structures that play a central role in controlling respiration, and they are also involved in the oxidation of pyruvate and acetyl CoA, which are produced through the catabolism of sugars into lipids, and in oxidative phosphorylation through the electron transport chain. In this way, mitochondria play a central role in the oxidation of sugars and fatty acids and the oxidative phosphorylation that accompanies them.

[0078] The "uncoupling protein 2 (UCP2)" of the present invention is mitochondrial uncoupling protein 2, a protein belonging to the mitochondrial anion transport protein family. UCP uncouples oxidative phosphorylation from ATP synthesis by dissipating mitochondrial membrane potential as heat, called mitochondrial proton leak. UCP promotes the transfer of protons from the inner mitochondrial membrane to the outer mitochondrial membrane, and reduces the mitochondrial membrane potential of mammalian cells, thereby reducing the production of reactive oxygen species. Unlike UCP1 and UCP3, which are mainly expressed in adipose and smooth muscle, UCP2 is expressed in various tissues, including the kidney, liver, gastrointestinal tract, brain, and skeletal muscle. Although the precise mechanism of UCP2 is unknown, its main function is known to be related to the control of mitochondrial-derived reactive oxygen species.

[0079] According to one embodiment of the present invention, the UCP2 may increase mitochondrial function derived from iPSCs.

[0080] According to one embodiment of the present invention, increasing the mitochondrial function may be regulating an immune cell, and the immune cell may be a cell selected from the group consisting of Th1, Th2, Th17, Treg, plasma B, Breg, Tfh, tissue-resident memory (TRM) T cell, Tc1, Tc2, and Tc17, and regulating the immune cell may be reducing the expression of a cell selected from the group consisting of Th1, Th2, Th17, plasma B, Tfh, tissue-resident memory (TRM) T cell, Tc1, Tc2, and Tc17, and increasing the expression of Treg or Breg.

[0081] According to one embodiment of the present invention, the mitochondria may decrease the expression of IFN-γ or IL-17 and increase the expression of IL-10.

[0082] In addition, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0083] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0084] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0085] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0086] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0087] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0088] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0089] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0090] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0091] According to one embodiment of the present invention, the iPSC-derived mitochondria may increase mitochondrial function within a cell, and the increase in mitochondrial function may be an increase in ATP synthesis or an increase in mitochondrial membrane potential.

[0092] According to one embodiment of the present invention, the mitochondria may increase colon length.

[0093] According to one embodiment of the present invention, the composition may inhibit intestinal tissue damage.

[0094] According to one embodiment of the present invention, the mitochondria may reduce the amount of autoantibodies, and the autoantibodies may be total IgG, IgG2a or IgM.

[0095] According to one embodiment of the present invention, the mitochondria may suppress renal damage, and the suppression of renal damage may be a reduction of a factor selected from the group consisting of a vasculitis index, a glomerulitis index, creatinine in urine, and albumin in urine.

[0096] According to one embodiment of the present invention, the mitochondria may reduce an allo response, and the allo response may be an increase in T cell proliferation.

[0097] According to one embodiment of the present invention, the mitochondria may reduce psoriasis disease activity, and the psoriasis disease activity may be an index selected from the group consisting of skin thickness, scaling, or redness, and the skin thickness may be epidermis thickness.

[0098] According to one embodiment of the present invention, the mitochondria may suppress the expression of a cartilage metabolic factor, and the cartilage metabolic factor may be a factor selected from the group consisting of MMP3, MMP9, MMP13, and MCP1.

[0099] According to one embodiment of the present invention, the autoimmune disease is inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfeiter's syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthropathy, pernicious anemia, scleroderma, graft-versus-host disease, and mitochondrial-related syndrome. It may be selected from a group consisting of:

[0100] According to one embodiment of the present invention, the autoimmune disease may be accompanied by metabolic abnormalities.

[0101] In addition, the present invention provides a food composition for preventing or improving autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0102] The term "improvement" as used herein means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0103] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0104] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0105] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0106] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving autoimmune diseases. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0107] In addition, the present invention provides an immunomodulatory pharmaceutical composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0108] In addition, the present invention provides an immune-modulating food composition comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0109] In addition, the present invention provides a method for treating an autoimmune disease, comprising the step of administering a pharmaceutically effective amount of the above mitochondria to a subject.

[0110] The treatment method of the present invention comprises administering to a subject a therapeutically effective amount of the iPSC-derived UCP2-overexpressing mitochondria. It is preferred that the specific therapeutically effective amount for a specific subject be applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration of each active ingredient should be such that the content of each active ingredient is not excessively high and side effects are not caused. Therefore, it is preferred that the effective amount of a composition suitable for the purpose of the present invention be determined in consideration of the aforementioned matters.

[0111] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0112] The iPSC-derived UCP2-overexpressing mitochondria of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are conceivable, and for example, administration can be by oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0113] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0114] <Example 1> Confirmation of the correlation between UCP2 expression and activity in iPSC-derived mitochondria.

[0115] <1-1> Confirmation of UCP2 expression according to cell type

[0116] Mitochondria derived from induced pluripotent stem cells (iPSCs) of the present invention express uncoupling protein 2 (UCP2), and in order to confirm the correlation between the expression of UCP2 and the activity of mitochondrial transplantation, the expression of UCP2 was confirmed according to cell types. Specifically, mitochondria were isolated from muscle cells (L6) and iPSCs (isolated using Mitochondria Isolation Kit, Thermo, Cat # 89874), and proteins were extracted from each mitochondria, and the expression of UCP2 was confirmed by Western blotting. In addition, to isolate iPSC-derived mitochondria, mitochondria were isolated from the WTC11 cell line, a human-derived iPSC cell line, and 1 × 10 of the WTC11 cell line was isolated. 6 8 to 10 μg of mitochondria were obtained from the cells.

[0117] As a result, as shown in Fig. 1, compared to the muscle cell line L6 cells, the iPSC-derived mitochondria of the present invention showed a 4 to 6-fold increase in the expression of UCP2, confirming that the expression of UCP2 was significantly increased.

[0118] <1-2> Confirmation of the correlation between UCP2 expression and immune-modulatory effects in iPSC-derived mitochondria.

[0119] We confirmed that mitochondria derived from induced pluripotent stem cells (iPSCs) of the present invention overexpress UCP2, and thus examined whether UCP2 expression correlates with the immune-regulatory capacity of iPSC-derived mitochondria. Specifically, iPSCs were treated with UCP2 siRNA, cultured, and mitochondria were collected. As a control, mitochondria isolated from iPSCs treated with control siRNA were used. UCP2 expression in the obtained mitochondria was then analyzed by Western blot.

