Composition for inhibiting alpha-synuclein aggregation and method for inhibiting aggregation

Abstract

The present invention relates to a composition for inhibiting alpha-synuclein aggregation and a method for inhibiting aggregation, more specifically, to a technology for inhibiting alpha-synuclein aggregation and phosphorylation by introducing Nurr1 and Foxa2 genes into brain cells and expressing them together. The composition according to the present invention has excellent effects of inhibiting the aggregation and phosphorylation of alpha-synuclein and can be used for the treatment and prevention of Parkinson's disease.

Description

[Technical Field]

[0001] The present invention relates to a composition and method for inhibiting alpha-synuclein aggregation, and more particularly to a technology for inhibiting alpha-synuclein aggregation and phosphorylation by introducing and inducing expression of Nurr1 and Foxa2 genes. [Background technology]

[0002] Parkinson's disease is a neurodegenerative disorder that causes movement disorders such as muscle tremors and muscle rigidity. Parkinson's disease primarily affects the elderly, and the risk of developing Parkinson's disease increases with age. In Korea, it is estimated that approximately 1-2 people per 1,000 people suffer from Parkinson's disease. Most cases of Parkinson's disease in the elderly are known to have little genetic influence. Parkinson's disease is known to develop as dopamine cells in the substantia nigra of the midbrain die. However, the exact cause of dopamine cell destruction in the substantia nigra remains unknown. As the average human lifespan continues to increase, the incidence of Parkinson's disease is expected to increase.

[0003] Furthermore, the management and treatment of Parkinson's disease requires enormous costs and causes considerable mental distress to patients, so there is a need for effective methods of preventing and treating Parkinson's disease.

[0004] In recent years, much research has focused on the alpha-synuclein protein in relation to Parkinson's disease. Alpha-synuclein is a protein abundant in the human brain and is primarily found at the ends of nerve cells in specialized structures called presynaptic terminals. Recent research has revealed that Parkinson's disease is associated with the formation of Lewy bodies, which are formed when the balance between the production and clearance of alpha-synuclein within neurons is disrupted, resulting in the formation of alpha-synuclein aggregates. Lewy bodies are known to affect the pathogenesis of Parkinson's disease by altering neuronal membrane permeability, allowing calcium ions to infiltrate, inducing oxidative stress through mitochondrial damage, and disrupting normal microtubule formation, leading to neuronal death. Therefore, despite ongoing efforts to inhibit alpha-synuclein, no therapeutic agents or methods have been developed to date that can effectively inhibit alpha-synuclein aggregation. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present inventors experimentally demonstrated that the transcription factors Nurr1 and Foxa2, when introduced and expressed in brain cells, inhibit alpha-synuclein protein aggregation, leading to the completion of the present invention. In particular, they confirmed that the combined expression of Nurr1 and Foxa2, a coactivator, results in a stronger synergistic inhibitory effect on alpha-synuclein protein aggregation than the effect of Nurr1 expression alone.

[0006] Therefore, one object of the present invention is to provide a composition for inhibiting alpha-synuclein protein aggregation, which comprises a gene carrier containing Nurr1 and Foxa2 genes.

[0007] Therefore, one object of the present invention is to provide an alpha-synuclein protein aggregation inhibitor, which comprises a gene carrier containing Nurr1 and Foxa2 genes.

[0008] Another object of the present invention is to provide a composition for inhibiting alpha-synuclein protein aggregation, which comprises a vector carrying Nurr1 and Foxa2 genes.

[0009] It is yet another object of the present invention to provide an alpha-synuclein protein aggregation inhibitor comprising a vector carrying Nurr1 and Foxa2 genes.

[0010] It is yet another object of the present invention to provide a composition for inhibiting alpha-synuclein protein aggregation, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0011] Yet another object of the present invention is to provide an agent for inhibiting alpha-synuclein protein aggregation, which comprises brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0012] It is yet another object of the present invention to provide a composition for inhibiting alpha-synuclein protein phosphorylation, which comprises a gene carrier containing Nurr1 and Foxa2 genes.

[0013] It is yet another object of the present invention to provide an agent for inhibiting alpha-synuclein protein phosphorylation, which comprises a gene carrier containing the Nurr1 and Foxa2 genes.

[0014] It is yet another object of the present invention to provide a composition for inhibiting alpha-synuclein protein phosphorylation, which comprises a vector carrying Nurr1 and Foxa2 genes.

[0015] It is yet another object of the present invention to provide an agent for inhibiting alpha-synuclein protein phosphorylation, which comprises a vector carrying Nurr1 and Foxa2 genes.

[0016] Yet another object of the present invention is to provide a composition for inhibiting alpha-synuclein protein phosphorylation, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0017] It is yet another object of the present invention to provide an agent for inhibiting alpha-synuclein protein phosphorylation, which comprises brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0018] It is yet another object of the present invention to provide a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising a gene carrier containing Nurr1 and Foxa2 genes.

[0019] It is yet another object of the present invention to provide a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising a vector carrying Nurr1 and Foxa2 genes.

[0020] It is yet another object of the present invention to provide a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced. [Means for solving the problem]

[0021] The present inventors have conducted extensive research into methods for inhibiting alpha-synuclein protein aggregation, which is known to be a major cause of Parkinson's disease. As a result, they have discovered that the introduction and expression of Nurr1 and Foxa2 genes can more effectively inhibit the aggregation and phosphorylation of alpha-synuclein protein than the introduction and expression of Nurr1 gene alone.

[0022] One aspect of the present invention is a composition for inhibiting alpha-synuclein protein aggregation, which comprises a gene carrier containing Nurr1 and Foxa2 genes.

