Kindlin-2-expressing gene therapy composition for osteoarthritis using non-viral bioresorbable delivery vehicle

Abstract

The present invention relates to a Kindlin-2-expressing gene therapy composition for osteoarthritis, using a non-viral bioresorbable delivery vehicle. It was confirmed that CaP prepared in the present invention is a non-viral delivery vehicle that can be stably introduced into cells and can deliver an expression vector into cells. It was also confirmed that, when a Kindlin-2 vector was introduced onto the surface of CaP particles and administered to an osteoarthritis animal model, osteoarthritis-induced pain was reduced and cartilage was protected from damage. Furthermore, it was confirmed that, when the Kindlin-2 vector was introduced onto the surface of CaP particles, delivery ability was increased, thereby enhancing osteoarthritis amelioration effects, compared to administration of Kindlin-2 alone.

Description

Kindlin-2 expression osteoarthritis gene therapy composition using a non-viral bioreabsorbable delivery vehicle

[0001] The present invention relates to a kindlin-2 expression osteoarthritis gene therapy composition using a non-viral bioreabsorbable delivery vehicle.

[0002] Osteoarthritis, also known as degenerative arthritis, is a type of arthritis characterized by degenerative changes in the cartilage and surrounding bone within synovial joints. Specifically, osteoarthritis is a disease characterized by the gradual loss of articular cartilage, along with hypertrophy of the bone located beneath the cartilage, bone formation at the joint margins, and non-specific synovial inflammation. Osteoarthritis is a condition caused by damage to cartilage resulting from aging or excessive physical pressure (e.g., obesity, trauma). Consequently, osteoarthritis manifests as severe pain and impaired movement in weight-bearing joints, such as the knee and hip, and if left untreated for a long period, it can lead to joint deformation.

[0003] Osteoarthritis progresses through the following stages: the chondrogenic stage (Stage 1), in which the water content within the cartilage increases and causes edema; the fibrillation stage (Stage 2), in which the cartilage is destroyed, causing the cartilage surface to crack and tear, resulting in damage that exposes the bone and narrows the joint cavity; the stage (Stage 3), in which cartilage cells begin to produce cartilage to restore it, but the destruction of cartilage occurs faster than the production of cartilage, leading to an overall reduction in cartilage; the bone deformity stage (Stage 4), in which the bone becomes deformed, causing joint deformity and dysfunction; and the joint soft tissue deformity stage (Stage 5), in which the soft tissue thickens.

[0004] Rheumatoid arthritis, which belongs to a different classification from osteoarthritis, is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells; unlike osteoarthritis, it causes osteoporosis and bone erosion of the bones surrounding the joints. Rheumatoid arthritis progresses through stages: stage 1, where inflammation of the synovial membrane spreads to the joint capsule, ligaments, and tendons; stage 2, where the joint space narrows and tension in the joint capsule and ligaments is lost due to the gradual destruction of joint cartilage; stage 3, where inflammation invades the bone and causes partial bone erosion; and stage 4, where joint function is lost. Therefore, osteoarthritis and rheumatoid arthritis differ completely in their causes and stages of progression, and their treatment methods are also different.

[0005] Currently, drugs such as acetaminophen, tramadol, nonsteroidal anti-inflammatory drugs (NSAIDs), diacerin, and glucosamine are used to treat osteoarthritis. Among these, NSAIDs are cited as problematic for gastrointestinal side effects such as gastric and duodenal ulcers. Therefore, when administering the above drugs to osteoarthritis patients who have risk factors for gastrointestinal side effects, cytoprotective agents such as rebamipide, H2 receptor antagonists such as cimetidine and ranitidine, and proton pump inhibitors such as omeprazole are prescribed simultaneously.

[0006] Meanwhile, gene therapy generally refers to a method of treating or preventing genetic defects such as cancer, infectious diseases, and autoimmune diseases through genetic modification of human cells by using genetic manipulation, such as DNA recombination, to transfect normal and therapeutic genes into the lesion, i.e., the patient's cells, in order to correct defective genes or add new functions to the cells. A drug consisting of genetic material manufactured for administration to the human body for the purpose of treating diseases, or of cells into which genetic material has been introduced, is called a gene therapy agent.

[0007] The fundamental technique of gene therapy lies in gene transfection. This technique has evolved to verify the function of genes cloned in molecular biology, but it has recently made even greater strides with the introduction of gene therapy. The essential requirements to be considered in gene transfection are the selection and manipulation of ① the gene, ② the carrier (vector), and ③ the target. Regarding the gene portion of these requirements, in addition to simply normal genes or repressor genes, genes for various purposes, such as antisense RNA or siRNA, can be used to inhibit the expression of target genes.

