KR20230011839A - Composition for inhibiting α-synuclein aggregation and method for inhibiting aggregation - Google Patents

Abstract

The present invention relates to a composition for inhibiting α-synuclein aggregation and a method for inhibiting aggregation. More particularly, the present invention relates to a technology for inhibiting α-synuclein aggregation and phosphorylation by introducing Nurr1 and Foxa2 genes into brain cells and expressing the same together. The composition according to the present invention has an excellent effect of inhibiting aggregation and phosphorylation of α-synuclein, and thus can be used for the treatment and prevention of Parkinson's disease.

Description

Composition for inhibiting alpha-synuclein aggregation and method for inhibiting aggregation {Composition for inhibiting α-synuclein aggregation and method for inhibiting aggregation}

The present invention relates to a composition for inhibiting alpha-synuclein aggregation and a method for inhibiting aggregation, and more particularly, to inhibit alpha-synuclein aggregation and phosphorylation by introducing Nurr1 and Foxa2 genes to induce expression. It's about technology.

Parkinson's disease (Parkinson's disease) is one of the neurodegenerative diseases that appear movement disorders such as muscle tremors and muscle stiffness at the onset. Parkinson's disease mainly develops in the elderly, and it is known that the risk of developing Parkinson's disease increases as the target age increases. In Korea, it is estimated that about 1 to 2 people per 1000 people are affected, and it is known that most Parkinson's diseases that occur in the elderly have little effect on genetic factors. Parkinson's disease is known to be caused by the death of dopamine cells in a region called the substantia nigra of the midbrain. Recently, as the average lifespan of humans increases, the frequency of Parkinson's disease is also expected to increase.

In addition, huge costs are incurred in managing and treating Parkinson's disease, and the patient's mental suffering is also considerable. Therefore, there is a need for an effective method for preventing and treating Parkinson's disease.

Recently, many studies have focused on the alpha-synuclein protein in relation to Parkinson's disease. Alpha-synuclein is a protein abundant in the human brain and is mainly found at the ends of nerve cells with special structures called presynaptic terminals. According to the results of ongoing research, it has been found that Parkinson's disease is related to the formation of Lewy bodies and the formation of aggregation of alpha-synuclein as the balance between the production and removal of alpha-synuclein inside neurons is disrupted. Lewy bodies alter the membrane permeability of neurons, influx calcium ions, induce oxidative stress due to mitochondrial damage, and interfere with normal microtubule formation, leading to neuron death and affecting the pathogenesis of Parkinson's disease. known to give Against this background, attempts have been made to inhibit alpha-synuclein, but until now, no treatment or method capable of effectively inhibiting aggregation of alpha-synuclein has been developed.

Accordingly, the present inventors completed the present invention by experimentally identifying that the aggregation of α-synuclein protein is inhibited when the transcription factors Nurr1 and Foxa2 genes are introduced and expressed in brain cells. In particular, it was confirmed that there is a strong alpha-synuclein protein accumulation inhibitory effect through a synergistic effect when Foxa2, a coactivator, is expressed in combination rather than a single expression of Nurr1.

Accordingly, an object of the present invention is to provide a composition for inhibiting α-synuclein protein aggregation comprising a vector loaded with Nurr1 and Foxa2 genes.

Another object of the present invention is to provide an α-synuclein protein aggregation inhibitor comprising a vector loaded with Nurr1 and Foxa2 genes.

Another object of the present invention is to provide a composition for inhibiting alpha-synuclein protein aggregation comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another object of the present invention is to provide an alpha-synuclein protein aggregation inhibitor comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another object of the present invention is to provide a composition for inhibiting alpha-synuclein protein phosphorylation comprising a vector loaded with Nurr1 and Foxa2 genes.

Another object of the present invention is to provide an alpha-synuclein protein phosphorylation inhibitor comprising a vector loaded with Nurr1 and Foxa2 genes.

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.

Another object of the present invention is to provide an alpha-synuclein protein phosphorylation inhibitor comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another object of the present invention is to provide a composition for preventing or treating diseases caused by alpha-synuclein protein aggregation, including vectors loaded with Nurr1 and Foxa2 genes.

Another object of the present invention is to provide a composition for preventing or treating diseases caused by alpha-synuclein protein aggregation, including brain cells into which Nurr1 and Foxa2 genes have been introduced.

The present inventors have intensively researched a method for inhibiting α-synuclein protein aggregation, which is known to be a major cause of Parkinson's disease. As a result, it was found that when the Nurr1 and Foxa2 genes were introduced and expressed, the aggregation and phosphorylation of the alpha-synuclein protein could be better inhibited than when the Nurr1 gene was introduced and expressed alone.

One aspect of the present invention is a composition for inhibiting aggregation of alpha-synuclein protein, including a vector loaded with Nurr1 and Foxa2 genes.

Another aspect of the present invention is an alpha-synuclein protein aggregation inhibitor comprising a vector loaded with Nurr1 and Foxa2 genes.

Another aspect of the present invention is a composition for inhibiting aggregation of alpha-synuclein protein, including brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is an alpha-synuclein protein aggregation inhibitor comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a vector loaded with Nurr1 and Foxa2 genes; And a composition for inhibiting α-synuclein protein aggregation, comprising any one selected from the group consisting of brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a viral vector loaded with Nurr1 and Foxa2 genes; And an alpha-synuclein protein aggregation inhibitor comprising any one selected from the group consisting of brain cells into which the Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a composition for inhibiting phosphorylation of alpha-synuclein protein, including a vector loaded with Nurr1 and Foxa2 genes.