[0120] To confirm the immunomodulatory effect, 1 × 10 spleen cells obtained from wild-type mice were cultured. 6 The cells were seeded in 24-well plates at a concentration of 0.5 μg / ml anti-CD3 and 100 ng / ml LPS as cell stimulation conditions, and then 5 μg of isolated mitochondria were treated for 24 hours. After the reaction was completed, the cultured spleen cells were analyzed by flow cytometry to confirm the phenotype of the immune cells. The mitochondrial and drug treatment conditions of each group are shown in Table 1.

[0121] [Table 1]

[0122]

[0123] As a result, as shown in Fig. 2, it was confirmed that treatment with UCP2 siRNA significantly reduced the expression of UCP2 in iPSC-derived mitochondria.

[0124] In addition, in iPSC-derived mitochondria, it was confirmed that when UCP2 was reduced, the expression of Th17 cells, which are autoimmune pathogenic immune cells, increased and the amount of IL-17, a cytokine associated with autoimmune diseases, significantly increased (Fig. 3).

[0125] <Example 2> Confirmation of increased intracellular mitochondrial function in iPSC-derived mitochondria

[0126] It was confirmed whether the UCP2-overexpressing mitochondria derived from induced pluripotent stem cells (iPSCs) of the present invention, when transplanted into cells, increased mitochondrial function within the cells. Specifically, after obtaining mouse splenocytes, 1 × 10 were seeded in a 96-well plate coated with anti-CD3 at a concentration of 0.5 μg / ml. 5 The cells were seeded at a cell density of 100 μg and cultured. 0.125 μg of mitochondria isolated from iPSCs (isolated using the Mitochondria Isolation Kit, Thermo, Cat # 89874) were added and cultured for 24 hours (iPSC group). After culture, the intracellular ATP concentration was measured using ATPlite (PerkinElmer, Cat # 6016943). In addition, 2 mM ADP was added to the cells and cultured at 37°C for 45 minutes. ATP synthesis ability was evaluated using an ATP assay kit, and mitochondrial membrane potential was analyzed by flow cytometry using JC-1 staining. An untreated control group was used as a control.

[0127] As a result, as shown in Figure 4, compared to the control group, in the iPSC-treated group, there was no significant difference in intracellular ATP content, but a significant increase in ATP synthesis was confirmed (Figures 4A and 4B). In addition, when mitochondrial membrane potential was confirmed by JC-1 staining, the JC-1 red / green ratio was significantly increased in the iPSC-treated group compared to the control group, confirming an increase in membrane potential (Figure 4C). Therefore, it was confirmed that iPSC-derived mitochondria enhance intracellular mitochondrial function.

[0128] <Example 3> Confirmation of immune cell regulation by iPSC-derived mitochondria

[0129] <3-1> Confirmation of immune cell regulation in mouse spleen cells

[0130] We confirmed whether the UCP2-overexpressing mitochondria derived from induced pluripotent stem cells (iPSCs) of the present invention regulate immune cells. Specifically, after obtaining mouse spleen cells, 1 × 10 were seeded in a 24-well plate coated with anti-CD3 at a concentration of 0.5 μg / ml. 6 The cells were seeded and cultured at a cell concentration of 100 μg. 0.5 μg of iPSC-derived mitochondria, obtained in the same manner as in Example 1, were added and cultured for 72 hours. IFN-γ and IL-17-positive cells were identified by flow cytometry, and FoxP3-positive cells, a marker for Treg, an immune regulatory cell, were also identified. The Th17 / Treg ratio was then analyzed, and the amounts of IFN-γ and IL-17 in the culture supernatant were measured by ELISA.

[0131] As a result, compared to the control group, the number of IFN-γ and IL-17 positive cells was significantly reduced in the iPSC group (Figures 5A and 5B), and the number of FoxP3 positive cells, a marker of Treg, an immune regulatory cell, was significantly increased (Figure 5C). In addition, the ratio of Th17 / Treg was reduced, confirming that the expression of Treg, an immune regulatory cell, was increased (Figure 5D).

[0132] In addition, when the expression of IFN-γ and IL-17 in the culture supernatant was measured by ELISA, it was confirmed that the amount of IFN-γ and IL-17 in the culture supernatant was significantly reduced in the iPSC group compared to the control group (Figures 6A and 6B).

[0133] <3-2> Confirmation of the combined effect of metformin and mitochondria in mouse spleen cells.

[0134] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention, when used in combination with metformin, increased the immune cell regulatory effect. Specifically, cells obtained from mouse spleen were seeded at 1 × 10 in a 24-well plate coated with aCD at a concentration of 0.5 μg / ml. 6 The cells were seeded and cultured at a cell concentration of 10 μg / mL. Afterwards, 0.5 μg of mitochondria isolated from iPSCs cultured with 0.5 mM metformin for 3 days were added, and cultured for 72 hours (Metformin group). After completion of culture, IFN-γ and IL-17-positive cells were analyzed by flow cytometry. A group treated only with iPSC-derived mitochondria served as a control group.

[0135] As a result, compared to the control group, the Metformin group treated with metformin showed a decrease in IFN-γ positive cells (Fig. 7A) and a significant decrease in IL-17 positive cells (Fig. 7B), confirming that the combined use of iPSC-derived mitochondria and metformin enhanced the immune cell regulation effect.

[0136] <Example 4> Confirmation of the pathological regulation of iPSC-derived mitochondria in an animal model of inflammatory bowel disease.

[0137] <4-1> Creation of an animal model of inflammatory bowel disease

[0138] The iPSC-derived mitochondria of the present invention were confirmed to have an effect on improving the symptoms of autoimmune diseases. Specifically, an animal model of inflammatory bowel disease, an autoimmune disease, was created, and 7-week-old male B6 mice were fed 3% dextran sodium sulfate (DDS). The water was replaced with regular drinking water on the 5th day, and 10 μg of iPSC-derived mitochondria were injected intraperitoneally on days 2, 6, and 9 of the experiment (iPSC mito group). Thereafter, the body weights of the mice were measured once daily until the end of the experiment, at which point the mice were humanely sacrificed, and small intestinal tissues were obtained and the intestinal length was measured. The control group consisted of wild-type mice, the Nomal group, and the Vehicle group, which was injected with an equal volume of saline.

[0139] As a result, as shown in Figure 8A, compared to the vehicle group, the group injected with iPSC-derived mitochondria showed a significant recovery in body weight at the end of the experiment. Furthermore, the intestinal length of the mice significantly increased compared to the vehicle group (Figure 8B), confirming an improvement in inflammatory bowel disease.