[0008] Two typical vectors, namely viral and non-viral vectors, have been used as gene carriers. While viral vectors have demonstrated higher transfection efficiency in most cells, safety concerns have been raised in some clinical trials. Non-viral vectors have recently garnered significant attention due to their ease of synthesis and modification, low immunoreactivity, and controllable size. It has been reported that non-viral delivery systems utilizing cationic liposomes and polymers containing polyethyleneimine (PEI) and poly(L-lysine) (PLL) are used to form nanoparticles by condensing plasmid DNA or siRNA. Although polymer-gene conjugates prepared from PEI and DNA have shown high transfection efficiency, they exhibit significant in vivo toxicity, limiting their clinical approach; therefore, there is a need for biocompatible and safe non-viral vectors.

[0009] Accordingly, the inventors established a unique therapeutic mechanism of the Kindlin-2 gene that targets STAT3 to mitochondria in order to repair mitochondrial dysfunction, which is the root cause of osteoarthritis. Furthermore, in order to safely and efficiently deliver this innovative therapeutic gene into target cells, they combined calcium phosphate (CaP)-based nanoparticles, which overcome the toxicity of the aforementioned existing vector, as a bioreabsorbable delivery vehicle, thereby completing a gene therapy composition with maximized therapeutic effects for osteoarthritis.

[0010] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

[0011] Another objective of the present invention is to provide a gene therapy drug for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

[0012] Another objective of the present invention is to provide a method for treating arthritis comprising the step of administering a pharmaceutically effective amount of calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface to an individual.

[0013] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of arthritis comprising, as an active ingredient, calcium phosphate nanoparticles (CaP) having a Kindlin-2 expression vector loaded on their surface.

[0014] In addition, the present invention provides a gene therapy drug for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

[0015] In addition, the present invention provides a method for treating arthritis comprising the step of administering a pharmaceutically effective amount of calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface to an individual.

[0016] The gene therapy agent according to the present invention effectively transports STAT3 into mitochondria via the mitochondrial targeting sequence (MTS) of Kindlin-2, thereby inhibiting the accumulation of reactive oxygen species (ROS) in chondrocytes and defending against apoptosis, resulting in an excellent effect of fundamentally improving the pathological mechanism of osteoarthritis. Furthermore, the CaP nanoparticles, which are non-viral carriers adopted in the present invention, are stably transported into cells, significantly increasing the delivery capacity of the Kindlin-2 vector loaded on their surface. Consequently, when administered to an animal model of osteoarthritis, it lowers the expression of cartilage destruction factors and inflammatory cytokines, reduces pain, and effectively protects against cartilage damage, making it useful for related industries.

[0017] Figure 1 illustrates the process of fabricating a non-viral bio-reabsorbable calcium phosphate delivery system according to the present invention.

[0018] Figure 2 is a figure confirming the structure of the non-viral bio-reabsorbable calcium phosphate delivery system of the present invention by TEM.

[0019] Figure 3 is a figure showing the hydration size distribution histogram of the non-viral bio-reabsorbable calcium phosphate carrier of the present invention.

[0020] Figure 4 is a figure confirming the intracellular uptake of the non-viral bio-reabsorbable calcium phosphate delivery system of the present invention using a fluorescent dye.

[0021] Figure 5 is a figure confirming the expression vector delivery ability of the non-viral bioreabsorbable calcium phosphate delivery system of the present invention with EGFP.

[0022] Figure 6 illustrates the Kindlin-2 and STAT3 protein-protein interaction model of the present invention.

[0023] Figure 7 is a figure showing the molecular docking results of the Kindlin-2 and STAT3 protein-protein interaction 1st model of the present invention.

[0024] Figure 8 is a figure showing the molecular docking results of the Kindlin-2 and STAT3 protein-protein interaction second model of the present invention.

[0025] Figure 9 is a figure showing the molecular docking results of the Kindlin-2 and STAT3 protein-protein interaction triad of the present invention.

[0026] Figure 10 is a figure showing the molecular docking results of the Kindlin-2 and STAT3 protein-protein interaction 4th model of the present invention.

[0027] Figure 11 is a figure showing the molecular docking results of the Kindlin-2 and STAT3 protein-protein interaction 5th-rank model of the present invention.

[0028] Figure 12 is a figure showing the results of comparing GRIM19 and MTS to confirm the mitochondrial targeting effect of Kindlin-2 of the present invention (A: analysis process and GRIM19 results, B: quantification of analysis results and Kindlin-2 results).

[0029] Figure 13 is a figure confirming the ability to regulate apoptosis and mitochondrial reactive oxygen species according to Kindlin-2 overexpression of the present invention (A: Confirmation of mitochondrial Kindlin-2 expression, B: Flow cytometry results and quantification).

[0030] Figure 14 is a Western blot analysis of the mitochondrial STAT3 regulatory effect according to Kindlin-2 overexpression of the present invention (A: Western blot result, B: quantification of result).

