Composition for preventing or treating Duchenne muscular dystrophy comprising miR-499-5p activator

Abstract

The present invention relates to a composition for preventing or treating Duchenne muscular dystrophy (DMD), which contains a miR-499-5p activator. The miR-499-5p activator promotes the expression of miR-499-5p by expressing MMP-1 in myotube cells, and restores fibrous muscle by down-regulating the activation of TGF-β receptors and phosphorylation of smad2 / 3, and therefore can be used for the prevention or treatment of Duchenne muscular dystrophy (DMD).

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2021-0186584, filed on December 23, 2021, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing or treating Duchenne muscular dystrophy, comprising an miR-499-5p activator. [Background technology]

[0003] Duchenne muscular dystrophy (DMD) is an X-linked dystrophic disorder characterized by a progressive loss of muscle mass and function. It occurs in approximately 1 in 3,500 males and is caused by a mutation in the dystrophin gene. It is characterized by progressive weakening of skeletal and cardiac muscles, and usually leads to early death around age 20. Currently, there is no effective treatment for Duchenne muscular dystrophy (DMD), only treatments that alleviate the progression of the disease. Various therapeutic approaches for Duchenne muscular dystrophy (DMD) have been attempted in preclinical and clinical studies, but no treatments have been reported to date that have demonstrated effective clinical outcomes. Moreover, most currently developed treatments are merely gene therapies that target a single target against dystrophin. Because DMD involves various pathogenic mechanisms such as muscle degeneration, inflammation, and fibrosis, multi-target drug therapy may be more effective.

[0004] Recently, many clinical trials have demonstrated that mesenchymal stem cells are a safe and beneficial therapeutic approach for a variety of pathologies. Mesenchymal stem cells are mesodermal tissue-derived cells that can be obtained from autologous or allogeneic sources, and can differentiate into fat, cartilage, and bone-forming lineages, engraft in muscle tissue, and differentiate into muscle cells. In addition, various proteins secreted from mesenchymal stem cells have therapeutic effects, and paracrine factors regulate inflammation and immune responses at the lesion site and maintain homeostasis, allowing for multi-targeted therapy.

[0005] Therefore, there is a need to develop new therapies that can regulate inflammation and immune responses at the lesion site and effectively treat Duchenne muscular dystrophy (DMD). Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect is to provide a pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD), comprising a miR-499-5p activator.

[0007] Another aspect is to provide a cell therapy for treating Duchenne muscular dystrophy (DMD), comprising a miR-499-5p activator.

[0008] Yet another aspect is to provide a composition for diagnosing Duchenne muscular dystrophy (DMD), comprising a preparation for measuring the expression level of miR-499-5p.

[0009] Yet another aspect is to provide a kit for diagnosing Duchenne muscular dystrophy (DMD) comprising the composition.

[0010] Yet another aspect is to provide an informative method for the diagnosis of Duchenne muscular dystrophy (DMD) comprising measuring the expression level of miR-499-5p in an individual sample. [Means for solving the problem]

[0011] One embodiment provides a pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD), comprising a miR-499-5p activator. Another embodiment provides a method for preventing or treating Duchenne muscular dystrophy (DMD), comprising administering a miR-499-5p activator to an individual in need thereof. Yet another embodiment provides a pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD), comprising a miR-499-5p activator.

[0012] As used herein, the term "microRNA (or miRNA, miR)" refers to a single-stranded RNA molecule of 21 to 25 nt that binds to the 3'UTR of mRNA and regulates gene expression in eukaryotes. That is, the microRNA is involved in cell development, proliferation and death, fat metabolism, tumor formation, and the like, by regulating the expression of a target protein. In one embodiment, the miR-499-5p includes variants having one or more substitutions, insertions, deletions, and combinations thereof, which are functional equivalents having changes that do not reduce the activity of the miR-499-5p. In one embodiment, it was confirmed that the miR-499-5p promotes the recovery of fibrotic muscle by down-regulating the TGF-β receptor. Thus, the miR-499-5p can specifically bind to the TGF-β receptor. The TGF-β receptor can be, for example, TGFβR1, TGFβR3, and the like. That is, the miR-499-5p can have an anti-fibrotic effect by specifically binding to the TGF-β receptor.

[0013] In one embodiment, the miR-499-5p activator may be a mesenchymal stem cell. The mesenchymal stem cell may be derived from the umbilical cord, umbilical cord blood, bone marrow, placenta, fat, or the like. The umbilical cord means a "cord" that connects the mother and the embryo so that the mammalian fetus can grow in the placenta, and generally means a tissue composed of three blood vessels, i.e., two umbilical arteries and one umbilical vein, surrounded by Wharton's jelly. Thus, in one embodiment, the mesenchymal stem cell may be an umbilical cord-derived or umbilical cord blood-derived mesenchymal stem cell, specifically, Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). In one embodiment, it was confirmed that the expression level of miR-499-5p is increased by MMP-1 induced in mesenchymal stem cells in Mdx mice, thereby exhibiting an anti-fibrotic effect. Thus, in another embodiment, the mesenchymal stem cells may be genetically engineered to express MMP-1 or to overexpress MMP-1 relative to the parent cells.

