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

Method for reprogramming reactive astrocytes into neurons in spinal cord injury model using neurogenin-2 (ngn2) Download PDF

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WO2021015352A1
WO2021015352A1 PCT/KR2019/010453 KR2019010453W WO2021015352A1 WO 2021015352 A1 WO2021015352 A1 WO 2021015352A1 KR 2019010453 W KR2019010453 W KR 2019010453W WO 2021015352 A1 WO2021015352 A1 WO 2021015352A1
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ngn2
astrocytes
protein
reprogramming
spinal cord
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Korean (ko)
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이창준
안희영
오수진
하윤
이혜란
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한국과학기술연구원
연세대학교 산학협력단
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0618Cells of the nervous system
    • C12N5/0619Neurons
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    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C12N2506/00Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2510/00Genetically modified cells

Definitions

  • 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.
  • 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.
  • SCI spinal cord injury
  • 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.
  • astrogliosis reactive astrocyte
  • 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.
  • Ngn2 neurogenin-2
  • 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.
  • the astrocytes when Ngn2 (neurogenin-2) expression is specifically increased in reactive astrocytes, the astrocytes can be directly reprogrammed into neurons.
  • 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-EF1 ⁇ -df-Ngn2-IRES-GFP used for astrocyte-specific Ngn2 expression in mice.
  • FIG. 1B shows the plasmid vector pAAV-EF1 ⁇ -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-EF1 ⁇ -df-Ngn2-IRES-GFP used for astrocyte-specific Ngn2 expression for cynomolgus monkey.
  • Figure 2b shows the plasmid vector pAAV-EF1 ⁇ -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.
  • 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.
  • FIG. 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.
  • SCI spinal cord injury
  • FIG. 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.
  • BMS Basso Mouse Score
  • 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.
  • 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).
  • 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
  • cells that are reprogrammed into neurons may be reactive astrocytes.
  • the reactive astrocytes may be generated by spinal cord injury, but are not limited thereto.
  • the reactive astrocytes may be accompanied by destruction of neurons or may be generated prior to destruction of neurons, but is not limited thereto.
  • GFAP Glial Fibrillary Acidic Protein
  • Tnf Tumor necrosis factor
  • Il1b Interleukin 1 beta
  • A1 type reactive astrocyte overexpressing C1q Chil3 (Chitinase-like 3)
  • Fzd1 Fluzled class receptor 1
  • Arg1 Reactive astrocytes overexpressing Arginase 1
  • 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.
  • Ngn2 neurogenin-2
  • NEUROG2 is a protein encoded by the NEUROG2 gene, and is one of the neurogenin subfamily of basic helix-loop-helix (bHLH) transcription factor genes.
  • Ngn2 may be composed of the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • an amino acid sequence showing homology a protein showing substantially the same or similar activity as Ngn2 may be included without limitation.
  • 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.
  • 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.
  • 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.
  • 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-EF1 ⁇ -df-Ngn2-IRES-GFP was used and pAAV-iNOS-Ccre for cynomolgus monkeys; pAAV-GFAP-Ncre; By injection of pAAV-EF1 ⁇ -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.
  • 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.).
  • 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.
  • the term "reprogramming” refers to a method of converting a target cell by controlling the global gene expression pattern of a specific cell.
  • 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.
  • 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-EF1 ⁇ -df-Ngn2-IRES-GFP and AAV-iNOS-Ccre; AAV-GFAP-Ncre; As a result of injecting AAV-EF1 ⁇ -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.
  • Ngn2 The Ngn2, astrocytes, neurons and reprogramming are as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a commonly used filler It can be prepared using diluents or excipients such as extenders, binders, wetting agents, disintegrants, and surfactants.
  • 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.
  • 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.
  • injectable ester such as ethyl oleate
  • witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
  • 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.
  • 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.
  • 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.
  • viruses (AAV-EF1 ⁇ -df-Ngn2-IRES-GFP, AAV-Lcn2-Ncre and AAV-GFAP-Ccre) containing the above three recombinant vectors for spinal cord injury model mice
  • AAV-EF1 ⁇ -df-Ngn2-IRES-GFP, AAV-Lcn2-Ncre and AAV-GFAP-Ccre viruses containing the above three recombinant vectors for spinal cord injury model mice
  • 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.
  • 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.
  • Example 1 Preparation of brain sample for electrophysiology test
  • 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 .
  • a 300 ⁇ m 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
  • FIG. 5a 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).
  • Ngn2 expression in reactive astrocytes can transform and differentiate reactive astrocytes into neurons.
  • Example 3 Direct differentiation into neurons in in vivo conditions (mouse)
  • the mouse brain was sliced to a thickness of 300 ⁇ m 3 weeks after injection, and whole cell patch-clamp recording was performed on GFP-expressing cells.
  • 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.
  • EPCs excitatory post-synaptic currents
  • Example 4 in vivo Direct differentiation into neurons under conditions ( cynomolgus monkey)
  • 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.
  • 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.
  • SCI spinal cord injury
  • the BMS score of the SCI model was measured every week after injury.
  • 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 ⁇ m with a cryostat microtome, and EC staining was performed on the sliced tissue.
  • 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.
  • the following experiment was conducted to confirm whether cells expressing fluorescence in the SCI/Ngn2 group exhibit electrophysiological characteristics of neurons including action potentials.
  • 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
  • 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.
  • 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.
  • 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
  • mice 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 ⁇ m 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.
  • 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.
  • 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.
  • 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
  • mice 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 ⁇ m 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
  • the virus-injected portion is indicated by a green dotted line.
  • 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.
  • the newly regenerated cells were differentiated from stem cells or transdifferentiated from astrocytes through BrdU staining with astrocyte and neuronal markers.
  • mice 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 ⁇ m 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.

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

[규칙 제26조에 의한 보정 13.09.2019] Ngn2(뉴로제닌-2)를 이용하여 척수 손상 모델에서 반응성 성상세포를 신경세포로 리프로그래밍하는 방법[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)
본 발명은 (a) 성상세포(astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계 및 (b) 상기 (a) 단계의 성상세포를 배양하는 단계를 포함하는, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 방법; Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 성상세포의 신경세포로의 리프로그래밍 유도용 조성물; 퇴행성 신경질환의 예방 또는 치료용 약학적 조성물; 상기 방법에 의해 제조된 신경세포 및 이를 포함하는 세포치료제에 관한 것이다.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.
특정 세포의 전환분화(transdifferentiation) 또는 직접 리프로그래밍(direct-reprogramming)을 통해 목적하는 세포의 제조는 장기이식(transplantation)을 대체할 유망한 치료 방법으로 활용될 수 있는바, 주목을 받고 있다. 즉, 이러한 재생의학 분야에서 전환분화 또는 직접 리프로그래밍을 통한 손상 세포의 대체는 질환을 근본적으로 치료 가능한 대안으로 제시되고 있으며, 구체적인 질환 및 세포에 대한 임상 시험 또한 활발히 진행되고 있는 추세이다.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.