[0031] Figure 15 is a figure showing the chondroprotective effect according to Kindlin-2 overexpression of the present invention analyzed by RT-PCR (A: quantification of chondrocatabolic factors, B: quantification of apoptotic factors).

[0032] Figure 16 is a figure showing the amount of inflammatory cytokines according to the overexpression of Kindlin-2 of the present invention analyzed by ELISA.

[0033] Figure 17 is a figure quantifying the osteoarthritis pain reduction effect with and without CaP to confirm the delivery capacity of CaP-Kindlin-2 of the present invention (A: quantification of pain analysis, B: quantification of hind leg weight).

[0034] Figure 18 is a figure showing the cartilage protective effect according to the presence or absence of CaP by Safranin O staining to confirm the delivery capacity of CaP-Kindlin-2 of the present invention (A: staining result, B: quantification of staining result).

[0035] Figure 19 is a figure showing the expression of cartilage catabolic factors confirmed by immunohistochemical analysis to verify the delivery ability and osteoarthritis improvement effect of CaP-Kindlin-2 of the present invention (A: staining result, B: quantification of staining result).

[0036] Embodiments of the present invention will be described in detail below with reference to the attached drawings. In the following description, detailed descriptions of technologies well known 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 descriptions would unnecessarily obscure the essence of the present invention. Additionally, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs.

[0037] Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0038] The present invention provides a pharmaceutical composition for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

[0039] "Kindlin-2" of the present invention is a small gene family of adapter proteins within a FERM domain, and is classically known as a protein that regulates cell morphological changes and the activation of integrins. However, "Kindlin-2" in the present invention refers to a protein that performs a novel and key therapeutic function by including its own Mitochondrial Targeting Sequence (MTS), physically interacting (such as salt bridge binding) with the transcription factor STAT3 within the cell, and localizing it into the mitochondria to inhibit the production of reactive oxygen species in mitochondria and defend against apoptosis.

[0040] The term “prevention” as used in this invention refers to any act of suppressing the symptoms of a specific disease or delaying its progression through the administration of the composition of this invention.

[0041] The term “treatment” as used in this invention refers to any act of improving or beneficially altering the symptoms of a specific disease through the administration of the composition of this invention.

[0042] The pharmaceutical composition of the present invention may additionally include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete or incomplete adjuvant may be further included to increase the effect.

[0043] The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated into 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 are not limited to these, but may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0044] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods.

[0045] When formulating, the product may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and such solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Water-insoluble solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories may include Witepsol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc.

[0046] The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration may be anticipated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0047] The dosage of the pharmaceutical composition according to the present invention is selected by taking into consideration the age, weight, gender, physical condition, etc. of the individual. 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 preferably, it is 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 appear, and if it exceeds 5,000 μg / ml, it may exhibit toxicity to the human body.

[0048] In addition, the polynucleotide encoding Kindlin-2 of the present invention may be isolated from nature or artificially synthesized or modified, and the nucleotide sequence encoding Kindlin-2 may be modified by substitution, deletion, or insertion of one or more nucleic acid bases, and the protein expressed by such modification must not contain a significant change in its biological functionality. The above modification includes modification into a heterologous homologous gene.

[0049] Accordingly, the polynucleotide encoding Kindlin-2 is characterized by being represented by the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto, and is characterized by being represented by a nucleotide sequence having 70% or more, preferably 80% or more, and more preferably 90% or more homology with the above nucleic acid sequence.

[0050] Preferably, the polynucleotide encoding Kindlin-2 of the present invention may be provided in the form of a recombinant expression vector operably linked to a vector expressing it. Expression vectors comprising the Kindlin-2 polynucleotide include, but are not limited to, plasmids, phages, cosmids, viral vectors, or other mediators known in the art. The vector may self-replicate or be incorporated into host DNA.

[0051] The polynucleotide encoding Kindlin-2 can be combined with expression regulatory sequences, such as promoter / enhancer sequences, and other sequences required for transcription, translation, or processing. Regulatory sequences include not only sequences that direct the constitutive expression of nucleotides but also tissue-specific regulatory and / or inducible sequences. The design of the expression vector can be determined by factors such as the host cell to be transfected and the desired expression level.

[0052] The expression vector expressing Kindlin-2 of the present invention may use a non-viral vector or a viral vector.

[0053] Plasmids are representative of the above-mentioned non-viral vectors. Plasmid expression vectors are FDA-approved gene delivery methods for human use that directly deliver plasmid DNA to human cells, and unlike viral vectors, plasmid DNA has the advantage of being able to be homogeneously purified. As plasmid expression vectors that can be used in the present invention, mammalian expression plasmids known in the art may be used. For example, but not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91 / 08291), and pVL1392 (PharMingen) are representative examples.