[0140] <4-2> Confirmation of improvement in intestinal tissue damage

[0141] We examined whether the iPSC-derived mitochondria of the present invention improve intestinal tissue damage in inflammatory bowel disease. Specifically, intestinal tissue was obtained from the mice sacrificed in Example 4-1, and paraffin blocks were prepared. Sections were then obtained using a microtome and stained with hematoxylin & eosin (H&E). After staining, intestinal tissue was examined, and loss of epithelium, crypt damage, depletion of goblet cells, infiltration of inflammatory cells, and histological scores were quantified.

[0142] As a result, compared to the Vehicle group, in the iPSC mito group, it was confirmed that small intestinal tissue damage was improved (Fig. 9A), and loss of epithelium, crypt damage, depletion of goblet cells, infiltration of inflammatory cells, and histological score were each confirmed to be significantly reduced (Fig. 9B), confirming that iPSC-derived UCP2-overexpressing mitochondria improve inflammatory bowel disease.

[0143] <Example 5> Confirmation of immune cell regulation by iPSC-derived mitochondria in an animal model of inflammatory bowel disease.

[0144] We investigated whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate immune cells in inflammatory bowel disease. Specifically, spleen cells were obtained from the mice sacrificed in Example 4, and the obtained spleen cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 hours. Flow cytometry was then used to analyze the activity of Th1 (CD4 + IFN-γ +), Th2 (CD4 + IL-4 +), and Th17 (CD4 + IL-17 +). Confocal analysis was also used to confirm the expression of Th17 (CD4 + IL-17 +) and Treg (CD4 +, CD25 +, and Foxp3 +) cells.

[0145] As a result, as shown in Fig. 10, compared to the Vehicle group, the activity of Th1, Th2, and Th17 cells was significantly reduced in the iPSC mito group, confirming that iPSC-derived mitochondria regulate the expression of autoimmune cells.

[0146] In addition, as shown in Fig. 11, confocal analysis was performed on spleen sections at the end of the experiment, and compared to the Vehicle group, the expression of Th17 cells was decreased in the iPSC mito group, and the expression of Treg was significantly increased. This confirmed that iPSC-derived mitochondria regulate the expression of autoimmune cells, and confirmed that iPSC-derived UCP2-overexpressing mitochondria improves inflammatory bowel disease.

[0147] <Example 6> Confirmation of immune cell regulation by iPSC-derived mitochondria

[0148] <6-1> Confirmation of Th17 cell suppression effect

[0149] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention suppressed and regulated the expression of Th17 cells, which are pathogenic cells related to autoimmune diseases, in human peripheral blood mononuclear cells (PBMCs). Specifically, human PBMCs were stimulated with 10 ng / mL of LPS and plated in a 48-well plate coated with 2 μg / mL of aCD, and 5 × 10 5 The cells were seeded and cultured at a concentration of 100 μg. After culturing under stimulation conditions for 24 hours, 0.5, 1.5, and 2.5 μg of mitochondria isolated after MTDR staining were added to the iPSC cells for mitochondrial staining, and the cells were cultured for 72 hours. The expression of Th17 cells was then confirmed by flow cytometry. In addition, the expression of MTDR-negative Th17 cells and positive Th17 cells was confirmed by flow cytometry, and the amount of IL-17 in the culture supernatant was measured using ELISA. The untreated control group (Nil) and the negative control group, Vehicle, were used as controls.

[0150] As a result, as shown in Fig. 12, compared to the Nil group, the expression of Th17 was significantly increased in the Vehicle group, but in the group treated with the iPSC-derived mitochondria of the present invention, it was confirmed that the expression of Th17 cells decreased in a concentration-dependent manner.

[0151] In addition, there was no significant difference in the effect of suppressing Th17 expression under conditions where intracellular movement of iPSC-derived mitochondria did not occur, which is an MTDR-negative condition. However, under conditions where intracellular movement of iPSC-derived mitochondria was confirmed, which is an MTDR-positive condition, it was confirmed that iPSC-derived mitochondria suppressed Th17 expression (Fig. 13).

[0152] In addition, as a result of confirming the amount of IL-17 secreted by Th17 cells in the culture supernatant, compared to the Nil group, the amount of IL-17 in the culture supernatant was significantly increased in the Vehicel group, indicating good stimulation, and in the group treated with iPSC-derived mitochondria under this condition, the increased amount of IL-17 was confirmed to be significantly reduced (Fig. 14).

[0153] <6-2> Confirmation of the effect of increasing Treg cells

[0154] It was confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate the expression of Treg cells, which are immune regulatory cells in autoimmune diseases. In the same manner as in Example 6-1, human peripheral blood mononuclear cells (PBMCs) were stimulated with 0.5 μg / ml of anti-CD3 and 10 ng / ml of LPS. After 24 hours, 0.5, 1.5, and 2.5 μg of mitochondria isolated after MTDR staining for mitochondrial staining were added to iPSC cells and cultured for 72 hours. Thereafter, the expression of Treg cells was confirmed by flow cytometry, and the expression of Treg according to positive Treg conditions was confirmed by flow cytometry. The amount of IL-10 in the culture supernatant was measured using ELISA. The untreated control group (Nil) and the negative control group, Vehicle, were used as controls.

[0155] In the MTDR-negative condition, where intracellular movement of iPSC-derived mitochondria did not occur, no increase in Tregs was observed. However, in the MTDR-positive condition, where intracellular movement of iPSC-derived mitochondria was confirmed, it was confirmed that iPSC-derived mitochondria increased Tregs. As a result, as shown in Figure 15, compared to the Nil group, the Vehicle group showed increased Tregs expression, confirming that stimulation was successful. In the MTDR-negative condition, where intracellular movement of iPSC-derived mitochondria did not occur, no increase in Tregs was observed. However, in the MTDR-positive condition, where intracellular movement of iPSC-derived mitochondria was confirmed, it was confirmed that iPSC-derived mitochondria increased Tregs (Figure 16).

[0156] In addition, when the amount of IL-10 secreted by Treg cells in the culture supernatant was confirmed, it was confirmed that the amount of IL-10 significantly increased in the group treated with iPSC-derived mitochondria compared to the Vehicel group (Fig. 17).

[0157] <Example 7> Confirmation of the effect of iPSC-derived mitochondria on improving lupus associated with metabolic abnormalities.