[0023] One aspect of the present invention is an agent for inhibiting the aggregation of alpha-synuclein protein, which comprises a gene carrier containing Nurr1 and Foxa2 genes.

[0024] Another aspect of the present invention is a composition for inhibiting alpha-synuclein protein aggregation, which comprises a vector carrying Nurr1 and Foxa2 genes.

[0025] Yet another aspect of the present invention is an agent for inhibiting alpha-synuclein protein aggregation, which comprises a vector carrying Nurr1 and Foxa2 genes.

[0026] Yet another aspect of the present invention is a composition for inhibiting alpha-synuclein protein aggregation, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0027] Yet another aspect of the present invention is an agent for inhibiting the aggregation of alpha-synuclein protein, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0028] Yet another aspect of the present invention is a composition for inhibiting alpha-synuclein protein aggregation, comprising any one selected from the group consisting of a gene carrier containing Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0029] Yet another aspect of the present invention is a composition for inhibiting alpha-synuclein protein aggregation, comprising any one selected from the group consisting of a vector carrying Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0030] Yet another aspect of the present invention is an alpha-synuclein protein aggregation inhibitor, comprising any one selected from the group consisting of a gene carrier containing the Nurr1 and Foxa2 genes, and brain cells into which the Nurr1 and Foxa2 genes have been introduced.

[0031] Yet another aspect of the present invention is an alpha-synuclein protein aggregation inhibitor, comprising any one selected from the group consisting of a viral vector carrying Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0032] Yet another aspect of the present invention is a composition for inhibiting the phosphorylation of alpha-synuclein protein, comprising a gene carrier containing Nurr1 and Foxa2 genes.

[0033] Yet another aspect of the present invention is an inhibitor of alpha-synuclein protein phosphorylation, which comprises a gene carrier containing the Nurr1 and Foxa2 genes.

[0034] Yet another aspect of the present invention is a composition for inhibiting the phosphorylation of alpha-synuclein protein, comprising a vector carrying Nurr1 and Foxa2 genes.

[0035] Yet another aspect of the present invention is an inhibitor of alpha-synuclein protein phosphorylation, comprising a vector carrying Nurr1 and Foxa2 genes.

[0036] Yet another aspect of the present invention is a composition for inhibiting the phosphorylation of alpha-synuclein protein, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0037] Yet another aspect of the present invention is an inhibitor of the phosphorylation of alpha-synuclein protein, comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0038] Yet another aspect of the present invention is a composition for inhibiting alpha-synuclein protein phosphorylation, comprising any one selected from the group consisting of a gene carrier containing the Nurr1 and Foxa2 genes and brain cells into which the Nurr1 and Foxa2 genes have been introduced.

[0039] Yet another aspect of the present invention is a composition for inhibiting alpha-synuclein protein phosphorylation, comprising any one selected from the group consisting of a viral vector carrying the Nurr1 and Foxa2 genes and brain cells into which the Nurr1 and Foxa2 genes have been introduced.

[0040] Yet another aspect of the present invention is an alpha-synuclein protein phosphorylation inhibitor, comprising any one selected from the group consisting of a gene carrier containing the Nurr1 and Foxa2 genes, and brain cells into which the Nurr1 and Foxa2 genes have been introduced.

[0041] Yet another aspect of the present invention is an alpha-synuclein protein phosphorylation inhibitor, comprising any one selected from the group consisting of a viral vector carrying Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0042] As used herein, the term "alpha-synuclein protein" refers to a protein abundant in the brain, found primarily at the ends of nerve cells called presynaptic terminals. Alpha-synuclein is known to interact with phospholipids and proteins and to help regulate the release of the neurotransmitter dopamine. Human alpha-synuclein consists of over 140 amino acids and is encoded by the SNCA gene.

[0043] As used herein, "alpha-synuclein protein aggregation" refers to the aggregation of two or more alpha-synuclein proteins. In one embodiment of the present invention, the alpha-synuclein aggregate may have a larger molecular weight and / or size than non-aggregated alpha-synuclein protein.

[0044] As used herein, "inhibition of alpha-synuclein protein aggregation" may be understood to mean inhibiting the phenomenon in which each alpha-synuclein protein aggregates with surrounding alpha-synuclein proteins to form aggregates, or inhibiting the aggregation of already formed aggregates by decomposition. Alternatively, inhibition of alpha-synuclein protein aggregation may include, in addition to inhibiting aggregation, increasing the rate of degradation of alpha-synuclein protein or its aggregates, or adjusting the balance between the production and degradation rates of alpha-synuclein or its aggregates to a normal state.

[0045] As used herein, the term "gene delivery vehicle" refers to a means for delivering a nucleic acid sequence or a composition containing a nucleic acid sequence to a cell or tissue. For example, gene delivery vehicles may include, but are not limited to, viral or non-viral vectors (e.g., retroviruses, adenoviruses, adeno-associated viruses, and other nucleic acid-based delivery vehicles), injection or microinjection of naked nucleic acids, polymer-based delivery systems (e.g., liposomes and metallic particle systems), biolistic injection lipid nanoparticles (LNPs), etc.

[0046] In one embodiment of the present invention, the gene delivery vehicle may be a viral vector.

[0047] As used herein, the term "brain cells" refers to cells located in the brain, and brain cells may include neurons (neuron cells) and glial cells (glia).

[0048] As used herein, the term "neuron" refers to a cell of the nervous system. As used herein, the term "neuron" may be used interchangeably with "neuron" or "neuronal cell."

[0049] As used herein, the term "glial cells" refers to the cells that make up the largest proportion of cells present in the brain, and glial cells may include astrocytes or microglia cells. Astrocytes are involved in protecting and supplying nutrients to neurons and inflammation, while microglia are responsible for inflammation in the brain and are known to play an important role in brain diseases such as Alzheimer's disease.