[0054] The expression vector according to the present invention may be introduced into cells using methods known in the art. Examples include, but are not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection,

[0055] nucleic acids can be introduced into cells by electroporation, a gene gun, and other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

[0056] According to one embodiment of the present invention, the nanoparticles may comprise O-phospho-DL-serine, phosphocholine, or O-phosphorylethanolamine ligands.

[0057] According to one embodiment of the present invention, the nanoparticles may have a hydration size distribution of 120 to 300 nm.

[0058] According to one embodiment of the present invention, the Kindlin-2 may include the nucleotide sequence of SEQ ID NO. 1.

[0059] According to one embodiment of the present invention, the Kindlin-2 may inhibit apoptosis.

[0060] According to one embodiment of the present invention, the Kindlin-2 may regulate mitochondrial function, and regulating mitochondrial function may involve inhibiting mitochondrial reactive oxygen species.

[0061] According to one embodiment of the present invention, regulating the mitochondrial function may be regulating the expression of mitochondrial STAT3, and regulating the expression of mitochondrial STAT3 may be increasing the expression of total STAT3 or pSTAT3 (p705) and decreasing the expression of pSTAT3 (p727).

[0062] According to one embodiment of the present invention, the composition may improve arthritis, and improving arthritis may reduce joint pain.

[0063] According to one embodiment of the present invention, improving the arthritis may involve protecting against cartilage damage, and protecting against cartilage damage may involve reducing the Mankin index or the OARSI index.

[0064] According to one embodiment of the present invention, protecting the cartilage damage may involve reducing the expression of a chondrocatabolic factor or a cell death factor, wherein the chondrocatabolic factor may be MMP1, MMP3, or MMP13, and the cell death factor may be MLKL or RIPK1.

[0065] According to one embodiment of the present invention, protecting the cartilage damage may involve reducing the expression of inflammatory cytokines, and the inflammatory cytokines may be IL-6 or MCP-1.

[0066] According to one embodiment of the present invention, the Kindlin-2 may interact with mitochondrial STAT3, and the interaction may be a salt bridge interaction.

[0067] According to one embodiment of the present invention, the Kindlin-2 may include a mitochondrial targeting sequence (MTS), and the mitochondrial targeting sequence may induce mitochondrial localization.

[0068] According to one embodiment of the present invention, the arthritis may be osteoarthritis, but is not limited thereto.

[0069]

[0070] In addition, the present invention provides a gene therapy drug for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

[0071]

[0072] In addition, the present invention provides a method for treating arthritis comprising the step of administering a pharmaceutically effective amount of calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface to an individual.

[0073] The therapeutic method of the present invention comprises administering calcium phosphate nanoparticles (CaP), on which the Kindlin-2 expression vector is supported on the surface, to an individual in a therapeutically effective amount. It is preferable to apply a specific therapeutically effective amount for a specific individual differently depending on various factors and similar factors well known in the pharmaceutical field, including the specific composition (such as the type and degree of the response to be achieved and whether other agents are used in some cases), the individual's age, body weight, general health status, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used together or concurrently with the specific composition. The daily dosage is 0.0001 to 100 mg / kg based on the amount of the pharmaceutical composition of the present invention, preferably 0.01 to 100 mg / kg, and may be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or amount of each active ingredient must be such that it does not contain an excessively high content of each active ingredient to cause side effects. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention by taking into consideration the aforementioned matters.

[0074] The above-mentioned individual is applicable to any mammal, and said mammal includes not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

[0075] Calcium phosphate nanoparticles (CaP) with the Kindlin-2 expression vector of the present invention loaded on their surface can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration are expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural or intracerebroventricular injection.

[0076] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0077] <Preparation Example 1> Preparation of a non-viral bioreabsorbable delivery system

[0078] Calcium phosphate nanoparticles (CaP), which are the non-viral bioreabsorbable carriers of the present invention, were prepared. Specifically, 1 ml of calcium solution (Ca solution) was prepared by mixing 100 μl of Tris-HCl buffer (10 mM, pH 7.6) and 67.5 μl of CaCl2 (2 M) with 832.5 μl of triple-distilled water. Additionally, 1 ml of PO4 solution (PO4 solution) was prepared by mixing 100 μl of HEPES buffer (0.5 M, pH 7.1) containing 1.4 M NaCl, 50 μl of Na2HPO4 (60 mM), and triple-distilled water. Subsequently, the PO4 solution was mixed with the stirred Ca solution and reacted at room temperature for 5 minutes, followed by reaction in an autoclave (121°C, 0.12 MPa) for 20 minutes. After the reaction was completed, unreacted elements were removed using centrifugation (11,000 g, 20 min) or a centrifugal tube (MWCO 10). Subsequently, to ensure that the reactants were free of chloride, nine types of CaP (a to i) were synthesized using Tris buffer or Ca(NO3)2, or by combining ligands such as O-phospho-DL-serine, phosphocholine, and O-phosphorylethanolamine; the specific methods are described below. A general schematic diagram of the CaP synthesis method is shown in Fig. 1.