[0158] <7-1> Confirmation of the B cell regulatory effect of iPSC-derived mitochondria

[0159] We investigated whether iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate B cells and autoantibodies, which are immune cells associated with lupus disease. Specifically, wild-type BALB / c mice were humanely sacrificed, and spleen tissues were obtained. Splenocytes were isolated from the spleen tissues and treated with R848 (SML0196) at a concentration of 100 ng / ml, a lupus-inducing condition. Subsequently, under B cell activation conditions, anti-IgM was treated at a concentration of 10 μg / ml and soluble CD40 ligand at a concentration of 1 μg / ml, and stimulation was performed at 37°C for 4 days. iPSC-derived mitochondria were administered together with lupus-inducing and B cell activation conditions at a concentration of 1 or 10 μg / ml. After the reaction was completed, each cell and culture medium were obtained, and the expression of IgM plasma B cells and IgG2a plasma B cells, which are lupus-associated immune cells, and Breg, which are immune regulatory cells, was analyzed by flow cytometry. In addition, the amounts of total IgG, IgG2a, and IgM, which are lupus-associated autoantibodies, in the culture medium were measured by ELISA.

[0160] In addition, to further confirm the B cell regulatory effect of iPSC-derived UCP2-overexpressing mitochondria, A20 cell line, a mouse B lymphocyte cell line, was obtained from ATCC cell line, and each cell was seeded at 1 × 10 in a 24-well plate. 6 The cells were seeded at a cell concentration of 100 μg / ml and stimulated for 4 days with iPSC-derived mitochondria treated under lupus-specific environmental conditions and B cell stimulation conditions under the same conditions as above. The cells were then collected, and the expression of IgG2a and Breg cells was analyzed by flow cytometry.

[0161] As a result, when iPSC-derived mitochondria were treated in splenocytes derived from wild-type mice, the expression of IgM plasma B cells and IgG2a plasma B cells, which are lupus-associated B cells, was significantly reduced under lupus-associated environmental conditions, and the expression of Breg cells was significantly increased (Fig. 18A). In addition, it was confirmed that the amount of lupus-associated autoantibodies in the culture medium was significantly reduced by treatment with iPSC-derived mitochondria (Fig. 18B).

[0162] In addition, when iPSC-derived mitochondria were treated in the A20 cell line, a mouse B lymphocyte cell line, under lupus environmental conditions and B cell activation conditions, the expression of IgG2a cells, which are lupus-causing B cells, was significantly reduced (Fig. 19), and Breg, which are immune regulatory cells, was confirmed to increase in a concentration-dependent manner (Fig. 20), confirming that the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate lupus-related cells.

[0163] <7-2> Confirmation of improvement in lupus nephritis with iPSC-derived mitochondria

[0164] To determine whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve refractory lupus disease accompanied by metabolic abnormalities, lupus nephritis activity was assessed. Specifically, 8-week-old Balb / c mice were treated with 10 μl of R848 solution prepared in acetone at a concentration of 5 mg / ml on the back of the right ear three times a week for 6 weeks. In addition, high-fat diet (HFD) was administered to the mice to induce metabolic abnormalities (R848+HFD group). 10 μg of iPSC-derived mitochondria were intravenously administered to the mice once a week (R848+HFD+iPSC mito group). Wild-type mice (WT group) and typical lupus mice (R848 group) served as controls. Afterwards, at the end of the experiment, the mice were humanely sacrificed, and the kidney tissues were obtained from the mice, sectioned, and the obtained sections were stained with H&E to quantify the disease activity of lupus nephritis as the vasculitis score and the glomerulonephritis score (GN score). The basement membrane structure of the kidney tissues was confirmed using an electron microscope. In addition, urine was obtained from the mice at the 1st and 5th weeks of the experiment, and the amounts of creatinine and albumin in the urine were measured and quantified using the Mouse Creatinine Parameter Assay kit (KGE005) and Goat anti-Mouse Albumin antibody Affinity Purified (A90-134A).

[0165] As a result, as shown in Figure 21A, compared to the WT group, the vasculitis index and glomerulitis index increased in the R848 group, but the vasculitis index and glomerulitis index increased significantly in the R848+HFD group compared to the R848 group, confirming that the concomitant metabolic abnormality significantly increases lupus disease activity. However, when iPSC-derived mitochondria were administered to the R848+HFD group, it was confirmed that nephritis was histologically reduced, and the abnormal structure of the basement membrane was restored (Figure 21B).

[0166] In addition, there was no significant difference in the amount of creatinine and albumin in urine samples between the R848+HFD group and the R848+HFD+iPSC mito group in week 1 of the experiment, but in week 5 of the experiment, the amount of creatinine and albumin in urine increased in the R848 group, but in the group administered iPSC-derived mitochondria, the increased amount of creatinine and albumin in urine was significantly reduced (Figure 21C), confirming that renal damage was suppressed.

[0167] <7-3> Confirmation of immune cell regulation by iPSC-derived mitochondria

[0168] We investigated whether iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate immune cells in lupus erythematosus (LU) with refractory metabolic disorders. Specifically, spleen tissue was obtained from the mice sacrificed in Example 7-2, and the expression of lupus-associated B cells and lupus-associated T cells was analyzed by flow cytometry.

[0169] As a result, as shown in Figure 22, compared to the R484+HFD group, the group administered iPSC-derived mitochondria showed a significant decrease in the expression of Plasma B cells and IgG2a Plasma B cells, which are immune cells that cause lupus. In addition, a tendency for the expression of IL-10 Plasma B cells, which are immune regulatory cells, to increase was confirmed.

[0170] In addition, as a result of confirming the T cell regulatory effect, compared to the R484+HFD group, in the group administered iPSC-derived mitochondria, the expression of Tfh (IL-21+CD4+ T cells), Th1 (IFNg+CD4+ T cells), and Th17 (IL-17+CD4+ T cells), which are lupus pathogenic immune cells, was confirmed to be significantly reduced (Fig. 23), confirming that iPSC-derived mitochondria can regulate the activity of immune cells.

[0171] <7-4> Confirmation of the spontaneous lupus improvement effect of iPSC-derived mitochondria

[0172] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate immune cells in SKG mice, which are spontaneously induced with autoimmune diseases. Specifically, SKG mice, which are spontaneously induced with autoimmune diseases, were humanely sacrificed at the age of 15 weeks, and spleen cells were isolated from spleen tissue. Thereafter, lupus-specific environmental conditions and B cell activation conditions were treated in the same manner as in Example 7-1, and iPSC-derived mitochondria were simultaneously treated at a concentration of 2 or 10 μg / ml, and cultured at 37°C for 4 days. After completion of culture, cells were harvested, and the expression of Plasma B cells, which are lupus-pathogenic immune cells, and Breg, which are immune regulatory cells, was analyzed by flow cytometry.

