WO2016171448A1 - Pharmaceutical composition for preventing or treating cranial nerve diseases - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating cranial nerve diseases, comprising a nucleic acid molecule encoding four kinds of pluripotent factors (Oct-4, Sox2, c-Myc, and Klf4) as an active ingredient, and being used for in vivo gene therapy. According to the present invention, the four kinds of pluripotent factors are expressed in the brain tissue and induce proliferation of astrocytes and neural progenitor cells and angiogenesis, and thereby a damaged brain function is restored.

Description

Pharmaceutical composition for the prevention or treatment of cerebral nerve disease

The present invention comprises a nucleic acid molecule encoding four pluripotent factors (Oct-4, Sox2, c-Myc and Klf4) as an active ingredient, the prevention of cerebral neurological diseases used for in vivo gene therapy or The present invention relates to a therapeutic pharmaceutical composition.

Restoration or regeneration of damaged tissue is important for recovery of function from damage or disease. Mammalian brains have significantly lower regenerative capacity than other organs and tissues. Therefore, brain tissue damage often leads to semi-permanent dysfunction.

Since the cell types required for brain regeneration are not sufficiently provided by endogenous cell sources, supplementation of other types of cells helps with recovery. For example, bone marrow mononudlear cells (Iihoshi S, Honmou O, Houkin K, Hashi K, Kocsis JD. Brain Res . 2004; 1007: 1-9), bone marrow stromal cells (Chen J, Li Y, Wang L, Lu M, Zhang X, Chopp M. J Neurol Sci . 2001; 189: 49-57) and the brain through transplantation of exogenous cells such as neural stem cells (Park KI, Teng YD, Snyder EY. Nat Biotechnol . 2002; 20: 1111-7). It has been reported that it can improve tissue regeneration. However, and the engraftment of transplanted cells limits (Delcroix GJ, Schiller PC, Benoit JP, Montero-Menei CN Biomaterials 2010; 31:.. 2105-20), the nervous system in the transplanted cells (neural lineage) production of cells There was not enough problem to induce a functional recovery (Roitberg B. Transplantation for stroke. Neurol Res . 2004; 26: 256-64). In addition, transplantation of allogeneic or heterologous cells into the brain may be rejected by the immune system following cell infusion, or tumor production (Fong SP, Tsang KS, Chan AB, et al. J Neurosci Res . 2007; 85: 1851-62). It can have an inducible adverse effect.

Mammalian brains have endogenous neural stem cells and progenitor cells that are mobilized by injury or disease (Goldman SA. Clin ). Pharmacol Ther . 2007; 82: 453-60). Mobilization and activation of these cells can be enhanced by exogenously delivered humoral factors such as epidermal growth factor (EGF) and brain-derived neurotrophic factor (BDNF) (Cho SR, Benraiss A, Chmielnicki E). , Samdani A, Economides A, Goldman SA J Clin Invest 2007; 117:.. 2889-902). The intraventricular delivery of BDNF, noggin or EGF by an adenovirus vector or an osmotic pump is characterized by chronic hypoxic ischemic brain injury (Im SH, Yu JH, Park ES, et al. Neuroscience . 2010; 169: 259-). 68) and subventricular areas leading to striatum regeneration and functional enhancement in mouse models with Huntington's disease (Benraiss A, Toner MJ, Xu Q, et al. Cell Stem Cell . 2013; 12: 787-99). It has been reported to activate endogenous neural stem / progenitor cells in the zone. However, endogenous cells that can be directly affected by these humoral factors are limited to cells with receptors for humoral factors, which act as a limiting factor in the recovery of brain function. Thus, there is a need for more effective therapies that can restore impaired brain function.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop new therapies capable of treating brain function deterioration due to brain injury. As a result, transient in situ expression in brain tissue of four pluripotent factors (Oct-4, Sox2, c-Myc, and Klf4) in ischemic brain injury animal models restores functional impairment due to brain injury. By clarifying that the present invention can be accomplished, the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cerebral neurological diseases.

Another object of the present invention to provide a method of treating neurological diseases.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention is Oct-4 (Octamer-binding transcription factor 4), Sox2 (SRY-box containing gene 2), c-Myc (c-myelocytomatosis oncogene) and Klf4 (Kruppel-like factor 4 It provides a pharmaceutical composition for the prevention or treatment of cerebral neurological diseases, which comprises a single nucleic acid molecule encoding four pluripotent factors, which is) as an active ingredient, and is used for gene therapy in vivo.