[0050] As used herein, the term "transduction" refers to the phenomenon in which genetic traits are transferred from one cell to another via a bacteriophage. When a bacteriophage infects a certain type of bacterium, the phage DNA binds to the host DNA, and when the phage emerges through bacteriolysis, it may lose some of its own DNA but retain some of the host DNA. When such a phage infects another bacterium, it introduces new genes from the previous host, resulting in the emergence of a new trait. In biological research, the term "transduction" generally refers to the introduction and expression of a specific exogenous gene in a target cell using a viral vector.

[0051] As used herein, the term "cellular therapeutic agent" refers to a pharmaceutical product (as defined by the U.S. FDA) used for therapeutic, diagnostic, and preventive purposes, including cells and tissues isolated, cultured, and specially manipulated from humans, that is produced by expanding, selecting, or otherwise altering the biological properties of living autologous, allogeneic, or xenogeneic cells in vitro in order to restore the function of cells or tissues. Cellular therapeutic agents are broadly classified into somatic cell therapeutic agents and stem cell therapeutic agents, depending on the degree of cell differentiation.

[0052] As used herein, the term "transducing (transducing) Nurr1 and Foxa2" refers to the simultaneous introduction of nucleic acids encoding both genes into brain cells. The two genes may be introduced separately or together by a gene transfer vehicle. When a vector is used as the gene transfer vehicle, the two genes may be introduced separately in their own expression vectors or simultaneously in a single expression vector.

[0053] In one example of the present invention, we confirmed that the introduction of both Nurr1 and Foxa2 genes resulted in significantly greater inhibition of alpha-synuclein aggregation than the introduction of either Nurr1 or Foxa2 genes alone, due to the synergistic effect between Nurr1 and Foxa2. Specifically, we confirmed that the simultaneous introduction of Nurr1 and Foxa2 genes into cells significantly reduced both alpha-synuclein monomers and aggregates compared to the introduction of either Nurr1 or Foxa2 genes alone (see Figures 5 and 6).

[0054] As used herein, the term "transducing (transducing) Nurr1" means introducing a nucleic acid encoding the Nurr1 gene into brain cells.

[0055] To introduce genes encoding Nurr1 and / or Foxa2 into brain cells, intracellular introduction techniques using gene carriers known in the art may be used, and for example, viral vectors such as those using adeno-associated virus (AAV), retrovirus, and adenovirus may be used.

[0056] Viral vectors can be used to deliver Nurr1 and / or Foxa2. Examples of viral vectors that can be used include, but are not limited to, adeno-associated viruses (AAV), adenoviruses, retroviruses, and / or lentiviruses. Therefore, in one specific example, delivery of Nurr1 and / or Foxa2 according to the present invention may involve inserting nucleic acids encoding Nurr1 and / or Foxa2 into separate expression vectors or a single expression vector, and then introducing the vectors into brain cells.

[0057] Nucleic acids encoding Nurr1 and / or Foxa2 may be used without limitation as long as they have a nucleotide sequence encoding Nurr1 and / or Foxa2 known in the art. Furthermore, the nucleic acids may have a nucleotide sequence encoding a functional equivalent of Nurr1 and / or Foxa2. A functional equivalent refers to a polypeptide having 60% or more, 70% or more, or 80% or more sequence homology (i.e., identity) with the amino acid sequence of Nurr1 and / or Foxa2. For example, functional equivalents include polypeptides having 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Functional equivalents may be produced as a result of addition, substitution, or deletion of a portion of the amino acid sequence. The amino acid deletion or substitution may be located in a region not directly associated with the biological activity of the polypeptide of the present invention.

[0058] Nucleic acids encoding Nurr1 and / or Foxa2 may also be produced by recombinant DNA methods known in the art, for example, by PCR amplification to amplify nucleic acids from genomes, chemical synthesis, or techniques for producing cDNA sequences.

[0059] Nucleic acids encoding Nurr1 and / or Foxa2 may be operably linked to expression control sequences and inserted into an expression vector. The term "operably linked" means that one nucleic acid fragment binds to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. Furthermore, an expression control sequence refers to a DNA sequence that regulates the expression of an operably linked nucleic acid sequence in a particular host cell. Such regulatory sequences may include a promoter for initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosomal binding site, and a sequence for regulating the termination of transcription and translation. These may be collectively referred to as a "DNA construct comprising a nucleic acid encoding Nurr1 and / or Foxa2."

[0060] As used herein, the term "expression vector" refers to a viral vector or other vehicle known in the art into which a nucleic acid encoding a structural gene may be inserted and which can result in expression of the nucleic acid in a host cell.

[0061] In the present invention, the vector may be a viral vector, such as an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes viral vector, or an avipox viral vector, including, but not limited to, an adeno-associated viral vector.

[0062] Adeno-associated virus (AAV) vectors can be created by introducing material capable of producing viruses into specific cells. Lentiviral vectors can also be created through multiple steps to enable virus production in specific cell lines.

[0063] Expression vectors containing the nucleic acids of the present invention can be introduced into brain cells by methods known in the art, including, but not limited to, viral transduction, transient transfection, and microinjection, which are well known for introducing nucleic acids into cells. For example, Nurr1 and / or Foxa2 can be inserted into an adeno-associated virus (AAV) or lentiviral vector using genetic engineering to prepare an expression vector, which can then be transduced into packaging cells. The transduced packaging cells can then be cultured and separated and purified to obtain an AAV or lentiviral solution. The AAV or lentiviral solution can then be used to infect brain cells (neuronal and / or glial cells) to introduce the Nurr1 and / or Foxa2 genes into the brain cells. Subsequently, the desired brain cells can be isolated by confirming the sole or co-expression of Nurr1 and / or Foxa2 using a selection marker contained in the AAV or lentiviral vector.