[0014] In one embodiment, WJ-MSCs were intravenously injected into 3-5 month old Mdx mice at a specific cell number to confirm skeletal muscle therapeutic effects such as intracellular creatine kinase reduction, behavioral recovery, muscle regeneration, inhibition of cell death, and reduction of fibrosis. That is, the dose-dependent effect of WJ-MSCs and skeletal muscle treatment by single intravenous administration rather than local administration were confirmed in Mdx mice, and the optimal dose and method of administration of WJ-MSCs for the treatment of Duchenne muscular dystrophy (DMD) was clarified by changing miR-499-5p expression in the skeletal muscle of Mdx mice. Thus, the composition was administered intravenously at a concentration of 1x10 2 Or 1x10 8 For example, the composition may contain 1x10 mesenchymal stem cells. 2 Or 1x10 8 Cell count, 1x10 3 Or 1x10 7 Cell count, 5x10 3 Or 1x107 Cell count, 5x10 3 Or 5x10 6 Cell count, 1x10 4 Or 1x10 6 Cell count, 5x10 4 Or 1x10 6 The number of cells may include mesenchymal stem cells. If the content of the mesenchymal stem cells is below or above the above range, there are problems in that the effects of lowering creatine kinase level, increasing muscle strength, and alleviating fibrosis cannot be fully exerted.

[0015] In one embodiment, the composition may be administered intravenously.

[0016] In general, Duchenne muscular dystrophy (DMD) is the most common progressive muscular dystrophies, and in the early stages of the disease, the gastrocnemius muscle becomes stiff and enlarged, and the entire skeleton and cardiac muscles gradually degenerate and weaken. Intramuscular injection (IM) is a method of directly administering a drug to a muscle, and has the advantage of being quickly absorbed because the muscle has a rich supply of blood vessels. However, when the composition is directly administered to a muscle, the stem cells contained in the composition have limited mobility from the administration site to the surrounding area, so the composition must be administered several times at various sites. Meanwhile, intravenous injection (IV) is a method of delivering a drug to the necessary tissues of the body via the heart within 1 to 2 minutes, so that the drug has a rapid effect and a reliable response. In particular, stem cells have the ability to seek out damaged sites by themselves, i.e., a homing effect, and can migrate to the skeletal muscle site or diaphragm site where the disease is present, thereby exerting a therapeutic effect. In one embodiment, the distribution of WJ-MSCs in the gastrocnemius veins of normal control and Mdx mice administered WJ-MSCs intravenously was confirmed, and it was confirmed that more cells remained in the gastrocnemius muscles of Mdx mice compared to the normal control group. In other words, by injecting a composition containing a miR-499-5p activator (e.g., WJ-MSCs) into the veins of individuals with Duchenne muscular dystrophy (DMD), the activator can migrate into the gastrocnemius muscles and exhibit a therapeutic effect, which is effective in the early treatment of Duchenne muscular dystrophy (DMD).

[0017] The pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD) according to one embodiment can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols; topical preparations; suppositories; and sterile injection solutions by a conventional method, and may contain suitable carriers, excipients, or diluents that are commonly used in the manufacture of pharmaceutical compositions for formulation.

[0018] The carriers, excipients or diluents may include a variety of compounds or mixtures thereof including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0019] When the formulation is made, it may be prepared by using a diluent or excipient that is commonly used, such as a filler, weighting agent, binder, wetting agent, disintegrant, or surfactant.

[0020] A solid preparation for oral administration can be prepared by mixing the above-mentioned bean extract with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0021] Oral liquid preparations include suspensions, solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents.

[0022] Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol gelatin, and the like.

[0023] The desired dosage of the pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD) according to one embodiment varies depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and can be appropriately selected by those skilled in the art. However, to achieve the desired effect, it may be administered at 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg per day. The dosage may be administered once a day or in several divided doses. However, the scope of the present invention is not limited by the dosage.

[0024] The pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD) according to one embodiment can be administered to mammals such as rats, mice, livestock, and humans by various routes. All of the administration routes can be, for example, oral administration, rectal administration, or intravenous, intramuscular, subcutaneous, intrauterine dura or intracerebroventricular injection.

[0025] Another aspect provides a health functional food composition for preventing or improving Duchenne muscular dystrophy (DMD), comprising an miRNA-499-5p activator as an active ingredient. The specific content of the miRNA-499-5p activator is as described above.

[0026] In the health functional food for preventing or improving Duchenne muscular dystrophy (DMD) according to one embodiment, when the compound is used as an additive for the health functional food, it can be added as it is or used together with other foods or food ingredients, and can be used appropriately by a general method. The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

[0027] The dosage form of health functional foods can be any of powder, granule, pill, tablet, and capsule forms, as well as general food or beverage forms.

[0028] The type of food is not particularly limited, and examples of foods to which the substance can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, energy drinks, alcoholic beverages, and vitamin complexes, but may include any food in the general sense.

