Composition for preventing or treating neurodegenerative diseases

Abstract

The present invention relates to a composition for preventing or treating degenerative brain diseases. The pharmaceutical composition of the present invention comprises two or more selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same. The pharmaceutical composition of the present invention shows a better effect of improving and preventing degenerative brain diseases by administering the active ingredients in combination than by administering them alone, and can show the same or better effect even with a low concentration of the active ingredients. Therefore, the pharmaceutical composition of the present invention can be usefully used for preventing or treating degenerative brain diseases.

Description

[Technical field]

[0001] The present invention relates to Neurodegeneration Composition for preventing or treating disease and method for treating the disease Neurodegeneration It relates to a method for preventing or treating a disease. [Background technology]

[0002] Neurodegeneration This disease is currently one of the most serious social problems due to the increase in the elderly population. However, there is no effective treatment (=drug) for this disease, so Prevention and treatment of Attention is being paid to the development of treatments . Neurodegeneration The disease is a general term for illnesses caused by the gradual and constant death of nerve cells in one or various parts of the nervous system. Neurodegeneration The disease can also occur as a secondary symptom of adult diseases such as structural degeneration of brain nerve cells due to aging, circulatory system disorders, or when the brain is damaged by physical or mechanical factors such as traffic accidents, industrial accidents, or carbon monoxide poisoning, or when blood flow to the brain is reduced or cut off, causing irreparable damage to brain tissue; however, the exact cause is unknown to date. Neurodegeneration When the disease develops, depending on the site and mechanism of onset, it may cause progressive cognitive impairment, progressive ataxia, muscle weakness, and muscle atrophy.

[0003] Various genes are known to be effective in preventing neuronal death. treatment causes the death of nerve cells Enough Restraint In terms of not being able to Limitations of single gene therapy In treatment teeth, To obtain a therapeutic effect high Inject the dose Therefore, it was not free from the problem of side effects such as toxicity. Neurodegeneration Not only can diseases be prevented or treated, but also by administering gene therapy drugs in combination Neurodegeneration There is a need to discover therapeutic agents that can produce synergistic effects on the prevention or treatment of diseases. Summary of the Invention [Problem to be solved by the invention]

[0004] Under the above-mentioned background, the present inventors Neurodegeneration As a result of research and efforts to develop a therapeutic agent having a preventive or therapeutic effect on a disease, it has been found that the use of two or more selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same, is significantly more effective than the use of each of them alone. Neurodegeneration The present invention was completed by confirming the fact that the compound exhibits a preventive or therapeutic effect against a disease.

[0005] Thus, the present invention relates to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent Neurodegeneration The object is to provide a pharmaceutical composition for preventing or treating a disease.

[0006] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of a pulmonary artery disease comprising administering to said ... Neurodegeneration Another object of the present invention is to provide a health functional food for preventing or improving diseases.

[0007] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to an individual in need thereof at least two members selected from the group consisting of an interleukin 10 (IL-10) protein or a gene encoding the same, a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and a glutamate decarboxylase (GAD) protein or a gene encoding the same, Neurodegeneration It is yet another object to provide a method for preventing or treating a disease.

[0008] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: Neurodegeneration It is yet another object of the present invention to provide use of two or more members selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same, for the manufacture of a medicament for the prevention or treatment of a disease.

[0009] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention provides a method for the treatment of neurodegenerative diseases comprising administering to the patient a therapeutically effective amount of a therapeutically effective amount of a medicament for the treatment of neurodegenerative diseases, comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of neurodegenerative diseases, the method ... Neurodegeneration Pharmaceutical compositions for the prevention or treatment of disease are provided.

[0011] In one embodiment of the present invention, the pharmaceutical composition may comprise interleukin 10 (IL-10) protein or a gene encoding the same and glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same.

[0012] In another embodiment of the present invention, the pharmaceutical composition may further comprise a glutamate decarboxylase (GAD) protein or a gene encoding the same.

[0013] In yet another embodiment of the present invention, the gene may be contained in a vector.

[0014] In yet another embodiment of the present invention, the vector may be a viral or non-viral vector.

[0015] In yet another embodiment of the present invention, the viral vector may be one or more selected from the group consisting of Adenovirus, Adeno-associated virus (AAV), Herpes simplex virus, Lentivirus, Retrovirus, Cytomegalovirus, Baculovirus, and Poxvirus.

[0016] In yet another embodiment of the present invention, the non-viral vector may be at least one selected from the group consisting of a plasmid, a liposome, a cationic polymer, a micelle, an emulsion, and a solid lipid nanoparticle.

[0017] In yet another embodiment of the present invention, the interleukin-10 protein may have the amino acid sequence set forth in SEQ ID NO:1.

[0018] In yet another embodiment of the present invention, the gene encoding interleukin-10 may be the nucleotide sequence represented by SEQ ID NO:2 or SEQ ID NO:3.

[0019] In yet another embodiment of the present invention, the neuroglial cell-derived trophic factor protein may have the amino acid sequence represented by SEQ ID NO:4.

[0020] In yet another embodiment of the present invention, the gene encoding the neuroglial cell-derived trophic factor may have a nucleotide sequence represented by SEQ ID NO:5 or SEQ ID NO:6.

[0021] In yet another embodiment of the present invention, the glutamate decarboxylase may be glutamate decarboxylase 65 or glutamate decarboxylase 67.

[0022] In yet another embodiment of the present invention, the glutamic acid decarboxylase protein may have the amino acid sequence represented by SEQ ID NO:7 or SEQ ID NO:9.

[0023] In yet another embodiment of the present invention, the gene encoding glutamic acid decarboxylase may be the nucleotide sequence represented by SEQ ID NO:8 or SEQ ID NO:10.

[0024] In still another embodiment of the present invention, Neurodegeneration The disease may be one or more selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, frontotemporal lobar degeneration, dementia with Lewy bodies, vascular dementia, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, primary lateral sclerosis, spinal muscular atrophy, stroke, cerebral infarction, head trauma, spinal cord injury, cerebral arteriosclerosis, Lu Gehrig's disease, and multiple sclerosis.