[0064] The brain cells according to the present invention into which Nurr1 and Foxa2 are introduced and expressed may be prepared by a method comprising the steps of:

[0065] a manufacturing step of producing a recombinant gene carrier comprising a DNA construct containing nucleic acids encoding Nurr1 and Foxa2;

[0066] A transfection step in which brain cells are infected with gene carriers containing Nurr1 and Foxa2.

[0067] The brain cells according to the present invention into which Nurr1 and Foxa2 are introduced and expressed may be prepared by a method comprising the steps of:

[0068] a manufacturing step of producing a recombinant viral vector comprising a DNA construct containing nucleic acids encoding Nurr1 and Foxa2;

[0069] a production step in which the recombinant viral vector is transfected into a virus-producing cell line to produce recombinant viruses expressing Nurr1 and Foxa2; and

[0070] Transfection step to infect brain cells with Nurr1- and Foxa2-expressing recombinant viruses.

[0071] The DNA construct can be operably linked to an expression control sequence, such as a promoter, and inserted into a viral vector known in the art to produce a recombinant viral vector. The recombinant viral vector containing a nucleic acid encoding Nurr1 and / or Foxa2 is then introduced into a virus-producing cell line to produce a recombinant virus that expresses Nurr1 and / or Foxa2. The virus-producing cell line can be a cell line that produces a virus corresponding to the viral vector used. Brain cells can then be infected with the recombinant AAV or lentivirus that expresses Nurr1 and Foxa2 or Nurr1. The above process can be performed by methods known in the art.

[0072] Brain cells expressing Nurr1 and / or Foxa2 according to the present invention may be grown and cultured by methods known in the art.

[0073] The brain cells of the present invention may be cultured in a culture medium that supports the survival or proliferation of the desired cell type. The culture medium may be supplemented with additives developed for the sustained culture of brain cells. Examples include N2 medium and B27 additives, available from Gibco, and bovine serum. Brain cells can be cultured by changing the medium while observing the state of the medium and cells. If the brain cells continue to proliferate and aggregate to form neurospheres, they can be subcultured. Subculture can be performed approximately every 7 to 8 days, depending on the conditions.

[0074] The compositions of the present invention inhibit the aggregation and phosphorylation of alpha-synuclein, protecting brain cells, including neurons and glial cells, from damage and allowing neurons to be replenished (regenerated) or constructed (restored).

[0075] As used herein, the term "regeneration" refers to the phenomenon in which a formed organ or part of an individual is replenished when that part is lost. "Restoration" can also be called "reconstitution," which refers to the reconstruction of tissues, and refers to the re-construction of tissues or organs from dissociated cells or tissues.

[0076] The composition or cell therapy agent of the present invention may be prepared as a suitable formulation by including an acceptable carrier depending on the administration route. Formulations suitable for administration routes are known and typically include formulations that facilitate transport across membranes.

[0077] The compositions of the present invention may be used in the form of common pharmaceutical preparations. Parenteral preparations may be prepared in the form of sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, or lyophilized preparations. Oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, or wafers. Injectable preparations may be prepared in unit-dose ampoules or multi-dose forms. The therapeutic compositions of the present invention may also be administered together with a pharmaceutically acceptable carrier. For example, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, or flavorings may be used. Injectable preparations may be used in combination with buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc., while topical administration may use bases, excipients, lubricants, preservatives, etc.

[0078] Yet another aspect of the present invention is a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising any one selected from the group consisting of a gene carrier containing Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0079] Yet another aspect of the present invention is a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising any one selected from the group consisting of a vector carrying Nurr1 and Foxa2 genes and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0080] Yet another aspect of the present invention is a method for treating or alleviating a disease caused by alpha-synuclein protein aggregation, comprising the steps of:

[0081] A step of administering to the subject a composition containing any one selected from the group consisting of a gene carrier containing Nurr1 and Foxa2 genes, and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0082] Yet another aspect of the present invention is a method for treating or alleviating a disease caused by alpha-synuclein protein aggregation, comprising the steps of:

[0083] A step of administering to a subject a composition containing any one selected from the group consisting of a viral vector carrying Nurr1 and Foxa2 genes and brain cells into which Nurr1 and Foxa2 genes have been introduced.

[0084] As used herein, the term "subject" refers to a vertebrate, such as a cow, pig, horse, goat, dog, cat, rat, mouse, rabbit, guinea pig, human, etc., that has been the object of treatment, observation, or experiment.

[0085] As used herein, the term "treatment" refers to an approach to obtaining beneficial or favorable clinical results. For purposes of this invention, beneficial or favorable clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delaying or reducing the rate of disease progression, and improvement or palliation and reduction (partially or totally) of the disease state, whether detectable or not. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes treatment required for already occurring disorders as well as disorders being prevented. "Palliating" a disease means reducing the extent and / or adverse clinical signs of the disease state and / or slowing or prolonging the time course of progression compared to the absence of treatment.

[0086] In one embodiment of the present invention, the disease caused by alpha-synuclein may be a disease selected from the group consisting of Parkinson's disease and Dementia with Lewy Bodies, but is not limited thereto.

[0087] Furthermore, a method for treating a disease caused by alpha-synuclein using the therapeutic composition of the present invention may include administering the composition via a common route by which a given substance is introduced into a subject or patient by an appropriate method.