[0079] a: The lower layer of the reactant was obtained after centrifugation.

[0080] b: In the above method a, chloride ions were removed using Tris buffer or Ca(NO3)2, 487.5 μl of O-phospho-DL-serine ligand was added and reacted, then centrifuged to obtain the lower layer of the reaction product.

[0081] c: In the above method a, 487.5 μl of phosphocholine ligand was added and reacted, then centrifuged to obtain the reaction product of the upper layer.

[0082] d: In the above method a, 50 μl of O-phosphorylethanolamine ligand was added and reacted, then centrifuged to obtain the reaction product of the upper layer.

[0083] e: In the above method a, chloride ions were removed using Tris buffer or Ca(NO3)2, 50 μl of O-phosphorylethanolamine ligand was added and reacted, then centrifuged to obtain the reaction product of the upper layer.

[0084] f: In the above method a, 487.6 μl of O-phosphorylethanolamine ligand was added and reacted, then centrifuged to obtain the lower layer of the reaction product.

[0085] g: In the above method f, the reaction product of the upper layer was obtained after centrifugation.

[0086] h: In the above method a, chloride ions were removed using Tris buffer or Ca(NO3)2, 487.6 μl of O-phosphorylethanolamine ligand was added and reacted, then centrifuged to obtain the lower layer of the reaction product.

[0087] i: 25 ml of triple-distilled water was heated and stirred in a 55°C constant temperature water bath for 30 minutes. Then, 5 ml of CaCl2 (0.04 M, 0.05 mmol) solution was added to the triple-distilled water at that temperature at a rate of 30 ml / h, and the temperature was stabilized at 55°C for 1 hour. Afterward, 3 ml of O-phosphorylethanolamine (0.3 M, 0.225 mmol) was added at a rate of 30 ml / h and stirred for 23 hours, after which the temperature was lowered to room temperature to allow the reaction to proceed.

[0088] The particle structures of the CaP prepared by the above method were confirmed using a transmission electron microscope (TEM) (Fig. 2), and the hydration size distribution histograms of particles c and i are shown in Fig. 3.

[0089] <Example 1> Confirmation of CaP endocellular uptake

[0090] It was confirmed whether the CaP prepared in Preparation Example 1 above was incorporated into the cells. Fluorescent dyes Rhodamine 6G (Rho6G), Rhodamine B (RhoB), Flourescein (Fcein), and FITC were additionally treated to load the dyes onto the surface of the CaP. Specifically, after mixing the CaP and fluorescent dyes at a mass ratio of 10:1, the unreacted fluorescent dyes were removed by centrifugation. Subsequently, the CaP loaded with fluorescent dyes was treated to HeLa cell lines at a concentration of 100 μg / ml and co-cultured for 5 hours at 37°C under 5% CO2 conditions. Afterward, the remaining CaP was washed away, and the CaP incorporated into the cells was confirmed using a fluorescence microscope.

[0091] As a result, as shown in Figure 4, it was confirmed that particle c could be loaded with RhoB or Fcein dyes and was absorbed into the cell. In addition, it was confirmed that particle e could be loaded with Rho6G or FITC dyes and was absorbed into the cell.

[0092] <Example 2> Verification of the vector transfer effect of CaP

[0093] It was confirmed whether the non-viral bioreabsorbable CaP of the present invention could be used as a delivery vehicle for an actual vector. The CaP delivery vehicle of the present invention can introduce a plasmid onto the particle surface, and the vector delivery effect was confirmed using particles d and e. Specifically, CaP and an EGFP plasmid vector were mixed at a mass ratio of 700:1. Subsequently, HEK293T cells were treated and co-cultured for 5 hours at 37°C under 5% CO2 conditions. After the culture was completed, the cells were washed, and after inducing EGFP expression on day 2, the expression of EGFP was confirmed using a fluorescence microscope.

[0094] As a result, as shown in Figure 5, it was confirmed that fluorescence was emitted within the cells of cell lines treated with d and e nanoparticles having an EGFP plasmid vector introduced to the surface, confirming that the CaP particles of the present invention can be used as non-viral bioreabsorbable vector delivery vehicles.

[0095] As the excellent gene delivery ability of CaP nanoparticles was confirmed through the above examples, the mechanism of mitochondrial function restoration and osteoarthritis treatment performed by Kindlin-2, the core therapeutic gene of the present invention, within target cells was specifically verified below.