[0025] In yet another embodiment of the present invention, the pharmaceutical composition may further comprise a physiologically acceptable carrier.

[0026] In yet another embodiment of the present invention, the pharmaceutical composition may be an injection.

[0027] In yet another embodiment of the present invention, the pharmaceutical composition may have an effect of improving movement disorders caused by the death of nerve cells.

[0028] In still another embodiment of the present invention, the neuron may be a motor neuron, an inter neuron, or a sensory neuron.

[0029] In yet another embodiment of the present invention, the pharmaceutical composition may have the effect of improving cognitive ability and memory.

[0030] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of a pulmonary artery disease comprising administering to said ... Neurodegeneration To provide a functional health food for preventing or improving a disease.

[0031] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to an individual in need thereof at least two members selected from the group consisting of an interleukin 10 (IL-10) protein or a gene encoding the same, a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and a glutamate decarboxylase (GAD) protein or a gene encoding the same, Neurodegeneration Methods for preventing or treating disease are provided.

[0032] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: Neurodegeneration The present invention provides use of two or more members selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same, for the manufacture of a drug for the prevention or treatment of a disease. Effect of the Invention

[0033] The composition of the present invention comprises two or more selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same. Thus, the pharmaceutical composition provides a more effective therapeutic effect by administering active ingredients in combination than by administering them alone. Neurodegeneration Since the pharmaceutical composition of the present invention shows the effect of improving and preventing diseases, it can show the same or even better effect even at a low concentration of the active ingredient. Neurodegeneration They can be usefully employed in the alleviation, prevention or treatment of diseases.

[0034] However, it should be understood that the effects of the present invention are not limited to the above effects, but include all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief description of the drawings]

[0035] [Figure 1a]The results confirm the effect of combined administration of hIL-10 and hGDNF on improving neurodegenerative disease. Figure 1a shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve fibers than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1b shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve cells than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1c shows the results of a rotation test performed in the rat compacta region, and Figure 1d shows the results of a rotation test in which administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective at improving symptoms of reduced motor function than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with the test substance and administration of 6-OHDA. [Figure 1b]The results confirm the effect of combined administration of hIL-10 and hGDNF on improving neurodegenerative disease. Figure 1a shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve fibers than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1b shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve cells than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1c shows the results of a rotation test performed in the rat compacta region, and Figure 1d shows the results of a rotation test in which administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective at improving symptoms of reduced motor function than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with the test substance and administration of 6-OHDA. [Figure 1c]The results confirm the effect of combined administration of hIL-10 and hGDNF on improving neurodegenerative disease. Figure 1a shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve fibers than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1b shows the results of confirming in the striatum region of the brain that administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective in protecting dopaminergic nerve cells than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with a test substance and administration of 6-OHDA. Figure 1c shows the results of a rotation test performed in the rat compacta region, and Figure 1d shows the results of a rotation test in which administration of rAAV5-hIL-10 / hGDNF (combined administration) was more effective at improving symptoms of reduced motor function than administration of rAAV5-hIL-10 or rAAV5-hGDNF alone in an animal model of neurodegenerative disease induced by pretreatment with the test substance and administration of 6-OHDA. [Diagram 2] This is the result of confirming the long-term improvement of motor function in an animal model of neurodegenerative disease. rAAV5-hIL-10 / hGDNF was injected into a neurodegenerative disease model induced by administration of 6-OHDA, and the improvement of motor function was confirmed through a rotation test. [Figure 3a] This is a result of confirming the neuronal protection effect of the gene therapy of the present invention on dopaminergic neurons and fibers. After pretreatment with one of the gene therapies of the present invention (rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65), 6-OHDA was administered to induce a neurodegenerative disease animal model to confirm the dopaminergic neuronal protection effect. Specifically, Figure 3a shows the results confirmed in Striatum, and Figure 3b shows the results confirmed in SNpc (Substantia nigra pars compacta). [Figure 3b]This is a result of confirming the neuronal protection effect of the gene therapy of the present invention on dopaminergic neurons and fibers. After pretreatment with one of the gene therapies of the present invention (rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65), 6-OHDA was administered to induce a neurodegenerative disease animal model to confirm the dopaminergic neuronal protection effect. Specifically, Figure 3a shows the results confirmed in Striatum, and Figure 3b shows the results confirmed in SNpc (Substantia nigra pars compacta). [Figure 4a] The present invention's gene therapy was used to confirm the effect of preventing hypokinesia in a mouse model of neurodegenerative disease induced by pretreatment with one of the gene therapies of the present invention (rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65) and treatment with 6-OHDA. Specifically, Figures 4a and 4b show the results of a rotation test 4 and 10 weeks after induction of the animal disease model, respectively. Figure 4c shows the results of a Rota-rod test 2 weeks after induction of the animal disease model. [Figure 4b] The present invention's gene therapy was used to confirm the effect of preventing hypokinesia in a mouse model of neurodegenerative disease induced by pretreatment with one of the gene therapies of the present invention (rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65) and treatment with 6-OHDA. Specifically, Figures 4a and 4b show the results of a rotation test 4 and 10 weeks after induction of the animal disease model, respectively. Figure 4c shows the results of a Rota-rod test 2 weeks after induction of the animal disease model. [Figure 4c] The present invention's gene therapy was used to confirm the effect of preventing hypokinesia in a mouse model of neurodegenerative disease induced by pretreatment with one of the gene therapies of the present invention (rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65) and treatment with 6-OHDA. Specifically, Figures 4a and 4b show the results of a rotation test 4 and 10 weeks after induction of the animal disease model, respectively. Figure 4c shows the results of a Rota-rod test 2 weeks after induction of the animal disease model. [Figure 5a]The results show that the combined administration of hIL-10 and hGDNF supernatants or the simultaneous administration of hIL-10, hGDNF and hGAD65 supernatants has an anti-apoptotic effect against oxidative stress. The combined supernatants were produced by transfection with each therapeutic gene plasmid. Specifically, in the case of neuronal death caused by H2O2 (Figure 5a), and in the case of neuronal death caused by 6-OHDA (Figure 5b), it was confirmed that the combined administration of hIL-10 and hGDNF supernatants or the simultaneous administration of hIL-10, hGDNF and hGAD65 supernatants had an inhibitory effect against neuronal death. [Figure 5b] The results show that the combined administration of hIL-10 and hGDNF supernatants or the simultaneous administration of hIL-10, hGDNF and hGAD65 supernatants has an anti-apoptotic effect against oxidative stress. The combined supernatants were produced by transfection with each therapeutic gene plasmid. Specifically, in the case of neuronal death caused by H2O2 (Figure 5a), and in the case of neuronal death caused by 6-OHDA (Figure 5b), it was confirmed that the combined administration of hIL-10 and hGDNF supernatants or the simultaneous administration of hIL-10, hGDNF and hGAD65 supernatants had an inhibitory effect against neuronal death. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The inventors have Neurodegeneration As a result of research and efforts to develop a therapeutic agent having a preventive or therapeutic effect on a disease, it has been found that the use of two or more proteins selected from the group consisting of interleukin 10 (IL-10) protein or a gene encoding the same, glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and glutamate decarboxylase (GAD) protein or a gene encoding the same has a superior effect compared to using each protein alone. Neurodegeneration The fact that it has preventive or therapeutic effects on diseases was confirmed, which led to the completion of the present invention.