[0088] Methods of administration include, but are not limited to, intracerebral administration, midbrain administration, intraventricular administration, spinal cavity administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravascular administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0089] Furthermore, the composition of the present invention may be administered by any device capable of delivering the active substance to target cells. The administration route and formulation may be intracranial injection, substantia nigra injection, ventricular injection, cerebrospinal fluid injection, intravenous injection, subcutaneous injection, intravascular injection, intramuscular injection, or infusion injection using a stereotactic system. Injections may be prepared using aqueous solvents such as saline or Ringer's solution, or non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin). Injectable solutions may contain pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and preservatives to prevent microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0090] The composition of the present invention may be administered to a subject in a pharmaceutically effective amount, which may be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the age, weight, health, and sex of the subject (patient), the sensitivity of the subject (patient) to the drug, the administration route, the administration method, the number of doses, the treatment period, and any other drugs used in combination or concomitantly.

[0091] Brain cells into which the Foxa2 and / or Nurr1 gene of the present invention has been introduced may be directly transplanted in the form of a composition into the lesion site in a therapeutically effective amount.

[0092] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to halt or reduce physiological effects in a subject or patient induced by alpha-synuclein aggregation or phosphorylation. The therapeutically effective amount of cells used may be determined by the subject's (patient's) needs, the subject's (patient's) age, physiological condition, and health, the desired therapeutic effect, the size and area of ​​the tissue targeted for treatment, the extent of the lesion, and the selected delivery pathway. Alternatively, cells may be administered in multiple small grafts at low cell doses to one or more sites within a given target tissue. The cells of the present invention may be completely dissociated before transplantation, e.g., forming a single-cell suspension, or nearly completely dissociated before transplantation, e.g., forming small cell aggregates. The cells may be administered in a manner that allows them to be transplanted or migrated to a given tissue site and reconstitute or regenerate a functionally deficient area.

[0093] The appropriate range of cells that can be administered to achieve therapeutic effectiveness can be determined appropriately for each subject or patient within the ordinary skill in the art. For example, the number of cells that can be contained in the composition of the present invention may be, but is not limited to, about 10 to 1,000,000,000 cells.

[0094] The appropriate dosage of the composition of the present invention may be determined depending on factors such as the formulation method, administration method, age, weight, sex, severity of disease symptoms, diet, administration time, administration route, excretion rate, and reaction sensitivity of the subject (patient). A physician of ordinary skill can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention can be administered in an amount of 1×10 1 ~1×10 13 Virus genome (vg) / μl, 1 × 10 2 ~1×10 13 vg / μl, 1 × 10 3 ~1×10 13 vg / μl, 1 × 10 4 ~1×10 13 vg / μl, 1 × 10 5 ~1×10 13 vg / μl, 1 × 10 6 ~1×10 13 vg / μl, 1 × 10 7 ~1×1013 vg / μl、1×10 8 ~1×10 13 vg / μl、1×10 9 ~1×10 13 vg / μl、1×10 10 ~1×10 13 vg / μl、1×10 11 ~1×10 13 vg / μl、1×10 12 ~1×10 13 vg / μl、1×10 1 ~1×10 12 vg / μl、1×10 1 ~1×10 11 vg / μl、1×10 1 ~1×10 10 vg / μl、1×10 1 ~1×10 9 vg / μl、1×10 1 ~1×10 8 vg / μl、1×10 1 ~1×10 7 vg / μl、1×10 1 ~1×10 6 vg / μl、1×10 1 ~1×10 5 vg / μl、1×10 1 ~1×10 4 vg / μl、1×10 1 ~1×10 3 vg / μl、1×10 1 ~1×10 2 vg / μl、1×10 2 ~1×10 12 vg / μl、1×10 3 ~1×10 11 vg / μl、1×10 4 ~1×10 10 vg / μl、1×10 5 ~1×10 9 vg / μl、1×10 6 ~1×10 8 vg / μl、1×10 2 ~1×10 3 vg / μl、1×10 3 ~1×10 4 vg / μl、1×10 4 ~1×10 5vg / μl, 1 × 10 5 ~1×10 6 vg / μl, 1 × 10 6 ~1×10 7 vg / μl, 1 × 10 7 ~1×10 8 vg / μl, 1 × 10 8 ~1×10 9 vg / μl, 1 × 10 9 ~1×10 10 vg / μl, 1 × 10 10 ~1×10 11 vg / μl, 1 × 10 11 ~1×10 12 The amount of the viral vector or viral gene may be 1×10 vg / μl. 6 ~2×10 16 The vg / dose can be injected into the patient 1 to 5 times, and repeated in a similar manner after several months or years to maintain efficacy. [Effects of the Invention]

[0095] The present invention relates to a composition and method for inhibiting alpha-synuclein aggregation, and more particularly to a technology for inhibiting alpha-synuclein aggregation and phosphorylation by introducing and inducing expression of Nurr1 and Foxa2 genes. The composition of the present invention has excellent inhibitory effects on alpha-synuclein aggregation and phosphorylation and can be used for the treatment and prevention of Parkinson's disease. [Brief explanation of the drawings]

[0096] [Figure 1] 1 is a photograph showing the results of Western blotting performed on a control group (Cont) and dopamine neurons and glial cells (NF) transfected with Nurr1 and Foxa2 genes after treatment with alpha-synuclein PFF (preformed fibril), according to one embodiment.

[0097] [Figure 2]1 is a graph comparing the degree of alpha-synuclein aggregation between a control group and a group (NF) into which Nurr1 and Foxa2 genes were introduced after Western blotting, according to one embodiment.

[0098] [Figure 3] 1 is a graph showing phosphorylated alpha-synuclein monomer levels in a control group and a group (NF) transfected with Nurr1 and Foxa2 genes after Western blotting, according to one example.