[0096] <Example 3> Analysis of STAT3 Binding and Mitochondrial Targeting Effects of Kindlin-2

[0097] <3-1> Analysis of Kindlin-2 and STAT3 Protein-Protein Interactions

[0098] To determine whether Kindlin-2 of the present invention can interact with the STAT3 protein, which enhances mitochondrial function, protein-protein interactions were analyzed. Specifically, docking simulations were performed using the ClusPro tool and an FFT-based algorithm (PIPER). PDB files of Kindlin-2 and STAT3 proteins were downloaded from Uniprot and uploaded to the ClusPro tool. Subsequently, the top five derived models were identified, and the interactions between Kindlin-2 and STAT3 proteins were analyzed using PDBsum. The specific docking structure is shown in Figure 6.

[0099] As a result, as shown in Figures 7 to 11, the top five docking models were examined, and in the top 1 model, it was confirmed that Kindlin-2 and STAT3 proteins interact via a salt bridge, and that the residues of STAT3 (Arg417, Arg423, Lys615, Glu760, Arg729, Glu753) and Kindlin-2 (Glu354, Glu342, Lys107, Asp210, Arg133) participate in the major interaction (Figure 7). This interaction pattern implies that the electrostatic attraction between positively charged amino acids Arg and Lys and negatively charged amino acid Glu is the main binding mechanism.

[0100] As a result of performing analysis on the top 2 to 5 models, it was confirmed that Kindlin-2 and STAT3 proteins interact as a salt bridge, similar to the 1st model, and that the Arg729 and Glu760 residues of STAT3 are most significantly involved in binding with Kindlin-2, and that the Glu670 and Lys555 residues of Kindlin-2 are significantly involved in binding interaction with STAT3.

[0101] In addition, when we checked whether Kindlin-2 could regulate the phosphorylation of STAT3 based on the above results, the binding affinity to the y705 and s727 sites associated with STAT3 phosphorylation was not analyzed, confirming that Kindlin-2 interacts with STAT3 but does not directly regulate phosphorylation.

[0102] <3-2> Confirmation of Kindlin-2's Mitochondrial Targeting Function

[0103] To confirm the mitochondrial targeting effect of Kindlin-2 of the present invention, the mitochondrial targeting sequence (MTS) was analyzed, and the MTS region was predicted by comparing it with the existing mitochondrial targeting (localization) gene, GRIM19. Specifically, using the TargetP 2.0 tool, a comparative analysis was performed with GRIM19 (NDUFA13), a representative gene included in MitoCarta 3.0 that contains MLS.

[0104] As a result, as shown in Figure 12, amino acid sequences 1–8 (MALDGIRM), 45–54 (MLKLVEKLDV), 468–475 (MAACRLAS), 566–573 (MRFIQAWQ), and 671–680 (MFYKLTSGWV) of the Kindlin-2 protein were predicted to be MLS regions in high proportions on the sequence. These figures were confirmed to be significantly dominant when compared to the 20–30 (DYKRNLPRRGL), 40–50 (IGTLIYGHWSI), and 40–60 (IGTLIYGHWSIMKWNRERRRL) sequences, which are known as representative MLS regions of GRIM19 (Mol Biol Cell. 2008 May;19(5):1893-1902. doi:10.1091 / mbc.E07-07-0683). Therefore, it was confirmed that the Kindlin-2 protein can move into mitochondria with a very high probability.

[0105] From the results of Examples 3-1 and 3-2 above, it was confirmed that Kindlin-2 protein in the cytosol can be blocked from moving into the nucleus (functioning as a transcription factor) and moved into the mitochondria through docking with STAT3.

[0106] <Example 4> Verification of mitochondrial function and chondroprotective effects of Kindlin-2

[0107] <4-1> Confirmation of Cell Apoptosis and Mitochondria Regulation Effects

[0108] Based on the results of Example 3 above, it was confirmed that Kindlin-2 can target mitochondria due to the presence of MLS; thus, the mitochondrial function-regulating effect of Kindlin-2 in osteoarthritis was verified. Specifically, Kindlin-2 was overexpressed (kindlin-2 OVN) in chondrocytes derived from osteoarthritis patients, and the chondrocytes were stabilized for 24 hours. After the end of culture, cells were harvested, proteins were extracted, and Kindlin-2 expression was confirmed by Western blot. In addition, to confirm apoptosis and the regulation of mitochondrial reactive oxygen species, cells were stained with AnnexinV and MitoSOX and analyzed by flow cytometry. A group injected with the MOCK vector was used as a control.

[0109] As a result, as shown in Figure 13, it was confirmed that mitochondrial Kindlin-2 significantly increased when Kindlin-2 was overexpressed. In addition, flow cytometry results confirmed that Kindlin-2 overexpression significantly reduced early apoptosis and reduced mitochondrial reactive oxygen species.

