WO2021015352A1 - Method for reprogramming reactive astrocytes into neurons in spinal cord injury model using neurogenin-2 (ngn2) - Google Patents

Abstract

The present invention relates to: a method for reprogramming reactive astrocytes into neurons, comprising the steps of (a) introducing neurogenin-2 (Ngn2) protein or a nucleic acid molecule encoding Ngn2 protein into reactive astrocytes or increasing the expression of Ngn2 protein in astrocytes and (b) culturing the astrocytes of step (a); a composition for inducing reprogramming of astrocytes comprising Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein into neurons; a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases; and neurons prepared by the method and cell therapeutics comprising same. According to the present invention, when neurogenin-2 (Ngn2) expression is specifically increased in reactive astrocytes, the reactive astrocytes can be directly reprogrammed into neurons. In addition, it is confirmed that behavioral ability is improved by reprogramming reactive astrocytes into neurons in a spinal cord injury mouse model using the method of the present invention. Therefore, the method for reprogramming into neurons can be utilized as a pharmaceutical composition or cell therapeutics for the purpose of preventing or treating degenerative neurological diseases including spinal cord injury.

Description

[Correction 13.09.2019 according to Rule 26] 　 Method of reprogramming reactive astrocytes into neurons in a spinal cord injury model using Ngn2 (Neurogenin-2)

The present invention comprises the steps of (a) introducing a nucleic acid molecule encoding Ngn2 (neurogenin-2) protein or Ngn2 protein into astrocytes, or increasing the expression of Ngn2 protein in astrocytes, and (b) the (a) A method of reprogramming (reprogramming) astrocytes into neurons, comprising the step of culturing the astrocytes of the step); A composition for inducing reprogramming of astrocytes into neurons, including Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein; A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases; It relates to a nerve cell prepared by the above method and a cell therapy product comprising the same.

The production of target cells through transdifferentiation or direct-reprogramming of specific cells is attracting attention as it can be used as a promising treatment method to replace organ transplantation. That is, in the field of regenerative medicine, replacement of damaged cells through transdifferentiation or direct reprogramming has been suggested as a fundamentally treatable alternative for diseases, and clinical trials for specific diseases and cells are also actively progressing.

In particular, nerve cells constitute the nervous system and are essential for controlling sensory perception, learning, memory, and motor skills, but once damaged, it takes a long time to recover to a normal state, and unlike other cells, it is difficult to regenerate, so it is difficult to permanently damage. It can be said to be of higher value as a treatment target as it is highly likely. In this regard, differences in damage response and regenerative ability appear depending on which part of the same nerve cell is damaged. In the case of spinal cord injury (SCI), which is a central nerve, regeneration failure occurs more than other parts. There are many cases, and there is no other treatment method in clinical practice until now. In addition to spinal cord injury, diseases caused by neuronal degeneration, such as Parkinson's disease, Alzheimer's disease, and Pick's disease, cause movement disorders and cognitive dysfunction, and are treated continuously. However, it is becoming a personal and social problem as there is no fundamental treatment method.

On the other hand, astrocytes are glial cells that form packing tissue in the central nervous system, and are known to supply energy, regulate blood flow, homeostasis of extracellular fluid, homeostasis of ions and transporters, and synaptic function in healthy nervous tissue. However, when the destruction of nearby neurons occurs due to trauma, infection, ischemia, stroke, autoimmune reactions, and neurodegenerative diseases of the central nervous system, the number of astrocytes abnormally increases, which is called reactive astrocyte (astrogliosis). do. In this regard, studies aimed at repairing the damaged spinal cord to date have been conducted on embryonic stem cells (embryonic stem cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells (iPS cells) with pluripotency. Most of the treatment methods were related to transplantation of mesenchymal stem cells (MSCs) and neural stem cells (NSCs), or for side effects that occur after spinal cord injury (Korean Patent Registration No. 10-1892457), No attempt has been made to directly convert nerve cells from reactive astrocytes to transplantation.

Accordingly, the inventors of the present invention made diligent efforts on a method for producing a neuron by reprogramming astrocytes, which are non-neuronal cells, and as a result of increasing the expression of Ngn2 (neurogenin-2) specifically for astrocytes, astrocytes become neurons. It has been confirmed that symptoms are improved when reprogrammed directly to and applied to a spinal cord injury model. Eventually, this method of reprogramming neurons can be used for the purpose of preventing or treating degenerative neurological diseases including spinal cord injury. By confirming the present invention was completed.

One object of the present invention is (a) introducing Ngn2 (neurogenin-2) protein or a nucleic acid molecule encoding Ngn2 protein into astrocytes, or increasing the expression of Ngn2 protein in astrocytes; And (b) comprising the step of culturing the astrocytes of the step (a), to provide a method for reprogramming (reprogramming) astrocytes into nerve cells.

Another object of the present invention is to provide a composition for inducing reprogramming of astrocytes into neurons, including Ngn2 protein or a nucleic acid molecule encoding the Ngn2 protein.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, including Ngn2 protein or a nucleic acid molecule encoding the Ngn2 protein.