[0099] [Figure 4] 1 is a graph showing the levels of phosphorylated alpha-synuclein aggregates in a control group and a group (NF) transfected with Nurr1 and Foxa2 genes after Western blotting, according to one example.

[0100] [Figure 5] In one embodiment, photographs show the results of Western blotting performed on dopamine neurons and glial cells treated with alpha-synuclein preformed fibrils (PFFs) from a control group (Cont), a group transfected with the Nurr1 gene alone (N), a group transfected with the Foxa2 gene alone (F), and a group transfected with both the Nurr1 and Foxa2 genes (NF).

[0101] [Figure 6] FIG. 1 is a graph comparing the levels of alpha-synuclein aggregates and monomers in a control group (Con), a group transfected with Foxa2 alone (F), a group transfected with Nurr1 alone (N), and a group transfected with Nurr1 and Foxa2 genes (NF) after Western blotting according to one embodiment.

[0102] [Figure 7] 1 is a graph showing the results of a pole test for a wild-type (WT), a control group (Cont) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example.

[0103] [Figure 8] 1 shows photographs illustrating the results of a pole test for a wild-type (WT), a control group (Cont) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example.

[0104] [Figure 9] 1 is a graph showing the results of a beam test at week 8 in a wild-type (WT), a control group (PD) which is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one embodiment.

[0105] [Figure 10] 1 is a graph showing the results of a beam test at 12 weeks for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example.

[0106] [Figure 11] 1 shows photographs illustrating the results of a beam test for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one embodiment.

[0107] [Figure 12] FIG. 1 is a graph showing the total distance traveled in an open field test (OFT) in a wild-type (WT) mouse, a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 had been introduced.

[0108] [Figure 13] 1 is a graph showing the average speed of a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced in an open field test, according to one embodiment.

[0109] [Figure 14] FIG. 1 shows the results of an open field test for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example.

[0110] [Figure 15] 1 is a graph showing the results of a rotarod test at week 8 for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one embodiment.

[0111] [Figure 16] 1 is a graph showing the results of a rotarod test at 12 weeks for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example.

[0112] [Figure 17] 1 shows photographs illustrating the results of a rotarod test for a wild-type (WT), a control group (PD) that is a Parkinson's disease model, and an experimental group (NF) into which Nurr1 and Foxa2 were introduced, according to one example. DETAILED DESCRIPTION OF THE INVENTION

[0113] A viral vector carrying the Nurr1 and Foxa2 genes; and Brain cells transfected with Nurr1 and Foxa2 genes An alpha-synuclein protein aggregation inhibitor comprising any one selected from the group consisting of: [Example]

[0114] The present invention will be described in more detail below using the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0115] Example 1: Vector Production

[0116] Vectors were prepared for lentiviral transduction. Lentiviral vectors expressing Nurr1 or Foxa2 were generated by inserting the respective cDNAs into the multiple cloning site of pCDH (System Biosciences, Mountain View, CA) under the control of the CMV promoter. pGIPZ-shNurr1 and pGIPZ-shFoxa2 lentiviral vectors were purchased from Open Biosystems (Rockford, IL). Empty backbone vectors, pCDH and pGIPZ, were used as negative controls. Lentiviral titers were determined using QuickTiter. TM The HIV lentivirus quantification kit (Cell Biolabs, San Diego, CA) was used for each transduction reaction, with 200 μl / well (24-well dish) or 2 ml / 6 cm dish containing 10 transducing units (TU) / ml (60-70 ng / ml).

[0117] To induce in vivo expression by stereotaxic injection, AAV expressing Nurr1 or Foxa2 under the control of a CMV promoter was generated by subcloning the respective cDNA into the pAAV-MCS vector (Addgene, Cambridge, MA). To evaluate the efficiency of transduced gene expression, AAV expressing green fluorescent protein (GFP) was also generated. Production, isolation, and purification of AAV (serotype 9 or 2) were performed at the Korea Institute of Science and Technology (Seoul, Republic of Korea). AAV titers were measured using a QuickTiter™ kit. TM The expression levels were measured using an AAV quantification kit (Cell Biolabs). Co-expression studies were performed by infecting cells with a mixture (1:1, v:v) of the individual virus preparations.

[0118] Example 2: Cell culture

[0119] 2-1. Ventral midbrain Neural Progenitor Cell (VM NPC) culture

[0120] Neural progenitor cells with dopaminergic potential were cultured at 4 × 10 in a 6-well plate coated with poly-L-ornitine-fibronectin (PLO-FN). 5 After 24 hours, lentiviruses expressing Nurr1 and Foxa2 under the control of the synapsin promoter were injected at 10 / well. 6 The cells were transduced with 60-70 ng / ml of TU (transducing unit) / ml of lentivirus at a 1:1 ratio (v:v). As a control, the cells were treated with the same amount of GFP-expressing lentivirus. Then, the cells were cultured for 3 days to differentiate into neurons.

[0121] 2-2.Culture of ventral midbrain glia

[0122] For the culture of ventral midbrain glia, 3 × 10 rat midbrain glial cells were cultured in a 100 mm × 20 mm culture dish coated with poly-L-ornithine-fibronectin. 6 10 cells / ml, and 24 hours later, 10 cells / ml were seeded with lentiviruses expressing Nurr1 and Foxa2 individually under the control of a CMV promoter. 6 The cells were transduced with a 1:1 (v:v) mixture of TU / ml (60-70ng / ml) of the control virus. The control group was treated with the same amount of the control virus. The cells were then cultured for 5 days.