특히, 신경세포는 신경계를 구성하며 감각인지, 학습, 기억 및 운동 능력을 제어하는데 필수적이나, 한번 손상을 입으면 정상 상태로 회복되는데 오랜 시간이 걸릴 뿐만 아니라 다른 세포와 달리 재생되기 어려워 영구적인 손상에 이를 가능성이 높은 점에서 치료 대상으로서 더욱 가치가 높다고 할 수 있다. 이와 관련하여, 동일한 신경세포에서도 어떤 부위에 손상을 입었는지에 따라 손상 반응 및 재생 능력의 차이가 나타나게 되는데, 중추 신경인 척수 손상(Spinal Cord Injury, SCI)과 같은 경우에는 다른 부위보다 재생 실패에 이르는 경우가 많이 나타나며, 현재까지 임상에서도 별다른 치료 방법이 없는 실정이다. 또한, 척수 손상 이외에도 신경세포의 퇴행으로 인한 질환들, 예컨대 파킨슨씨병(Parkinson's disease), 알츠하이머(Alzheimer's disease), 피크병(Pick's disease) 등과 같은 질환들은 운동장애 및 인지기능 장애 등을 유발하여 지속적인 치료를 요구하나, 근본적인 치료방법이 존재하지 않는 점에서 개인적, 사회적 문제로도 자리잡고 있다.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.
한편, 성상세포(astrocyte)는 중추신경계에서 패킹 조직을 형성하는 신경교세포로, 건강한 신경 조직에서 에너지 공급, 혈류 조절, 세포 외액의 항상성, 이온 및 전송기의 항상성, 시냅스 기능 조절을 하는 것으로 알려져 있다. 그러나 중추신경계의 외상, 감염, 허혈, 뇌졸중, 자가 면역 반응 및 신경 퇴행성 질환 등으로 인해 인근 뉴런의 파괴가 일어날 경우, 성상세포의 수가 비정상적으로 증가하게 되는데 이를 반응성 성상세포(reactive astrocyte, astrogliosis)라고 한다. 이와 관련하여, 현재까지 손상된 척수를 회복시키고자 한 연구들은 전능성을 가진 배아줄기세포(embryonic stem cell)와 유도만능 줄기세포(induced pluripotent stem cell; iPS cell), 다능성을 가진 중간엽 줄기세포(mesenchymal stem cell; MSC) 및 신경줄기세포(neural stem cell; NSC) 등의 이식에 관한 것이거나, 척수 손상 이후에 발생되는 부작용 증상에 대한 치료방법이 대부분이었으나(한국 등록특허 제10-1892457호), 반응성 성상세포로부터 신경세포를 직접 전환시켜 이식하고자 하는 시도는 이루어지지 않았다.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.
이에, 본 발명의 발명자들은 비신경세포인 성상세포를 리프로그래밍하여 신경세포를 제조하는 방법에 대해 예의 노력한 결과, 성상세포 특이적으로 Ngn2(neurogenin-2) 발현을 증가시킬 경우 성상세포가 신경세포로 직접 리프로그래밍되고, 척수 손상 모델에 적용시 증상이 개선되는 것을 확인하였는바, 종국적으로 이러한 신경세포로의 리프로그래밍 방법이 척수 손상을 비롯한 퇴행성 신경질환에 대한 예방 또는 치료 목적으로 활용될 수 있음을 확인하여 본 발명을 완성하였다.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.
본 발명의 하나의 목적은 (a) 성상세포(astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계; 및 (b) 상기 (a) 단계의 성상세포를 배양하는 단계를 포함하는, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 방법을 제공하는 것이다.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.
본 발명의 다른 하나의 목적은 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 성상세포의 신경세포로의 리프로그래밍 유도용 조성물을 제공하는 것이다. 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.
본 발명의 또 다른 하나의 목적은 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 퇴행성 신경질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.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.
본 발명의 또 다른 하나의 목적은 상기 방법에 의해 제조된 신경세포를 제공하는 것이다.Another object of the present invention is to provide a nerve cell produced by the above method.
본 발명의 또 다른 하나의 목적은 상기 방법에 의해 제조된 신경세포를 유효성분으로 포함하는 세포치료제를 제공하는 것이다.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.
본 발명에 따라 반응성 성상세포에 특이적으로 Ngn2(neurogenin-2) 발현을 증가시킬 경우 상기 성상세포를 신경세포로 직접 리프로그래밍할 수 있다. 또한 마우스에서 GFAP-Lcn2 프로모터 조합을, 원숭이에서 GFAP-iNOS 프로모터 조합을 이용하여 반응성 성상세포 특이적으로 Ngn2 발현을 조절할 수 있다. 이러한 신경세포로의 리프로그래밍 방법은 척수 손상을 비롯한 퇴행성 신경질환에 대한 예방 또는 치료 목적의 약학적 조성물 또는 세포치료제 등으로 활용될 수 있다.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.
도 1의 a는 mouse에 대해 성상세포 특이적 Ngn2 발현을 위해 사용된 재조합 플라스미드 벡터 pAAV-GFAP-Ccre, pAAV-Lcn2-Ncre 및 pAAV-EF1α-df-Ngn2-IRES-GFP를 나타낸 것이다.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.
도 1의 b는 mouse에 대해 성상세포 특이적 Ngn2 발현의 대조군으로 사용된 플라스미드 벡터 pAAV-EF1α-df-GFP를 나타낸 것이다.FIG. 1B shows the plasmid vector pAAV-EF1α-df-GFP used as a control for expression of astrocyte-specific Ngn2 in mice.
도 2 a는 cynomolgus monkey에 대해 성상세포 특이적 Ngn2 발현을 위해 사용된 재조합 플라스미드 벡터 pAAV-iNOS-Ccre, pAAV-GFAP-Ncre 및 pAAV-EF1α-df-Ngn2-IRES-GFP를 나타낸 것이다.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.
도 2 b는 cynomolgus monkey에 대해 성상세포 특이적 Ngn2 발현의 대조군으로 사용된 플라스미드 벡터 pAAV-EF1α-df-GFP를 나타낸 것이다.Figure 2b shows the plasmid vector pAAV-EF1α-df-GFP used as a control for astrocyte-specific Ngn2 expression for cynomolgus monkey.
도 3의 a는 성상세포 특이적인 Ngn2 발현을 위한 재조합 벡터를 포함하는 바이러스를 mouse 및 cynomolgus monkey에 대해 injection하는 프로토콜을 나타낸 것이다. 3A shows a protocol for injecting a virus containing a recombinant vector for astrocyte-specific Ngn2 expression into mouse and cynomolgus monkeys.
도 3의 b 및 c는 각각 mouse 및 cynomolgus monkey의 손상된 선조체 조직에서 GFP, GFAP, DAPI 및 Lcn2/iNOS의 발현 여부를 확인한 면역화학염색(immunohistochemistry) 결과를 나타낸 것이다. 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.
도 4의 a는 mouse 및 cynomolgus monkey 각각에 대해 성상세포 특이적 Ngn2 발현을 위한 double floxed Split-Cre system의 도식을 나타낸 것이다.Figure 4a shows a schematic of a double floxed Split-Cre system for astrocyte-specific Ngn2 expression for each mouse and cynomolgus monkey.
도 4의 b는 in vitro 조건에서 반응성 성상세포의 배양 및 Ngn2 발현을 유도하는 프로토콜을 나타낸 것이다.4B shows a protocol for inducing the cultivation of reactive astrocytes and Ngn2 expression under in vitro conditions.
도 4의 c는 대조군과 Ngn2 발현 그룹에서 성상세포의 morphology 변화를 나타낸 것이다. 4C shows the change in morphology of astrocytes in the control group and the Ngn2 expression group.
도 4의 d는 EGFP 발현 세포와 함께 리프로그래밍된 세포의 전체 세포 patch-clamp recording 결과(in vitro)를 나타낸 것이다. 뉴런 유사 세포(Neuron-like cell)는 성공적으로 활동전위를 나타내었다.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.