Another object of the present invention is to provide a nerve cell produced by the above method.

Another object of the present invention is to provide a cell therapy product comprising nerve cells prepared by the above method as an active ingredient.

According to the present invention, when Ngn2 (neurogenin-2) expression is specifically increased in reactive astrocytes, the astrocytes can be directly reprogrammed into neurons. In addition, the GFAP-Lcn2 promoter combination in mice and the GFAP-iNOS promoter combination in monkeys can be used to specifically regulate Ngn2 expression in reactive astrocytes. Such a method of reprogramming nerve cells may be used as a pharmaceutical composition or cell therapy for the purpose of preventing or treating degenerative neurological diseases including spinal cord injury.

1A shows the recombinant plasmid vectors pAAV-GFAP-Ccre, pAAV-Lcn2-Ncre and pAAV-EF1α-df-Ngn2-IRES-GFP used for astrocyte-specific Ngn2 expression in mice.

FIG. 1B shows the plasmid vector pAAV-EF1α-df-GFP used as a control for expression of astrocyte-specific Ngn2 in mice.

Figure 2a shows the recombinant plasmid vectors pAAV-iNOS-Ccre, pAAV-GFAP-Ncre, and pAAV-EF1α-df-Ngn2-IRES-GFP used for astrocyte-specific Ngn2 expression for cynomolgus monkey.

Figure 2b shows the plasmid vector pAAV-EF1α-df-GFP used as a control for astrocyte-specific Ngn2 expression for cynomolgus monkey.

3A shows a protocol for injecting a virus containing a recombinant vector for astrocyte-specific Ngn2 expression into mouse and cynomolgus monkeys.

3 b and c show the results of immunohistochemistry confirming the expression of GFP, GFAP, DAPI and Lcn2/iNOS in damaged striatal tissues of mouse and cynomolgus monkey, respectively.

Figure 4a shows a schematic of a double floxed Split-Cre system for astrocyte-specific Ngn2 expression for each mouse and cynomolgus monkey.

4B shows a protocol for inducing the cultivation of reactive astrocytes and Ngn2 expression under in vitro conditions.

4C shows the change in morphology of astrocytes in the control group and the Ngn2 expression group.

4D shows the results of whole cell patch-clamp recording (in vitro) of cells reprogrammed with EGFP-expressing cells. Neuron-like cells successfully exhibited action potentials.

Figure 5a shows a protocol for confirming the reprogramming of astrocytes into neurons according to Ngn2 expression in mouse striatum.

5B shows the immunochemical staining results (in vivo) confirming the expression patterns of GFP, GFAP and NeuN in the control group and the Ngn2 expression group.

5C shows the proportion of GFAP or NeuN-expressing cells among GFP-expressing cells.

5D shows a typical current of astrocytes as a result of patch-clamp recording of GFP-expressing cells in the control group.

Figure 5e shows that the result of patch-clamp recording of GFP-expressing cells in the Ngn2 expression group shows an action potential and a spontaneous post-synaptic current.

6A shows a protocol for confirming the reprogramming of astrocytes into neurons according to Ngn2 expression in cynomolgus monkey putamen.

6B shows the immunochemical staining results (in vivo) confirming the expression patterns of GFP, GFAP and NeuN in the control group and the Ngn2 expression group.

6C shows the scatter plot results according to the GFAP and NeuN intensities. In the control group, the NeuN intensity was mostly less than 1K, but the NeuN intensity was significantly increased in the Ngn2 expression group.

FIG. 6D summarizes the results of the above scatter plot and shows that the ratio of NeuN intensity of 1K or more (red area) increases.

6E shows that the GFAP and NeuN intensities were compared in the control group and the GFAP intensity was higher.

Figure 6f shows that the intensity of NeuN in the Ngn2 expression group increased to a similar degree to that of GFAP.

6G shows that the NeuN intensity was significantly higher in the Ngn2 expression group by comparing the NeuN intensity in the control and the Ngn2 expression group.

6h shows that the GFAP intensity was significantly higher in the control group by comparing the GFAP intensity in the control group and the Ngn2 expression group.

7A shows an experimental protocol according to virus injection including a recombinant vector for astrocyte-specific Ngn2 expression in a spinal cord injury (SCI) model.

7B shows a process of creating a spinal cord injury model due to compression injury and injecting a virus for gene expression 2 weeks later.

FIG. 7C shows the change in BMS (Basso Mouse Score) score for measuring paraparesis in the PBS group, the control group, and the Ngn2 group among the sham group and the spinal cord injury model. Compared with the PBS group and the control group, it was shown that the improvement of behavioral ability occurred significantly in the Ngn2 expression group.

Fig. 7D is a result of patch clamp recording of GFP-expressing cells in the Ngn2 expression group, confirming that the action potential is displayed.

7E is a patch clamp recording of GFP-expressing cells in the Ngn2 expression group to confirm the current caused by the activity of the Na channel.

Fig. 7 f shows that a current is generated after spontaneous synapses as a result of patch clamp recording GFP-expressing cells in the Ngn2 expression group.