[0037] Thus, the present invention relates to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent comprising administering to said patient a therapeutic agent Neurodegeneration Pharmaceutical compositions for the prevention or treatment of disease are provided.

[0038] In the present invention, the term "prevention" means Neurodegeneration It means any action of administering the pharmaceutical composition provided in the present invention to an individual who is expected to develop a disease, thereby suppressing or delaying the onset of the disease.

[0039] In the present invention, the term "treatment" refers to any clinical intervention aimed at altering the natural processes of the individual or cells to be treated, which may be performed during or to prevent the progression of a clinical pathological condition. The intended therapeutic effect includes preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or palliating the disease state, improving the amelioration or prognosis of the disease state. For the purposes of the present invention, said treatment is defined as: Neurodegeneration A pharmaceutical composition of the present invention is administered to a patient suffering from a disease, Neurodegeneration It is understood to include any action that improves the symptoms of a disease, but is not limited to this.

[0040] The combination of two or more of the above may be interleukin-10 (IL-10) and glial cell-derived trophic factor (GDNF); interleukin-10 (IL-10) and glutamic acid decarboxylase (GAD); glial cell-derived trophic factor (GDNF) and glutamic acid decarboxylase (GAD); interleukin-10 (IL-10), glial cell-derived trophic factor (GDNF) and glutamic acid decarboxylase (GAD), and the IL-10, GDNF and GAD may each be a protein individually or a gene encoding the same.

[0041] The gene may be DNA or RNA (specifically, mRNA) and may be operably contained in a vector. Specifically, the gene may be operably contained in a viral vector or a non-viral vector.

[0042] The gene encoding IL-10 may be operably contained in a first vector, the gene encoding GDNF may be operably contained in a second vector, and the gene encoding GAD may be operably contained in a third vector, or two or more genes may be contained in one vector.

[0043] The two or more genes selected from the group consisting of a gene encoding IL-10, a gene encoding GDNF, and a gene encoding GAD are vector In this case, the above vector may be a viral vector or a non-viral vector such as a plasmid or liposome, etc. Also, a part of the gene may be contained in a viral vector, and the remaining gene may be contained in a non-viral vector.

[0044] In one specific example, IL-10 may be contained in a viral vector, and GDNF may be contained in a non-viral vector. GDNF may be contained in a viral vector, and IL-10 may be contained in a non-viral vector. Furthermore, IL-10 may be contained in a viral vector, and GAD may be contained in a non-viral vector. IL-10 may be contained in a viral vector, and GDNF and GAD may be contained in a non-viral vector. IL-10 and GDNF may be contained in a viral vector, and GAD may be contained in a non-viral vector. IL-10 and GAD may be contained in a viral vector, and GDNF may be contained in a non-viral vector. IL-10 may be contained in a viral vector, and GDNF and GAD may be contained in a non-viral vector. IL-10 may be contained in a viral vector, and GDNF and GAD may be contained in a non-viral vector. GDNF and GAD may be contained in a viral vector, and IL-10 may be contained in a non-viral vector. Also, GDNF may be contained in a viral vector, and IL-10 and GAD may be contained in a non-viral vector. Also, IL-10, GDNF and GAD may all be contained in a viral vector. IL-10, GDNF and GAD may all be contained in a non-viral vector.

[0045] The viral vector may be one or more selected from the group consisting of, but not limited to, adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, and poxvirus. Preferably, the viral vector may be an adeno-associated virus.

[0046] The non-viral vector may be at least one selected from the group consisting of a plasmid, a liposome, a cationic polymer, a micelle, an emulsion, and a solid lipid nanoparticle.

[0047] In the present invention, the term "plasmid" refers to a circular DNA fragment that exists separately from the bacterial chromosome. Plasmids do not contain genes essential for bacterial survival, but contain genes essential for resistance to certain antibiotics and gene exchange between bacteria. Plasmids can also replicate independently of the chromosome and can contain a selectable marker.

[0048] In the present invention, the term "liposome" refers to a vesicle formed by a bilayer of hydrophilic and hydrophobic parts when a molecule having both hydrophobic and hydrophilic parts in one molecule, such as phospholipids, is suspended in an aqueous solution. Liposomes are isolated from the outer membrane by a membrane composed of a lipid bilayer, and liposomes containing DNA, mRNA, etc. can be used as a medium for genetic information.

[0049] The term "cationic polymer" as used in the present invention refers to a cationic lipid or polymer compound that forms a complex with anionic DNA through ionic bonds and is delivered into cells.