According to another aspect of the present invention, the present invention comprises a plurality of nucleic acid molecules separately encoding the four pluripotent factors as an active ingredient, the pharmaceutical for the prevention or treatment of cerebral neurological diseases used for gene therapy in vivo To provide a composition.

According to another aspect of the present invention, the present invention provides a gene therapy in vivo comprising administering to a subject in need thereof a pharmaceutical composition comprising a single nucleic acid molecule encoding the four pluripotent factors. It provides a method of treating cranial nerve disease through.

According to yet another aspect of the present invention, the present invention provides gene therapy in vivo, comprising administering to a subject in need thereof a pharmaceutical composition comprising a plurality of nucleic acid molecules encoding the four pluripotent factors separately. It provides a method of treating neurological diseases through.

The present inventors have tried to develop new therapies capable of treating brain function deterioration due to brain injury. As a result, transient in situ expression in brain tissues of four pluripotent factors (Oct-4, Sox2, c-Myc and Klf4) in ischemic brain injury animal models can restore the functional impairment due to brain injury. It was clarified.

Gene therapy is divided into ex-vivo gene therapy and in-vivo gene therapy according to gene delivery methods, and in vitro gene therapy is a method of treating by injecting cells cultured outside the body again, In vivo gene therapy is a method of injecting genes directly into a patient without the need to extract cells from the patient.

The pharmaceutical composition of the present invention is an in vivo gene therapy agent for cerebral neurological diseases, and one or more nucleic acid molecules encoding the four pluripotency factors are used for gene therapy using the present invention. The nucleic acid molecule may be a single polynucleotide encoding four pluripotent factors together, or may be a plurality of polynucleotides encoding four pluripotent factors separately.

The plurality of polynucleotides separately encoding the four pluripotent factors is (i) four polynucleotides encoding each pluripotent factor, or (ii) one of the four pluripotency factors It may be a combination of a polynucleotide encoding a pluripotent factor and a polynucleotide encoding together two or three pluripotent factors.

Since the nucleotide sequence of the pluripotency factor of the present invention is disclosed in the National Institute of Biological Information (NCBI) website and the like, a person skilled in the art can easily prepare the nucleic acid molecule of the present invention based on such a known nucleotide sequence.

As used herein, the term "prevention" refers to any action that inhibits the onset or delays the progression of cerebral neuropathy by administration of a composition of the invention, and "treatment" refers to the inhibition of the development of cerebral neuropathy, Symptomatic relief or recovery of impaired brain function.

For example, the composition of the present invention can exhibit a therapeutic effect on neurological diseases by restoring cognitive decline, a symptom of neurological diseases. In addition, the composition of the present invention can exhibit a therapeutic effect on the neurological disorder by restoring the motor function, which is another symptom of the neurological disorder.

According to one embodiment of the present invention, the composition of the present invention may exhibit a therapeutic effect on cerebral neuropathy by inducing angiogenesis in brain tissues or by inducing proliferation of astrocytes or neural progenitor cells.

The present invention can be applied to the prevention and treatment of various cranial nerve diseases. Examples of neurological diseases to which the present invention is applicable include stroke, cerebral palsy, ischemic brain injury, traumatic brain injury, delirium and degenerative neurological disease.

Examples of the degenerative neurological disease to which the present invention is applicable include dementia, Parkinson's disease, and Huntington's disease. The dementia includes senile dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, anterior temporal lobe dementia, Parkinson's dementia, Huntington's disease dementia, dementia due to normal pressure hydrocephalus, dementia due to head trauma, and substance-induced dementia. It is not limited.

The compositions of the present invention can be applied in vivo through various delivery methods commonly known in the art of gene therapy.

According to one embodiment of the present invention, the nucleic acid molecule of the present invention is contained in a gene carrier or is naked DNA (naked DNA). When using a gene carrier comprising a nucleic acid molecule encoding the pluripotent factor as an active ingredient, the pluripotent factor-encoding nucleic acid molecule is preferably present in a suitable expression construct.