Another aspect of the present invention is an alpha-synuclein protein phosphorylation inhibitor comprising a vector loaded with Nurr1 and Foxa2 genes.

Another aspect of the present invention is a composition for inhibiting phosphorylation of alpha-synuclein protein, including brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is an alpha-synuclein protein phosphorylation inhibitor comprising brain cells into which Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a viral vector loaded with Nurr1 and Foxa2 genes; And a composition for inhibiting alpha-synuclein protein phosphorylation, comprising any one selected from the group consisting of brain cells into which the Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a viral vector loaded with Nurr1 and Foxa2 genes; And an alpha-synuclein protein phosphorylation inhibitor comprising any one selected from the group consisting of brain cells into which the Nurr1 and Foxa2 genes have been introduced.

The term "alpha-synuclein protein" in this specification is one of the proteins abundant in the brain and is mainly found at the end of a nerve cell called the presynaptic terminal. Alpha-synuclein is known to interact with phospholipids and proteins and is known to help regulate the release of the neurotransmitter dopamine. In the case of human alpha-synuclein, it consists of about 140 amino acids and is encoded by the SNCA gene.

“Aggregation of alpha-synuclein proteins” in the present specification means the aggregation of two or more alpha-synuclein proteins. In one embodiment of the present invention, alpha-synuclein aggregates may have a larger molecular weight and/or size than non-aggregated alpha-synuclein proteins.

“Inhibiting protein aggregation of alpha-synuclein” in the present specification means suppressing the phenomenon of each alpha-synuclein protein aggregating with surrounding alpha-synuclein proteins to form an aggregate or decomposing and inhibiting an aggregate that has already been formed. can be understood Alternatively, inhibition of protein aggregation of alpha-synuclein does not mean only inhibition of aggregation, but either increases the rate of degradation of alpha-synuclein protein or aggregates thereof, or balances the rate of production and degradation of alpha-synuclein or aggregates thereof in a normal manner. It can include all things that are adjusted to the state.

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

The term "nerve cell" used herein is a cell of the nervous system. In this specification, the term “nerve cell” may be used interchangeably with “neuron” or “neuron cell”.

The term "glial cells" in the present specification is a cell that occupies the largest portion of cells present in the brain, and the glial cells may include astrocytes or microglia cells. Astrocytes are involved in nerve cell protection, nutrient supply, and inflammation, and microglial cells are cells responsible for inflammation in the brain and are known to be a cell group that plays an important role in brain diseases such as Alzheimer's disease.

The term "transduction" in this specification refers to a phenomenon in which genetic traits are transferred from one cell to another by the medium of a bacteriophage and the genetic traits are introduced. When phages bind to host DNA and come out through lysis, they may come out with part of the host DNA instead of losing part of their own DNA. When these phages infect other bacteria, they introduce new genes from the previous host, resulting in new traits. 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.

As used herein, the term "cell therapy product" refers to cells and tissues manufactured from human isolation, culture, and special chewing, which are used for the purpose of treatment, diagnosis, and prevention (according to US FDA regulations), and are used to restore the functions of cells or tissues. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro, or changing the biological characteristics of cells in other ways. Cell therapy is largely classified into somatic cell therapy and stem cell therapy depending on the degree of cell differentiation.

The term “transduction (transduction) of Nurr1 and Foxa2” as used herein means introducing nucleic acids encoding the two genes into brain cells together. The two genes can be introduced separately from each expression vector or simultaneously into one expression vector.

In one embodiment of the present invention, when both Nurr1 and Foxa2 genes are introduced, it is confirmed that the ability to inhibit alpha-synuclein protein aggregation is significantly superior to that when Nurr1 or Foxa2 genes are introduced alone due to the synergistic effect between Nurr1 and Foxa2. did Specifically, when the Nurr1 and Foxa2 genes were simultaneously introduced into cells, it was confirmed that both alpha-synuclein protein monomers and aggregates were significantly reduced compared to when the Nurr1 or Foxa2 genes were introduced alone (see FIGS. 5 and 6).

The term "introducing (transducing) Nurr1" as used herein means introducing a nucleic acid encoding the Nurr1 gene into brain cells.

In order to introduce a gene encoding Nurr1 and/or Foxa2 into brain cells, a method known in the art such as a method using an adeno-associated virus (AAV), a retrovirus, or an adenovirus may be used.

Viral vectors can be used when loading Nurr1 and/or Foxa2. As the viral vector, adeno-associated virus (AAV), adenovirus, retrovirus, and/or lentivirus may be used, but is not limited thereto. Therefore, the introduction of Nurr1 and/or Foxa2 according to the present invention, in one embodiment, involves inserting nucleic acids encoding Nurr1 and/or Foxa2 into separate expression vectors or into one expression vector and then introducing them into brain cells. can include

Each nucleic acid encoding Nurr1 and/or Foxa2 may be used without limitation as long as it has a nucleotide sequence encoding Nurr1 and Foxa2 known in the art. In addition, the nucleic acid may have a nucleotide sequence encoding each functional equivalent of Nurr1 and/or Foxa2. A functional equivalent refers to a polypeptide having at least 60% or more, 70% or more, or 80% or more sequence homology (i.e., identity) to the amino acid sequence of Nurr1 and/or Foxa2. For example, functional equivalents are 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%, 100% sequence homology. Functional equivalents may be those that result from additions, substitutions or deletions of some of the amino acid sequences. Amino acid deletion or substitution may be located in a region not directly related to the physiological activity of the polypeptide of the present invention.