[0050] The term "micelle" used in the present invention means a thermodynamically stable colloidal aggregate formed by the association of molecules consisting of polar and non-polar hydrophobic groups, such as surfactants and lipid molecules, in a solution due to van der Waals forces, etc. Micelles encapsulating DNA, mRNA, etc. can be used as a medium for genetic information.

[0051] The term "emulsion" as used in the present invention means that when two solutions of different phases are mixed, one liquid forms fine particles and is dispersed in the other liquid. DNA, mRNA, etc. can be encapsulated in the center of the emulsion particles and used as a carrier of genetic information.

[0052] The term "solid lipid nanoparticles" as used in the present invention refers to a formulation in which a drug is encapsulated in nano-sized particles made of solid lipids instead of liquid lipids.

[0053] The present invention relates to a method for treating a pulmonary arthritis, comprising administering to a patient a therapeutically effective amount of a pulmonary arthritis or a pulmonary arthritis. vector 1 (e.g., the first vector) and vector Contains one or more genes selected from the remaining group of genes not included in 1 vector 2 (for example, the second vector) may be mixed at a ratio of 1:1 to 100 or 1 to 100:1. vector 1 and vector The mixing ratio of the two VG standards may be 1:1 to 100 or 1 to 100:1.

[0054] The present invention relates to a method for the preparation of a polypeptide comprising the IL-10 encoding gene of the present invention. vector 1 (e.g., the first vector), which contains the coding gene for GDNF vector 2 (e.g., the second vector) and a gene encoding GAD vector 3 (e.g., a third vector) may be mixed in various ratios, for example, but not limited to, the mixing ratio may be 1:1:1 to 1:1:100; 1:1:1 to 1:100:1; 1:1:1 to 100:1:1; 1:1:1 to 1:100:100; 1:1:1 to 100:100:1; or 1:1:1 to 100:1:100.

[0055] In the present invention, the term "operably" means that the transgene is linked to a regulatory sequence in such a manner that the transgene can be expressed in a host cell. The regulatory sequence is a DNA base sequence that regulates the expression of a gene, and may include a promoter and other regulatory elements such as an enhancer and polyadenylation. The regulatory sequence may also provide a binding site for a transcription factor that regulates the expression of the transgene, and may affect the complex structure with the transcription factor to determine the function of the transcription factor.

[0056] In the present invention, the term "IL-10" refers to an anti-inflammatory cytokine belonging to class II cytokines (Renauld, Nat Rev Immunol, 2003). The IL-10 is a homodimer consisting of two subunits each having a length of 178 amino acids, and is also known as a human cytokine synthesis inhibitory factor (CSIF). IL-10 functions to suppress the activity of NK (Natural Killer) cells in immune responses, and forms a complex with an IL-10 receptor to participate in signal transduction. The IL-10 may be, but is not limited to, a protein derived from human, mouse, rat, dog, cat, or horse, or a base sequence encoding the same. The IL-10 may be an amino acid sequence of SEQ ID NO: 1 of NCBI accession no. NP_000563.1, and the gene encoding the same may be a base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.

[0057] The IL-10-encoding gene may be a nucleotide sequence encoding an IL-10 mutant that maintains the activity of IL-10. The nucleotide sequence encoding the IL-10 mutant may be a nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity with the IL-10 amino acid sequence shown, and most preferably, an amino acid sequence having 95% or more sequence identity.

[0058] Furthermore, the nucleotide sequence encoding the IL-10 mutant may be a nucleotide sequence having at least 60% or more, 70% or more, 80% or more, or 90% or more sequence homology to the IL-10 nucleotide sequence shown above, and most preferably a nucleotide sequence having 95% or more sequence homology.

[0059] The "percentage of sequence identity" is determined by comparing the comparison regions when the two sequences are optimally aligned, and a portion of the base sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0060] In the present invention, the term "GDNF" refers to a nerve growth factor that constitutes the GDNF ligand family, which is composed of GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN). The GDNF is a protein that promotes the survival of many types of nerve cells, and transmits signals via GFRα1 (GDNF receptor alpha-1), GFRα2 (GDNF receptor alpha-2), and GFRα3 (GDNF receptor alpha-3). The GDNF may be, but is not limited to, a protein derived from human, mouse, rat, dog, cat, or horse, or a base sequence encoding the same. The GDNF may be composed of the amino acid sequence of SEQ ID NO: 4 of NCBI accession no. NCBI NP_954701.1, and the gene encoding the same may be a base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.

[0061] The GDNF-encoding gene may be a nucleotide sequence encoding a GDNF variant that maintains the activity of GDNF. The nucleotide sequence encoding the GDNF variant may be a nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 80%, 90% or more sequence identity with the indicated GDNF amino acid sequence, and most preferably, an amino acid sequence having 95% or more sequence identity.

[0062] In addition, the base sequence encoding the GDNF mutant may be a base sequence having at least 60%, 70%, 80%, or 90% sequence homology to the GDNF base sequence shown above, and most preferably a base sequence having 95% or more sequence homology.

[0063] In the present invention, the term "GAD" refers to an enzyme that decarboxylates glutamic acid to produce GABA (gamma-aminobutyric acid). The GAD may be GAD65 or GAD67. Specifically, the GAD65 may be, but is not limited to, a protein derived from humans, mice, rats, dogs, cats, or horses, or a base sequence encoding the same. The GAD65 may have the amino acid sequence of SEQ ID NO: 7 of NCBI accession no. NP_000809.1, and the gene encoding the same may have the base sequence of SEQ ID NO: 8. The GAD67 may be, but is not limited to, a protein derived from humans, mice, rats, dogs, cats, or horses, or a base sequence encoding the same. The GAD67 may have the amino acid sequence of SEQ ID NO: 9 of NCBI accession no. NP_000808.2, and the gene encoding the same may have the base sequence of SEQ ID NO: 10.

[0064] The GAD-encoding gene may also be a nucleotide sequence encoding a GAD mutant that maintains the activity of GAD and is capable of producing GABA. The GAD mutant includes all sequences that maintain the property of GAD to produce GABA, Those Although not limited to, preferably, the nucleotide sequence encoding the GAD variant may be a nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity to the displayed GAD amino acid sequence, and most preferably, a nucleotide sequence encoding an amino acid sequence having 95% or more sequence identity.