[0123] 2-3.Co-culture

[0124] Dopaminergic midbrain neurons (VM neurons) cultured in Example 2-1 were seeded with VM glia cultured in Example 2-2 at a ratio of 2:1 (VM neurons:VM glia = 2:1). The next day, alpha-synuclein preformed fibrils (PFFs) were added to a final concentration of 2 μg / ml. Seven days later, the amount of alpha-synuclein protein was determined by Western blotting.

[0125] Example 3: Western Blot

[0126] Proteins were extracted from plated cells using a 1% Triton X-100 / PBS solution containing a protease inhibitor (Roche) and a phosphatase inhibitor cocktail (Sigma). After centrifugation, the pellet was dissolved in 1% SDS sample buffer, and 15 μg of protein was loaded onto an SDS-PAGE gel (4-16% gradient gel). After transfer to a membrane, the cells were blocked with 5% BSA / TBST and incubated with a primary antibody overnight at 4°C, followed by a secondary antibody at room temperature for 1 hour. The primary antibodies used were α-syn (BD Biosciences, 610787) and pS129-α-syn (Bio Legend, 825701). The Western blot results are shown in Figure 1. Western blot results were quantitatively analyzed using the ImageJ program and are shown in Figures 2-4 and Tables 1-3.

[0127] [Table 1]

[0128] [Table 2]

[0129] [Table 3]

[0130] Example 4: Confirmation of Nurr1 and Foxa2 inhibition of alpha-synuclein aggregation and phosphorylation

[0131] As shown in Figure 1, Western blot results showed that when alpha-synuclein PFFs were treated in neurons and glial cells transfected with Nurr1 and Foxa2 genes, alpha-synuclein protein aggregation was reduced compared to the control group, compared to neurons and glial cells not treated with Nurr1 or Foxa2. Furthermore, the levels of phosphorylated alpha-synuclein monomers and aggregates were also significantly reduced compared to the control group.

[0132] Specifically, as can be seen from Figure 2 and Table 1, when Nurr1 and Foxa2 genes were transduced, the amount of aggregated alpha-synuclein protein and aggregation were reduced compared to the control group. Furthermore, as can be seen from Figures 3 and 4 and Tables 2 and 3, when Nurr1 and Foxa2 genes were transduced, the level of phosphorylated alpha-synuclein aggregates was reduced to about 60% of the control level, and the level of monomers was reduced to about 30% of the control level.

[0133] Example 5: Comparison of the inhibitory effect of alpha-synuclein aggregate formation between administration of Nurr1 alone and administration of Nurr1 and Foxa2 in combination

[0134] Neurons and glial cells transfected with Nurr1 alone, Foxa2 alone, or both Nurr1 and Foxa2 were plated, and proteins were extracted in 1% Triton X-100 / PBS containing a protease inhibitor (Roche) and a phosphatase inhibitor cocktail (Sigma).

[0135] Control cells were used without Nurr1 or Foxa2 transfection. After centrifugation, the pellet was dissolved in 1% SDS sample buffer, and 15 μg of protein was loaded onto an SDS-PAGE gel (4-16% gradient gel). After transfer to a membrane, the gel was blocked with 5% BSA / TBST and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 hour at room temperature. The primary antibodies used were α-syn (BD Biosciences, 610787) and pS129-α-syn (Bio Legend, 825701). The Western blot results are shown in Figure 5. Western blot results were quantitatively analyzed using the ImageJ program and are shown in Figure 6 and Table 4.

[0136] [Table 4]

[0137] As can be seen from Figure 6 and Table 4, the experimental results showed that the group transduced with both Nurr1 and Foxa2 genes (N+F) showed a significant reduction in alpha-synuclein aggregation compared to the groups transduced with either Nurr1 or Foxa2 alone (N or F). Specifically, the group transduced with both Nurr1 and Foxa2 genes showed a 48% or greater reduction in alpha-synuclein aggregation compared to the control group, and in particular, a 32% or greater reduction in alpha-synuclein aggregation compared to the group transduced with Nurr1 alone. These results demonstrate that transducing both Nurr1 and Foxa2 genes significantly reduces alpha-synuclein aggregation compared to transducing with Nurr1 alone. Furthermore, transducing both Nurr1 and Foxa2 genes is expected to be more effective in treating Parkinson's disease.

[0138] Example 6: Confirmation of behavioral improvement by introduction of Nurr1 and Foxa2 in a Parkinson's disease model

[0139] 6-1. Generation of Parkinson's disease model (PD model) mice

[0140] A mixture of AAV2-CMV-α-syn-HA and 5 μg of α-syn PFF was injected into the substantia nigra (SN) of ICR mice. Specifically, the mice were immobilized using a stereotaxic instrument, and an approximately 1 cm midline incision was made in the skin of the head to identify the bregma. An electric drill was used to drill an opening in the skull at a position -3.3 mm anterior and 1.2 mm posterior to the bregma, and AAV2-CMV-α-syn-HA (1.3 x 10 13 2 μl of α-syn PFF (5 mg / mL) and 2 μl of α-syn PFF (5 mg / mL) were loaded into a stereotaxic injector, which was inserted 4.6 mm deep from the skull and injected 2 μl into each side of the substantia nigra (the dose of each vector per injection site was 1.3 × 10 13 The mixture was gradually administered at a rate of 0.5 μl / min. 20 minutes after administration into both substantia nigra sites, the injector was withdrawn by 1.5 mm every 10 minutes to minimize leakage of the administered mixture. The skin on the skull of the mouse was sutured using a medical skin stapler, disinfected with povidone, and placed in a cage after recovery.