도 5의 a는 mouse 선조체에서 Ngn2 발현에 따른 성상세포의 신경세포로의 리프로그래밍을 확인하기 위한 프로토콜을 나타낸 것이다.Figure 5a shows a protocol for confirming the reprogramming of astrocytes into neurons according to Ngn2 expression in mouse striatum.
도 5의 b는 대조군과 Ngn2 발현 그룹에서 GFP, GFAP 및 NeuN 발현 양상을 확인한 면역화학염색 결과(in vivo)를 나타낸 것이다.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.
도 5의 c는 GFP 발현 세포 중 GFAP 또는 NeuN 발현 세포의 비율을 나타낸 것이다.5C shows the proportion of GFAP or NeuN-expressing cells among GFP-expressing cells.
도 5의 d는 대조군에서 GFP 발현세포를 patch-clamp recording한 결과 성상세포의 전형적인 current를 보이는 것을 나타낸 것이다.5D shows a typical current of astrocytes as a result of patch-clamp recording of GFP-expressing cells in the control group.
도 5의 e는 Ngn2 발현 그룹에서 GFP 발현 세포를 patch-clamp recording한 결과 활동 전위 및 자발적인 시냅스후 전류를 보이는 것을 나타낸 것이다.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.
도 6의 a는 cynomolgus monkey 피곡(putamen)에서 Ngn2 발현에 따른 성상세포의 신경세포로의 리프로그래밍을 확인하기 위한 프로토콜을 나타낸 것이다.6A shows a protocol for confirming the reprogramming of astrocytes into neurons according to Ngn2 expression in cynomolgus monkey putamen.
도 6의 b는 대조군과 Ngn2 발현 그룹에서 GFP, GFAP 및 NeuN 발현 양상을 확인한 면역화학염색 결과(in vivo)를 나타낸 것이다.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.
도 6의 c는 GFAP 및 NeuN 강도에 따른 scatter plot 결과를 나타낸 것이다. 대조군의 경우 NeuN 강도가 대부분 1K 미만이었으나, Ngn2 발현 그룹에서는 NeuN 강도가 유의하게 증가하였다.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.
도 6의 d는 위 scatter plot의 결과를 요약하여 NeuN 강도가 1K 이상 (붉은색 영역)인 비율이 증가하는 것을 나타낸 것이다. 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.
도 6의 e는 대조군에서 GFAP 및 NeuN 강도를 비교한 결과 GFAP의 강도가 더 높은 것을 나타낸 것이다.6E shows that the GFAP and NeuN intensities were compared in the control group and the GFAP intensity was higher.
도 6의 f는 Ngn2 발현 그룹에서 NeuN의 강도가 GFAP의 강도와 비슷한 정도로 증가한 것을 나타낸 것이다.Figure 6f shows that the intensity of NeuN in the Ngn2 expression group increased to a similar degree to that of GFAP.
도 6의 g는 대조군과 Ngn2 발현 그룹에서 NeuN 강도를 비교하여 Ngn2 발현 그룹에서 유의미하게 NeuN 강도가 높은 것을 나타낸 것이다.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.
도 6의 h는 대조군과 Ngn2 발현 그룹에서 GFAP 강도를 비교하여 대조군에서 유의미하게 GFAP 강도가 높은 것을 나타낸 것이다.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.
도 7의 a는 척수 손상(Spinal Cord Injury, SCI) 모델에서 성상세포 특이적인 Ngn2 발현을 위한 재조합 벡터를 포함하는 바이러스 injection에 따른 실험 프로토콜을 나타낸 것이다.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.
도 7의 b는 압박 손상에 의한 척수 손상 모델을 만들고 2주 후 유전자 발현을 위해 바이러스 injection을 하는 과정을 나타낸 것이다.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.
도 7의 c는 모의대조군(sham) 그룹과 척수 손상 모델 중 PBS 그룹, 대조군 및 Ngn2 그룹에서 시간 경과에 따라 하반신 마비 (paraparesis)를 측정하는BMS (Basso Mouse Score) 점수 변화를 나타낸 것이다. PBS 그룹 및 대조군과 비교했을 때 Ngn2 발현 그룹에서 유의하게 행동 능력의 개선이 일어난 것을 나타낸다. 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.
도 7의 d는 Ngn2 발현 그룹에서 GFP 발현 세포를 patch clamp recording한 결과 활동 전위를 나타내는 것을 확인한 것이다. 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.
도 7의 e는 Ngn2 발현 그룹에서 GFP 발현 세포를 patch clamp recording하여 Na channel의 활동에 의한 전류를 확인한 것이다. 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.
도 7의 f는 Ngn2 발현 그룹에서 GFP 발현 세포를 patch clamp recording한 결과 자발적 시냅스 후 전류가 발생함을 나타낸 것이다. 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.
도 8의 b는 sham 그룹과 척수손상 모델 중 PBS 그룹, 대조군, Ngn2 그룹의 척수 조직에서 GFAP, NeuN, ist1을 면역염색한 결과를 나타낸 것이다. 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.
도 8의 c는 도 8의 a의 면역염색 결과에서 MAP2의 강도를 측정하여 비교한 것이다. PBS 그룹 및 대조군과 비교하여 Ngn2 그룹에서 유의하게 MAP2 강도가 증가하였다. 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.
도 8의 d는 도 8의 b의 면역염색 결과에서 ist1과 GFAP의 강도를 측정하여 비교한 것이다. PBS 그룹 및 대조군과 비교하여 Ngn2 그룹에서 유의하게 ist1 강도가 증가하고, GFAP 강도가 감소하였다.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.
도 8의 e는 sham 그룹과 척수 손상 모델 중 PBS 그룹, 대조군, Ngn2 그룹에서 수득한 척수 조직의 coronal, longitudinal section의 EC 염색 결과를 나타낸 것이다. PBS 그룹 및 대조군과 달리 Ngn2 그룹에서는 손상된 척수 조직의 회복이 유의한 수준으로 나타났다.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.
도 8의 f는 도 8의 e의 염색 결과에서 백색질(white matter)과 회색질(gray matter)의 면적을 측정하여 비교한 것이다. Ngn2 발현 그룹에서 백색질과 회색질, 그리고 총 면적이 모두 유의하게 증가하였다. 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.
도 9의 a는 sham 그룹과 척수손상 모델 중 PBS 그룹, 대조군, Ngn2 그룹의 척수 조직에서 BrdU와 함께 GFAP 또는 NeuN 또는 MBP(Myelin binding protein)을 염색한 결과를 나타낸 것이다. 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.
도 9의 b는 척수손상모델에 유전자 발현을 위하여 바이러스 inection 및 BrdU injection 하는 것에 대한 프로토콜을 나타낸 것이다. 9B shows a protocol for viral inection and BrdU injection for gene expression in a spinal cord injury model.
도 9의 c는 도 9의 a의 면역염색 결과를 분석하여 요약한 것으로써, GFP를 발현하면서 GFAP 또는 NeuN 또는 MBP에 의해 염색된 세포에서 BrdU가 염색되지 않는 것을 나타낸 것이다.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.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.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.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 (a) 성상세포(astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계; 및 (b) 상기 (a) 단계의 성상세포를 배양하는 단계를 포함하는, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 방법을 제공한다.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).
본 발명에서 용어 "성상세포(astrocyte)"는 중추신경계에서 패킹 조직을 형성하는 신경교세포로, 건강한 신경 조직에서 에너지 공급, 혈류 조절, 세포 외액의 항상성, 이온 및 전송기의 항상성, 시냅스 기능 조절을 하는 것으로 알려져 있다. 구체적으로, 상기 성상세포는 반응성 성상세포(reactive astrocyte)일 수 있다. 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.