[0029] In general, when producing food or beverage, the compound may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, the amount may be less than the above range, and since it is a fraction of a natural product, there is no problem in terms of safety, and it may also be used in an amount greater than the above range.

[0030] The beverage of the health functional food according to one embodiment may contain various flavorings or natural carbohydrates as additional ingredients, like ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0031] In addition to the above, the health functional food for preventing or improving Duchenne muscular dystrophy (DMD) according to one embodiment may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated drinks. In addition, the sleep improving composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. Such ingredients may be used independently or in mixtures. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food of the present invention.

[0032] Another aspect provides a cell therapy agent for treating Duchenne muscular dystrophy (DMD), comprising a miR-499-5p activator. The specific details of the miR-499-5p activator are as described above.

[0033] As used herein, the term "cellular therapeutic agent" refers to a pharmaceutical agent for therapeutic, diagnostic or preventive purposes that involves the expansion, selection or, in various ways, alteration of the biological properties of living autologous, allogenic or xenogenic cells in vitro in order to restore the functions of cells and tissues.

[0034] As described above, in one embodiment, the miR-499-5p activator promotes the expression of miR-499-5p by expressing MMP-1 in myotube cells, and restores fibrous muscle by down-regulating TGF-β receptor activation and smad2 / 3 phosphorylation, and can be used to prevent or treat Duchenne muscular dystrophy (DMD).

[0035] (blank)

[0036] Another embodiment provides a composition for diagnosing Duchenne muscular dystrophy (DMD), comprising a preparation capable of specifically detecting the expression level of miR-499-5p. The specific content of the miR-499-5p is as described above.

[0037] As used herein, the term "preparation capable of specifically detecting the expression level of miRNA" refers to a substance that can be used to specifically confirm or detect the miRNA in an individual sample. The preparation can be, for example, a primer, a probe, or an antisense nucleic acid.

[0038] In yet another embodiment, a kit for diagnosing Duchenne muscular dystrophy (DMD) is provided, comprising the composition. Specific contents of the composition are as described above.

[0039] The kit may include a primer, a probe, or an antisense nucleic acid capable of specifically detecting the expression level of miR-499-5p, and may further include one or more other component compositions, solutions, or devices suitable for analysis. In one embodiment, the kit may be an RT-PCR kit, a microarray chip kit, or a protein kit.

[0040] Yet another embodiment provides a method for providing information for the diagnosis of Duchenne muscular dystrophy (DMD), comprising measuring the expression level of miR-499-5p in an individual biological sample. The specific content of the miR-499-5p is as described above. In one embodiment, the method may comprise determining that the subject has Duchenne muscular dystrophy (DMD) when the expression level of miR-499-5p is decreased compared to a control group.

[0041] The individual refers to a patient diagnosed with Duchenne muscular dystrophy (DMD). The individual may be a vertebrate, such as a mammal, amphibian, reptile, bird, or mammal, such as a human (Homo sapiens) (Korean).

[0042] The biological sample may include samples such as tissue isolated from an individual, tumor tissue, lung tumor tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

[0043] The expression level of miR-499-5p can be measured by Western blotting, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), real-time reverse transcription polymerase reaction (real-time RT-PCR), RNase protection assay (RPA), Northern blotting, DNA chip, etc.

[0044] Yet another embodiment provides a method for screening a therapeutic agent for Duchenne muscular dystrophy (DMD), comprising the steps of contacting miR-499-5p with a test substance, and selecting a test substance that increases the expression level of the miR-499-5p compared to a non-treated control group with a Duchenne muscular dystrophy (DMD) therapeutic agent. Yet another embodiment provides a method for screening a therapeutic agent for Duchenne muscular dystrophy (DMD), comprising the steps of contacting a test substance with muscle cells in which the expression of miR-499-5p0 is reduced compared to a normal control group, measuring the level of pibronectin or p-smad2 / 3 in the muscle cells, and selecting a test substance that reduces the level of pibronectin or p-smad2 / 3 compared to a control group not treated with the test substance with a Duchenne muscular dystrophy (DMD) therapeutic agent. The specific content of the miR-499-5p is as described above. The test substance, e.g., a drug candidate, a test compound, or a test composition, may include a small molecule compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, a peptide, or other extract or natural product. The contacting may be performed in vitro. Effect of the Invention

[0045] In one embodiment, a composition in which a miR-499-5p activator promotes expression of miR-499-5p by expressing MMP-1 in myotube cells, thereby restoring fibrous muscle by down-regulating TGF-β receptor activation and smad2 / 3 phosphorylation, may be used to prevent or treat Duchenne muscular dystrophy (DMD). [Brief description of the drawings]

[0046] [Figure 1A] FIG. 1A shows the results of measuring mouse muscle strength after intravenous injection of WJ-MSCs into an Mdx mouse model.

[0047] [Figure 1B] FIG. 1B shows the results of measuring serum creatine kinase levels after intravenous injection of WJ-MSCs into Mdx mice.

[0048] [Figure 1C] FIG. 1C shows the results of examining the fibrotic area, myosin heavy chain (MHC) and annexin V expression levels after intravenous injection of WJ-MSCs into Mdx mice.