FIG. 8B shows the results of immunostaining with GFAP, NeuN, and ist1 in spinal cord tissues of the PBS group, the control group, and the Ngn2 group among the sham group and the spinal cord injury model.

FIG. 8C is a comparison of the intensity of MAP2 measured in the immunostaining result of FIG. 8A. Compared with the PBS group and the control group, the MAP2 intensity was significantly increased in the Ngn2 group.

FIG. 8D is a comparison of the intensity of ist1 and GFAP measured in the immunostaining result of FIG. 8B. Compared to the PBS group and the control group, the ist1 intensity was significantly increased and the GFAP intensity was decreased in the Ngn2 group.

8E shows the results of EC staining of coronal and longitudinal sections of spinal cord tissue obtained from the PBS group, the control group, and the Ngn2 group among the sham group and the spinal cord injury model. Unlike the PBS group and the control group, the Ngn2 group showed a significant level of recovery of damaged spinal cord tissue.

FIG. 8 f is a comparison of the areas of white matter and gray matter in the staining result of FIG. 8 e. In the Ngn2 expression group, both white and gray matter and total area were significantly increased.

9A shows the results of staining GFAP or NeuN or MBP (Myelin binding protein) with BrdU in spinal cord tissues of the PBS group, the control group, and the Ngn2 group among the sham group and the spinal cord injury model.

9B shows a protocol for viral inection and BrdU injection for gene expression in a spinal cord injury model.

FIG. 9C is a summary of the immunostaining results of FIG. 9A, and shows that BrdU is not stained in cells stained with GFAP or NeuN or MBP while expressing GFP.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

One aspect of the present invention for achieving the above object is to (a) introduce Ngn2 (neurogenin-2) protein or a nucleic acid molecule encoding Ngn2 protein into astrocytes, or express Ngn2 protein in astrocytes. Increasing; And (b) it provides a method of reprogramming (reprogramming) astrocytes into neurons, comprising the step of culturing the astrocytes of step (a).

In the present invention, the term "astrocyte" is a glial cell that forms a packing tissue in the central nervous system, which supplies energy, regulates blood flow, and regulates extracellular fluid homeostasis, ion and transporter homeostasis, and synaptic function. It is known. Specifically, the astrocyte may be a reactive astrocyte.

The "reactive astrocytes" change the properties of astrocytes in response to a toxic substance accumulated due to trauma, infection, ischemia, stroke, autoimmune reaction and neurodegenerative diseases of the central nervous system, or a response to a pathological environment. It is a cell produced by For the purposes of the present invention, cells that are reprogrammed into neurons may be reactive astrocytes. Specifically, the reactive astrocytes may be generated by spinal cord injury, but are not limited thereto. Alternatively, the reactive astrocytes may be accompanied by destruction of neurons or may be generated prior to destruction of neurons, but is not limited thereto.

As an example of a method for identifying reactive astrocytes, when the expression level of GFAP (Glial Fibrillary Acidic Protein) is higher than that of normal cells, it may be determined as reactive astrocytes. In addition, Tnf (Tumor necrosis factor), Il1b (Interleukin 1 beta) and/or A1 type reactive astrocyte overexpressing C1q, Chil3 (Chitinase-like 3), Fzd1 (Frizzled class receptor 1) and/or Arg1 ( Reactive astrocytes overexpressing Arginase 1) are also included in the scope of the present invention.

The astrocytes may be derived from the brain or spinal cord of an individual, and more specifically, may be derived from striatum or putamen, but is not limited thereto.

In the present invention, the term "Ngn2 (neurogenin-2)" is a protein encoded by the NEUROG2 gene, and is one of the neurogenin subfamily of basic helix-loop-helix (bHLH) transcription factor genes. In the present invention, Ngn2 may be composed of the amino acid sequence of SEQ ID NO: 1. Specifically, in the present invention, the Ngn2 is 70% or more of the sequence of SEQ ID NO: 1, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more. As an amino acid sequence showing homology, a protein showing substantially the same or similar activity as Ngn2 may be included without limitation. In addition, the NEUROG2 gene may be composed of the nucleotide sequence of SEQ ID NO: 2, and specifically, the sequence of SEQ ID NO: 2 and 70% or more, specifically 80% or more, more specifically 90% or more, more specifically It may be a base sequence exhibiting 95% or more, and most specifically 99% or more homology, but is not limited thereto.

In the present invention, the term "introduction" refers to any activity that naturally or artificially causes the activity of a specific protein or a gene encoding it to appear naturally or artificially, or to increase its expression, which was not originally possessed by a cell, tissue or individual, and the protein is It may be Ngn2, and the gene may be a NEUROG2 gene.

Specifically, in the present invention, the cleavage map disclosed in Fig. 1a or Fig. 2a to increase the expression of Ngn2 protein in astrocytes or introducing a nucleic acid molecule encoding Ngn2 protein or Ngn2 protein specifically for reactive astrocytes It may be carried out by transferring the having a recombinant vector or a virus containing the recombinant vector to astrocytes, but is not particularly limited thereto.