[0173] In addition, in order to confirm the cell regulatory effect according to metabolic abnormality, serum obtained from SKG mice fed HFD for 6 weeks was treated together with the lupus environmental conditions and B cell activation conditions, and after the culture was completed, cells were separated under the same conditions as above, and the expression of Plasma B cells and Breg was analyzed by flow cytometry.

[0174] As a result, as shown in Figure 24, under lupus-stimulating conditions, the expression of Plasma B cells, which are lupus-pathogenic cells, was significantly increased in SKG mouse-derived spleen cells. However, when iPSC-derived mitochondria were treated, the increased Plasma B cells were significantly reduced. In addition, when iPSC-derived mitochondria were treated under lupus-stimulating conditions, the expression of Breg was confirmed to increase in a concentration-dependent manner.

[0175] Furthermore, when the serum of SKG mice fed a HFD and under lupus-stimulating conditions was treated, the expression of Plasma B cells, which are lupus-pathogenic cells, in the spleen cells of SKG mice significantly increased. However, when iPSC-derived mitochondria were treated, the increased Plasma B cells were significantly reduced. Furthermore, when iPSC-derived mitochondria were treated under lupus-stimulating conditions, the expression of Breg was significantly increased (Fig. 25), confirming that iPSC-derived UCP2-overexpressing mitochondria improve lupus.

[0176] <Example 8> Confirmation of the effect of iPSC-derived mitochondria on improving transplant rejection disease.

[0177] <8-1> Confirmation of the homologous response regulation effect of iPSC-derived mitochondria

[0178] In order to confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve graft-versus-host disease (GVHD), a transplant rejection disease, it was confirmed whether they regulate cell proliferation in allogeneic response. Specifically, 2 × 10 mitochondria were seeded per well in a 96-well plate. 5 2 × 10 spleen cells were cultured with CD4+ T cells from normal recipients (Bclb / c, responder) and T cells from irradiated recipients (allogeneic) or donors (C57BL / 6, stimulator, allogeneic). 5The cells were co-cultured with dogs. During co-culture, iPSC-derived mitochondria were administered to the allogeneic model at a concentration of 0.5 μl / ml. The allogeneic Nil group and the allogeneic transplantation group (Allo-response) were used as controls. Afterwards, T cell proliferation response (allogeneic response) was confirmed in the cultured cells.

[0179] As a result, as shown in Figure 26, in the homologous reaction group, T cell proliferation was significantly increased compared to the Nil group, but when iPSC-derived mitochondria were treated, it was confirmed that the increased T cell proliferation was significantly reduced.

[0180] <8-2> Confirmation of increased IFN-r and IL-10 in iPSC-derived mitochondria

[0181] It was confirmed that the iPSC-derived UCP2-overexpressing mitochondria of the present invention specifically increase IFN-r and IL-10, which are immune regulatory factors in graft-versus-host disease (GVHD), a transplant rejection disease. Specifically, in the cell culture solution cultured in Example 8-1, the amounts of IFN-r and IL-10, and IL-17, an autoimmune disease interleukin, were confirmed by ELISA.

[0182] As a result, as shown in Figure 27, compared to the Allo response group, it was confirmed that the immune regulatory factors IFN-r and IL-10 were specifically increased in the group treated with iPSC-derived mitochondria.

[0183] <8-3> Confirmation of improvement in GVHD with iPSC-derived mitochondria

[0184] It was confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention reduce the disease activity of graft-versus-host disease (GVHD), a transplant rejection disease. Specifically, to create a GVHD model, recipient mice Balb / c (H-2k / d) were subjected to 690 cGy of total body irradiation (TBI), and hematopoietic stem cells and spleen cells were isolated from the femur and tibia of donor mice C57BL / 6 (H-2k / b), and hematopoietic stem cells (BM, bone marrow) were 5 × 10 6 and 5 × 10 splenocytes 6 were transplanted into Balb / c (H-2k / d) mice. After the occurrence of aGVHD, iPSC-derived mitochondria were intravenously injected once a week at a concentration of 10 μg / ml (iPSC mito group). The disease activity and body weight of each group were then measured once a week. As controls, wild-type mice (Syngenic group) and GVHD-induced vehicle group were used, and the same amount of solvent was administered only to the vehicle group.

[0185] As a result, as shown in Figure 28A, compared to the Syngenic group, the disease activity in the Vehicle group continued to increase until the end of the experiment, but the increased disease activity in the iPSC mito group significantly decreased. In addition, no weight loss was observed in the Syngenic group until the end of the experiment, whereas the Vehicle group showed a tendency for weight loss to decrease, and the iPSC mito group showed a tendency for weight loss to recover (Figure 28B).

[0186] <8-4> Confirmation of GVHD immune cell regulation by iPSC-derived mitochondria

[0187] We investigated whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate immune cells in graft-versus-host disease (GVHD), a transplant rejection disease. Specifically, the mice of Example 8-3 were humanely sacrificed at the end of the experiment, and cells were isolated from blood and spleen tissue. Thereafter, the expression of Th17 cells, which are autoimmune pathogenic cells, and CD8 TRM+IL-17+ cells, which are tissue-resident memory (TRM) cells, in the blood and CD8 TRM+IL-17+ cells in the spleen were analyzed by flow cytometry.

[0188] As a result, as shown in Figure 29, compared to the Syngenic group, the Vehicle group showed a significant increase in Th17 cells and CD8 TRM+IL-17+ cells in serum and spleen, but the group administered iPSC-derived mitochondria showed a significant decrease in the increased Th17 cells and CD8 TRM+IL-17+ cells, confirming that iPSC-derived UCP2-overexpressing mitochondria improves GVHD.

[0189] <Example 9> Confirmation of the psoriasis improvement effect of iPSC-derived mitochondria.

[0190] <9-1> Confirmation of the IL-17 expression and immune cell regulation effect of iPSC-derived mitochondria

[0191] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve psoriasis, a skin immune disease, we examined whether they regulate a subtype of immune cells expressing IL-17. Specifically, BALB / C mice were humanely sacrificed, spleen tissue was obtained, and single cells were isolated. The cells were then seeded at 1 × 10 in a 24-well plate. 6The cells were seeded at a cell concentration of 100 μg / ml, and the expression of inflammatory T cells expressing IL-17 was stimulated with 2 μg / ml of CD3 and 100 ng / ml of LPS. Simultaneously with stimulation, 0.4, 2, or 10 μg of iPSC-derived mitochondria were treated. Then, inflammatory T cells, Th17 and Tc17 cells, and tissue-resident memory (TRM) inflammatory T cells were analyzed by flow cytometry. As controls, the Nil group, an untreated control group, and the negative (-) control group, which stimulated the expression of inflammatory T cells, were used.