[0049] [Figure 1D] FIG. 1D shows the results of measuring the fibrotic area, myosin heavy chain (MHC) and annexin V expression levels using Image J after intravenous injection of WJ-MSCs into Mdx mice.

[0050] [Figure 2A] FIG. 2A is a heat map showing miRNAs commonly expressed in humans, Mdx mice, and healthy mice.

[0051] [Figure 2B] FIG. 2B shows the relative miRNA-499 expression before and after intravenous injection of WJ-MSCs into Mdx mice.

[0052] [Figure 2C] FIG. 2C shows the results of confirming the expression level of miR-499-5p by MMP-1 protein.

[0053] [Figure 3A] FIG. 3A shows the relative TGFβR1 expression levels before and after intravenous injection of WJ-MSCs into Mdx mice.

[0054] [Figure 3B] FIG. 3B shows the relative expression levels of TGFβR3 before and after intravenous injection of WJ-MSCs into Mdx mice.

[0055] [Figure 4A] 4A and 4B show the results of confirming the anti-fibrotic effect in skeletal muscle after intravenous injection of WJ-MSCs into Mdx mice. [Figure 4B] Same as above.

[0056] [Figure 4C] Figures 4C and 4D show the anti-fibrotic effect in the diaphragm after intravenous injection of WJ-MSCs into Mdx mice. [Figure 4D] Same as above.

[0057] [Figure 5A] 5A and 5B show the results of measuring the phosphorylation level of Smad2 / 3 after intravenous injection of WJ-MSCs into Mdx mice. [Figure 5B] Same as above.

[0058] [Figure 6A] FIG. 6A shows the results of quantification of DNA in organs after intravenous injection of WJ-MSCs into normal control mice and Mdx mice.

[0059] [Figure 6B]6B and 6C show the results of measuring fibrosis over time after intravenous injection of WJ-MSCs into Mdx mice. [Figure 6C] Same as above.

[0060] [Figure 7A] FIG. 7A shows the results of inducing oxidative stress in mouse myotubes, followed by injection of miR-499-5p transfection or treatment with a TGF-β inhibitor.

[0061] [Figure 7B] FIG. 7B shows the results of inducing oxidative stress in mouse myotubes, followed by transfection with miR-499-5p, and confirming collagen accumulation in the myotubes.

[0062] [Figure 7C] FIG. 7C shows the results of inducing oxidative stress in mouse myotubes, transfected with miR-499-5p, and confirming collagen I deposition in the myotubes.

[0063] [Figure 7D] Figure 7D shows the results of inducing oxidative stress in mouse myotubes, then injecting them with miR-499-5p or treating them with a TGF-β inhibitor, and examining the expression of myosin heavy chain (MHC), pibronectin, and p-smad2 / 3.

[0064] [Figure 8] FIG. 8 shows the anti-fibrotic effect of miR-499-5p in skeletal muscle of Duchenne muscular dystrophy (DMD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0066] (blank) EXAMPLES

[0067] Working Example

[0068] Example 1. Confirmation of the therapeutic effect of Wharton's jelly-derived mesenchymal stem cells (WJ-MCS) on muscular dystrophy

[0069] 1-1.Grip strength measurements

[0070] To confirm the therapeutic effect of mesenchymal stem cells on muscular dystrophy, muscle strength of mice was measured in the forelimbs and hindlimbs. First, Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) were isolated by a known method and cultured according to standard operating procedures at the Good Manufacturing Practice Facility of Samsung Seoul Medical Center. Then, the WJ-MSCs were injected at various doses (5x10) into the caudal side of 3-5 month old Duchenne muscular dystrophy (DMD) C57BL / 10ScSn-Dmdmdx / J (Mdx) mice (The Jackson Laboratory, USA). 3 (dose 1), 1x10 4 (dose 2), 5x10 4 (dose 3), 1x10 5 (dose 4), 5x10 5 The WJ-MSCs were administered intravenously at a dose of 5 mg / kg / day (dose 5). Because clumped cells may induce intravascular embolism, the WJ-MSCs were mixed with 100 μL of PBS to form a suspension before injection, and then injected as slowly as possible. The muscle strength of the mouse's forelimbs and / or hindlimbs was then measured using a grip strength measuring device (BIO GS3, BIOSEB, Vitrolles, France). C57BL / 10ScSnJ mice (The Jackson Laboratory, USA) were used as a control group.

[0071] FIG. 1A shows the results of measuring mouse muscle strength after intravenous injection of WJ-MSCs into an Mdx mouse model.

[0072] As a result, as shown in Figure 1A, it was confirmed that muscle strength was increased in the forelimb (left) and forelimb and hindlimb (right) of Mdx mice administered with WJ-MSCs, compared to the control group not administered with WJ-MSCs. In particular, muscle strength increased in a concentration-dependent manner at doses 1 to 3, and tended to decrease at doses 4 and above. In other words, administration of WJ-MSCs at a specific dose can have a muscle strength-increasing effect.