More specifically, the recombinant vector may be used for the purpose of expressing Ngn2 specifically for astrocytes and confirming the expression, and in an embodiment of the present invention, pAAV-GFAP-Ccre; pAAV-Lcn2-Ncre; pAAV-EF1α-df-Ngn2-IRES-GFP was used and pAAV-iNOS-Ccre for cynomolgus monkeys; pAAV-GFAP-Ncre; By injection of pAAV-EF1α-df-Ngn2-IRES-GFP, Ngn2 was specifically expressed in astrocytes, and whether or not it was confirmed by immunohistochemistry.

The recombinant vector is a promoter Lcn2 or iNOS for mouse and cynomolgus monkey, respectively; And a GFAP promoter, specifically, in the recombinant vector, a vector containing a Gfap promoter for a mouse; And a vector containing the Lcn2 promoter, and a vector containing a Gfap promoter for monkey; And a vector containing the iNOS promoter can be used. A sequence encoding N-terminal Cre and C-terminal Cre fragments, respectively, may be included under the two types of recombinant vector promoters. The N-terminal Cre and C-terminal Cre mean that Cre recombinase is cut and divided into N-terminal and C-terminal regions. By using the promoter combination, gene expression can be specifically regulated in reactive astrocytes. have. That is, by using the combination of promoters, gene expression can be selectively controlled only in specific cells (reactive astrocytes), and by using this, only reactive astrocytes can be transformed into neurons.

The GFAP promoter may be represented by the nucleotide sequence of SEQ ID NO: 3, the Lcn2 promoter is the nucleotide sequence of SEQ ID NO: 4, and the iNOS promoter may be represented by the nucleotide sequence of SEQ ID NO: 5, but is not limited thereto. It can be obtained from a known database (NCBI genbank, etc.).

In the present invention, the term "culture" refers to growing cells under appropriately controlled environmental conditions, and the culturing process of the present invention may be performed according to a suitable medium and culture conditions known in the art. This culture process can be adjusted and used by a person skilled in the art depending on the cells to be selected.

In the present invention, the term "reprogramming" refers to a method of converting a target cell by controlling the global gene expression pattern of a specific cell. Specifically, in the present invention, reprogramming refers to a method of artificially manipulating specific cells to convert them into cells having completely different characteristics, and for the purposes of the present invention, the reprogramming is a foreign gene or nucleic acid in astrocytes, which are non-neuronal cells. It may be performed by introducing a recombinant vector containing a molecule. More specifically, the reprogramming may be transdifferentiation or direct-reprogramming, but is not limited thereto.

In one embodiment of the present invention, virus AAV-GFAP-Ccre, which delivers a recombinant plasmid vector, respectively, for astrocyte-specific Ngn2 expression for mouse and cynomolgus monkey; AAV-Lcn2-Ncre; AAV-EF1α-df-Ngn2-IRES-GFP and AAV-iNOS-Ccre; AAV-GFAP-Ncre; As a result of injecting AAV-EF1α-df-Ngn2-IRES-GFP into reactive astrocytes and culturing it, the Ngn2 expression group showed an action potential in patch clamp recording, and the GFP-expressing cells (reactive astrocytes) were neuron-like cells. -like cell).

Another aspect of the present invention for achieving the above object provides a composition for inducing reprogramming of astrocytes into neurons, including Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein.

The Ngn2, astrocytes, neurons and reprogramming are as described above.

In addition, the present invention provides the use of a Ngn2 protein or a nucleic acid molecule encoding an Ngn2 protein for inducing reprogramming of reactive astrocytes into neurons.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, including Ngn2 protein or a nucleic acid molecule encoding the Ngn2 protein.

The Ngn2, astrocytes and neurons are as described above.

In addition, the present invention provides the use of a Ngn2 protein or a nucleic acid molecule encoding an Ngn2 protein for the prevention or treatment of neurodegenerative diseases.

In the present invention, the term "degenerative neurological disease" refers to an abnormal motor control ability, cognitive function, perceptual function, sensory function and autonomic nerve function due to a decrease or loss of neuronal function, and progressive cognitive function disorder according to the main symptoms , Progressive ataxia, muscle weakness, and muscular atrophy group. The neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic brain disease (stroke), and dehydration. It may be selected from the group consisting of demyelinating disease, multiple sclerosis, epilepsy, neurodegenerative disease, and spinal cord injury (SCI), and may be more specifically spinal cord injury, but is not particularly limited thereto.

In the present invention, the term "spinal cord injury" means that the performance of sensory signals and motor signals passing through the damaged site is affected by damage to the spinal cord due to an acquired accident, and thus sensory neuron or movement It refers to a disease that causes a transmission disorder of a motor neuron, leading to a partial or complete paralysis of human body functions.

Specifically, the spinal cord injury may be caused by trauma such as a traffic accident or a fall. The spinal cord injury can be divided into complete damage and incomplete damage according to the degree of damage. Spinal cord injury may be accompanied by symptoms of loss of function mainly due to paralysis of motor nerves below the damaged site. Specifically, complete damage may result in complete loss of motor and sensory functions of the spinal cord by a complete transverse cut of the spinal cord. Incomplete damage, unlike complete damage, may appear as a state in which some sensory and motor functions below the damaged area are preserved. Symptoms may vary depending on the injured part, and for example, quadriplegia, blood pressure, pulse, body temperature, and respiratory rate may all drop due to hard water injury. Paralysis of the lower extremities due to subpleural effusion damage, loss of sensory function, loss of colon and bladder and sexual function may be seen.