[0192] As a result, as shown in Figure 30, compared to the Nil group, the negative control group showed a significant increase in inflammatory T cells, Th17 and Tc17 cells, but when iPSC-derived mitochondria were treated, the increased Th17 cells were significantly reduced, and it was confirmed that Tc17 cells decreased in a concentration-dependent manner with iPSC-derived mitochondria treatment.

[0193] In addition, compared to the Nil group, in the negative control group, CD4 positive, CD8 positive, CD4 positive IL-17 positive, and CD8 positive IL-17 positive TRM subtypes among TRMs were significantly increased, but when iPSC-derived mitochondria were treated, it was confirmed that the increased CD4 positive, CD8 positive, CD4 positive IL-17 positive, and CD8 positive IL-17 positive TRM subtypes were significantly decreased (Fig. 31).

[0194] <9-2> Confirmation of the effect of iPSC-derived mitochondria on improving psoriasis pathological activity

[0195] We investigated whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention had a psoriasis-improving effect in a psoriasis animal model. Specifically, SKG mice were depilated using a depilatory cream on the back and buttocks, and 1 day later, 62.5 mg of 5% imiquimod (IMQ) cream was topically applied once daily to the back skin to induce acute psoriasis. Three days after psoriasis induction, 10 μg of iPSC-derived mitochondria were injected intraperitoneally, and the mice were humanely sacrificed on day 7 (Fig. 32A). After psoriasis induction, clinical indices such as back skin thickness, scaling, and erythema were measured daily and indexed.

[0196] As a result, as shown in Figure 32B, compared to mice in which psoriasis was induced (SKG+IMQ), the mitochondria-injected group (iPSC mito) showed a reduction in skin damage due to psoriasis induction. In addition, significant reductions in skin thickness, scaling, and redness indices were confirmed (Figure 32C).

[0197] <9-3> Confirmation of the regulatory effect of iPSC-derived mitochondria on psoriasis-related immune cells.

[0198] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate subtypes of inflammatory immune cells and tissue-resident memory cells in a psoriasis animal model. Specifically, mice from each group of Example 9-2 were humanely sacrificed at the end of the experiment, and spleen tissues were obtained. Thereafter, single cells were isolated from the spleen, and the isolated cells were seeded at 1 × 10 in a 24-well plate. 6The cells were seeded at a cell concentration of 100 μg / ml and stimulated with PMA-lonomycin for 2 hours to induce T cell activation and cytokine secretion. Afterwards, Golgi apparatus stop was performed for 2 hours to block T cell activation and cytokine secretion. The cells were then harvested and the expression of Th1, Th2, and Th17 cells, which are CD4 lineage among inflammatory immune cells, and Tc1, Tc2, and Tc 17 cells, which are CD8 lineage, as well as Treg cells, which are immunoregulatory cells, were analyzed by flow cytometry. TRM of CD4 and CD8 lineage among TRM T cells were analyzed.

[0199] As a result, as shown in Figure 33A, compared to mice induced with psoriasis (SKG+IMQ), the mitochondria-injected group (iPSC mito) showed a significant decrease in CD4-type Th1 and Th17 cell subtypes, while Th2 showed a decreasing trend. In addition, Tc1, Tc2, and Tc17 of the CD8-type were significantly decreased (Figure 33B), and a significant increase in Treg expression was confirmed (Figure 33C).

[0200] In addition, as a result of analyzing the subtypes of TRM, it was confirmed that the expression of CD4 TRM1 and CD4 TRM17 of the CD4 lineage was significantly reduced in the group injected with mitochondria (iPSC mito) compared to mice induced with psoriasis (SKG+IMQ), and that CD8 TRM1 and CD8 TRM17 were also significantly reduced in the CD8 lineage (Fig. 34).

[0201] <9-4> Confirmation of the histological protective effect of iPSC-derived mitochondria

[0202] We investigated whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention protect against psoriasis-induced skin tissue changes. Specifically, skin tissue was obtained from each group of mice sacrificed in Example 9-3 and sectioned. The tissue was then stained with hematoxylin & eosin (H&E), and epidermal thickness was quantified.

[0203] As a result, as shown in Figure 35, compared to mice in which psoriasis was induced (SKG+IMQ), the thickness of the epidermal layer was confirmed to be reduced in the group injected with mitochondria (iPSC mito), confirming that iPSC-derived UCP2-overexpressing mitochondria improves psoriasis.

[0204] <Example 10> Confirmation of the anticancer effect of iPSC-derived mitochondria.

[0205] <10-1> Confirmation of the regulation of gastric cancer patient immune cells by iPSC-derived mitochondria

[0206] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention have an anticancer effect on solid cancer. Specifically, 1 × 10 PBMCs derived from gastric cancer patients were injected 6 The cells were seeded in 6-well plates at a cell concentration of 10 μg and cultured with 0.1, 0.3, or 0.5 μg of iPSC-derived mitochondria for 72 hours, and T cell subtypes among immune cells were analyzed by flow cytometry. The Vegicle group, which is a negative (-) control group and an untreated control group, was used as a control.

[0207] As a result, as shown in Fig. 36, compared to the negative control group, the expression of immune regulatory cells, CD4 positive IFN-r positive Th1 cells, CD8 positive IFN-r positive Tc1 cells, and CD8 positive GZMB positive cells, increased with the treatment of iPSC-derived mitochondria, and the expression of CD8 positive IL-17 positive Tc17 cells, CD4 positive CTLA-4 positive cells, and CD8 positive CTLA-4 positive cells was confirmed to be significantly decreased.

[0208] Additionally, as shown in Figure 37, it was confirmed that the expression of CD8-positive IFN-r-positive TRM was significantly increased by treatment of mitochondria compared to the control group.