[0073] 1-2. Measurement of serum creatine kinase (CK) levels

[0074] In order to confirm the symptom alleviation effect according to the dose of mesenchymal stem cells administered, the level of creatine kinase (CK) in mouse serum was measured. Specifically, retro-orbital plexus bleeding was induced in Mdx mice administered with WJ-MSC in Example 1-1, and the mouse blood was collected, incubated at 22-24°C for 1 hour or more, centrifuged at 15,000xg for 10 minutes, and the upper layer of the clear serum was collected. Then, the creatine kinase (CK) activity in serum was measured using a Creatine Kinase Activity Assay Kit (Colorimetric).

[0075] FIG. 1B shows the results of measuring serum creatine kinase (CK) levels after intravenous injection of WJ-MSCs into Mdx mice.

[0076] As a result, as shown in Figure 1B, creatine kinase (CK) levels were significantly decreased in Mdx mice administered WJ-MSCs compared to the control group not administered WJ-MSCs. In particular, administration of doses 3 to 5 showed a significant decrease in creatine kinase (CK) levels compared to lower doses.

[0077] 1-3. Confirmation of intramuscular fibrosis level, muscle cell regeneration and anti-cell death effects

[0078] The degree of fibrosis, muscle cell regeneration, and anti-cell death effects of Mdx mouse muscle by administration of mesenchymal stem cells were confirmed. Specifically, the Mdx mouse administered with WJ-MSC in Example 1-1 was euthanized, the skin was removed, and the gastrocnemius muscle and diaphragm were removed and fixed in 4% paraformaldehyde for 24 hours. The muscle was then embedded in paraffin, cut to 4 μm, and stained with Sirius Red to observe muscle fibrosis. Muscle cell regeneration was detected by incubating fixed gastrocnemius sections with myosin heavy chain (MHC) antibody (#MAB4470, diluted 1 / 1,000, R&D Systems, MN, USA) at 4°C for 18 hours, followed by incubation with secondary antibody Alexa Fluor® 488 AffiniPure Goat Anti-mouse IgG (H+L) (A10680, Thermo Fisher Scientific, Waltham, MA, USA). The gastrocnemius sections were then counterstained with Hoechst 33342 (H1339, Thermo Fisher Scientific).

[0079] FIG. 1C shows the results of examining the fibrotic area and the expression levels of myosin heavy chain (MHC) and annexin V after intravenous injection of WJ-MSCs into Mdx mice.

[0080] FIG. 1D shows the results of measuring the fibrotic area, myosin heavy chain (MHC) and annexin V expression levels using Image J after intravenous injection of WJ-MSCs into Mdx mice.

[0081] As a result, as shown in FIG. 1C and FIG. 1D, the Sirius red stained area was significantly reduced in Mdx mice administered with WJ-MSCs compared to the control group not administered with WJ-MSCs, indicating the effect of alleviating fibrosis at dose 3. In addition, the intensity of myosin heavy chain (MHC) was significantly increased in Mdx mice administered with WJ-MSCs compared to the control group not administered with WJ-MSCs, and the intensity of myosin heavy chain (MHC) was significantly increased at dose 3, indicating the effect of muscle cell regeneration. In addition, in Mdx mice administered with WJ-MSCs, the Annexin V expression level was concentration-dependently decreased at doses 1 to 3 of WJ-MSCs, but the Annexin V expression level was increased at dose 4, compared to the control group not administered with WJ-MSCs. From the above results, it was confirmed that WJ-MSC dose 3 showed a therapeutic effect at the lowest dose. That is, since the effects of fibrosis alleviation, muscle regeneration and anti-cell death were not significantly increased at higher doses, dose 3 was determined as the optimal dose for treating muscular dystrophy.

[0082] (blank)

[0083] Example 2. Sequencing and analysis of miRNAs expressed in muscular dystrophy

[0084] 2-1. Identification of miRNAs differentially expressed in muscular dystrophy

[0085] To identify miRNAs that are differentially expressed in muscular dystrophies, miRNA sequencing and data analysis were performed. Specifically, total RNA was extracted from the mouse gastrocnemius skeletal muscle of Example 1-3 using TRIzol Reagent (Invitrogen, Waltham, MA, USA). Then, RNA quality was evaluated using Agilent 2100 Bioanalyzer and RNA 6000 Pico Chips (Agilent Technologies, Amsterdam, Netherlands), and RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). miRNA library construction was performed using NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., Ipswich, MA, USA), and high-throughput sequences were generated using single-end sequencing of 75 bp reads on the NextSeq500 system (Illumina, San Diego, CA, USA). Sequence reads were analyzed by using a Bowtie 2 and used mature miRNA sequences as reference. Read counts mapped to mature miRNA sequences were extracted from alignment files using Bioconductor using bedtools (v2.25.0) and the R statistical programming language (version 3.2.2 (R development Core Team (2011)). Comparisons between samples were made using the quantile normalization method and heatmap clustering was performed using MeV (version 4.9.0).

[0086] FIG. 2A shows a heat map of commonly expressed miRNAs in humans, Mdx mice, and healthy mice.

[0087] As a result, as shown in Figure 2A, it was found that several miRNAs were down-regulated in muscular dystrophy mice compared with healthy mice, and among them, the expression of miR-499-5p was most significantly reduced.