In the present invention, the term "prevention" means any action that inhibits the progression of or delays the onset of neurodegenerative diseases including spinal cord injury by the composition of the present invention.

In the present invention, the term "treatment" refers to any action in which symptoms of neurodegenerative diseases including spinal cord injury are improved or advantageously changed by the composition of the present invention.

The "pharmaceutical composition" of the present invention means that it is prepared for the purpose of preventing or treating diseases, and can be administered in various oral and parenteral dosage forms at the time of actual clinical administration. In the case of formulation, a commonly used filler, It can be prepared using diluents or excipients such as extenders, binders, wetting agents, disintegrants, and surfactants. In addition, pharmaceutically acceptable additives may be further included according to each formulation, and in this case, pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , Lactose, mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol, talc, and the like can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and water in the mixed extract of the present invention. It can be prepared by mixing sucrose, lactose, or gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. I can.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The composition of the present invention can be administered orally or parenterally according to a desired method, and when administered parenterally, external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method You can choose. The dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

In addition, the composition of the present invention can be administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health condition, type of disease, severity, The activity of the drug, its sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. In addition, the pharmaceutical composition of the present invention may be used alone or in combination with other pharmaceutically active compounds exhibiting an effect of preventing, treating, or improving cancer, or in a suitable set.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

In one embodiment of the present invention, viruses (AAV-EF1α-df-Ngn2-IRES-GFP, AAV-Lcn2-Ncre and AAV-GFAP-Ccre) containing the above three recombinant vectors for spinal cord injury model mice As a result of the injection, it was confirmed that the damaged spinal cord tissue recovered to a significant level compared to the control group and the PBS group, thereby improving symptoms of paraplegia and increasing the BMS score. From this, the composition of the present invention can be used for the purpose of preventing or treating neurodegenerative diseases including spinal cord injury.

The "spinal cord injury model" refers to an animal model that induces spinal cord injury. The "spinal cord injury" is as described above.

Another aspect of the present invention for achieving the above object is to provide a nerve cell prepared by the above method. In addition, another aspect of the present invention for achieving the above object is to provide a cell therapy product comprising nerve cells prepared by the above method as an active ingredient.

The "cell therapy agent" of the present invention can be used for the prevention or treatment of neurodegenerative diseases, which introduces Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein into astrocytes, or increases the expression of Ngn2 protein in astrocytes. It may include a neuron prepared through the step and culturing the same, and for the purposes of the present invention, in addition to the neuron, a substance capable of replacing the damaged neuron in degenerative neurological diseases including spinal cord injury is not limited and may be included therein. have.

Another aspect of the present invention is a nucleic acid molecule encoding Ngn2 (neurogenin-2) protein or Ngn2 protein in (a) reactive astrocytes for achieving the above object. Introducing or increasing the expression of Ngn2 protein in astrocytes; And (b) culturing the astrocytes of the step (a), and reprogramming the astrocytes into nerve cells. It provides a method for preventing or treating neurodegenerative diseases comprising.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of brain sample for electrophysiology test

To prepare a brain sample, each animal (mouse and cynomolgus monkey) was anesthetized with halothane. After decapitation, the brain was rapidly excised from the skull, and it was ice-cold cutting solution (130 NaCl, 24 NaHCO ₃ , 1.25 NaH ₂ PO ₄ , 3.5 KCl, 1.5 CaCl ₂ , 1.5 MgCl ₂ , and 10 D(+)-glucose. , pH 7.4). The entire solution was gassed with 95% O ₂ /5% CO ₂ . Subsequently, after trimming the cerebellum, a 300 μm coronal slice was cut using a blade (DORCO, Seoul, Korea) and a vibratome (DSK linear slicer, Kyoto, Japan). ₃ , 1.25, NaH ₂ PO ₄ , 3.5 KCl, 1.5 CaCl ₂ , 1.5 MgCl ₂ , and 10, D(+)-glucose, pH 7.4). The pipette is an internal solution for current measurement (in mm, 135 CsCl, 4 NaCl, 0.5 CaCl2, 10 HEPES, 5 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, 10 QX-314, pH adjusted to 7.2) with CsOH (278-285 mOsmol)) and an internal solution (mM unit, 140 K-gluconate, 10 HEPES, 7 NaCl, and 2 MgATP adjusted to pH 7.4 with CsOH) for voltage measurement.