[0065] In addition, the base sequence encoding the GAD mutant may be a base sequence having at least 60% or more, 70% or more, 80% or more, or 90% or more sequence homology to the GAD base sequence shown above, and most preferably, a base sequence having 95% or more sequence homology.

[0066] The pharmaceutical composition of the present invention comprises: treatment Gene or containing vector in Neurodegeneration The compositions of the present invention have different mechanisms for preventing the disease. Therapeutic Agents By administering these drugs together, the effects were even more excellent than when administered alone. Neurodegeneration It is possible to achieve preventive or therapeutic effects on diseases.

[0067] In particular, according to the present invention, when two or more genes selected from the group consisting of genes encoding IL-10, GDNF, and GAD65 are administered in combination, a synergistic effect is observed. Neurodegeneration The pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect against a disease. Neurodegeneration The present invention can be useful for the prevention or treatment of diseases, specifically, diseases induced by neuronal cell death. Neurodegeneration It can improve the movement disorder of the disease, Neurodegeneration It can improve cognitive and memory decline, which is a major symptom of the disease.

[0068] In the present invention, each vector containing IL-10, GDNF and GAD65 may be administered alone or in combination of two or more kinds, or in a form in which one vector contains two or more genes. The administration site is not limited thereto, but IL-10 may be administered to the striatum (ST), GDNF may be administered to the striatum (ST), and GAD65 may be administered to the subthalamic nucleus (SN). The administration site of rAAV5-hGDNF / hIL-10, which is one of the above examples, may be, but is not limited thereto, the striatum (ST).

[0069] In the present invention, the nerve cells are signal These are distributed in the central nervous system through motor neurons, which transmit signals to the brain and spinal cord, and then to effector organs such as muscle cells and secretory organs. signal from sensory neurons to motor neurons signal The neurons may be sensory neurons that transmit signals to the brain and spinal cord, and then to effector organs such as muscle cells and secretory organs.

[0070] In the present invention, Neurodegeneration The disease may be, but is not limited to, one or more selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, frontotemporal lobar degeneration, dementia with Lewy bodies, vascular dementia, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, primary lateral sclerosis, spinal muscular atrophy, stroke, cerebral infarction, head trauma, spinal cord injury, cerebral arteriosclerosis, Lu Gehrig's disease, and multiple sclerosis, and is preferably Parkinson's disease, dementia, or Alzheimer's disease.

[0071] Based on specific examples, the present inventors have demonstrated that the pharmaceutical composition of the present invention Neurodegeneration It was confirmed that it can be useful for the prevention or treatment of diseases.

[0072] In one embodiment of the present invention, rAAV5-hIL-10 / hGDNF Neurodegeneration When administered to a mouse model of induced disease, Neurodegeneration Motor dysfunction, a symptom induced by the disease, Statistically significant (See Example 1).

[0073] In another embodiment of the present invention, For 6-OHDA-induced neurodegenerative disease models Pretreatment with rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 By doing , Neurodegenerative diseases Symptoms include dopaminergic neuron death and motor dysfunction Changed It was confirmed that it was improved The improvement of this neurodegenerative condition is induced by administering the pharmaceutical composition of the present invention. (See Example 2).

[0074] In yet another embodiment of the present invention, Each gene Plasmid-produced hIL-10 and hGDNF Supernatant or Each gene was produced by plasmid hIL-10, hGDNF and hGAD65 Supernatant It was confirmed that pretreatment with H2O2 or 6-OHDA suppressed the death of nerve cells induced by H2O2 or 6-OHDA (see Example 3).

[0075] Based on the above results, the present inventors have confirmed that the pharmaceutical composition of the present invention Neurodegeneration caused by It has been confirmed that the disease can be prevented or treated.

[0076] The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent that is conventionally used in the manufacture of pharmaceutical compositions. Group ofThe composition may be in various oral or parenteral dosage forms. When preparing the formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant that is commonly used. The solid preparation for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.

[0077] In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. can also be used. As liquid preparations for oral administration, suspensions ,milk Examples of the diluent include aqueous solutions, syrups, etc., and in addition to water and liquid paraffin, which are frequently used simple diluents, various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, and suppositories. Examples of non-aqueous solvents and suspension solvents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of the base for suppositories include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0078] The pharmaceutical composition of the present invention may be in the form of a tablet, a pill, a powder, a granule, a capsule, a suspension, or the like. ,milk The pharmaceutical composition may have any one of the dosage forms selected from the group consisting of a pharmaceutical preparation, a syrup, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, and a suppository.

[0079] In yet another aspect of the invention, the method includes administering the pharmaceutical composition to an individual. Neurodegeneration Methods for preventing or treating disease are provided.

[0080] In the present invention, the term "individual" refers to Neurodegeneration This refers to any animal, including humans, that may develop or has developed a disease. By administering the composition of the present invention to an individual, Neurodegeneration The symptoms caused by the disease can be alleviated or cured.

[0081] In the present invention, the term "alleviation" refers to the administration of a composition according to the present invention. Neurodegeneration It refers to any action that improves the symptoms of a disease or provides a beneficial effect.

[0082] The compositions of the present invention are administered in a pharma- ceutically effective amount.

[0083] In the present invention, the term "administration" refers to administration in any suitable manner. individual introducing the pharmaceutical composition of the present invention into Pharmaceutical Compositions of the Present Invention can reach the destination organization History Administration can be via a variety of routes, including orally or parenterally.

[0084] The pharmaceutical composition of the present invention can be appropriately administered to an individual according to the purpose or need by a conventional method, administration route, and dosage used in the art. Examples of administration routes include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0085] In addition, an appropriate dosage and frequency of administration can be selected by a method known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention to be actually administered can be appropriately determined depending on various factors such as the type of symptoms to be treated, the administration route, sex, health condition, diet, age and weight of the individual, and the severity of the disease.