[0088] 2-2. Confirmation of miR-499-5p expression levels in muscular dystrophy

[0089] To confirm the miRNA sequencing results of Example 2-1, the expression level of miR-499-5p was analyzed in muscular dystrophy. TM miRNA was isolated using the miRNA Isolation Kit (Invitrogen). The miRNA concentration was then measured using the Qubit microRNA Assay kit and Qubit 4 fluorometer (Invitrogen). TM The isolated miRNA was reverse transcribed into cDNA at a concentration of 5 ng / μL using the Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Waltham, MA, USA). TM Fast Advanced Master Mix (Applied Biosystems) was used for QuantStudio TM qPCR was performed using the Taqman Flex Real-Time PCR System (Applied Biosystems). TM The levels of hsa-miR-499-5p (Assay ID: 478139_mir) were measured using Advanced miRNA Assay (Invitrogen). All reactions were performed in triplicate, and comparative quantification of each target gene was performed based on the cycle critical value (CT) normalized to miR-26a-5p using the DDCT method proposed by Livak and Schmittgen.

[0090] FIG. 2B shows the relative miRNA-499 expression before and after intravenous injection of WJ-MSCs into Mdx mice.

[0091] As a result, as shown in Figure 2B, the expression of miR-499-5p was relatively reduced in Mdx mice compared to the normal control group, but when WJ-MSCs were injected into Mdx mice, the expression of miR-499-5p was significantly increased. In other words, miR-499-5p is a miRNA that is down-regulated in Duchenne muscular dystrophy (DMD), and its expression level was increased by intravenous injection of mesenchymal stem cells.

[0092] 2-3. Confirmation of increase in miR-499-5p expression level by MMP-1 protein

[0093] In a previous study, it was confirmed that WJ-MSCs alleviate skeletal muscle fibrosis in Mdx mice and in vitro fibrosis models through MMP-1 in myotubes. Based on the results, we confirmed whether the expression level of miR-499-5p was increased by MMP-1 protein. Specifically, 20 ng of human recombinant MMP-1 was injected into the gastrocnemius muscle of Mdx mice, and the mice were sacrificed on the 7th day. Then, the expression level of miR-499-5p was confirmed by the same method as in Example 2-2 above.

[0094] FIG. 2C shows the results of confirming the expression level of miR-499-5p by MMP-1 protein.

[0095] As a result, as shown in Figure 2C, the expression of miR-499-5p was reduced in Mdx mice compared with the control group, but the expression level of miR-499-5p in Mdx mice was restored to the level of miR-499-5p in the control group by MMP-1 protein. That is, WJ-MSCs can induce MMP-1 and increase the expression level of miR-499-5p in muscular dystrophy, and show anti-fibrotic effects.

[0096] (blank)

[0097] Example 3. Confirmation of target receptor of miRNA-499-5p

[0098] It was confirmed that the increase in miR-499-5p by mesenchymal stem cells acts in the treatment of muscular dystrophy through what mechanism. First, the target receptor of miRNA-499-5p was predicted by a bioinformatics method (TargetScan). As a result, highly supported miR-466-5p target sites were detected in mouse TGFβR1 and mouse TGFβR3. Thereby, the expression levels of TGFβR1 and TGFβR3 mRNA were measured to confirm whether they directly bind to miRNA-499-5p. Specifically, total RNA was isolated from the mouse gastrocnemius skeletal muscle of Example 1-3 using TRIzol Reagent. Then, the RNA was reverse transcribed into cDNA using SuperScript IV Reverse Transcriptase (Invitrogen). PCR amplification was performed using the primers in Table 1 below and 2x Power SYBR Green Master Mix (Applied Biosystems). After initial denaturation at 95°C for 10 minutes, a cycle of 95°C for 15 seconds and 55°C for 55 seconds was repeated 40 times.

[0099] [Table 1]

[0100] FIG. 3A shows the relative TGFβR1 expression levels before and after intravenous injection of WJ-MSCs into Mdx mice.

[0101] FIG. 3B shows the relative expression levels of TGFβR3 before and after intravenous injection of WJ-MSCs into Mdx mice.

[0102] As a result, as shown in Figures 3A and 3B, the expression of TGFβR1 and TGFβR3 was significantly increased in Mdx mice compared with the control group, but after WJ-MSC injection, the expression of TGFβR1 and TGFβR3 was decreased to levels similar to those of the normal control group.

[0103] That is, it can be seen that miR-499-5p targets TGFβR1 and TGFβR3, which are key factors in fibrosis.

[0104] (blank)

[0105] Example 4. Confirmation of therapeutic effect on muscular dystrophy

[0106] 4-1. Confirmation of anti-fibrotic effects in skeletal muscle and diaphragm

[0107] The anti-fibrotic effect of mesenchymal stem cells in muscular dystrophy was confirmed. Specifically, the skeletal muscle and diaphragm of the Mdx mice (injected at a dose of 3) obtained in Example 1-3 were stained with H&E and Sirius Red in the same manner as in Example 1-3, and muscle fibrosis was observed. In addition, immunohistochemistry was performed in the same manner as in Example 1-3, except that fibronectin antibody (ab2413 (at 1 / 200 dilution) (Abcam)) was used as the primary antibody, and Alexa Fluor® 594 AffiniPure Goat Anti-rabbit IgG (H+L) (A11037 (Thermo Fisher Scientific)) was used as the secondary antibody.