상기 "반응성 성상세포"는 중추신경계의 외상, 감염, 허혈, 뇌졸중, 자가 면역 반응 및 신경 퇴행성 질환 등으로 인해 유입축적된 독성물질에 대한 반응 또는 병리적 환경에 대한 반응으로 성상세포의 성질이 변화하여 생성되는 세포이다. 본 발명의 목적상, 신경세포로 리프로그래밍되는 세포는 반응성 성상세포 일 수 있다. 구체적으로, 상기 반응성 성상세포는 척수 손상에 의해 생성되는 것일 수 있으나, 이에 제한되지 않는다. 또는, 상기 반응성 성상세포는 뉴런의 파괴를 동반하거나, 뉴런의 파괴에 선행하여 생성되는 것일 수 있으나, 이에 제한되지 않는다.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.
반응성 성상세포를 확인하는 방법의 일 예시로, GFAP(Glial Fibrillary Acidic Protein) 발현 수준이 정상 세포보다 높은 경우, 반응성 성상세포로 판별할 수 있다. 뿐만 아니라, Tnf(Tumor necrosis factor), Il1b(Interleukin 1 beta) 및/또는 C1q를 과발현하는 A1 type의 반응성 성상세포, Chil3(Chitinase-like 3), Fzd1(Frizzled class receptor 1) 및/또는 Arg1(Arginase 1)를 과발현하는 반응성 성상세포 역시 본 발명의 범위에 포함된다.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.
상기 성상세포는 개체의 뇌 또는 척수로부터 유래된 것일 수 있으며, 보다 구체적으로 선조체 또는 피곡(putamen)으로부터 유래된 것일 수 있으나, 이에 제한되지 않는다. 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.
본 발명에서 용어 "Ngn2(neurogenin-2)"는 NEUROG2 유전자에 의해 코딩되는 단백질로, basic helix-loop-helix (bHLH) 전사인자 유전자의 neurogenin 서브 패밀리의 하나이다. 본 발명에서 상기 Ngn2는 서열번호 1의 아미노산 서열로 구성된 것일 수 있다. 구체적으로, 본 발명에서 상기 Ngn2는 서열번호 1의 서열과 70% 이상, 구체적으로는 80% 이상, 보다 구체적으로는 90%이상, 보다 더 구체적으로는 95%이상, 가장 구체적으로는 99% 이상의 상동성을 나타내는 아미노산 서열로서 실질적으로 Ngn2와 동일하거나 유사한 활성을 나타내는 단백질의 경우, 이에 제한없이 포함될 수 있다. 또한, 상기 NEUROG2 유전자는 서열번호 2의 염기서열로 구성된 것일 수 있으며, 구체적으로 서열번호 2의 서열과 70% 이상, 구체적으로는 80% 이상, 보다 구체적으로는 90%이상, 보다 더 구체적으로는 95%이상, 가장 구체적으로는 99% 이상의 상동성을 나타내는 염기서열일 수 있으며, 이에 제한되지 않는다. 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.
본 발명에서 용어 "도입"은 세포, 조직 또는 개체에서 본래 가지고 있지 않았던 특정 단백질 또는 이를 코딩하는 유전자의 활성이 자연적 혹은 인위적으로 나타나게 하거나, 이의 발현을 증가시키는 모든 행위를 의미하는 것으로, 상기 단백질은 Ngn2일 수 있으며, 상기 유전자는 NEUROG2 유전자일 수 있다. 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.
구체적으로, 본 발명에서 반응성 성상세포 특이적으로 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입, 또는 성상세포에서 Ngn2 단백질의 발현을 증가는 도 1의 a 또는 도 2의 a에 개시된 개열지도를 갖는 재조합 벡터, 또는 상기 재조합 벡터를 포함하는 바이러스를 성상세포에 전달함으로써 수행될 수 있으며, 특별히 이에 제한되는 것은 아니다. 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.
보다 구체적으로, 상기 재조합 벡터는 성상세포 특이적으로 Ngn2를 발현시키고 발현 여부를 확인하는 목적으로 사용될 수 있으며, 본 발명의 일 실시예에서는 mouse에 대해 pAAV-GFAP-Ccre; pAAV-Lcn2-Ncre; pAAV-EF1α-df-Ngn2-IRES-GFP를 사용하고, cynomolgus monkey에 대해 pAAV-iNOS-Ccre; pAAV-GFAP-Ncre; pAAV-EF1α-df-Ngn2-IRES-GFP를 injection하여 성상세포에 특이적으로 Ngn2를 발현시키고, 그 여부를 면역화학염색(immunohistochemistry)을 통해 확인하였다. 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.
상기 재조합 벡터는 프로모터로 mouse와 cynomolgus monkey에 대해 각각 Lcn2 또는 iNOS; 및 GFAP 프로모터를 사용한 것으로, 구체적으로, 상기 재조합 벡터에 있어서, mouse에 대해서는 Gfap 프로모터를 포함하는 벡터; 및 Lcn2 프로모터를 포함하는 벡터를 이용하고, monkey에 대해서는 Gfap 프로모터를 포함하는 벡터; 및 iNOS 프로모터를 포함하는 벡터를 이용할 수 있다. 상기 2종의 재조합 벡터 프로모터 하단에는 각각 N-terminal Cre와 C-terminal Cre 단편을 코딩하는 서열이 포함되어 있을 수 있다. 상기 N-terminal Cre와 C-terminal Cre는, Cre recombinase를 절단하여 N-말단 및 C-말단 부위로 나눈 것을 의미한다.. 상기 프로모터 조합을 사용함으로써, 반응성 성상세포 특이적으로 유전자 발현을 조절할 수 있다. 즉, 상기 프로모터 조합을 이용하여, 특정 세포(반응성 성상세포)에서만 선택적으로 유전자 발현을 조절할 수 있는 것으로, 이를 이용하여 반응성 성상세포만을 신경세포로 전환분화시킬 수 있다. 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.
상기 GFAP 프로모터는 서열번호 3의 염기서열로, Lcn2 프로모터는 서열번호 4의 염기서열로, iNOS 프로모터는 서열번호 5의 염기서열로 표시되는 것일 수 있으나, 이에 제한되지 않으며, 당업자는 상기 프로모터 서열을 공지된 데이터베이스(NCBI genbank 등)에서 얻을 수 있다.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.).
본 발명에서 용어 "배양"은 세포를 적당히 조절된 환경 조건에서 생육시키는 것을 의미하며, 본 발명의 배양 과정은 당업계에 알려진 적당한 배지와 배양 조건에 따라 이루어질 수 있다. 이러한 배양 과정은 선택되는 세포에 따라 당업자가 조정하여 사용할 수 있다.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.
본 발명에서 용어 "리프로그래밍(reprogramming)"은 특정 세포가 가지는 전체 유전자 발현 패턴(global gene expression pattern)을 조절하여, 목적하는 세포로 전환시키는 방법을 의미한다. 구체적으로, 본 발명에서 리프로그래밍은 특정 세포를 인위적으로 조작하여 전혀 다른 특성을 가지는 세포로 전환시키는 방법을 의미하며, 본 발명의 목적상 상기 리프로그래밍은 비신경세포인 성상세포에 외래 유전자 혹은 핵산분자를 포함하는 재조합벡터를 도입함으로써 수행되는 것일 수 있다. 보다 구체적으로, 상기 리프로그래밍은 전환분화(transdifferentiation) 또는 직접분화(direct-reprogramming)일 수 있으나, 이에 제한되지 않는다.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.