[0086] The frequency of administration may be, in the case of a viral vector, once or more per year, and in the case of repeated administration, at intervals of one day to one month, or one month to one year. In addition, in the case of a non-viral vector, the pharmaceutical composition may be administered once or more per year, and in the case of repeated administration, at intervals of 12 to 24 hours, or one day to 14 days. In addition, in the case of a protein, the pharmaceutical composition may be administered once or more per year, and in the case of repeated administration, at intervals of one day to one month, or one month to one year.

[0087] In the present invention, the term "pharmacologically effective amount" refers to a reasonable benefit / risk ratio applicable to medical use. Neurodegeneration It means an amount sufficient to suppress or alleviate a disease, and the effective dose level can be determined depending on factors including the type and severity of the individual, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0088] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or in multiple doses. Taking all of the above factors into consideration, it is possible to minimize side effects and to minimize side effects. valid It is important to administer an amount that will provide the maximum effect, and this can be easily determined by one skilled in the art.

[0089] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of a pulmonary artery disease comprising administering to said ... Neurodegeneration To provide a functional health food for preventing or improving a disease.

[0090] The above-mentioned "interleukin-10", "glial cell-derived trophic factor", "glutamic acid decarboxylase", "protein", "gene", Neurodegeneration "Disease," "prevention," etc. may be within the scope described above.

[0091] The term "improvement" refers to any action that at least reduces the parameters associated with the condition being treated, for example, the severity of symptoms. In this case, the health functional food is Neurodegeneration They can be used simultaneously or separately with therapeutic agents before or after the onset of the disease to prevent or ameliorate the disease.

[0092] In the health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately by a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use (for prevention or improvement). In general, when producing a food or beverage, the health functional food can be added to the raw material in an amount of about 15% by weight or less, more specifically about 10% by weight or less. 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.

[0093] The health functional food may further include one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which the compound according to the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0094] Specific examples of the carrier, excipient, diluent and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0095] In addition to containing the active ingredient, the health functional food may contain other ingredients as essential ingredients without any special restrictions. For example, it may contain various flavorings or natural carbohydrates as additional ingredients, like ordinary beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as ordinary sugars, such as dextrin, cyclodextrin, etc., and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. As flavorings other than those mentioned above, natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

[0096] In addition to the above, the health functional food according to one aspect may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. Such ingredients may be used independently or in combination, and the proportions of such additives may also be appropriately selected by those skilled in the art.

[0097] The present invention also relates to a method for treating a pulmonary artery disease comprising administering to an individual in need thereof at least two members selected from the group consisting of an interleukin 10 (IL-10) protein or a gene encoding the same, a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same, and a glutamate decarboxylase (GAD) protein or a gene encoding the same, Neurodegeneration Methods for preventing or treating disease are provided.

[0098] The above-mentioned "interleukin-10", "glial cell-derived trophic factor", "glutamic acid decarboxylase", "protein", "gene", Neurodegeneration "Disease", "prevention", "treatment", "individual" and the like may fall within the scope as previously mentioned.

[0099] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: Neurodegeneration A method for producing a drug for preventing or treating a disease, comprising the steps of: producing an interleukin 10 (IL-10) protein or a gene encoding the same; ...GDNF) protein or a gene encoding the same; and producing an interleukin 10 (GAD) protein or a gene encoding the same, wherein the interleukin 10 (IL-10) protein or a gene encoding the same is selected from the group consisting of an interleukin 10 (IL-10) protein or a gene encoding the same; and producing an interleukin 10 (GL-10) protein or a gene encoding the same. preparation to provide.

[0100] The above-mentioned "interleukin-10", "glial cell-derived trophic factor", "glutamic acid decarboxylase", "protein", "gene", Neurodegeneration "Disease", "prevention", "treatment" and the like may be within the scope described above.

[0101] 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 the contents of the present invention are not limited to the following examples.

[0102] [Example] Example 1. Confirmation of synergistic effect of combined administration of hIL-10 and hGDNF on improving neurodegenerative diseases The present invention The cure One of the therapeutic agents is rAAV5-hIL-10 / hGDNF. Neurodegeneration To confirm the effect of saline, rAAV5-hIL-10, rAAV5-hGDNF and rAAV5-hIL-10 / hGDNF on the disease, Neurodegenerative diseases Administered to animal models , D Inhibition of paminergic neuronal cell death and improvement of motor function Goodness Confirmed.

[0103] 1-1. Preparation of animal model Neurodegeneration Animals affected by the disease and Human In either case, brain In nerve Transmittal Quality It is known that changes occur in the brain and cause a decline in motor ability. Neurodegeneration Animal models to confirm disease improvement effects 、C 57BL / 6 Induction with mouse did. Neurodegeneration To create a mouse model of the disease, 6-hydroxydopamine (6-OHDA), which has been known to induce dopaminergic neuronal death and abnormal excitatory neurotransmission in the subthalamic nucleus (SN), was injected into the left striatum (ST) of mice. injection Specific Neurodegeneration Disease mouse models Induction The method is as follows.

[0104] The mice were treated with alfaxan-sedator mixtureThe animals were anesthetized by intraperitoneal administration of 100 mg / kg / day, and the head of the anesthetized animal was fixed using the nose ring and earrings of a stereotaxic instrument (RWD life science). The heights of Bregma and lambda of the mouse were then measured, and the head was adjusted so that it could be level. Target area After checking the coordinates, use the micromotor drill (Seashin) to drill the location. Skull A hole was made in the bone. Hamilton syringe (10μL syringe was used, but not limited to this) Approximately 2 μL of test substance was used. Substances (which may be saline, 0.1% ascorbic acid, 6-hydroxydopamine (OHDA), or rAAV5-GFP, which may vary from experiment to experiment) were added. 、s Using a stereotaxic injection pump (DONGBANG HITECH trading company), the rate was 0.5μL / min. Throw it Granted. After injection , using black silk Brain cortex After suturing, disinfect with povidone. Home After returning the animal to its cage, anesthesia was administered. Wake up from to Using infrared heaters Body temperature Stabilize Ta.