[0108] 4A and 4B show the results of confirming the anti-fibrotic effect in skeletal muscle after intravenous injection of WJ-MSCs into Mdx mice.

[0109] Figures 4C and 4D show the anti-fibrotic effect in the diaphragm after intravenous injection of WJ-MSCs into Mdx mice.

[0110] As a result, as shown in Figures 4A to 4D, the Sirius red stained area and pybronectin detection intensity were relatively increased in Mdx mice compared to the control group, but were significantly reduced by injection of WJ-MSCs. In particular, the pybronectin detection intensity in muscle cells and diaphragm tissue was similar to or even lower than that of the normal control group. In other words, mesenchymal stem cells are effective in preventing or treating muscular dystrophy by reducing muscle fibrosis.

[0111] 4-2. Confirmation of anti-fibrotic effect of TGF-β signal transduction

[0112] To confirm the anti-fibrotic effect of TGF-β signaling, the phosphorylation level of Smad2 / 3 was measured by Western blot. Specifically, mouse myoblast cell line C2C12 (ACTC CRL-1772) was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Biowest SA S, Nuaille, France) supplemented with 10% fetal bovine serum (Gibco BRL, MA, USA), 100 U / mL penicillin, and 100 ug / mL streptomycin (Gibco BRL) at 5% CO2 and 37°C. The culture medium was then replaced with differentiation medium supplemented with 5% horse serum (Gibco BRL) for 5 days to differentiate into myotubes. Myotubes were scraped onto culture dishes and dissolved in ice-cold radioimmunoprecipitation buffer (9.8 mol / L UREA, 4% CHAPS, 130 mmol / L dithiothreitol, 40 mmol / L Tris HCl, 0.1% sodium dodecyl sulfate, 1 mmol / L EDT, and Protease / Phosphatase Inhibitor Cocktail) to extract total protein. Protein was then quantified using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Identical amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories).After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies against pibronectin (ab2413, diluted 1 / 10,000 (Abcam)), p-Smad2 / 3 (8828S, diluted 1 / 1,000 (Cell Signaling Technology, Danvers, MA, USA)), and smad2 / 3 (3102S, diluted 1 / 1,000 (Cell Signaling Technology)). The membrane was then washed three times with TBST and incubated overnight with goat anti-rabbit IgG HRP-conjugated antibody (GTX213110-01, diluted 1 / 10,000 (GeneTex, Irvine, CA, USA)) or goat anti-mouse IgG HRP-conjugated antibody (GTX213110-01, diluted 1 / 10,000 (GeneTex, Irvine, CA, USA)). The sections were then incubated with a secondary antibody (GTX213111-01 (diluted at 1 / 10,000) (GeneTex)) for 1 hour at room temperature.

[0113] 5A and 5B show the results of measuring the phosphorylation level of Smad2 / 3 after intravenous injection of WJ-MSCs into Mdx mice.

[0114] As a result, as shown in Figure 5A and Figure 5B, the expression of pibronectin and p-Smad2 / 3 was significantly increased in the Mdx group compared to the control group, but it was significantly decreased by WJ-MSC compared to the normal control group. In other words, Smad2 / 3 phosphorylation shows a similar pattern to the expression of pibronectin, but it can suppress fibrosis by activating the TGF-β signaling pathway.

[0115] 4-3. Confirmation of tissue distribution of WJ-MSCs

[0116] The distribution of mesenchymal stem cells in tissues was confirmed. Specifically, in Example 1-1, WJ-MSCs present in the gastrocnemius muscle, heart, liver, lung, and spleen of Mdx mice intravenously injected with WJ-MSCs were quantified. PCR was performed using 2x Power SYBR TMPCR was performed in 20 μL reactions containing 10 μL Green PCR Master Mix (4367659, Thermo Fisher Scientific), 1 μL of each primer at 10 pmol / μL, and 8 μL of DNA at various concentrations. The PCR was performed using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) in MicroAmp Optical 384-Well Reaction Plates (4326270, Thermo Fisher Scientific). After initial denaturation at 95°C for 10 min, 40 cycles of 95°C for 15 s and 68°C for 30 s were performed.

[0117] [Table 2]

[0118] FIG. 6A shows the results of quantification of DNA in organs after intravenous injection of WJ-MSCs into normal control mice and Mdx mice.

[0119] As a result, as shown in Table 2 and Figure 6A, more cells were found to remain in the gastrocnemius muscle of Mdx mice (right) compared to the control group (left). In other words, it is clear that WJ-MCS administered intravenously to individuals with muscular dystrophy migrates to the muscle.