[0209] <10-2> Confirmation of the anticancer effect of iPSC-derived mitochondria on gastric cancer

[0210] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention exhibit anticancer effects against solid tumors, the tumor size suppression effect in a gastric cancer transplantation animal model was confirmed. Specifically, 1 × 10 PBMCs from gastric cancer patients were injected into 6-week-old male NSG mice, which are immunodeficient mice. 6 was injected intraperitoneally at a cell concentration of 5 × 10. After one week, the gastric cancer cell line AGS cell line was injected at a concentration of 5 × 10 6 The mice were injected subcutaneously at a cell concentration of 10 μg. From the 4th week of the experiment, 10 μg of iPSC-derived mitochondria were injected intraperitoneally once a week, and the mice were humanely sacrificed at the 7th week of the experiment. The specific experimental schedule is shown in Figure 38. The control group was the HC group injected with PBMCs derived from healthy adults, the group injected with PBMCs derived from gastric cancer patients and AGS cell lines (GC Veh), and the AGS group injected with only the AGS cell line. Tumor tissues were obtained from the sacrificed mice, tumor volumes were measured, and spleen cells were isolated and analyzed for immune cell subtypes.

[0211] As a result, as shown in Figure 39, in the GC Veh group, the tumor volume continuously increased, reflecting the immune status of the gastric cancer patient, but in the group injected with iPSC-derived mitochondria, the tumor volume was confirmed to have significantly decreased, and it was confirmed that the tumor volume at the end of the experiment was also significantly decreased (Figure 40).

[0212] In addition, when the immune cell subtypes were identified in the spleen cells isolated from the mice of each group, it was confirmed that, compared to the GC Veh group, the group injected with iPSC-derived mitochondria had an increase in total CD8 positive cells, a significant increase in Treg expression, and a marked decrease in Th17 expression (Fig. 41).

[0213] <10-3> Confirmation of the anticancer effect of iPSC-derived mitochondria against liver cancer

[0214] In order to confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention exhibit an anticancer effect against solid tumors, the tumor proliferation inhibitory effect in a liver cancer animal model was confirmed. Specifically, an animal model was prepared in the same manner as in Example 9-2, and the HepG2 cell line, a hepatocellular carcinoma cell line, was transplanted instead of the AGS cell line as the cancer cell line to be transplanted. The specific experimental process is shown in Figure 42. The volume of the transplanted tumor was confirmed until the end of the experiment, and as a control group, PBMC and the HepG2 cell line of a liver cancer patient were treated, and a vehicle group that was administered an equal amount of solvent was used.

[0215] As a result, as shown in Figure 43, compared to the vehicle group, the group treated with iPSC-derived mitochondria showed a continuous decrease in tumor volume, and it was confirmed that the tumor volume was significantly reduced until the end of the experiment.

[0216] <Example 11> Confirmation of the osteoarthritis improvement effect of iPSC-derived mitochondria.

[0217] <11-1> Confirmation of the effect of iPSC-derived mitochondria on regulating cartilage metabolism factors

[0218] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve osteoarthritis, the purified quality of the mitochondria and the transplantation efficiency of chondrocytes were analyzed, and the effect of regulating cartilage metabolic factors was confirmed. Specifically, after isolating iPSC-derived mitochondria, the protein expression of tubulin, a cytosolic factor, and COX4, a mitochondrial factor, was analyzed by Western blotting to measure the quality of the mitochondria. Thereafter, mitochondria isolated from chondrocytes were stained with MTDR, and the intrachondrocyte delivery efficiency according to the amount of mitochondria (0.5 or 1 μg) was analyzed by flow cytometry. The Nil group, an untreated control group, and the Vehicle group, which was treated with the same amount of solvent, were used as controls.

[0219] Afterwards, chondrocytes obtained from mice were stimulated with IL-1β, treated with 1 ng of iPSC-derived mitochondria, and co-cultured. As controls, the Nil group, which was an untreated control group, and the Vehicle group, which was treated with the same amount of solvent instead of mitochondria, were used. After 1 day of culture, cells were harvested, and the expression of cartilage metabolic factors MMP3, MMP9, and MMP13 was analyzed by quantitative PCR (q-PCR), and the expression of MCP1, a cartilage metabolic factor in the culture medium, was analyzed by ELISA.

[0220] Additionally, cells were harvested after 2 days of culture, and the protein expression and phosphorylation of RIP3 and MLKL, which are inflammatory cell death factors, were analyzed by Western blot.

[0221]

[0222] As a result of checking the quality of mitochondria, as shown in Figure 44, iPSC-derived mitochondria were well isolated, and protein expression of tubulin and COX4 was confirmed, and chondrocytes 1 × 10 5We confirmed that 60% of iPSC-derived mitochondria were transferred when 1 μg of iPSCs were transferred per cell.

[0223] As a result, as shown in Figure 45, compared to the Nil group, the expression of cartilage metabolic factors MMP3, MMP9, and MMP13 significantly increased in the Vehicle group, but the increased expression of cartilage metabolic factors significantly decreased in the group treated with iPSC-derived mitochondria. In addition, it was confirmed that the expression of MCP1, a cartilage metabolic factor in the culture medium, also significantly decreased.

[0224] In addition, when protein expression and phosphorylation of inflammatory cell death factors were confirmed on the second day of culture, the expression of RIP3, an inflammatory cell death factor, was significantly increased in the Vehicle group compared to the Nil group, and the expression and phosphorylation of MLKL were also increased. However, when iPSC-derived mitochondria were treated, both the increased expression of RIP3 and the expression and phosphorylation of MLKL were significantly decreased (Figure 46).

[0225] <11-2> Confirmation of improvement in osteoarthritis with iPSC-derived mitochondria

[0226] To determine whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve osteoarthritis, the improving effect was confirmed in a MIA-induced osteoarthritis animal model. Specifically, 6-week-old male Wistar rats were maintained at a temperature of 21-22°C with a light-dark cycle of 12-hour intervals and supplied with sterilized water and food. To induce osteoarthritis, monosodium iodoacetate (MIA, Sigma, St. Louis, MO) was administered to the right knee of the rats at a dose of 3 mg / 50 μl. MIA was dissolved in saline and administered. The rats were then divided into two groups: the MIA group, which received no drug but a drug vehicle, and the mIPSC group, which received intra-articular administration of the iPSC-derived mitochondria of the present invention. The specific drug administration and experimental schedule are shown in Figure 47A. After that, pain due to osteoarthritis was measured using a Dynamic plantat aesthesiometer (Ugo Basile, Comerio, Italy). The pain measurement method of the machine was as follows: a mesh plate was placed on the measuring machine, and the rat was placed in an acrylic animal fixation frame on top of the mesh plate. Then, the right paw injected with the drug was pricked with the measuring machine. After the prick, the time (s, seconds) taken by the machine to automatically remove the paw and the weight (g) required to remove the paw were measured, and the time and weight at that time were confirmed. Using this, the pain assessment (Paw Withdrawal Latency) was used to measure the degree of pain.