본 발명의 일 실시예에서는 mouse 및 cynomolgus monkey에 대해 성상세포 특이적인 Ngn2 발현을 위해 각각 재조합 플라스미드 벡터를 전달하는 바이러스 AAV-GFAP-Ccre; AAV-Lcn2-Ncre; AAV-EF1α-df-Ngn2-IRES-GFP 및 AAV-iNOS-Ccre; AAV-GFAP-Ncre; AAV-EF1α-df-Ngn2-IRES-GFP를 반응성 성상세포에 injection하고 이를 배양한 결과, Ngn2 발현 그룹에서 patch clamp recording에서 활동전위를 나타내며, GFP 발현 세포(반응성 성상세포)가 뉴런 유사 세포(neuron-like cell)로 분화되었음을 확인하였다.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).
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 성상세포의 신경세포로의 리프로그래밍 유도용 조성물을 제공한다.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.
상기 Ngn2, 성상세포, 신경세포 및 리프로그래밍은 전술한 바와 같다. The Ngn2, astrocytes, neurons and reprogramming are as described above.
또한, 본 발명은 반응성 성상세포의 신경세포로의 리프로그래밍 유도를 위한, Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자의 용도를 제공한다.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.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 퇴행성 신경질환의 예방 또는 치료용 약학적 조성물을 제공한다. 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.
상기 Ngn2, 성상세포 및 신경세포는 전술한 바와 같다.The Ngn2, astrocytes and neurons are as described above.
또한, 본 발명은 퇴행성 신경질환의 예방 또는 치료하기 위한, Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자의 용도를 제공한다.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.
본 발명에서 용어 "퇴행성 신경질환"은 신경세포의 기능 감소 또는 소실에 의해 운동조절능력, 인지기능, 지각기능, 감각기능 및 자율신경의 기능 이상을 의미하는 것으로, 주요 증상에 따라 진행성 인지기능 장애, 진행성 운동실조, 근력저하 및 근위축 그릅으로 나뉠 수 있다. 상기 퇴행성 신경질환은 파킨슨씨병(Parkinson's disease), 알츠하이머(Alzheimer's disease), 피크병(Pick's disease), 헌팅톤병(Huntington's disease), 근위축성 측색 경화증(amyotriophic lateral sclerosis), 허혈성 뇌질환(stroke), 탈수초질환(demyelinating disease), 다발성 경화증, 간질, 퇴행성 신경질환 및 척수손상(Spinal Cord Injury, SCI)으로 구성된 군으로부터 선택되는 것 일 수 있으며, 보다 구체적으로 척수손상일 수 있으나, 이에 특별히 제한되지 않는다.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.
본 발명에서 용어 "척수손상(Spinal cord injury)"은, 후천적인 사고로 인하여 척수에 손상이 가해져 손상된 자리를 통과하는 감각신호와 운동신호의 수행이 영향을 받게 되어 감각신경(sensory neuron)이나 운동신경(motor neuron)의 전달 장애를 초래하여 인체기능을 부분적 또는 완전 마비 상태에 이르게 하는 질환을 의미한다. 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.
구체적으로, 상기 척수 손상은 교통사고, 추락 등의 외상에 의해 발생하는 것일 수 있다. 상기 척수 손상은 손상 정도에 따라 완전손상과 불완전 손상으로 나눌 수 있다. 척수손상은 주로 손상부위 이하에서 운동 신경의 마비로 기능을 소실하게 되는 증상을 수반할 수 있다. 구체적으로 완전손상은 척수의 완전 횡 절단에 의해 척수의 운동, 감각 기능을 완전 소실하게 될 수 있다. 불완전 손상은 완전손상과 달리 손상 부위 이하의 일부 감각, 운동 기능이 보전된 상태로 나타날 수 있다. 손상부에 따라 증상이 달라질 수 있으며, 일 예로, 경수 손상에 의해 사지마비와 혈압, 맥박, 체온, 호흡수가 모두 떨어지는 증상이 나타날 수 있으며. 흉수 이하 손상에 의한 하지마비, 감각기능의 소실, 대장과 방광 및 성기능의 소실을 보일 수 있다.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.
본 발명에서 용어 "예방"은 본 발명의 조성물에 의해 척수 손상을 비롯한 신경 퇴행성 질환의 진행을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.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.
본 발명에서 용어 "치료"는 본 발명의 조성물에 의해 척수 손상을 비롯한 신경 퇴행성 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.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.
본 발명의 "약학적 조성물"은 질병의 예방 또는 치료를 목적으로 제조된 것을 의미하며, 실제 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 또한, 각각의 제형에 따라 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 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.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 혼합 추출물에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose), 락토오스 (Lactose) 또는 젤라틴 등을 섞어 조제할 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데, 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 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.
비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.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.
본 발명의 조성물은 목적하는 방법에 따라 경구투여하거나 비경구투여할 수 있으며, 비경구투여시 피부 외용 또는 복강 내 주사, 직장 내 주사, 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 선택할 수 있다. 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양할 수 있다.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.
또한, 본 발명의 조성물은 약학적으로 유효한 양으로 투여할 수 있다. 상기 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효 용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 또한, 본 발명의 약학적 조성물은 단독으로 또는 암의 예방, 치료, 또는 개선 효과를 나타내는 기타 약학적 활성 화합물과 결합하여 또는 적당한 집합을 이루어 사용될 수 있다.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.
본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여할 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용을 유발하지 않으면서 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.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.
본 발명의 일 실시예에서는, 척수손상 모델 mouse에 대해 상기 3종의 재조합벡터를 포함하는 바이러스(AAV-EF1α-df-Ngn2-IRES-GFP, AAV-Lcn2-Ncre 및 AAV-GFAP-Ccre)를 injection한 결과, 대조군 및 PBS 그룹 대비 손상된 척수 조직이 유의한 수준으로 회복되며, 그에 따라 하반신 마비의 증상이 개선되고 BMS 점수 또한 증가된 수치를 나타내는 것을 확인하였다. 이로부터 본 발명의 조성물은 척수 손상을 비롯한 퇴행성 신경질환의 예방 또는 치료 목적으로 활용 될 수 있다.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.
상기 "척수손상 모델"은 척수 손상을 유도한, 동물 모델을 의미한다. 상기 "척수 손상"에 대해서는 전술한 바와 같다.The "spinal cord injury model" refers to an animal model that induces spinal cord injury. The "spinal cord injury" is as described above.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 상기 방법에 의해 제조된 신경세포를 제공한다. 또한, 상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 상기 방법에 의해 제조된 신경세포를 유효성분으로 포함하는 세포치료제를 제공한다.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.
본 발명의 "세포치료제"는 퇴행성 신경질환의 예방 또는 치료용으로 사용될 수 있는 것으로, 성상세포에 Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계 및 이를 배양하는 단계를 통해 제조된 신경세포를 포함할 수 있으며, 상기 신경세포 이외에도 본 발명의 목적상 척수 손상을 비롯한 퇴행성 신경질환에서 손상된 신경세포를 대체 가능한 물질은 제한되지 않고 여기에 포함될 수 있다.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.
본 발명의 또 다른 하나의 양태는 상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 (a) 반응성 성상세포(reactive astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계; 및 (b) 상기 (a) 단계의 성상세포를 배양하여, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 단계;를 포함하는 퇴행성 신경질환의 예방 또는 치료 방법을 제공한다. 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.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다. 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.