[0110] <4-2> Confirmation of Mitochondrial STAT3 Regulatory Effect

[0111] To determine whether Kindlin-2 regulates mitochondrial function, it was determined whether it regulates mitochondral STAT3. Specifically, in the same manner as in Example 4-1 above, Kindlin-2 was overexpressed in chondrocytes derived from osteoarthritis patients, and then mitochondria and cytoplasm were fractionated and harvested separately. Subsequently, protein expression of Kindlin-2, pSTAT3 (p705), pSTAT3 (p727), total STAT3, and COX IV was analyzed using Western blotting.

[0112] As a result, as shown in Fig. 14, it was confirmed that Kindlin-2 is overexpressed in mitochondria, and it was confirmed that the expression of total STAT3 and pSTAT3 (p705) in mitochondria increases due to Kindlin-2 overexpression, thereby supporting the results of Example 4.

[0113] <4-3> Confirmation of Regulation of Chondrocatabolic Factors and Apoptotic Factors

[0114] We confirmed whether Kindlin-2 regulates cartilage metabolism and apoptosis in osteoarthritis. Specifically, Kindlin-2 was overexpressed in chondrocytes derived from osteoarthritis patients using the same method as in Example 4-1, and the expression of cartilage catabolic factors MMP1, MMP9, and MMP13, as well as the expression of apoptotic factors MLKL and RIPK1, were analyzed by RT-PCR. In addition, after obtaining the cell culture supernatant, the amounts of inflammatory cytokines IL-6 and MCP-1 were analyzed by ELISA.

[0115] As a result, as shown in Figure 15, compared to the MOCK group, it was confirmed that the expression of cartilage catabolic factors MMP1, MMP9, and MMP13, and the expression of apoptotic factors MLKL and RIPK1 were significantly reduced in the group with overexpression of Kindlin-2.

[0116] In addition, when the amount of inflammatory cytokines secreted by the cells was examined, the amount of inflammatory cytokines IL-6 and MCP-1 in the cell culture medium was significantly reduced in chondrocytes overexpressing Kindlin-2 compared to the control group MOCK (Fig. 16), confirming that the overexpression of Kindlin-2 can protect chondrocytes.

[0117] <Example 5> Confirmation of the therapeutic effect of CaP-Kindlin-2 on osteoarthritis

[0118] <5-1> Osteoarthritis Pain Assessment

[0119] To confirm the therapeutic effect of CaP-Kindlin-2, which is loaded with Kindlin-2 (Sequence No. 1) of the present invention on its surface, on osteoarthritis, pathological activity was assessed by pain evaluation. Specifically, 5-week-old male Wistar rats weighing 200–250g were reared at a temperature of 21–22°C in a light-dark cycle at 12-hour intervals, supplied with sterilized water and feed. Subsequently, to induce osteoarthritis, MIA (Monosodium iodoacetate, Sigma, ST. Louis, MO), dissolved in physiological saline at a dose of 3 mg / 50 μl, was administered to the right knee of the rats to induce osteoarthritis. Afterward, CaP-Kindlin-2 was injected into the joint cavity using the d CaP of Preparation Example 1. Subsequently, pain was evaluated using a Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio, Italy). The measurement method involved placing a mesh plate on the measuring device, placing a rat inside an acrylic animal restraint, and then pricking the right paw, into which the drug had been injected, with the measuring device. Afterward, the time taken to lift the paw (Paw Withdrawal Latency, s, seconds) and the weight required to lift the paw (Paw Withdrawal Threshold, g) were measured. To verify changes in weight balance due to arthritis induction, the weight on the right hind rim was measured to determine the weight relative to body weight. As controls, a Vehicle group injected with an equal amount of physiological saline instead of CaP-Kindlin-2 and a CaP-MOCK group loaded with the MOCK vector were used.

[0120] As a result, as shown in Figure 17, compared to the CaP-MOCK group and the Kindlin-2 OVN group, it was confirmed that in the group administered CaP-Kindlin-2, pain caused by arthritis was reduced, the Paw Withdrawal Latency time increased, and the Paw Withdrawal Threshold weight was the highest. In addition, it was confirmed that the weight of the right hind leg increased relative to body weight compared to the CaP-MOCK group, confirming that CaP-Kindlin-2 improved osteoarthritis and increased Kindlin-2 delivery capacity.

[0121] <5-2> Confirmation of Histological Protective Effect

[0122] To confirm the therapeutic effect of CaP-Kindlin-2 loaded with Kindlin-2 of the present invention on its surface on osteoarthritis, a histological analysis was performed. Specifically, mice from each group of Example 5-1 were humanely sacrificed at the end of the experiment, and joint cavity tissues were obtained. Afterward, the joint cavity tissues were sectioned, stained with Safronin O, and the degree of cartilage damage was quantified using the Total Mankin score (comprehensive analysis of cartilage and inflammation infiltration) and the OARSI score (cartilage-specific analysis).

