WO2021015352A1 - Procédé de reprogrammation d'astrocytes réactifs en neurones dans un modèle de lésion de la moelle épinière à l'aide de neurogénine-2 (ngn2) - Google Patents

Procédé de reprogrammation d'astrocytes réactifs en neurones dans un modèle de lésion de la moelle épinière à l'aide de neurogénine-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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이창준
안희영
오수진
하윤
이혜란
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한국과학기술연구원
연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically 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.

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Abstract

La présente invention concerne: un procédé de reprogrammation d'astrocytes réactifs en neurones, comprenant les étapes suivantes: (a) introduction de protéine neurogénine-2 (Ngn2) ou d'une molécule d'acide nucléique codant pour la protéine Ngn2 dans des astrocytes réactifs ou pour augmenter l'expression de la protéine Ngn2 dans les astrocytes, et (b) culture des astrocytes de l'étape (a) ; une composition pour induire la reprogrammation d'astrocytes comprenant une protéine Ngn2 ou une molécule d'acide nucléique codant pour la protéine Ngn2 dans les neurones ; une composition pharmaceutique pour la prévention ou le traitement de maladies neurodégénératives ; et des neurones préparés par le procédé et des agents thérapeutiques cellulaires les comprenant. Selon la présente invention, lorsque l'expression de la neurogénine-2 (Ngn2) est spécifiquement augmentée dans les astrocytes réactifs, les astrocytes réactifs peuvent être directement reprogrammés en neurones. De plus, il est confirmé que la capacité comportementale est améliorée par la reprogrammation d'astrocytes réactifs en neurones dans un modèle murin de lésion de la moelle épinière à l'aide du procédé de la présente invention. Par conséquent, le procédé de reprogrammation en neurones peut être utilisé en tant que composition pharmaceutique ou en thérapie cellulaire dans le but de prévenir ou de traiter des maladies neurologiques dégénératives comprenant une lésion de la moelle épinière.
PCT/KR2019/010453 2019-07-25 2019-08-16 Procédé de reprogrammation d'astrocytes réactifs en neurones dans un modèle de lésion de la moelle épinière à l'aide de neurogénine-2 (ngn2) WO2021015352A1 (fr)

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