실시예 1: Electrophysiology test를 위한 brain 시료의 준비Example 1: Preparation of brain sample for electrophysiology test
Brain 시료를 준비하기 위해 각각의 동물(mouse 및 cynomolgus monkey)을 할로탄(halothane)으로 마취하였다. Decapitation 후, brain을 두개골로부터 빠르게 절제하고, 이를 얼음처럼 차가운 절삭 용액(130 NaCl, 24 NaHCO3, 1.25 NaH2PO4, 3.5 KCl, 1.5 CaCl2, 1.5 MgCl2, 및 10 D(+)-glucose, pH 7.4)에 담구었다. 전체 용액은 95 % O2 / 5 % CO2로 가스 처리하였다. 이어서 소뇌를 trimming한 후, blade(DORCO, 서울, 한국)와 vibratome(DSK 선형 슬라이서, 교토, 일본)를 사용하여 300μm의 coronal slice를 절단하였고, 이를 세포 외 ACSF 솔루션(mM, 130 NaCl, 24 NaHCO3, 1.25, NaH2PO4, 3.5 KCl, 1.5 CaCl2, 1.5 MgCl2, 및 10, D(+)-glucose, pH 7.4)로 옮겼다. 피펫은 전류 측정(current measurement)을 위한 internal solution(mM 단위, 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)) 및 전압 측정(voltage measurement)을 위한 internal solution(mM 단위, 140 K-gluconate, 10 HEPES, 7 NaCl, and 2 MgATP adjusted to pH 7.4 with CsOH)로 충진하였다.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.
실시예 2: in vitro 조건에서 신경세포로의 직접분화Example 2: Direct differentiation into nerve cells under in vitro conditions
Split-Cre/df system을 이용하여 반응성 성상세포에서 Ngn2의 특이적 발현을 유도하고자 하였다. Mouse에 대해 pAAV-GFAP-Ccre, pAAV-mLcn2-Ncre, pAAV-EF1a-df-Ngn2-IRES-GFP, pAAV-EF1a-df-GFP를 클로닝하고, cynomolgus monkey에 대해 pAAV-GFAP-Ncre, pAAV-iNOS-Ccre, pAAV-EF1a-df-Ngn2-IRES-GFP, pAAV-EF1a-df-GFP를 클로닝하였다. 반응성 성상세포에 3종의 DNA로 된 발현 mixture를 전기천공법(electroporation)으로 도입하였다. 각각의 발현 mixture는 아래 도 5의 a 및 도 6의 a에 나타내었다. 3일 동안 성상세포 배지(10% Horse serum, 10% FBS, 1% P/S in DMEM)에서 세포를 배양하였고, 전기천공 후 4일째 배지를 신경세포 배양 배지(2% B-27, 1% Glutamax, 1%P/S in F-12 media)로 교체하였다.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).
그 결과, 전기천공 후 10일 째 Ngn2 그룹의 세포는 극성 및 신경세포와 같은 형태를 나타냄을 확인하였으며, Ngn2 그룹에서 GFP 발현 세포는 patch clamp recording 결과 활동전위를 나타냄을 확인하였다. 이는 반응성 성상세포에서 Ngn2의 발현이 반응성 성상세포를 신경세포로 전환분화시킬 수 있음을 나타내는 결과이다.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.
실시예 3: in vivo 조건에서 신경세포로의 직접분화 (mouse)Example 3: Direct differentiation into neurons in in vivo conditions (mouse)
Naive mouse(7주령, 수컷)의 선조체에 3종의 바이러스 mixture 2μl을 micro injection하였다. Injection 후 3주 째에 mouse를 희생시켜 1일내 fixation시키고, 2일 동안 dehydration시켜 면역조직화학검사(immunohistochemistry)를 수행하였다. 냉동 brain 조직을 크라이오스탯(cryostat) 마이크로톰으로 30μm 두께로 얇게 슬라이스하고, NeuN (신경세포 핵, 신경세포 마커) 및 GFAP(Glial Fibrillary Acidic Protein, 성상세포 마커)로 염색하였다. DAPI는 대비 염색하였으며, GFP는 항체로 염색되지 않았다. Imaging은 confocal microscope Nikon A1를 이용하여 수행하였다.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.
선조체에서 GFP 발현 세포로부터 NeuN 양성 세포와 GFAP 양성 세포 수를 측정한 결과, 도 5의 b로부터 알 수 있듯이, GFP 발현 세포의 약 70%가 대조군에서 GFAP 양성이며, GFP 발현 세포의 약 70%가 Ngn2 그룹에서 NeuN 양성임을 나타내었다. 이로부터 GFP 발현 세포(반응성 성상세포)가 뉴런 유사 세포(neuron-like cell)로 분화되었음을 알 수 있었으며, 이는 반응성 성상세포에서 Ngn2의 발현이 반응성 성상세포를 신경세포로 전환분화시킬 수 있음을 나타내는 결과이다.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.
또한, mouse brain을 injection 3주 후에 300 μm 두께로 슬라이스하고 GFP 발현 세포에 대해 전체 세포 patch-clamp recording을 수행하였다. 그 결과, 대조군의 GFP 발현 세포는 전압 클램프(voltage clamp)에 대해 수동적 컨덕턴스를 나타내며, Ngn2 그룹의 GFP 발현 세포는 신경세포 특성으로 활동 전위 및 miniature 흥분성 후시냅스 전류(EPSC)를 나타냄을 확인하였다. 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.
실시예Example 4: in 4: in vivovivo 조건에서 신경세포로의 직접분화 ( Direct differentiation into neurons under conditions ( cynomolguscynomolgus monkey) monkey)
stereotaxic surgery를 통해 Cynomolgus monkeys(2-4년)의 피곡(putamen)에 3종의 바이러스 mixture 25μl을 micro injection하였다. Injection 후 3주 째에 monkey를 희생시켜 1일내 fixation시키고, 2일 동안 dehydration시켜 면역조직화학검사(immunohistochemistry)를 수행하였다. 냉동 brain 조직을 크라이오스탯(cryostat) 마이크로톰으로 50μm 두께로 얇게 슬라이스하고, NeuN (신경세포 핵, 신경세포 마커) 및 GFAP(Glial Fibrillary Acidic Protein, 성상세포 마커)로 염색하였다. DAPI는 대비 염색하였으며, GFP는 항체로 염색되지 않았다. Imaging은 confocal microscope Nikon A1를 이용하여 수행하였다. 그 결과, 대조군은 GFP와 GFAP(흰색)의 colocalization를 나타내며, Ngn2 그룹은 GFP와 NeuN(황색)의 colocalization을 나타내었다.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).
피곡(putamen)에서 GFP 발현 세포로부터 NeuN 양성 세포와 GFAP 양성 세포 수를 측정한 결과(각 세포의 ROI는 DAPI 영역으로 정의), scatter plot으로부터 대조군의 GFP 발현 세포로부터 GFAP 강도가 높고 NeuN 강도는 1K 미만임을 확인하였으며, Nhn2 그룹에서는 NeuN 강도가 4K까지 증가한 것을 확인하였다. 또한, 도 6의 e 내지 h로부터 알 수 있듯이, 대조군에서 GFAP 강도가 NeuN 강도보다 유의하게 높고, Ngn2 그룹에서는 별다른 차이가 없음을 확인하였다. 아울러, NeuN의 강도를 보면 Ngn2 군이 대조군보다 높았고, 대조군은 Ngn2 군보다 GFAP 강도가 높음을 확인하였다. 이는 반응성 성상세포에서 Ngn2의 발현이 반응성 성상세포를 신경세포로 전환분화시킬 수 있음을 나타내는 결과이다.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.