As used herein, the term “gene delivery” means that a gene is carried into a cell, and has the same meaning as the cell's intraduction of a gene. At the tissue level, the term gene delivery has the same meaning as spread of a gene.

In the expression construct, the pluripotent factor-encoding nucleic acid molecule can be operably linked to a promoter. The term "operably linked" means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is To regulate transcription and / or translation of said other nucleic acid sequences.

In the present invention, a promoter bound to a pluripotent factor-encoding nucleic acid molecule is capable of operating in an animal cell (eg, a mammalian cell) to regulate transcription of the pluripotent factor-encoding nucleic acid molecule, which is derived from a mammalian virus. Promoters and promoters derived from the genome of mammalian cells, such as, for example, cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha Promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and promoter of human GM-CSF gene Including but not limited to.

The expression construct used in the present invention may comprise a poly adenylation sequence (eg, a glial growth hormone terminator and a SV40 derived poly adenylation sequence).

According to one embodiment of the present invention, the pluripotent factor-encoding nucleic acid molecule used in the present invention has a structure of “promoter-pluripotent factor-encoding nucleic acid molecule-polyadenylation sequence”.

Nucleic acid molecules of the present invention can be prepared in various forms, for example (i) naked recombinant DNA molecules using plasmids as carriers, (ii) viral vectors, (iii) liposomes or niosomes, (iv) Lipid-gene complexes (Lipid-DNA complex) or (v) polymer-gene complexes (Polymer-DNA complex) can be produced in the form of.

Pluripotent factor-encoding nucleic acid molecules can be applied to all gene delivery systems used in conventional gene therapy, such as plasmids, adenoviruses (Lockett LJ, et al., Clin . Cancer Res. 3: 2075-2080 (1997) ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors) Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), lentivirus (Wang G. et al., J. Clin . Invest . 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl . Acad . Sci USA 92: 1411-1415 (1995)), vassin virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)) , Liposomes ( Methods in Molecular Biology , Vol 199, SC Basu and M. Basu (Eds., Humana Press 2002) or niosomes.

According to one embodiment of the present invention, the gene carrier of the present invention may be implemented as a viral vector.

The method of introducing the gene delivery system of the present invention described above into a cell may be carried out through various methods known in the art.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to a pharmaceutically effective amount of an active ingredient. The term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-mentioned effective ingredient.

The pharmaceutical composition of the present invention is preferably parenteral, and may be administered using, for example, intravenous, subcutaneous or topical administration. According to one embodiment of the invention, the composition of the invention is administered to brain tissue, four pluripotent factors are expressed in brain tissue.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment.

The pharmaceutical compositions of the present invention are prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.

According to one embodiment of the invention, the subject is a mammal, including a human.

The features and advantages of the present invention are summarized as follows:

(i) The present invention includes a nucleic acid molecule encoding four pluripotent factors (Oct-4, Sox2, c-Myc and Klf4) as an active ingredient, and prevents or treats brain neurological diseases used for in vivo gene therapy. It provides a pharmaceutical composition.

(ii) According to the present invention, the four pluripotent factors are expressed in brain tissue to induce angiogenesis and proliferation of astrocytes and neural progenitor cells, thereby restoring impaired brain function.

Figure 1 shows a schematic of the experimental process of the present invention. A is an experimental procedure using a non-transgenic mouse, and B is an experimental procedure using a transgenic mouse.

2A-2B show the recovery of motor function by the expression of in situ pluripotency factors in brain hull mice expressing pluripotency factor with Sendai virus. In Fig. 2, □ indicates Sendai virus (GFP) administration group, and ■ indicates Sendai virus (pluripotent factor + GFP) administration group.

3A-3B show motor function recovery by expression of in situ pluripotency factors in pluripotency factor gene-transplanted ischemic mice.

Figure 4 shows the results of confirming the expression of pluripotent factor by Sendai virus using immunohistochemical staining (A-C = 200 ㎛).

5 shows no dysplasia by expression of in situ pluripotent factors in pluripotent factor gene-transplanted ischemic mice (A-C = 200 μm).

6A-6C show immune tissue showing cell proliferation (BrdU ⁺ cells) and predominant differentiation into neural stem cells (Nestin ⁺ cells) and astrocytes (GFAP ⁺ cells) in the subventricular region of pluripotent factor gene transplant-brain ischemic mice. It is the result of chemical staining.