Example 2: Direct differentiation into nerve cells under in vitro conditions

We tried to induce specific expression of Ngn2 in reactive astrocytes using the Split-Cre/df system. Cloning pAAV-GFAP-Ccre, pAAV-mLcn2-Ncre, pAAV-EF1a-df-Ngn2-IRES-GFP, pAAV-EF1a-df-GFP for mouse, and pAAV-GFAP-Ncre, pAAV- for cynomolgus monkey iNOS-Ccre, pAAV-EF1a-df-Ngn2-IRES-GFP, pAAV-EF1a-df-GFP were cloned. An expression mixture consisting of three kinds of DNA was introduced into reactive astrocytes by electroporation. Each expression mixture is shown in Figure 5a and Figure 6a below. Cells were cultured in astrocyte medium (10% Horse serum, 10% FBS, 1% P/S in DMEM) for 3 days, and on the fourth day after electroporation, the medium was used as a neuronal cell culture medium (2% B-27, 1%). Glutamax, 1%P/S in F-12 media).

As a result, 10 days after electroporation, it was confirmed that the cells of the Ngn2 group showed polarity and the same morphology as neurons, and the GFP-expressing cells in the Ngn2 group showed an action potential as a result of patch clamp recording. This is a result indicating that Ngn2 expression in reactive astrocytes can transform and differentiate reactive astrocytes into neurons.

Example 3: Direct differentiation into neurons in in vivo conditions (mouse)

Naive mouse (7 weeks old, male) was micro-injected with 2 μl of a mixture of three viruses into the striatum. At 3 weeks after injection, the mice were sacrificed and fixed within 1 day, followed by dehydration for 2 days to perform immunohistochemistry. The frozen brain tissue was sliced thinly to a thickness of 30 μm with a cryostat microtome, and stained with NeuN (neural cell nucleus, nerve cell marker) and GFAP (Glial Fibrillary Acidic Protein, astrocyte marker). DAPI was counter-stained, and GFP was not stained with antibodies. Imaging was performed using a confocal microscope Nikon A1.

As a result of measuring the number of NeuN-positive cells and GFAP-positive cells from GFP-expressing cells in the striatum, as can be seen from b of FIG. 5, about 70% of the GFP-expressing cells are GFAP-positive in the control, and about 70% of the GFP-expressing cells are The Ngn2 group showed NeuN positive. From this, it was found that GFP-expressing cells (reactive astrocytes) differentiated into neuron-like cells, indicating that the expression of Ngn2 in reactive astrocytes can transform and differentiate reactive astrocytes into neurons. It is the result.

In addition, the mouse brain was sliced to a thickness of 300 μm 3 weeks after injection, and whole cell patch-clamp recording was performed on GFP-expressing cells. As a result, it was confirmed that the GFP-expressing cells of the control group exhibited passive conductance to a voltage clamp, and the GFP-expressing cells of the Ngn2 group exhibited action potentials and miniature excitatory post-synaptic currents (EPSCs) as neuronal characteristics.

Example 4: in vivo Direct differentiation into neurons under conditions ( cynomolgus monkey)

Through stereotaxic surgery, 25 μl of a mixture of three viruses was micro-injected into the putamen of Cynomolgus monkeys (2-4 years). At 3 weeks after injection, the monkey was sacrificed and fixed within 1 day, followed by dehydration for 2 days to perform immunohistochemistry. The frozen brain tissue was thinly sliced to a thickness of 50 μm with a cryostat microtome, and stained with NeuN (neural cell nucleus, nerve cell marker) and GFAP (Glial Fibrillary Acidic Protein, astrocyte marker). DAPI was counter-stained, and GFP was not stained with antibodies. Imaging was performed using a confocal microscope Nikon A1. As a result, the control group showed colocalization of GFP and GFAP (white), and the Ngn2 group showed colocalization of GFP and NeuN (yellow).

As a result of measuring the number of NeuN-positive cells and GFAP-positive cells from GFP-expressing cells in putamen (ROI of each cell is defined as the DAPI region), GFAP intensity is high and NeuN intensity is 1K from GFP-expressing cells of the control group from the scatter plot. It was confirmed that it was less than, and in the Nhn2 group, it was confirmed that the NeuN strength increased to 4K. In addition, as can be seen from e to h of Figure 6, it was confirmed that the GFAP intensity in the control group was significantly higher than the NeuN intensity, and there was no significant difference in the Ngn2 group. In addition, when looking at the intensity of NeuN, it was confirmed that the Ngn2 group was higher than the control group, and the control group had a higher GFAP intensity than the Ngn2 group. This is a result indicating that Ngn2 expression in reactive astrocytes can transform and differentiate reactive astrocytes into neurons.

Example 5: Direct differentiation into nerve cells in a spinal cord injury (SCI) model

The following experiment was conducted to confirm whether cells expressing fluorescence in the SCI/Ngn2 group exhibit electrophysiological characteristics of neurons including action potentials.

Naive mice (7 weeks old, male) were used to create a spinal cord injury (SCI) model due to compression injury at spinal cord T10. Two weeks after injury, 2 μl of a mixture of three viruses was micro-injected into two areas 1 mm from the damaged area of the SCI model through stereotaxic surgery. In addition, the BMS score of the SCI model was measured every week after injury. At 5 weeks after injection, the mice were sacrificed and fixed within 1 day, followed by dehydration for 2 days to perform immunohistochemistry. The frozen tissue was thinly sliced to a thickness of 20 μm with a cryostat microtome, and EC staining was performed on the sliced tissue.