In addition, nucleic acids encoding Nurr1 and/or Foxa2 can be prepared by genetic recombination methods known in the art. For example, nucleic acids encoding Nurr1 and/or Foxa2 can be prepared by PCR amplification to amplify nucleic acids from the genome, chemical synthesis, or techniques to prepare cDNA sequences.

A nucleic acid encoding Nurr1 and/or Foxa2 can be inserted into an expression vector operably linked to an expression control sequence. The term “operably linked” means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. In addition, expression control sequence (expression control sequence) refers to a DNA sequence that controls the expression of operably linked nucleic acid sequences in a specific host cell. Such regulatory sequences may include promoters to initiate transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences to control termination of transcription and translation. In total, these may be expressed as 'DNA constructs containing nucleic acids encoding Nurr1 and/or Foxa2'.

The term "expression vector" used herein refers to a viral vector or other vehicle known in the art into which a nucleic acid encoding a structural gene can be inserted and capable of expressing the nucleic acid in a host cell.

In the present invention, the vector may be a viral vector. The viral vector may be an adeno-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector, an avipoxvirus vector, and the like, and may be, for example, an adeno-associated viral vector, but is limited thereto It is not.

Adeno-associated virus (AAV) vectors can be constructed by introducing materials capable of making viruses into specific cells. Lentiviral vectors can also be constructed in several steps to produce viruses in specific cell lines.

The expression vector containing the nucleic acid according to the present invention is prepared using methods known in the art, such as, but not limited to, viral transduction, transient transfection, and microinjection. nucleic acids can be introduced into brain cells by known methods for introducing nucleic acids into cells. For example, after constructing an expression vector by inserting Nurr1 and/or Foxa2 into an adeno-associated virus (AAV) or lentiviral vector by genetic recombination technology, the vector is transduced into packaging cells, and the transduced After culturing the pavement cells, an AAV or lentivirus solution may be obtained by separation and purification. In addition, Nurr1 and/or Foxa2 genes can be introduced into brain cells by infecting brain cells (nerve cells and/or glial cells) using the same. Next, after confirming that Nurr1 and/or Foxa2 are expressed singly or simultaneously using a selectable marker included in AAV or lentiviral vector, desired brain cells can be obtained.

Brain cells in which Nurr1 and Foxa2 are introduced and expressed according to the present invention can be prepared by a manufacturing method comprising the following steps:

Preparing a recombinant viral vector containing a DNA construct containing nucleic acids encoding Nurr1 and Foxa2;

A production step of transfecting the recombinant viral vector into a virus-producing cell line to produce Nurr1 and Foxa2 expressing recombinant viruses; and

Transfection step of infecting brain cells with Nurr1 and Foxa2 expressing recombinant viruses.

Recombinant viral vectors can be prepared by operably linking the DNA construct to expression control sequences, such as promoters, and inserting into viral vectors known in the art. Thereafter, a recombinant virus vector containing nucleic acids encoding Nurr1 and/or Foxa2 is introduced into a virus-producing cell line to prepare a recombinant virus expressing Nurr1 and/or Foxa2. Virus producing cell line A cell line producing a virus corresponding to the used viral vector may be used. Then, recombinant AAV or lentivirus expressing Nurr1 and Foxa2 or Nurr1 can be infected with brain cells. This process may be performed by a method known in the art.

Brain cells expressing Nurr1 and/or Foxa2 according to the present invention may be proliferated and cultured according to methods known in the art.

Brain cells according to the present invention can be cultured in a culture medium that helps the survival or proliferation of the target cell type. Additives developed for the continuous culture of brain cells can be supplemented with the culture medium. For example, N2 medium and B27 additive, bovine serum, etc. commercially available from Gibco. Brain cells can be cultured by exchanging the medium while observing the conditions of the medium and the cells during culture. At this time, if the brain cells continue to proliferate and aggregate with each other to form neurospheres, subculture can be performed. Subcultures can be performed approximately every 7-8 days depending on circumstances.

The composition according to the present invention inhibits aggregation and phosphorylation of alpha-synuclein to protect brain cells, including nerve cells and glial cells, from damage, so that nerve cells can be replenished (regenerated) or rebuilt (restored). can

The term "regeneration" in this specification refers to a phenomenon in which a part of a formed organ or object is supplemented when it is lost. In addition, "restoration" may also be referred to as "reconstitution", which means the reconstruction of a tissue, which means reconstructing a tissue or organ from once dissociated cells or tissues.

The composition or cell therapy agent of the present invention may be prepared in an appropriate formulation including an acceptable carrier depending on the administration method. Agents suitable for the mode of administration are known and may include agents that facilitate movement, typically across membranes.