실시예Example 5: 척수 손상(Spinal Cord Injury, SCI) 모델에서 신경세포로의 직접분화 5: Direct differentiation into nerve cells in a spinal cord injury (SCI) model
SCI/Ngn2 그룹에서 형광을 발현하는 세포가 활동전위를 비롯한 신경세포의 전기생리학적 특징을 나타내는 지 확인하고자 다음의 실험을 실시하였다.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 마우스(7주령, 수컷)를 이용하여 척수 T10에서 압박 손상에 의한 척수 손상(SCI) 모델을 제작하였다. 손상 후 2주 째에 stereotaxic surgery를 통해 SCI 모델의 손상 부위로부터 1mm 거리의 2개 부위에 3종의 바이러스 mixture 2μl을 micro injection하였다. 아울러, 손상 후 매주 SCI 모델의 BMS 점수를 측정하였다. Injection 후 5주 째 mouse를 희생시켜 1일 내 fixation시키고, 2일 동안 dehydration시켜 면역조직화학검사(immunohistochemistry)를 수행하였다. 냉동된 조직을 크라이오스탯(cryostat) 마이크로톰으로 20μm 두께로 얇게 슬라이스 하고, 슬라이스된 조직에 대해 EC 염색을 수행하였다. 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.
그 결과, 대조군 및 PBS 그룹은 척수 손상이 회복되지 않았으나, Ngn2 그룹에서는 회복된 조직을 확인하였다. 또한, BMS 점수의 평가 결과는 Ngn2 그룹의 BMS 점수가 injection 후 2주부터 대조군 및 PBS 그룹과는 유의한 차이가 있음을 나타낸다. 이는 반응성 성상세포에서 Ngn2의 발현이 반응성 성상세포를 신경세포로 전환분화시킬 수 있음을 나타내며, 나아가 이를 척수 손상 또는 신경 퇴행성 질환에 대한 치료 목적으로 활용할 수 있음을 나타내는 결과이다.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.
실시예 6: SCI/Ngn2 그룹의 신경세포로의 전기생리학적 특징 확인Example 6: Confirmation of electrophysiological characteristics of SCI/Ngn2 group into neurons
SCI/Ngn2 그룹에서 형광을 발현하는 세포가 활동전위를 비롯한 신경세포의 전기생리학적 특징을 나타내는 지 확인하고자 다음의 실험을 실시하였다.The following experiment was conducted to confirm whether cells expressing fluorescence in the SCI/Ngn2 group exhibit electrophysiological characteristics of neurons including action potentials.
먼저, 마취된 생쥐에서 척수 조직을 꺼내어 vibratome을 이용해 가볍게 얼린 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-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)에서 350~400um 두께로 절편하였다. 절편 후 32-34℃ 챔버에서 10-15분간 인큐베이션하고, 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))으로 교체하여 1시간 정도 안정화 시켰다.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.
그런 다음, Whole-cell patch clamp recording은 Internal solution (mM): 120 potassium gluconate, 10 KCl, 1 MgCl2, 0.5 EGTA, 40 HEPES (pH 7.2 was adjusted with KOH)을 사용하였다. 전류 또는 전압 신호 측정은 DigiDATA 1550B1(10kHz)으로 디지털화하여 측정한 후 pCLAMP10 software로 분석하였다.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.
그 결과, 도 7의 d 내지 f로부터 알수 있듯이, SCI/Ngn2 그룹의 GFP를 발현하는 세포에서 활동전위를 확인하였으며, voltage clamp 모드로 레코딩하여 Na 이온 채널의 활동으로 확인되는 3-4nA크기의 큰 전류신호를 측정하였다. 또한, 아무 자극을 주지 않았을 때, synapse에서 자발적으로 보내는 시냅스 후 전류(spontaneous EPSC)가 나오는 것을 확인함으로써, SCI/Ngn2 그룹이 기존의 신경세포와 시냅스를 구성하고 있음을 확인할 수 있었다.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.
실시예Example 7: 7: 성상세포와With astrocytes 신경세포 Nerve cell 마커로With a marker 염색 후 신경세포로의 재생 확인 Confirmation of regeneration into nerve cells after staining
네 그룹의 쥐 (Sham, SCI/PBS, SCI/Ctrl, SCI/Ngn2)를 saline perfusion 후 4% PFA를 관류하여 pre-fixation하였다. 척수 조직을 분리하여 PBS 중의 30% sucrose에 하룻동안 보관하여 탈수한 후 OCT compound에 embedding하여 얼렸다. 얼린 조직을 동결조직절편기를 이용하여 20um두께로 절편하고, 1시간 블로킹 후 (2% donkey serum, 2% goat serum, 0.3% T-x 100 in PBS), GFAP,(1:500) MAP2(1:500) 안티바디를 이용하여 염색하였다. Primary Ab는 4℃에서 O/N처리 후 다음날 PBS로 10분간 3회 워시하고, Secondary Ab (Jacson laboratory, 1:500)는 상온에서 2시간 처리 후 PBS에 10분간 3회 워시하였다. 2회째 워시에서 DAPI cross staining하였고, 염색한 조직은 Nikon A1 confocal microscope 또는 LSM700, Carl Zeiss에서 확인하였다.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.
모든 그룹에서 일정한 threshold를 지정하고 imageJ 사용하여 강도를 측정하였고, GFP를 발현하는 두 그룹에서 GFP발현 세포에 염색된 MAP2 시그널을 측정한 결과, SCI/Ngn2 그룹에서 SCI/PBS, SCI/Ctrl 두 그룹 모두와 비교했을 때 MAP2 강도가 유의미하게 증가하였고, GFP 발현 세포에서도 MAP2 강도가 유의미하게 증가함을 확인하였다.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.
또한, 성상세포와 신경세포 및 운동신경세포 마커로 염색 후 운동신경세포로의 재생 확인하기 위해, 상기 실시예 6과 동일한 방법으로 실험을 실시하였다.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.
모든 그룹에서 일정한 threshold를 지정하여 ist1 강도 측정, GFP를 발현하는 두 그룹에서 GFP발현 세포에 염색된 ist1 시그널을 측정 및 성상세포를 나타내는 GFAP의 강도를 측정하여 SCI/Ngn2 그룹에서 SCI/PBS 및 SCI/Ctrl 그룹과 비교하였다.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.
그 결과, SCI/Ngn2 그룹에서 SCI/PBS, SCI/Ctrl 두 그룹 모두와 비교했을 때 ist1 강도가 유의미하게 증가하였고, GFP 발현 세포에서도 ist1 강도가 유의미하게 증가함을 확인하였다. 또한, 성상세포를 나타내는 GFAP의 강도를 측정하여 SCI/Ngn2 그룹에서 SCI/PBS 및 SCI/Ctrl 그룹과 비교했을 때 GFAP강도가 줄어드는 것을 확인하였다.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.
실시예Example 8: EC( 8: EC( EriochromeEriochrome CyanineCyanine ) staining으로 ) by staining 수초화된Myelinated 영역 및 전체 조직의 면적 확인 Check the area and area of the entire organization
상기 네 그룹의 쥐를 saline perfusion 후 4% PFA를 관류하여 pre-fixation하였다. 척수 조직을 분리하여 PBS 중의 30% sucrose에 하룻동안 보관하여 탈수한 후 OCT compound에 embedding하여 얼렸다. 얼린 조직을 동결조직절편기를 이용하여 20um두께로 절편하였다. 그런 다음, 2시간 상온에서 탈수 후 acetone에 5분간 처리하고, 상온에서 10분간 기다린 후, EC solution을 상온에서 30분간 처리하였다. 흐르는 물과 5% iron alum에서 회백질이 드러날때까지 씻어내고, Boraxferricyanide solution을 처리한 후 70%, 90%, 100% ethanol에서 순차적으로 탈수하였다. Injury site를 검은 점선으로 표시하고, 바이러스가 인젝션된 부분을 녹색 점선으로 표시하였다.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.