[0227] As a result, as shown in Figure 47B, compared to the MIA group, the group administered iPSC-derived mitochondria showed a decrease in pain and an increase in the pain assessment (Paw Withdrawal Latency) index.

[0228] <11-3> Confirmation of the cartilage protective effect of iPSC-derived mitochondria

[0229] To determine whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention improve osteoarthritis, cartilage tissue damage was assessed. Specifically, the animal models from each group of Example 11-2 were humanely sacrificed at the end of the experiment, and cartilage tissue was obtained and sectioned. The sectioned cartilage tissue was then stained with Safranin O to determine the Mankin score for cartilage tissue damage, and immunohistochemical staining confirmed the expression of MMP3 in cartilage.

[0230] As a result, as shown in Figure 48, compared to the MIA group, the Mankin index was significantly reduced in the group administered iPSC-derived mitochondria, and it was confirmed that the expression of MMP13 in cartilage tissue was significantly reduced.

[0231] <11-4> Confirmation of immune cell regulation in osteoarthritis by iPSC-derived mitochondria

[0232] We confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present invention regulate immune cells in osteoarthritis. Specifically, spleen tissue was obtained from the mice sacrificed in Example 11-4, and T cell subtypes were analyzed using flow cytometry in cells isolated from the spleen tissue.

[0233] As a result, as shown in Figure 49, in the MIA group, the expression of Th1, Th2, and Th17 cells, which are autoimmune pathogenic immune cells, was significantly increased, but in the group administered with iPSC-derived mitochondria, the increased expression of Th1, Th2, and Th17 was significantly decreased, and it was confirmed that the expression of Treg, which is an immune regulatory cell, was significantly increased.

[0234] Therefore, it was confirmed that the iPSC-derived mitochondria of the present invention overexpress UCP2, and that the expression of UCP2 is related to the immune-modulatory effect. In addition, it was confirmed that the administration of iPSC-derived mitochondria enhances mitochondrial function, increasing ATP synthesis and mitochondrial membrane potential. In addition, it was confirmed that it regulates the expression of immune cells related to autoimmune diseases, and when used in combination with metformin, the synergistic effect of immune cell regulation was confirmed. In addition, it was confirmed that it reduces the pathological activity of autoimmune diseases and suppresses the expression of autoimmune-related cells. In addition, it was confirmed that when iPSC-derived mitochondria are treated with peripheral blood mononuclear cells, the expression of Th17 is suppressed and the expression of Treg is increased, and it was confirmed that it improves immune regulation and the pathological activity in various immune diseases.

Claims

1. Mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs).

2. In paragraph 1, The above UCP2 increases the function of iPSC-derived mitochondria.

3. In paragraph 2, The above mitochondrial function increases, which regulates immune cells, mitochondria.

4. In paragraph 3, The above immune cells are selected from the group consisting of Th1, Th2, Th17, Treg, plasma B, Breg, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2 and Tc17, mitochondria.

5. In paragraph 3, Regulating the above immune cells is by reducing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma B, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2 and Tc17, mitochondria.

6. In paragraph 3, Mitochondria, which regulate the above immune cells, increase the expression of Treg or Breg.

7. In paragraph 1, The above mitochondria are mitochondria that reduce the expression of IFN-γ or IL-17.

8. In paragraph 1, The above mitochondria are mitochondria that increase the expression of IL-10.

9. A pharmaceutical composition for preventing or treating an autoimmune disease, comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

10. In paragraph 9, The above mitochondria are a composition that increases mitochondrial function within a cell.

11. In paragraph 10, A composition wherein the increase in mitochondrial function is an increase in ATP synthesis or an increase in mitochondrial membrane potential.

12. In paragraph 9, The above mitochondria are a composition that regulates immune cells.

13. In paragraph 12, A composition wherein the above immune cells are cells selected from the group consisting of Th1, Th2, Th17, Treg, plasma B, Breg, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2, and Tc17.

14. In paragraph 12, A composition that regulates the above immune cells by reducing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma B, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2 and Tc17.

15. In paragraph 12, A composition that regulates the above immune cells by increasing the expression of Treg or Breg.

16. In paragraph 9, A composition wherein the above mitochondria increase colon length.

17. In paragraph 9, The composition above is a composition that inhibits damage to intestinal tissue.

18. In paragraph 9, A composition wherein the above mitochondria reduce the amount of autoantibodies.

19. In paragraph 18, A composition wherein the autoantibody is total IgG, IgG2a or IgM.

20. In paragraph 9, A composition wherein the above mitochondria inhibit kidney damage.

21. In paragraph 20, A composition that suppresses the above renal damage by reducing a factor selected from the group consisting of vasculitis index, glomerulitis index, creatinine in urine and albumin in urine.

22. In paragraph 9, The above mitochondria are a composition that reduces allo-response.

23. In paragraph 22, The composition wherein the above homologous reaction is an increase in T cell proliferation.

24. In paragraph 9, A composition wherein the above mitochondria reduce psoriasis disease activity.

25. In paragraph 24, A composition wherein the above psoriasis disease activity is an index selected from the group consisting of skin thickness, scaling, or redness.

26. In paragraph 25, A composition wherein the above skin thickness is the epidermis thickness.

27. In paragraph 9, A composition wherein the above mitochondria inhibit the expression of cartilage metabolic factors.

28. In paragraph 27, A composition wherein the cartilage metabolic factor is a factor selected from the group consisting of MMP3, MMP9, MMP13, and MCP1.

29. In paragraph 9, The above autoimmune disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfeiter's syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthropathy, pernicious anemia, scleroderma, graft-versus-host disease, cancer, and mitochondrial-related syndromes. It is a composition.

30. In paragraph 29, A composition wherein the above autoimmune disease is accompanied by metabolic abnormalities.

31. A food composition for preventing or improving autoimmune diseases, comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

32. A pharmaceutical composition for immunomodulation comprising mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

33. In paragraph 32, The above mitochondria are a composition that regulates immune cells.

34. In paragraph 32, A composition wherein the above immune cells are cells selected from the group consisting of Th1, Th2, Th17, Treg, plasma B, Breg, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2, and Tc17.

35. In paragraph 32, A composition that regulates the above immune cells by reducing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma B, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2 and Tc17.

36. In paragraph 32, A composition that regulates the above immune cells by increasing the expression of Treg or Breg.

37. A food composition for immune regulation containing mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

38. A method for treating an autoimmune disease, comprising the step of administering to a subject a pharmaceutically effective amount of the mitochondria of paragraph 1.

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