As a result, spinal cord injury was not recovered in the control group and the PBS group, but the recovered tissue was confirmed in the Ngn2 group. In addition, the evaluation result of the BMS score indicates that the BMS score of the Ngn2 group is significantly different from the control group and the PBS group from 2 weeks after injection. This indicates that the expression of Ngn2 in reactive astrocytes can transform and differentiate reactive astrocytes into neurons, and furthermore, it is a result that it can be used for the purpose of treatment for spinal cord injury or neurodegenerative diseases.

Example 6: Confirmation of electrophysiological characteristics of SCI/Ngn2 group into neurons

The following experiment was conducted to confirm whether cells expressing fluorescence in the SCI/Ngn2 group exhibit electrophysiological characteristics of neurons including action potentials.

First, remove spinal cord tissue from anesthetized mice and lightly frozen NMDG dissection solution (92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na) using a vibratome. -ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2·4H2O and 10 mM MgSO4·7H2O.Titrate pH to 7.3-7.4 with concentrated hydrochloric acid, oxygenation with 95% O2 and 5% CO2) to a thickness of 350-400um. . After sectioning, incubate for 10-15 minutes in a chamber at 32-34°C, recording solution (in mM, 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1 CaCl2, 3 MgCl2 and 10 glucose, pH 7.4, room temperature with oxygenation ( 95% O2 and 5% CO2)) and stabilized for about 1 hour.

Then, for whole-cell patch clamp recording, internal solution (mM): 120 potassium gluconate, 10 KCl, 1 MgCl2, 0.5 EGTA, 40 HEPES (pH 7.2 was adjusted with KOH) was used. Current or voltage signal measurements were digitized with DigiDATA 1550B1 (10 kHz) and analyzed with pCLAMP10 software.

As a result, as can be seen from d to f of FIG. 7, the action potential was confirmed in the cells expressing GFP of the SCI/Ngn2 group, and the large size of 3-4nA confirmed by the activity of the Na ion channel by recording in voltage clamp mode The current signal was measured. In addition, by confirming that the spontaneous EPSC spontaneously sent from the synapse when no stimulation was applied, it was confirmed that the SCI/Ngn2 group constitutes a synapse with the existing neurons.

Example 7: With astrocytes Nerve cell With a marker Confirmation of regeneration into nerve cells after staining

Four groups of mice (Sham, SCI/PBS, SCI/Ctrl, SCI/Ngn2) were pre-fixed by perfusion with 4% PFA after saline perfusion. The spinal cord tissue was separated, stored in 30% sucrose in PBS for one day, dehydrated, and then embedding in OCT compound and frozen. The frozen tissue was sectioned into a thickness of 20 μm using a frozen tissue slicer, and after blocking for 1 hour (2% donkey serum, 2% goat serum, 0.3% Tx 100 in PBS), GFAP, (1:500) MAP2 (1:500) ) It was dyed using an antibody. Primary Ab was treated with O/N at 4° C. and then washed three times with PBS for 10 minutes the next day, and Secondary Ab (Jacson laboratory, 1:500) was washed three times with PBS for 10 minutes after 2 hours treatment at room temperature. DAPI cross staining was performed in the second wash, and the stained tissue was confirmed with a Nikon A1 confocal microscope or LSM700, Carl Zeiss.

In all groups, a certain threshold was designated and intensity was measured using imageJ. As a result of measuring MAP2 signal stained on GFP-expressing cells in two groups expressing GFP, two groups were SCI/PBS and SCI/Ctrl in SCI/Ngn2 group. Compared with all, MAP2 intensity was significantly increased, and MAP2 intensity was also significantly increased in GFP-expressing cells.

In addition, in order to confirm the regeneration into motor neurons after staining with astrocytes, neurons and motor neuron markers, an experiment was performed in the same manner as in Example 6.

SCI/PBS and SCI in SCI/PBS and SCI in SCI/Ngn2 group by measuring the intensity of ist1 by designating a certain threshold in all groups, measuring ist1 signal stained in GFP-expressing cells in two groups expressing GFP, and measuring the intensity of GFAP representing astrocytes. Compared with the /Ctrl group.

As a result, it was confirmed that the ist1 intensity was significantly increased in the SCI/Ngn2 group when compared to both SCI/PBS and SCI/Ctrl groups, and the ist1 intensity was also significantly increased in GFP-expressing cells. In addition, by measuring the intensity of GFAP representing astrocytes, it was confirmed that the GFAP intensity decreased in the SCI/Ngn2 group when compared with the SCI/PBS and SCI/Ctrl groups.

Example 8: EC( Eriochrome Cyanine ) by staining Myelinated Check the area and area of the entire organization

The four groups of mice were pre-fixed by perfusion with 4% PFA after saline perfusion. The spinal cord tissue was separated, stored in 30% sucrose in PBS for one day, dehydrated, and then embedding in OCT compound and frozen. The frozen tissue was sectioned to a thickness of 20 μm using a frozen tissue slicer. Then, after dehydration at room temperature for 2 hours, acetone was treated for 5 minutes, waited at room temperature for 10 minutes, and EC solution was treated at room temperature for 30 minutes. Washed with running water and 5% iron alum until gray matter was revealed, treated with Boraxferricyanide solution, and then dehydrated in 70%, 90%, and 100% ethanol sequentially. The injury site is indicated by a black dotted line, and the virus-injected portion is indicated by a green dotted line.