사진의 모든 그룹에서 흰색으로 염색된 부분 (gray matter)의 영역을 일정한 threshold를 지정하여 면적을 측정하였고, 푸른색으로 염색된 부분 (white matter)의 영역을 일정한 threshold를 지정하여 면적을 측정한 후, 총 면적의 합을 그룹별로 비교하였다.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.
그 결과, SCI/Ngn2 그룹에서 SCI/PBS, SCI/Ctrl 두 그룹 모두와 비교했을 때 white matter와 gray matter의 면적이 유의미하게 증가하였고, 총 면적 또한 유의미하게 증가함을 확인하였다.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.
실시예Example 9: 9: Ngn2Ngn2 유전자 발현 후 새로 재생된 세포가 Newly regenerated cells after gene expression 성상세포에서In astrocytes 전환분화된 것인지 확인 Confirmation of conversion
Ngn2 유전자 발현 후 성상세포 및 신경세포 마커와 함께 BrdU 염색을 통해서 새로 재생된 세포가 줄기세포에서 분화된 것인지 성상세포에서 전환분화된 것인지 확인하고자 하였다.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.
상기 네 그룹의 쥐를 saline perfusion 후 4% PFA를 관류하여 pre-fixation하였다. 척수 조직을 분리하여 PBS 중의 30%sucrose에 하룻동안 보관하여 탈수한 후, OCT compound에 embedding하여 얼렸다. 얼린 조직을 동결조직절편기를 이용하여 20um두께로 절편하고, 1시간 블로킹 후 (2% donkey serum, 2% goat serum, 0.3% T-x 100 in PBS), GFAP(1:500) NeuN(1:500), BrdU(1:2000), MBP(1:400) 안티바디를 이용하여 염색하였다. Primary Ab는 4℃에서 O/N처리 후 다음날 PBS로 10분간 3회 워시하고, Secondary Ab (Jacson laboratory, 1:500)는 상온에서 2시간 처리 후 PBS에 10분간 3회 워시하였다. 2회 째 워시에서 DAPI로 cross staining하고, 염색한 조직은 Nikon A1 confocal microscope 또는 LSM700, Carl Zeiss 에서 확인하였다. 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.
그런 다음, SCI/Ctrl과 SCI/Ngn2그룹의 GFP를 발현하는 세포와 그렇지 않은 세포 중 BrdU 시그널이 있는 세포를 카운팅하고, GFP를 발현하면서 GFAP으로 염색되는 세포에서 BrdU가 있는 지 확인하였다. 그런 다음, SCI/Ctrl과 SCI/Ngn2그룹의 GFP를 발현하는 세포와 그렇지 않은 세포 중 BrdU 시그널이 있는 세포를 카운팅하고, GFP를 발현하면서 NeuN으로 염색되는 세포에서 BrdU가 있는 지 확인하였다. 마지막으로, SCI/Ctrl과 SCI/Ngn2그룹의 GFP를 발현하는 세포와 그렇지 않은 세포 중 BrdU 시그널이 있는 세포를 카운팅하고, GFP를 발현하면서 MBP으로 염색되는 세포에서 BrdU가 있는 지 확인하였다.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.
그 결과, 세 가지 염색된 그룹 모두에서 BrdU 신호가 GFP를 발현하지 않는 세포에 대부분 나타나는 것을 확인하였다.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.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.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) 반응성 성상세포(reactive astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계; 및(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) 상기 (a) 단계의 성상세포를 배양하는 단계를 포함하는, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 방법.(b) A method for reprogramming astrocytes into nerve cells, comprising the step of culturing the astrocytes of step (a).
  2. 제1항에 있어서, 상기 Ngn2 단백질을 코딩하는 핵산분자는 서열번호 2의 염기서열로 구성된 것인, 방법.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. 제1항에 있어서, 상기 리프로그래밍은 전환분화(transdifferentiation) 또는 직접분화(direct-reprogramming)인 것인, 방법. The method of claim 1, wherein the reprogramming is transdifferentiation or direct-reprogramming.
  4. 제1항에 있어서, 상기 (a) 단계에서 Ngn2 단백질을 코딩하는 핵산분자의 도입은 도 1의 a 또는 도 2의 a에 개시된 개열지도를 갖는 재조합 벡터, 또는 상기 재조합 벡터를 포함하는 바이러스를 성상세포에 전달하는 것인, 방법.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. 제4항에 있어서, 상기 성상세포는 마우스(mouse) 성상세포이고, Cre 재조합 효소의 일부를 코딩하는 서열을 포함하는 재조합 벡터 2종의 프로모터는 각각 Lcn2 및 GFAP 프로모터인 것인, 방법. 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. 제4항에 있어서, 상기 성상세포는 원숭이(monkey) 성상세포이고, Cre 재조합 효소의 일부를 코딩하는 서열을 포함하는 재조합 벡터 2종의 프로모터는 각각 iNOS 및 GFAP 프로모터인 것인, 방법.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. 제1항에 있어서, 상기 성상세포는 개체의 뇌 또는 척수로부터 유래된 것인, 방법.The method of claim 1, wherein the astrocytes are derived from the brain or spinal cord of an individual.
  8. 제1항에 있어서, 상기 반응성 성상세포는 척수 손상 개체로부터 유래된 것인, 방법.The method of claim 1, wherein the reactive astrocyte is derived from a spinal cord injury individual.
  9. Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 반응성 성상세포의 신경세포로의 리프로그래밍 유도용 조성물.A composition for inducing reprogramming of reactive astrocytes into neurons, including Ngn2 protein or a nucleic acid molecule encoding Ngn2 protein.
  10. Ngn2 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 포함하는, 퇴행성 신경질환의 예방 또는 치료용 약학적 조성물.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. 제10항에 있어서, 상기 퇴행성 신경질환은 파킨슨씨병(Parkinson's disease), 알츠하이머(Alzheimer's disease), 피크병(Pick's disease), 헌팅톤병(Huntington's disease), 근위축성 측색 경화증(amyotriophic lateral sclerosis), 허혈성 뇌질환(stroke), 탈수초질환(demyelinating disease), 다발성 경화증, 간질, 퇴행성 신경질환 및 척수손상(Spinal Cord Injury, SCI)으로 구성된 군으로부터 선택되는 것인, 약학적 조성물.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. 제1항 내지 제8항 중 어느 한 항의 방법에 의해 제조된 신경세포.A nerve cell produced by the method of any one of claims 1 to 8.
  13. 제1항 내지 제8항 중 어느 한 항의 방법에 의해 제조된 신경세포를 유효성분으로 포함하는, 세포치료제.A cell therapy agent comprising as an active ingredient nerve cells prepared by the method of any one of claims 1 to 8.
  14. 동물에서,In animals,
    (a) 반응성 성상세포(reactive astrocyte)에 Ngn2(neurogenin-2) 단백질 또는 Ngn2 단백질을 코딩하는 핵산분자를 도입하거나, 또는 성상세포에서 Ngn2 단백질을 발현을 증가시키는 단계; 및(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) 상기 (a) 단계의 성상세포를 배양하여, 성상세포를 신경세포로 리프로그래밍(reprogramming)하는 단계를 포함하는, 퇴행성 신경질환의 예방 또는 치료 방법.(b) culturing the astrocytes of the step (a), and reprogramming the astrocytes to nerve cells, preventing or treating neurodegenerative diseases.
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