[0123] As a result, as shown in Figure 18, compared to the CaP-MOCK group and the Kindlin-2 group, it was confirmed that cartilage damage was protected in the group injected with CaP-Kindlin-2, and that the Total Mankin score and OARSI score were significantly reduced, confirming that Kindlin-2 delivery by CaP was increased.

[0124] <5-3> Confirmation of Regulation of Cartilage Catabolism Factor Expression

[0125] To confirm the therapeutic effect of CaP-Kindlin-2 loaded with Kindlin-2 of the present invention on its surface on osteoarthritis, it was determined whether it regulates the expression of cartilage catabolic factors. Specifically, the expression of cartilage catabolic factors MMP1, MMP3, and MMP13 was confirmed using immunohistochemical staining on the joint tissue section obtained in Example 5-2.

[0126] As a result, as shown in Figure 19, compared to the CaP-MOCK group, the expression of cartilage catabolic factors MMP1, MMP3, and MMP13 was significantly reduced in the Kindlin-2 group, but it was confirmed that the expression of cartilage catabolic factors MMP1, MMP3, and MMP13 was significantly reduced in the group injected with CaP-Kindlin-2, confirming that CaP increased the delivery of Kindlin-2, thereby increasing the effect of improving osteoarthritis.

[0127] Therefore, it was confirmed that the CaP prepared in this invention can be stably introduced into cells and is a non-viral carrier capable of delivering expression vectors into cells. Furthermore, it was confirmed that introducing the Kindlin-2 vector onto the surface of CaP particles and administering them to an animal model of osteoarthritis reduces osteoarthritis-induced pain and protects against cartilage damage. Additionally, compared to the administration of Kindlin-2 alone, it was confirmed that introducing the Kindlin-2 vector onto the surface of CaP particles increases delivery capacity, thereby enhancing the effect of improving osteoarthritis.

Claims

1. A pharmaceutical composition for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

2. In Paragraph 1, A composition in which the above nanoparticles comprise an O-phospho-DL-serine, phosphocholine, or O-phosphorylethanolamine ligand.

3. In Paragraph 1, A composition in which the above nanoparticles have a hydration size distribution of 120 to 300 nm.

4. In Paragraph 1, A composition wherein the above Kindlin-2 comprises the nucleotide sequence of SEQ ID NO.

1.

5. In Paragraph 1, The above Kindlin-2 is a composition that inhibits apoptosis.

6. In Paragraph 1, The above Kindlin-2 is a composition that regulates mitochondrial function.

7. In Paragraph 6, A composition that regulates the above mitochondrial function by inhibiting mitochondrial reactive oxygen species.

8. In Paragraph 6, A composition in which the above-mentioned mitochondrial function is regulated by the expression of mitochondrial STAT3.

9. In Paragraph 8, A composition that regulates the expression of the above mitochondrial STAT3 by increasing the expression of total STAT3 or pSTAT3 (p705).

10. In Paragraph 8, A composition that regulates the expression of the above mitochondrial STAT3 by reducing the expression of pSTAT3 (p727).

11. In Paragraph 1, The above composition is a composition that improves arthritis.

12. In Paragraph 11, A composition that improves the above arthritis and reduces joint pain.

13. In Paragraph 11, A composition that improves the above arthritis and protects against cartilage damage.

14. In Paragraph 13, A composition that protects against the above-mentioned cartilage damage by reducing the Mankin index or OARSI index.

15. In Paragraph 13, A composition that protects against the above-mentioned cartilage damage by reducing the expression of cartilage catabolic factors or cell death factors.

16. In Paragraph 15, A composition in which the above-mentioned cartilage catabolic factor is MMP1, MMP3, or MMP13.

17. In Paragraph 15, A composition in which the above-mentioned cell death factor is MLKL or RIPK1.

18. In Paragraph 13, A composition that protects against the above-mentioned cartilage damage by reducing the expression of inflammatory cytokines.

19. In Paragraph 18, A composition in which the above-mentioned inflammatory cytokine is IL-6 or MCP-1.

20. In Paragraph 1, A composition in which the above Kindlin-2 interacts with mitochondrial STAT3.

21. In Paragraph 20, A composition in which the above interaction is a salt bridge interaction.

22. In Paragraph 1, The above Kindlin-2 is a composition comprising a mitochondrial targeting sequence (MTS).

23. In Paragraph 22, The above-mentioned mitochondrial targeting sequence is a composition that induces mitochondrial localization.

24. A gene therapy drug for the prevention or treatment of arthritis comprising calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface as an active ingredient.

25. A method for treating arthritis comprising the step of administering a pharmaceutically effective amount of calcium phosphate nanoparticles (CaP) with a Kindlin-2 expression vector loaded on their surface to an individual.

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