In all groups in the picture, the area of the gray matter area was measured by designating a certain threshold, and the area of the area dyed in blue (white matter) was measured by designating a certain threshold. , The sum of the total area was compared for each group.

As a result, it was confirmed that the areas of white matter and gray matter increased significantly, and the total area increased significantly when compared with both groups of SCI/PBS and SCI/Ctrl in the SCI/Ngn2 group.

Example 9: Ngn2 Newly regenerated cells after gene expression In astrocytes Confirmation of conversion

After Ngn2 gene expression, the newly regenerated cells were differentiated from stem cells or transdifferentiated from astrocytes through BrdU staining with astrocyte and neuronal markers.

The four groups of mice were pre-fixed by perfusion with 4% PFA after saline perfusion. The spinal cord tissue was separated, stored in 30% sucrose in PBS for one day, dehydrated, and then embedding in an OCT compound and frozen. The frozen tissue was sectioned into a thickness of 20 μm using a frozen tissue slicer, and after 1 hour blocking (2% donkey serum, 2% goat serum, 0.3% Tx 100 in PBS), GFAP (1:500) NeuN (1:500) , BrdU (1:2000), MBP (1:400) was stained using an antibody. Primary Ab was treated with O/N at 4° C. and then washed three times with PBS for 10 minutes the next day, and Secondary Ab (Jacson laboratory, 1:500) was washed three times with PBS for 10 minutes after 2 hours treatment at room temperature. Cross staining with DAPI in the second wash, and stained tissues were confirmed with a Nikon A1 confocal microscope or LSM700, Carl Zeiss.

Then, among the cells expressing GFP of the SCI/Ctrl and SCI/Ngn2 groups, cells with a BrdU signal were counted, and the presence of BrdU in the cells stained with GFAP while expressing GFP was confirmed. Then, among the cells expressing GFP of the SCI/Ctrl and SCI/Ngn2 groups, cells with a BrdU signal were counted, and the presence of BrdU in the cells stained with NeuN while expressing GFP was confirmed. Finally, cells expressing GFP of the SCI/Ctrl and SCI/Ngn2 groups and cells that did not have a BrdU signal were counted, and the presence of BrdU in the cells stained with MBP while expressing GFP was confirmed.

As a result, it was confirmed that most of the BrdU signals appear in cells that do not express GFP in all three stained groups.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (14)

1. (a) introducing Ngn2 (neurogenin-2) protein or a nucleic acid molecule encoding Ngn2 protein into reactive astrocytes, or increasing the expression of Ngn2 protein in astrocytes; And

   (b) A method for reprogramming astrocytes into nerve cells, comprising the step of culturing the astrocytes of step (a).
2. The method of claim 1, wherein the nucleic acid molecule encoding the Ngn2 protein is composed of the nucleotide sequence of SEQ ID NO: 2.
3. The method of claim 1, wherein the reprogramming is transdifferentiation or direct-reprogramming.
4. The method of claim 1, wherein the introduction of the nucleic acid molecule encoding the Ngn2 protein in step (a) is performed by using a recombinant vector having a cleavage map disclosed in Fig. 1a or 2a, or a virus containing the recombinant vector. Delivery to cells.
5. The method of claim 4, wherein the astrocytes are mouse astrocytes, and the promoters of the two kinds of recombinant vectors including a sequence encoding a part of a Cre recombinase are Lcn2 and GFAP promoters, respectively.
6. The method of claim 4, wherein the astrocytes are monkey astrocytes, and the promoters of the two kinds of recombinant vectors comprising a sequence encoding a part of a Cre recombinase are iNOS and GFAP promoters, respectively.
7. The method of claim 1, wherein the astrocytes are derived from the brain or spinal cord of an individual.
8. The method of claim 1, wherein the reactive astrocyte is derived from a spinal cord injury individual.
9. A composition for inducing reprogramming of reactive astrocytes into neurons, including Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein.
10. A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising a Ngn2 protein or a nucleic acid molecule encoding the Ngn2 protein.
11. The method of claim 10, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic brain Disease (stroke), demyelinating disease (demyelinating disease), multiple sclerosis, epilepsy, neurodegenerative disease and spinal cord injury (Spinal Cord Injury, SCI) that is selected from the group consisting of, the pharmaceutical composition.
12. A nerve cell produced by the method of any one of claims 1 to 8.
13. A cell therapy agent comprising as an active ingredient nerve cells prepared by the method of any one of claims 1 to 8.
14. In animals,

    (a) introducing Ngn2 (neurogenin-2) protein or a nucleic acid molecule encoding Ngn2 protein into reactive astrocytes, or increasing the expression of Ngn2 protein in astrocytes; And

    (b) culturing the astrocytes of the step (a), and reprogramming the astrocytes to nerve cells, preventing or treating neurodegenerative diseases.

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