7A-7D show cell proliferation (BrdU ⁺ cells) and predominant differentiation into astrocytic cells (GFAP ⁺ cells) and glial scars (CS-56 ⁺ cells) in the striatum of pluripotent factor gene transplant-brain ischemic mice. Immunohistochemical staining showing no.

8A and 8B are immunoassay results showing expression levels of angiogenesis factors in astrocytes and neuronal cells. The increase in blood vessels in mice expressing pluripotent factor compared to the control group was quantified and shown graphically.

9A-9C show immunohistochemical staining showing an increase in angiogenesis (CD31 ⁺ cells) in striatum of pluripotent factor transgenic-ischemic mice (AC = 100 μm, EM = 25 μm).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Methods

2-vessel occlusion mouse model

Birateral common carotid artery of mice was temporarily ligated and released 20 minutes later to induce ischemic brain injury.

Versatility  Factor Induction-Using Viral Vectors

Sendai virus expressing pluripotent factors (Oct-4, Sox2, c-Myc, and Klf4) into lateral ventricle immediately after ischemic brain injury in cerebral ischemic mice (CytoTume ^™ -IPS Sendai Reprogramming Kit, Life Technologies Carlsbad, CA). As a control group, Sendai virus (CytoTume ^™ -EmGFP Sendai Fluorescence Reporter, Life Technologies, Carlsbad, Calif.) Expressing only a green fluorescent reporter was used. The experimental procedure is shown in A of FIG. 1.

Versatility  Derivation of arguments- Doxycycline  Osmotic pump

Reprogrammable mice (Gt (ROSA) 26Sor ^{tm1 ( rtTA * M2 ) Jae} Col1a1 ^{tm3 ( tetO - Pou5f1 , -Sox2, -Klf4, -Myc) Jae} prepared to express transplanted pluripotent factor gene upon doxycycline administration / J), immediately after ischemic brain injury, an osmotic pump containing doxycycline was implanted into the right lateral ventricle to express pluripotency factor for about 7 days. At this time, two concentrations (1 μg / ml (low concentration group) and 100 μg / ml (high concentration group)) were used to compare the effects of doxycycline concentration, and the control group contained phosphate buffered saline. Osmotic pumps were implanted. All mice were injected intraperitoneally with BrdU (5-bromo-2-deoxyuridine, 50 mg / kg) for 2 weeks from the day after ischemic brain injury to label newly generated cells. The experimental procedure is shown in B of FIG.

Behavioral assessment

In order to evaluate motor coordination function, a rotarod test was conducted at two-week intervals using a Rotarod (Cat. No. 47600, UGO Basile, VA, Italy). Specifically, during the 5-minute run at a fixed speed of 48 rpm and an acceleration of up to 4-80 rpm, the time taken for the subject animal to fall off the rod was measured. The evaluation was conducted twice, and the mean latency was analyzed.

Ladder pedestrian test is a video analysis of the number of slips between the ladder crosses and four walks of the mouse on a 1 m horizontal ladder with iron core crosses at various distances. Measured through.

Experimental results are shown in Figures 2a-2b and 3a-3b.

Immunochemical staining ( Immunohistochemistry ; IHC)

In order to perform immunochemical staining, once 8 weeks after breeding in an environment and a floating environment, brain tissues of the animals were extracted, cut at intervals of 16 μm, and brain tissue sections were attached on microglass slides. To confirm the expression of the pluripotent factor, the brain tissues of mice 3 days after transplantation were observed under confocal microscopy after immunochemical staining using Oct4 antibody. The experimental results are shown in FIG. 4.

To confirm dysplasia by in situ pluripotency factor expression, hematoxylin-eosin staining was used and stained under a microscope. The experimental results are shown in FIG. 5.

Cell proliferation by in situ pluripotency factor expression (BrdU antibody) and neural stem cells (Nestin antibody) and astrocytic cells (GFAP antibody) were identified using confocal microscopy after immunochemical staining. In addition, glial cell scars (CS-56 antibodies) were identified to determine whether scars of proliferated astrocytes formed. Experimental results are shown in Figures 6a-6c and 7a-7d.