[0120] 4-4. Check the duration of WJ-MSC

[0121] To confirm the therapeutic effect of mesenchymal stem cells on muscular dystrophy, the duration of anti-fibrotic effect in muscle was examined. Specifically, the skeletal muscle of Mdx mice obtained in Example 1-3 was stained with Sirius Red in the same manner as in Example 1-3, and muscle fibrosis was observed.

[0122] 6B and 6C show the results of measuring fibrosis over time after intravenous injection of WJ-MSCs into Mdx mice.

[0123] As a result, as shown in Figures 6B and 6C, the fibrotic area was significantly increased in the muscles of Mdx mice compared to the control group, and was found to be continuously reduced for 4 days to 4 weeks after WJ-MSC injection.

[0124] 4-5. Confirmation of therapeutic effect on fibrosis caused by oxidative stress

[0125] The therapeutic effect of mesenchymal stem cells on fibrosis induced by oxidative stress was confirmed. Specifically, C2C12 cell line was differentiated into myotubes in the same manner as in Example 4-2, and then 2 mM H2O2 (Sigma, MO (USA)) was added to the growth medium, and the cells were further cultured for 24 hours to induce fibrosis. Then, the myotubes were transfected with 30 nmol / L human miR-499-5p mimic (GenePharma, Shanghai (China)) in serum-free medium. In addition, the myotubes induced to undergo fibrosis in serum-free medium were treated with 10 μM galunisertib (LY2157299, S2230 (Selleck Chemicals, Houston, TX (USA))) for 24 hours. Then, Sirius red staining was performed in the same manner as in Example 1-3 to confirm collagen accumulation, and immunohistochemistry was performed to confirm collagen II deposition. In addition, to confirm the expression of the protein in the myotubes, Western blotting was performed in the same manner as in Example 4-2, except that a primary antibody against myosin heavy chain (#MAB4470 (diluted at 1 / 10,000) (R&D Systems)) was additionally used.

[0126] FIG. 7A shows the results of inducing oxidative stress in mouse myotubes, followed by injection of miR-499-5p transfection or treatment with a TGF-β inhibitor.

[0127] (blank)

[0128] As a result, as shown in Figure 7A, myotube injury was induced by H2O2, but cell death was not induced. In addition, it was found that galunisertib and miR-499-5p induced the recovery of damaged myotubes.

[0129] FIG. 7B shows the results of inducing oxidative stress in mouse myotubes, followed by transfection with miR-499-5p, and confirming collagen accumulation in the myotubes.

[0130] FIG. 7C shows that collagen I deposition was confirmed in mouse myotubes after inducing oxidative stress and transfecting them with miR-499-5p.

[0131] As a result, as shown in Figures 7B and 7C, it was found that collagen accumulation and collagen I deposition were significantly increased by H2O2, and were restored to a lower level than the normal control group by miR-499-5p transfection. In other words, miR-499-5p may have a therapeutic effect on fibrosis by suppressing collagen accumulation and collagen I deposition.

[0132] Figure 7D shows the results of inducing oxidative stress in mouse myotubes, then injecting them with miR-499-5p or treating them with a TGF-β inhibitor, and examining the expression of myosin heavy chain (MHC), pibronectin, and p-smad2 / 3.

[0133] As a result, as shown in Figure 7D, it was found that the expression of pibronectin and p-Smad2 / 3 was significantly increased by H2O2, and the expression levels of pibronectin and p-Smad2 / 3 were decreased by injection of miR-499-5p or treatment with galunisertib compared to the normal control group. In addition, it was found that the expression of myosin heavy chain (MHC) was decreased by H2O2, but was significantly increased by injection of miR-499-5p or treatment with galunisertib. In other words, miR-499-5p can exhibit anti-fibrotic effects by suppressing the TGF-β signaling pathway, and can be used for the prevention or treatment of Duchenne muscular dystrophy (DMD).

[0134] (blank)

[0135] The above description of the present invention is for illustrative purposes only, and a person having ordinary skill in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A pharmaceutical composition for preventing or treating Duchenne muscular dystrophy (DMD), comprising an miR-499-5p activator.

2. The pharmaceutical composition of claim 1, wherein the miR-499-5p activator is Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs).

3. 3. The pharmaceutical composition of claim 2, wherein the mesenchymal stem cells are genetically engineered to express MMP-1 or to overexpress MMP-1 compared to parent cells.

4. The pharmaceutical composition of claim 1, wherein the miR-499-5p binds to a TGFβ receptor.

5. The pharmaceutical composition of claim 1 , which is administered intravenously.

6. A cell therapy agent for treating Duchenne muscular dystrophy (DMD), comprising an miR-499-5p activator.

7. A composition for diagnosing Duchenne muscular dystrophy (DMD), comprising a preparation for measuring the expression level of miR-499-5p.

8. A kit for diagnosing Duchenne muscular dystrophy (DMD), comprising the composition of claim 7.

9. An informative method for the diagnosis of Duchenne muscular dystrophy (DMD), comprising the step of measuring the expression level of miR-499-5p in an individual sample.

10. The method for providing information according to claim 9, comprising a step of determining that the subject has Duchenne muscular dystrophy (DMD) when the expression level of miR-499-5p is decreased as compared to a control group.

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