In addition, the composition of the present invention can be used in the form of general pharmaceutical preparations. For parenteral preparations, it can be prepared in the form of sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions or lyophilized preparations, and for oral administration, tablets, troches, capsules, elixirs, suspensions, syrups or wafers. It can be prepared in unit dose ampoules or multi-dose form. In addition, the therapeutic composition of the present invention may be administered together with a pharmaceutically acceptable carrier. For example, for oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant or a flavoring agent may be used. In the case of injection, a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used.

Another aspect of the present invention is a vector loaded with Nurr1 and Foxa2 genes; And a composition for preventing or treating a disease caused by alpha-synuclein protein aggregation, comprising any one selected from the group consisting of brain cells into which the Nurr1 and Foxa2 genes have been introduced.

Another aspect of the present invention is a method for treating or alleviating a disease caused by alpha-synuclein protein aggregation comprising the following steps.

Viral vectors loaded with Nurr1 and Foxa2 genes; and administering to a subject a composition comprising any one selected from the group consisting of brain cells into which Nurr1 and Foxa2 genes have been introduced.

The term "subject" in the present specification may be a vertebrate animal that is a subject of treatment, observation, or experiment, for example, cow, pig, horse, goat, dog, cat, rat, mouse, rabbit, guinea pig, human, and the like.

The term "treatment" as used herein refers to an approach to obtain beneficial or desirable clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state improvement or palliation and relief (partial or total), detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or prophylactic methods. Treatments include treatment required for disorders that have already occurred as well as disorders that are prevented. "Palliating" a disease means reducing the extent and/or undesirable clinical signs of the disease state and/or slowing or prolonging the time course of the disease compared to no treatment. means to lose

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.

In addition, a method of treating a disease caused by alpha-synuclein using the therapeutic composition of the present invention may include administering to a subject or patient in an appropriate manner through a general route through which a predetermined substance is introduced.

Methods of administration include intracerebral, midbrain, intraventricular, spinal, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration. My administration includes, but is not limited to.

In addition, the composition according to the present invention can be administered by any device capable of transporting an active substance to a target cell. The administration method and formulation may be a brain midbrain injection, brain substantia nigra injection, ventricle injection, cerebrospinal fluid injection, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or drip injection using a stereotactic system. Injections are formulated with aqueous solvents such as physiological saline or IV, non-aqueous solvents such as vegetable oils, higher fatty acid esters (eg, oleic acid ethyl, etc.), alcohols (eg, ethanol, benzyl alcohol, propylene glycol or glycerin, etc.). can be produced using Injections contain a stabilizer to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), an emulsifier, a buffer to adjust pH, and a preservative to prevent microbial growth (e.g., nitric acid). phenylmercury, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) and the like).

A composition according to the present invention can be administered to a subject in a pharmaceutically effective amount. The pharmaceutically effective amount is the type of disease, the age, weight, health, sex of the subject (patient), the sensitivity of the subject (patient) to the drug, the route of administration, the method of administration, the number of times of administration, the treatment period, drugs used in combination or concurrently, etc. It can be readily determined by a person skilled in the art according to factors well known in the medical arts.

The brain cells into which the Foxa2 and/or Nurr1 genes according to the present invention have been introduced can be directly transplanted into the lesion site according to a therapeutically effective amount in the form of a composition.

As used herein, the term "therapeutically effective amount" means an amount sufficient to stop or alleviate the physiological effects of a subject or patient caused by aggregation or phosphorylation of alpha-synuclein. The therapeutically effective amount of the cells used depends on the need of the subject (patient), the age, physiological state and health of the subject (patient), the desired therapeutic effect, the size and area of the tissue to be targeted for treatment, the extent of the lesion and the selected delivery It can be determined according to the route. Alternatively, the cells can be administered to more than one site in a given target tissue in multiple mini-grafts at low cell doses. The cells of the present invention may be completely isolated prior to transplantation, eg forming a suspension of single cells, or may be almost completely isolated prior to transplantation, eg forming small aggregates of cells. Cells can be administered by transplanting or migrating them to a given tissue site and reconstructing or regenerating functionally deficient areas.

An appropriate range of cells that can be administered to achieve a therapeutic effect can be appropriately determined according to the subject or patient within the common knowledge of those skilled in the art. For example, the number of cells that may be included in the composition according to the present invention may be about 10 to 1,000,000,000, but is not limited thereto.