[0141] Four weeks after administration, Nurr1 and Foxa2 were transduced using viral vectors expressing Nurr1 and Foxa2. Specifically, the PD mouse model was immobilized using a stereotaxic instrument, and a 1-cm midline incision was made in the skin of the head to identify the bregma. The skull was drilled using an electric drill at positions -3.3 mm anterior and 1.2 mm posterior to the bregma, and AAV9-hNurr1 and AAV9-hFoxa2 were transduced at a concentration of 1 × 10 for each vector. 10 The vector was loaded into a stereotaxic injector at a concentration of 1.0 × 10 s. The injector was inserted 4.6 mm deep into the skull and injected 2 μl into each side of the substantia nigra (1.0 × 10 s.). 10The mixture was gradually administered at a rate of 0.5 μl / min. 20 minutes after administration into both substantia nigra sites, the injector was withdrawn 1.5 mm every 10 minutes to minimize leakage of the administered mixture. The skin on the skull was sutured using a medical skin stapler, disinfected with povidone, and the rats were placed in cages after recovery.

[0142] 6-2. Pole test

[0143] Pole tests were performed 4, 8, and 12 weeks after Nurr1 and Foxa2 transduction. The mice were trained and adapted for the test 2–3 days prior to the test. The time it took wild-type (WT), Parkinson's disease model control (Cont), and Nurr1 and Foxa2 transduced (NF) mice to descend from the top of the pole was measured. If mice fell or slipped during the experiment, a negative value was entered, and the data was generated by setting the value equal to the maximum value for that week. Six wild-type mice and eight control and Nurr1 and Foxa2 transduced mice were used for the experiment. Significant effects were determined for the measured times using one-way analysis of variance (ANOVA).

[0144] [Table 5]

[0145] As can be seen from Figures 7 and 8 and Table 5, the control mice took longer to climb down the pole and fell or slipped off the pole more frequently than the wild-type and Nurr1 and Foxa2 transduced (NF) mice.

[0146] 6-3.Beam test

[0147] Mice transduced with Nurr1 and Foxa2 were subjected to beam tests at 8 and 12 weeks. The tests were conducted after 2–3 days of training and adaptation. The beam used for the test was rectangular and 10 mm thick. The time it took for wild-type (WT), Parkinson's disease model control (PD), and Nurr1 and Foxa2 transduced (NF) mice to travel from one end of the beam to the other was measured twice. If mice fell or slipped off the beam during the experiment, a negative value was entered, and the maximum value for that week was used to generate the data. Six wild-type, control (PD), and Nurr1 and Foxa2 transduced mice were used for each group. Significant effects were determined for the measured times using one-way analysis of variance (ANOVA).

[0148] [Table 6]

[0149] As can be seen from Figures 7 and 8 and Table 6, the experimental results showed that the control mice took longer to pass through the beam than the wild-type and Nurr1 and Foxa2 transduced (NF) mice.

[0150] 6-4. Open Field Test (OFT)

[0151] Eight weeks after Nurr1 and Foxa2 transduction, an open field test was performed. Wild-type (WT), Parkinson's disease model control (PD), and Nurr1 and Foxa2 transduced (NF) mice were placed in the open field, and the movement path, speed, and distance traveled by each group were measured for 5 minutes. Six wild-type, control (PD), and Nurr1 and Foxa2 transduced mice were used. Significant effects were determined for the measured times using one-way analysis of variance (ANOVA).

[0152] [Table 7]

[0153] [Table 8]

[0154] As can be seen from Figures 12 to 14 and Tables 7 and 8, the behavior of the Nurr1 and Foxa2 transduced (NF) mice was improved to the extent that there was almost no difference in the total distance traveled and average speed compared to the wild-type mice. Furthermore, the total distance traveled and average speed during movement were significantly increased in the Nurr1 and Foxa2 transduced (NF) mice compared to the control (PD) group.

[0155] 6-5.Rotarod Test

[0156] The rotarod test was performed 8 and 12 weeks after Nurr1 and Foxa2 transduction. The mice were trained and adapted for the test 2–3 days before the test. The speed was gradually increased from 4 rpm to 40 rpm. The time it took wild-type (WT), Parkinson's disease model control (PD), and Nurr1 and Foxa2 transduced (NF) mice to fall from the cylinder was measured twice. Six wild-type, control (PD), and Nurr1 and Foxa2 transduced mice were used. Significant effects were determined using one-way analysis of variance (ANOVA).

[0157] [Table 9]

[0158] [Table 10]

[0159] As can be seen from Figures 15 to 17 and Tables 9 and 10, the Nurr1 and Foxa2 transduced mice (NF) took significantly longer to fall off the cylinder than the control group at 8 and 12 weeks.

[0160] 6-6. Small knot

[0161] These results demonstrate that the motor abilities and behaviors of the Nurr1 and Foxa2 transduced (NF) mice were significantly improved in the pole test, beam test, open field test, and rotarod test compared with the control group, a Parkinson's disease model. These results demonstrate that Nurr1 and Foxa2 gene transfer can reverse the motor impairments characteristic of Parkinson's disease, such as rigidity, bradykinesia, and postural instability. Therefore, Nurr1 and Foxa2 gene transfer is expected to be useful in the treatment of Parkinson's disease. [Industrial Applicability]

[0162] The present invention relates to a composition and method for inhibiting alpha-synuclein aggregation, and more specifically to a technology for inhibiting alpha-synuclein aggregation and phosphorylation by introducing and inducing expression of Nurr1 and Foxa2 genes.

Claims

1. A composition for inhibiting the phosphorylation of alpha-synuclein protein, comprising: A composition comprising a gene carrier containing the Nurr1 and Foxa2 genes.

2. The composition of claim 1 , wherein the gene delivery vehicle is a viral vector.

3. The viral vector is The composition according to claim 2, which is one selected from the group consisting of an adeno-associated virus vector, a retrovirus vector, and an adenovirus vector.

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