In order to confirm the formation of blood vessels by in situ pluripotency factor expression, it was confirmed by confocal microscopy after immunochemical staining using CD31, a vascular endothelial cell labeled antibody. Experimental results are shown in Figures 8a-8b.

Cell culture

To determine which cells express angiogenesis factors, mouse astrocyte cell lines (C8-D1A; Astrocyte Type 1 clone, ATCC, CRL-2541) and neuroblastoma cell lines (N1E-115, ATCC, CRL-2263) After culturing for 2 days in serum free culture, cells were harvested and used for the experiment.

Immunoassay

For immunoassay, proteins extracted using the lysis solution from the obtained cells were fractionated by SDS polyacrylamide gel electrophoresis, and the location of the electrophoretic fraction was transferred to a filter paper as it was. The protein transferred to the filter paper was analyzed by radioimmunoassay using ANGPT1 (angiopoietin-1), vascular endothelial growth factor-A (VEGFA), fibroblast growth factor-2 (FGF2) antibody, and GAPDH antibody, an anti-zone gene protein. radioimmunoassay). Experimental results are shown in Figures 9a-9c.

Experiment result

Exercise function test result

In order to confirm the change of motor function caused by in situ pluripotency factor expression in cerebral ischemic mice, behavioral tests were performed with rotarod and ladder walking tests. As a result, as shown in Figure 2a, in the ischemic mice administered Sendai virus, mice with pluripotency factor expression at the acceleration (4-80 rpm) rate of rotarod were significantly longer in duration after 2 weeks than in the control group. Increased (graph on the right). As a result of the ladder walking test, the degree of slipping of the forelimbs was decreased at 4 weeks in the mice expressing pluripotent factor compared to the control group (FIG. 2B).

In addition, as shown in FIG. 3A, when pluripotent factor expression was induced by doxycycline administration in brain ischemic mice transplanted with pluripotent factor gene, rotarod was temporarily fixed in a high concentration group of doxycycline (100 μg / ml). At 48 rpm), the duration began to increase significantly from 2 weeks compared to the control and low concentration groups (1 μg / ml), and this behavior was maintained until 4 weeks (left graph). Even at the acceleration of rotarod (4-80 rpm), the high concentration group (100 μg / ml) of doxycycline began to increase significantly compared to the control and low concentration groups (1 μg / ml) at 2 weeks, and at 4 weeks. The duration increased significantly only compared to the control group (right graph). As a result of ladder walking test, the degree of slipping of the forelimb was decreased in 2 weeks in the high concentration group (100 ㎍ / ml) and low concentration group (1 ㎍ / ml) of the mice compared to the control group. Only (100 μg / ml) showed a significant decrease (FIG. 3b).

Immunochemical Staining Results

To confirm the in situ pluripotency factor expression, the brain tissues of mice 3 days after the Sendai virus transplantation were confirmed by confocal microscopy after immunochemical staining. As a result, as shown in FIG. 4, 3 days after the Sendai virus implantation into the ventricle, the result was confirmed by immunohistochemical staining using Oct4 antibody in mouse brain tissue. As shown in panel A, staining was not performed in the control group. In panel B, Oct4 expression by Sendai virus was observed, and in panel C, green fluorescent expression by Sendai virus expressing only green fluorescent reporter was shown.

In order to confirm dysplasia by in situ pluripotency factor expression, hematoxylin-eosin staining was performed after staining under a microscope. As a result, as shown in FIG. 5, it was confirmed that pluripotency factor expression by high concentration (100 μg / ml) and low concentration (1 μg / ml) of doxycycline in the injured brain did not cause dysplasia. . It was confirmed that both the control group of panel A, the low concentration group of panel B (1 μg / ml) and the high concentration group of panel C (100 μg / ml) did not have dysplasia.