A suitable dosage of the composition of the present invention depends on factors such as formulation method, administration method, subject's (patient's) age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity. can be determined A ordinarily skilled physician can readily determine and prescribe effective dosages for the desired treatment. The pharmaceutical composition of the present invention contains 1 × 10 ¹ to 1 × 10 ¹³ virus genome (vg) / μl, 1 × 10 ² to 1 × 10 ¹³ vg / μl, 1 × 10 ³ to 1 × 10 ¹³ vg / μl, 1 × 10 ⁴ to 1 × 10 ¹³ vg/µl, 1 × 10 ⁵ to 1 × 10 ¹³ vg/µl, 1 × 10 ⁶ to 1 × 10 ¹³ vg/µl, 1 × 10 ⁷ to 1 × 10 ¹³ vg/µl μl, 1 × 10 ⁸ to 1 × 10 ¹³ vg/μl, 1 × 10 ⁹ to 1 × 10 ¹³ vg/μl, 1 × 10 ¹⁰ to 1 × 10 ¹³ vg/μl, 1 × 10 ¹¹ to 1 × 10 ¹³ vg/μL, 1 × 10 ¹² to 1 × 10 ¹³ vg/μL, 1 × 10 ¹ to 1 × 10 ¹² vg/μL, 1 × 10 ¹ to 1 × 10 ¹¹ vg/μL, 1 × 10 ¹ to 1 × 10 ¹⁰ vg/μL, 1 × 10 ¹ to 1 × 10 ⁹ vg/μL, 1 × 10 ¹ to 1 × 10 ⁸ vg/μL, 1 × 10 ¹ to 1 × 10 ⁷ vg/μL, 1 × 10 ¹ to 1 × 10 ⁶ vg/μL, 1 × 10 ¹ to 1 × 10 ⁵ vg/μL, 1 × 10 ¹ to 1 × 10 ⁴ vg/μL, 1 × 10 ¹ to 1 × 10 ³ vg/μL, 1 × 10 ¹ to 1 × 10 ² vg/μL, 1 × 10 ² to 1 × 10 ¹² vg/μL, 1 × 10 ³ to 1 × 10 ¹¹ vg/μL, 1 × 10 ⁴ to 1 × 10 ¹⁰ vg/μL, 1 × 10 ⁵ to 1 × 10 ⁹ vg/μL, 1 × 10 ⁶ to 1 × 10 ⁸ vg/μL, 1 × 10 ² to 1 × 10 ³ vg/μL, 1 × 10 ³ to 1 × 10 ⁴ vg/μL , 1 × 10 ⁴ to 1 × 10 ⁵ vg/μL, 1 × 10 ⁵ to 1 × 10 ⁶ vg/μL, 1 × 10 ⁶ to 1 × 10 ⁷ vg/μL, 1 × 10 ⁷ to 1 × 10 ⁸ vg /μl, 1 × 10 ⁸ to 1 × 10 ⁹ vg/μl, 1 × 10 ⁹ to 1 × 10 ¹⁰ vg/μl, 1 × 10 ¹⁰ to 1×10 ¹¹ vg/μl, 1×10 ¹¹ to 1×10 ¹² vg/μl of a viral vector or viral gene. Typically, 1 × 10 ⁶ to 2 × 10 ¹⁶ vg/dose can be injected into the patient 1 to 5 times. In order to maintain the effect, it can be injected again in a similar manner after several months or years.

The present invention relates to a composition for inhibiting alpha-synuclein aggregation and a method for inhibiting aggregation, and more particularly, to inhibit alpha-synuclein aggregation and phosphorylation by introducing Nurr1 and Foxa2 genes to induce expression. Regarding technology, the composition according to the present invention has an excellent effect of inhibiting aggregation and phosphorylation of alpha-synuclein, and thus can be used for the treatment and prevention of Parkinson's disease.

Figure 1 shows the results of western blotting after processing alpha-synuclein PFF (Preformed fibril) to a control group (Cont) and dopamine neurons and glial cells (NF) into which Nurr1 and Foxa2 genes were introduced. This is the picture shown
Figure 2 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.
Figure 3 is a graph showing the levels of phosphorylated alpha-synuclein monomers in a control group and a group (NF) into which Nurr1 and Foxa2 genes were introduced after western blotting according to an embodiment.
Figure 4 is a graph showing the levels of phosphorylated alpha-synuclein aggregates in a control group and a group (NF) into which Nurr1 and Foxa2 genes were introduced after Western blotting according to one embodiment.
5 shows alpha-synuclein in dopaminergic neurons and glial cells in which a control group (Cont), Nurr1 gene alone (N), Foxa2 gene alone (F), and both Nurr1 and Foxa2 genes are introduced (NF) according to an embodiment. After processing PFF (Preformed fibril), it is a photograph showing the result of Western blotting.
Figure 6 shows alpha-Synu of a control group (Con), a Foxa2-only introduction group (F), a Nurr1-only introduction group (N), and a Nurr1 and Foxa2 gene introduction group (NF) after Western blotting according to an embodiment. It is a graph comparing the levels of Klein aggregates and monomers.
7 is a graph showing the results of a pole test of a wild type (WT), a control group (Cont), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2, according to an embodiment.
8 is a photograph showing the poll test results of a wild type (WT), a control group (Cont), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.
9 is a graph showing beam test results at week 8 of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.
10 is a graph showing beam test results at week 12 of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.
11 is a photograph showing the beam test results of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.
12 is a total distance traveled by a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an open field test (OFT) according to an embodiment. It is a graph showing Total distance).
13 is a graph showing average speeds of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an open field test according to an embodiment.
14 is a view showing open field test results of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.
15 is a graph showing the results of a Rotarod test at week 8 of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2, according to one embodiment.
16 is a graph showing the results of the rotarod test at week 12 of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to one embodiment.
17 is a photograph showing the rotarod test results of a wild type (WT), a control group (PD), which is a Parkinson's disease model, and an experimental group (NF) introduced with Nurr1 and Foxa2 according to an embodiment.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Vector Preparation

Lentiviruses were prepared for transduction. Lentiviral vectors expressing Nurr1 or Foxa2 were generated by inserting each cDNA into the multiple cloning site of pCDH (System Biosciences, Mountain View, CA) under the control of the CMV promoter. The pGIPZ-shNurr1 and pGIPZ-shFoxa2 lentiviral vectors were purchased from Open Biosystems (Rockford, IL). Empty backbone vectors, pCDH or pGIPZ, were used as negative controls. The titer of lentivirus was measured using the QuickTiter™ HIV Lentivirus Quantification Kit (Cell Biolabs, San Diego, CA), and 200 μl containing 106 transducing units (TU)/ml (60 to 70 ng/ml) /well (24 well dish) or 2ml/6cm dish was used for each transduction reaction.