[0105] 1-2. Confirmation of the protective effect of combined administration of hIL-10 and hGDNF on dopaminergic neurons in animal models Specifically, to confirm the inhibitory effect of saline, rAAV5-hIL-10, rAAV5-hGDNF, and rAAV5-hIL-10 / hGDNF on dopaminergic neuronal cell death, mice were pretreated with saline, rAAV5-hIL-10, rAAV5-hGDNF, and rAAV5-hIL-10 / hGDNF, and then 6-OHDA was administered two weeks later. injection death, Neurodegenerative diseases Animal models Induction SNpc (Substantia nigra pars compacta) and SNpc Nerve Thin CellIn order to confirm the protective effect of dopaminergic neurons in the striatum (ST), where the axons of the dopaminergic neurons extend, 3,3'-diaminobenzidine staining (DAB) was performed. The experiment was carried out by 6 weeks after the administration of saline, rAAV5-hIL-10, rAAV5-hGDNF, and rAAV5-hIL-10 / hGDNF (4 weeks after 6-OHDA administration), and then the mice were sacrificed and the brain was Collect To prevent non-specific color development, the sections were treated with 1% H2O2 for 15 minutes, and then treated with anti-TH antibody to detect tyrosine hydroxylase (TH), a known representative dopaminergic neuron marker, and incubated at 4°C for more than 8 hours. Incubate The biotinylated antibody was applied to the tissue, and then attached to the anti-TH antibody. The avidin-biotin enzyme conjugate was bound to the biotinylated antibody using the ABC KIT, and the bound biotin was colored brown using the DAB KIT. Brightfield was observed under a microscope.

[0106] As a result, as shown in Figure 1a, the dopaminergic neuron death level in the striatum was significantly lower in the group pretreated with rAAV5-hIL-10 / hGDNF than in the groups pretreated with saline, rAAV5-hIL-10, or rAAV5-hGDNF alone. Furthermore, the dopaminergic neuron death level in the SNpc was significantly lower in the groups pretreated with rAAV5-hIL-10 / hGDNF than in the groups pretreated with saline, rAAV5-hIL-10, or rAAV5-hGDNF alone (Figure 1b).

[0107] 1-3.rAAV5-hIL-10 / hGDNF Neurodegeneration Confirmation of the effect of improving motor impairment symptoms in disease animal models In addition, a rotation test was performed to confirm the motor function improving effects of saline, rAAV5-hIL-10, rAAV5-hGDNF, and rAAV5-hIL-10 / hGDNF. Saline After dissolving in Neurodegeneration The drug was administered subcutaneously at 4 mg / kg to a diseased mouse model. After 5 minutes, the direction and extent of the animal's movements were recorded using a camera for 20 minutes. Drawing They then used the recorded video to analyze how much the animals rotated clockwise.

[0108] As a result, the gene therapy of the present invention Treatment( rAAV5-hIL-10 / hGDNF) teeth, Mice administered saline, rAAV5-hIL-10, or rAAV5-hGDNF Compared to In comparison Improved motor activity was observed , and improvement of motor function decline Certain recognition Be (Figure 1c).

[0109] 1-4.rAAV5-hIL-10 / hGDNF Long-term neurodegeneration Confirmation of disease treatment effectiveness The present invention 1 of 1 One of these is rAAV5-hIL-10 / hGDNF Long-term neurodegeneration Disease treatment efficacy Results To confirm, 6-OHDA Animals induced by Model In contrast, After a week has passed, This is one of the present invention Administration of rAAV5-hIL-10 / hGDNF did The specific experimental method is shown in Table 1 below.

[0110] [Table 1]

[0111] The same method as in Example 1-3 De Post-treatment with AAV5-hGDNF / hIL-10 , rotation test was performed If Neurodegeneration Disease abnormalityline Dynamic As a result of confirming the improvement effect, as shown in Figure 2, administration of rAAV5-hIL-10 / hGDNF alone did not improve the 6-OHDA-induced IL-10 / hGDNF ... abnormality line Movement It was confirmed that the effects were improved and that such effects were maintained for a long period of time even 5 weeks after administration of the rAAV5-hIL-10 / hGDNF.

[0112] Based on the above results, the present inventors concluded that when the gene therapy agent of the present invention (rAAV5-hIL-10 / hGDNF) is administered, Neurodegeneration It was confirmed that the motor impairment symptoms of the disease can be prevented as well as treated, and that such preventive and therapeutic effects are maintained for a long period of time.

[0113] Example 2. Confirmation of the preventive effect of neurodegenerative disease when rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 are administered simultaneously 2-1. Confirmation of the protective effect of rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 on dopaminergic neuronal cells in animal models In order to confirm the effect of rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 (simultaneous administration), which is one of the gene therapy agents of the present invention, on the suppression of dopaminergic neuronal death, mice were pretreated with rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65, and 6-OHDA was administered two weeks later. Induction of neurodegenerative diseases in mice SNpc (Substantia nigra pars compacta) and SNpc Nerve In order to confirm the protective effect on dopaminergic neurons in the striatum from which the axons of the cells extend, the same procedure as in Example 2-2 was carried out.

[0114] As a result, as shown in Figure 3a, the dopaminergic neuronal cell death level in Striatum was significantly increased by pretreatment with rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65. The crowd , not In a groupIt was also confirmed that the dopaminergic neuronal cell death levels in the SNpc were significantly lower in the rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 pretreatment groups compared to the 6-OHDA only treatment group, as shown in Figure 3b.

[0115] 2-2. rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 Neurodegeneration Confirmation of the effect of improving motor impairment symptoms in disease animal models In the method of Example 2-1 Diseased animals Mau Induce rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65 were pretreated 2 weeks before the experiment and prepared by the above method. Neurodegeneration The mouse model was subjected to a rotation test (apomorphine induced rotation) and a rota-rod test. The specific experimental method is shown in Table 2 below.