To induce in vivo expression by stereotaxic injection, AAV expressing Nurr1 or Foxa2 under the control of the CMV promoter was subcloned into the pAAV-MCS vector (Addgene, Cambridge, MA), respectively. Created. To evaluate the efficiency of gene expression delivered, AAVs expressing Green Fluorescence Protein (GFP) were also generated. Production and separation and purification of AAVs (serotype 9 or 2) were performed at the Korea Institute of Science and Technology (Seoul, Korea). AAV titers were measured with the QuickTiterTM AAV Quantification Kit (Cell Biolabs). Co-expression studies were performed by infecting cells with a mixture (1:1, v:v) of individual virus preparations.

Example 2: Cell culture

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

Neural progenitor cells with dopaminergic potential in 6-well plates coated with poly-L-ornitine-fibronectin (PLO-FN). ) was seeded with 4x10 ⁵ /well. After 24 hours, lentiviruses expressing Nurr1 and Foxa2 under the control of synapsin promoters were transfected at 10 ⁶ transducing unit (TU)/ml (60-70 ng/ml) and 1:1 (v:v), respectively. introduced. As a control group, lentivirus expressing GFP was treated at the same dose. Thereafter, they were cultured for 3 days and differentiated into neurons.

2-2. Culture of ventral midbrain glia

For the culture of ventral midbrain glia, 3x10 ⁶ mice were seeded on a 100 mm x 20 mm culture dish coated with poly-L-ornithine-fibronectin, and after 24 hours Lentiviruses individually expressing Nurr1 and Foxa2 under the control of the CMV promoter were mixed and transduced at a ratio of 1:1 (v:v) at 10 ⁶ transducing unit (TU)/ml (60-70 ng/ml). As a control group, a control virus was treated at the same dose. Thereafter, cultured for 5 days.

2-3. co-culture

Dopaminergic VM neurons cultured in Example 2-1 were seeded with VM glia cultured in Example 2-2 at a ratio of 2:1 (VM Neuron : VM glia = 2 : 1 ). The next day, alpha-synuclein PFF (Preformed fibril) was treated to a final concentration of 2 μg/ml, and after 7 days, the protein amount of alpha-synuclein was confirmed by Western blotting.

Example 3: Western blot

Protein was extracted by adding protease inhibitor (Roche) and phosphatase inhibitor cocktails (Sigma) to the plated cells in 1% Triton X-100/PBS solution. After centrifugation, the pellet was dissolved in 1% SDS sample buffer, and 15 μg of protein was loaded on an SDS-PAGE gel (4-16% gradient gel). After transfer to the membrane, the membrane was blocked in 5% BSA/TBST, the primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Primary antibodies were used as follows: α-syn (BD biosciences, 610787); pS129-α-syn (Bio Legend, 825701). And, the western blot test results are shown in FIG. 1 . After Western blotting, Western blotting results were quantitatively measured using the ImageJ program, which are shown in FIGS. 2 to 4 and Tables 1 to 3.

Cont NF Aggregated
α-syn/β-actin One 0.73

Cont NF Monomeric
Pα-syn/β-actin One 0.34

Cont NF Aggregated
Pα-syn/β-actin One 0.62

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

As a result of western blotting, as can be seen in FIG. 1, when alpha-synuclein PFF was treated in neurons and glial cells transduced with Nurr1 and Foxa2 genes, compared to neurons and glial cells in which Nurr1 and Foxa2 were not treated, alpha-synuclein It was confirmed that the aggregation of the clay protein was reduced compared to the control group. In addition, it was confirmed that the levels of phosphorylated monomers and aggregates of alpha-synuclein protein were significantly reduced compared to the control group.

Specifically, as can be seen in Figure 2 and Table 1, when the Nurr1 and Foxa2 genes were transduced, the amount and aggregation of aggregated alpha-synuclein protein were reduced compared to the control group. In addition, as can be seen in Figures 3 to 4 and Tables 2 to 3, when the Nurr1 and Foxa2 genes were transduced, the level of phosphorylated alpha-synuclein aggregates was reduced to about 60% of the control group, and the monomers In the case of , it was confirmed that it decreased to about 30% level.

Example 5: Comparison of alpha-synuclein aggregate formation inhibitory effect between single administration of Nurr1 and combined administration of Nurr1 and Foxa2

Neurons and glial cells into which Nurr1 alone, Foxa2 alone, or both Nurr1 and Foxa2 were introduced were plated and phosphatized with a protease inhibitor (Roche) in 1% Triton X-100/PBS solution. Enzyme inhibitor cocktails (phosphatase inhibitor cocktails, Sigma) were added to extract proteins.

As a control group, cells not introduced with Nurr1 or Foxa2 were used. After centrifugation, the pellet was dissolved in 1% SDS sample buffer, and 15 μg of protein was loaded on an SDS-PAGE gel (4-16% gradient gel). After transfer to the membrane, the membrane was blocked in 5% BSA/TBST, the primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Primary antibodies were used as follows: α-syn (BD biosciences, 610787); pS129-α-syn (Bio Legend, 825701). And, the western blot test results are shown in FIG. 5 . After Western blotting, Western blotting results were quantitatively measured using the ImageJ program, which are shown in FIG. 6 and Table 4.

Monomer Aggregates Non Con F N NF Non Con F N NF Aggregated
α-syn/β-actin 1.52 10.10 9.85 7.77 5.25 1.17 10.24 9.02 8.28 4.76

Experimental results, as can be seen in Figure 6 and Table 4, the group transduced with both Nurr1 and Foxa2 genes (N+F) was alpha-synuclein aggregation compared to the group transduced with Nurr1 or Foxa2 alone (N or F). It was confirmed that this was significantly reduced. Specifically, the group transduced with both Nurr1 and Foxa2 genes reduced alpha-synuclein aggregation by more than 48% compared to the control group, and in particular, alpha-synuclein aggregation was reduced by more than 32% compared to the Nurr1 alone input group. Confirmed. As a result, when both Nurr1 and Foxa2 genes were transduced, it was confirmed that alpha-synuclein aggregation could be significantly reduced compared to when Nurr1 was introduced alone, and furthermore, Nurr1 and Foxa2 genes were used in the treatment of Parkinson's disease. Incorporating them all is expected to be more effective.

Example 6: Confirmation of behavioral improvement according to introduction of Nurr1 and Foxa2 in Parkinson's disease model

6-1. Construction of Parkinson's disease model (PD model) mouse

A mixture of 5 μg of ICR mouse AAV2-CMV-αsyn-HA and α-syn PFF was injected into the substantia nigra (SN) of the mouse. Specifically, after fixing the mouse with a stereotaxic instrument, a midline incision of about 1 cm was made in the skin of the head region to confirm the bregma. Based on the vestibule, pierce the skull with an electric drill at -3.3mm anterior and 1.2mm lateral positions, and 2 μL of AAV2-CMV-α-syn-HA (1.3 x 10 ¹³ gc/μL) and α-syn PFF (5 mg/mL) ) Load 2 μL into a stereotaxic injector, enter 4.6 mm deep from the skull, and 2 microliters each on both sides of the substantia nigra (the dose of each vector for both administration sites is 1.3 x 10 ¹³ gc/site) 0.5 μl/min Administered slowly at a rapid rate. After 20 minutes of administration to both substantia nigra regions, the injector was pulled back 1.5 mm per 10 minutes to minimize leakage of the administered mixture. The mouse skull skin was sutured using a medical skin stapler, disinfected with povidone, and placed in a cage after recovery.

Four weeks after administration, Nurr1 and Foxa2 were transduced using a viral vector expressing Nurr1 and Foxa2. Specifically, after fixing the PD mouse model with a stereotaxic instrument, a midline incision of about 1 cm was made in the skin of the head region to confirm the bregma. Based on the vestibule, the skull was drilled with an electric drill at -3.3mm anterior and 1.2mm lateral positions, and AAV9-hNurr1 and AAV9-hFoxa2 were loaded into a stereotaxic injector at a concentration of 1 x 10 ¹⁰ gc/μL for each vector. After entering a depth of 4.6 mm from the skull, 2 microliters were slowly administered to each side of the substantia nigra region (the dose of each vector for both administration sites was 1.0 x 10 ¹⁰ gc/site) at a rate of 0.5 μl/min. After 20 minutes of administration to both substantia nigra regions, the injector was pulled back 1.5 mm per 10 minutes to minimize leakage of the administered mixture. The skin of the skull was sutured using a medical skin stapler, disinfected with povidone, and placed in a cage after recovery.

6-2. Pole test

A pole test was performed 4 weeks, 8 weeks, and 12 weeks after transduction of Nurr1 and Foxa2. The test was conducted after adapting the mice by training them 2 to 3 days before the test. During the test, the time taken for mice of the wild type (WT), Parkinson's disease model, control group (Cont), and Nurr1 and Foxa2 transduced group (NF) to move down from the upper end of the pole was measured. In the case of falling or slipping from the pole during the experiment, it was set as a negative value, and data was created by setting it to the same value as the maximum value in the state. At this time, 6 wild-type mice and 8 mice of the control and Nurr1 and Foxa2 transduced groups were tested. The measured time determined a significant effect through one-way ANOCA.

Before NF 4 weeks later 8 weeks later after 12 weeks wild type (WT) 7.84 4.77 4.62 4.92 Control (Cont) 10.26 10.20 8.86 9.02 Transgenic group (NF) 10.42 7.23 6.35 6.72

As a result of the experiment, as can be seen in FIGS. 7 to 8 and Table 5, the control mice took longer to go down the poles than the wild-type and mice of the Nurr1 and Foxa2 transduction group (NF) groups, and they did not fall or slip on the poles. frequency was high.