[0116] [Table 2]

[0117] (1) Rotation test results The rotation test was carried out in the same manner as in Example 1-3. 6-OHDA Infusion 4 weeks Compared with the saline injection group after 1 h The gene therapy agent of the present invention pre-injection did Neurodegenerative disease models , Significant exercise machine Noh Revised Goodness confirmation was done (Figure 4a) .Also ,As shown in Fig. 4b, Pretreatment for gene therapy was performed 6-OHDA -induced neurodegenerative diseases Model In , Modeling neurodegenerative diseases 10 weeks Even after a while , Saline injection Compared to the group Significant Hypokinetic treatment effect Certain Approval Be (Figure 4b).

[0118] Based on the above results, the present inventors concluded that when the gene therapy agent of the present invention (simultaneous administration of rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65) is administered, Neurodegeneration It was confirmed that the disease's hypokinetic symptoms can be prevented and that this preventive effect is maintained for a long period of time.

[0119] (2) Results of the Rota-rod study In the Rota-rod test, the mouse was placed on the cylinder of the Rota-rod behavioral experiment device, and the disk was rotated clockwise at a speed of 5 RPM for 2 minutes to allow the mouse to adapt to the device. The time that the mouse walked on the cylinder was measured while the cylinder was rotated at various RPMs. The experiment was repeated three times for each individual, and the average of the three measurements was shown as the representative value for the individual.

[0120] As a result, as shown in Figure 4c , Books The invention One When pretreated with rAAV5-hIL-10 / hGDNF and rAAV5-hGAD65, At the second week, when 6-OHDA injection significantly reduced motor function, 0.1% ascorbic acid only Administer S Ham control l and At a similar level Improved motor function observation Be Ta.

[0121] Example 3. Confirmation of the inhibitory effect of combined administration of hIL-10 and hGDNF supernatants produced by transfection of each therapeutic gene plasmid, or simultaneous administration of hIL-10, hGDNF, and hGAD65 supernatants on the death of nerve cells caused by oxidative stress hIL-10 and hGDNF Supernatant or combination of hIL-10, hGDNF and hGAD65 Supernatant When co-administered with Anti-apoptosis Check the effect The combined supernatant was generated by transfection of each therapeutic gene plasmid. 2 O 2 After inducing neuronal death by applying 6-OHDA, cell viability was confirmed, and each therapeutic gene plasmid was transfected to induce neuronal death. Produced hIL-10 and hGDNF Supernatant or hIL-10, hGDNF and hGAD65 Pretreat the supernatant did.

[0122] As a result, as can be seen from Figs. 5a and 5b, Each therapeutic gene was generated by plasmid transfection. hIL-10 and hGDNF Supernatant Concomitant administration of or each therapeutic gene plasmid transfection hIL-10, hGDNF and hGAD65 Supernatant It was confirmed that both administration of 1,2-dichlorophenylalanine and 1,2-dichlorophenylalanine suppressed neuronal cell death.

[0123] Through these results, it was revealed that not only the genes of hIL-10 and hGDNF, but also their respective proteins, Neurodegeneration It has been found to have preventive or therapeutic effects on various diseases, including Parkinson's disease, by having a neuroprotective effect on neurons regardless of the cause of neuronal death. Neurodegeneration It has been found to be effective against the disease.

[0124] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising an interleukin 10 (IL-10) protein or a gene encoding the same, and a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same.

2. The composition of claim 1, further comprising a glutamate decarboxylase (GAD) protein or a gene encoding the same.

3. The composition of claim 1 , wherein the gene is contained in a vector.

4. The composition of claim 3, wherein the vector is a viral or non-viral vector.

5. The composition according to claim 4, wherein the viral vector is at least one selected from the group consisting of adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, and poxvirus.

6. The composition according to claim 4, wherein the non-viral vector is at least one selected from the group consisting of a plasmid, a liposome, a cationic polymer, a micelle, an emulsion, and a solid lipid nanoparticle.

7. The composition of claim 1, wherein the interleukin-10 protein has the amino acid sequence represented by SEQ ID NO:

1.

8. The composition according to claim 1, wherein the gene encoding the interleukin-10 protein has a base sequence represented by SEQ ID NO:2 or SEQ ID NO:

3.

9. The composition of claim 1, wherein the neuroglial cell-derived trophic factor protein has an amino acid sequence represented by SEQ ID NO:

4.

10. The composition according to claim 1, characterized in that the gene encoding the neuroglial cell-derived trophic factor protein has a base sequence represented by SEQ ID NO:5 or SEQ ID NO:

6.

11. The composition of claim 2, wherein the glutamate decarboxylase is glutamate decarboxylase 65 or glutamate decarboxylase 67.

12. The composition according to claim 2, wherein the glutamic acid decarboxylase protein has an amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO:

9.

13. The composition according to claim 2, wherein the gene encoding the glutamic acid decarboxylase protein has a base sequence represented by SEQ ID NO: 8 or SEQ ID NO:

10.

14. The composition according to claim 1, characterized in that the neurodegenerative disease is one or more selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, frontotemporal lobar degeneration, dementia with Lewy bodies, vascular dementia, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, primary lateral sclerosis, spinal muscular atrophy, stroke, cerebral infarction, head trauma, spinal cord injury, cerebral arteriosclerosis, Lu Gehrig's disease and multiple sclerosis.

15. The composition of claim 1, wherein the pharmaceutical composition further comprises a physiologically acceptable carrier.

16. The composition according to claim 1, wherein the pharmaceutical composition is an injectable preparation.

17. The composition according to claim 1, characterized in that the pharmaceutical composition has an effect of improving movement disorders caused by neuronal death.

18. 18. The composition of claim 17, wherein the neuron is a motor neuron, a sensory neuron, or an inter neuron.

19. The composition according to claim 1, characterized in that the pharmaceutical composition has the effect of improving cognitive ability and memory.

20. A health functional food for preventing or improving a neurodegenerative disease, comprising an interleukin 10 (IL-10) protein or a gene encoding the same, and a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same.

21. Use of an interleukin 10 (IL-10) protein or a gene encoding the same and a glial cell line-derived neurotrophic factor (GDNF) protein or a gene encoding the same for the manufacture of a medicament for preventing or treating a neurodegenerative disease.

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