6-3. Beam test

Beam tests were performed 8 and 12 weeks after Nurr1 and Foxa2 were transduced. The test was conducted after adapting the mice by training them 2 to 3 days before the test. The beam used in the test was rectangular and had a thickness of 10 mm. During the test, the time for mice of the wild type (WT), Parkinson's disease model, control group (PD), and Nurr1 and Foxa2 transduced group (NF) groups to move from end to end of the beam was measured twice. In the case of falling or slipping from the beam during the experiment, it was set as a negative value, and data was created by setting it to the same value as the maximum value in the state. At this time, wild-type mice, control group (PD group), and Nurr1 and Foxa2 transduced mice were all 6 mice. The measured time determined a significant effect through one-way ANOCA.

week 8 week 12 WT PD NF WT PD NF Required time (Sec) 4.26 11.46 7.85 4.89 13.57 9.42

As a result of the experiment, as can be seen in FIGS. 7 to 8 and Table 6, control mice took more time to pass the beam than mice in the wild type and Nurr1 and Foxa2 transduced groups (NF).

6-4. Open Field Test (OFT)

Open field tests were conducted 8 weeks after transduction of Nurr1 and Foxa2. Mice of the wild type (WT), control (PD), and Nurr1 and Foxa2 transduced (NF) groups, which are Parkinson's disease models, were placed in an open field, and the movement, speed, and distance of each group's mice for 5 minutes, etc. was measured. At this time, wild-type mice, control group (PD group), and Nurr1 and Foxa2 transduced mice were all 6 mice. The measured time determined a significant effect through one-way ANOCA.

week 8 WT PD NF total distance (m) 12.84 5.91 12.95

week 12 WT PD NF Average speed (m/sec) 0.042 0.019 0.043

As a result of the experiment, as can be seen in FIGS. 12 to 14 and Tables 7 to 8, the behavior of the mice in the Nurr1 and Foxa2 transduction group (NF) groups was improved to the extent that there was almost no difference in the total distance and average speed moved from the wild type. Confirmed. In addition, the total distance traveled and the average movement speed of mice in the Nurr1 and Foxa2 transduced (NF) groups were significantly increased compared to the control group (PD).

6-5. Rotarod Test

Rotarod tests were performed 8 weeks and 12 weeks after Nurr1 and Foxa2 were transduced. The test was conducted after adapting the mice by training them 2 to 3 days before the test. In the test, while gradually increasing the speed from 4 rpm to 40 rpm, the time taken for wild type (WT), Parkinson's disease model control (PD), and Nurr1 and Foxa2 transduced (NF) group mice to fall from the cylinder was measured. It was carried out by measuring twice. At this time, wild-type mice, control group (PD group), and Nurr1 and Foxa2 transduced mice were all 6 mice. The measured time determined a significant effect through one-way ANOCA.

week 8 WT PD NF Required time (sec) 195.45 71.40 136.40

week 12 WT PD NF Required time (sec) 211.3 89.58 176.58

As a result of the experiment, as can be seen in FIGS. 15 to 17 and Tables 9 to 10, the Nurr1 and Foxa2 transduced group (NF) mice spent much more time away from the cylinder than the control group at 8 weeks and 12 weeks.

6-6. sintering

As such, it was confirmed that the mice of the Nurr1 and Foxa2 transduction group (NF) groups had significantly improved motor skills and behavior in all of the pole test, beam test, open field test and rotarod test compared to the control group, which is a Parkinson's disease model. Through the above results, it was confirmed that the introduction of the Nurr1 and Foxa2 genes can restore the decline in exercise capacity such as stiffness, bradykinesia, and postural instability characteristic of Parkinson's disease. expected to be usable.

Claims (10)

Viral vectors loaded with Nurr1 and Foxa2 genes; and
An alpha-synuclein protein aggregation inhibitor comprising any one selected from the group consisting of brain cells into which Nurr1 and Foxa2 genes have been introduced. The method of claim 1, wherein the viral vector,
An alpha-synuclein protein aggregation inhibitor, which is one selected from the group consisting of an adeno-associated virus vector, a retrovirus vector, and an adenovirus vector. The method of claim 1, wherein the brain cells,
An alpha-synuclein protein aggregation inhibitor that is neuron, and glia, including astrocytes or microglia. Viral vectors loaded with Nurr1 and Foxa2 genes; and
An alpha-synuclein protein phosphorylation inhibitor comprising any one selected from the group consisting of brain cells into which Nurr1 and Foxa2 genes have been introduced. The method of claim 4, wherein the viral vector,
An inhibitor of alpha-synuclein protein phosphorylation, which is one selected from the group consisting of an adeno-associated virus vector, a retrovirus vector, and an adenovirus vector. The method of claim 4, wherein the brain cells,
Neurons, and glia, including astrocytes or microglia, alpha-synuclein protein phosphorylation inhibitors. Viral vectors loaded with Nurr1 and Foxa2 genes; and
A composition for preventing or treating a disease caused by α-synuclein protein aggregation, comprising any one selected from the group consisting of brain cells into which Nurr1 and Foxa2 genes have been introduced. The method of claim 7, wherein the viral vector,
One selected from the group consisting of an adeno-associated virus vector, a retroviral vector, and an adenoviral vector, the composition. The method of claim 7, wherein the brain cells,
Neurons, and glial cells including astrocytes or microglia (microglia), the composition. The composition of claim 7, wherein the disease caused by aggregation of the alpha-synuclein protein is a disease selected from the group consisting of Parkinson's disease and Dementia with Lewy Bodies.

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