Cell proliferation and differentiation in brain tissue of pluripotent factor gene-transplanted ischemic mice was confirmed by immunohistochemical staining. As a result, as shown in FIGS. 6A-6B, cell proliferation (BrdU ⁺ cells) in the subventricular zone was confirmed (Panel A). Pluripotency factor expression by high concentrations (100 μg / ml) and low concentrations (1 μg / ml) of doxycycline increases production of new cells compared to controls and likewise induces differentiation into neural stem cells (Nestin ⁺ cells). In addition (panel B), it increases differentiation into astrocytic cells (GFAP ⁺ cells) (panel C). On the other hand, staining of young neurons (Tuj-1 ⁺ cells) showed that newly generated cells did not induce differentiation into neurons (Panel D). Panels E, H and K of FIG. 6C show neural stem cells (Nestin ⁺ cells), astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the control group. Panels F, I and J show neural stem cells (Nestin ⁺ cells), astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the low concentration (1 μg / ml) group, panel K, L and M represent neural stem cells (Nestin ⁺ cells), astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the high concentration (100 μg / ml) group of cerebral ischemic damaged mice (EM = 25 μm).

In addition, as shown in FIGS. 7A-7B, cell proliferation (BrdU ⁺ cells) of new cells occurred in the striatum (Panel A). Pluripotency factor expression by high concentrations (100 μg / ml) and low concentrations (1 μg / ml) of doxycycline in the injured brain increases the production of new cells compared to the control and likewise into astrocytes (GFAP ⁺ cells). Differentiation was increased (Panel B). On the other hand, it was shown that newly generated cells did not induce differentiation into neurons (Panel C). Panels E and H of FIGS. 7C-7D show astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the control group. Panels F and I show astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the low concentration (1 μg / ml) group, while panels G and J show high concentrations (100 μg / ml) Astrocytes (GFAP ⁺ cells) and young neurons (Tuj-1 ⁺ cells) in the group are shown (EJ = 25 μm). In addition, it was confirmed that the newly generated astrocytes formed glial scars (CS-56 ⁺ cells), it was confirmed that there is no difference between the groups (KP = 50 ㎛).

As shown in FIG. 8A, pluripotency factor expression by high concentrations (100 μg / ml) of doxycycline in the injured brain showed vascular proliferation (CD31 ⁺ cells) in the striatum. Panel A represents the control group, panel B represents the low concentration group (1 μg / ml), panel C represents the high concentration group (100 μg / ml), and significantly increased blood vessel proliferation (CD31 in the doxycycline high concentration group (100 μg / ml). ⁺ Cells) (Panel D). Panel EM of FIG. 8B shows the correlation of astrocytes (GFAP ⁺ cells) and blood vessels (CD31 ⁺ cells) in the control group, low concentration group (1 μg / ml) and high concentration group (100 μg / ml) (EM = 25 μm).

Immunoassay Results

In order to confirm the expression of angiogenesis factors in cells, mouse astrocytic cell lines (C8-D1A) and neuroblastoma cell lines (N1E-115) were cultured in serum-free culture for two days, and then cells were harvested to extract proteins. Immunoassay was carried out with factors ANGPT1 (angiopoietin-1), vascular endothelial growth factor-A (VEGFA) and fibroblast growth factor-2 (FGF2) and GAPDH antibody as anti-zone gene protein.

As a result, as shown in Figures 9a-9c, it was confirmed that the angiogenesis factor was expressed more in astrocytes than neurons as a result. Panel A shows ANGPT1, panel B shows VEGFA, panel C shows FGF2 increase in astrocytes.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (12)

1. Encoding four pluripotent factors: Octamer-binding transcription factor 4, Sox2 (SRY-box containing gene 2), c-Myc (c-myelocytomatosis oncogene) and Klf4 (Kruppel-like factor 4) A pharmaceutical composition comprising a single nucleic acid molecule as an active ingredient and used for the treatment of genes in vivo.
2. Separately encodes four pluripotent factors: Octamer-binding transcription factor 4, Sox2 (SRY-box containing gene 2), c-Myc (c-myelocytomatosis oncogene) and Klf4 (Kruppel-like factor 4) A pharmaceutical composition for preventing or treating cranial nerve disease, comprising a plurality of nucleic acid molecules as an active ingredient, and used for gene therapy in vivo.
3. The composition of claim 1 or 2, wherein the cerebral neurological disease is selected from the group consisting of stroke, cerebral palsy, ischemic brain injury, traumatic brain injury, delirium and degenerative neurological disease.
4. 4. The composition of claim 3, wherein the degenerative cranial nerve disease is dementia, Parkinson's disease or Huntington's disease.
5. The composition of claim 1 or 2, wherein the composition restores cognitive decline, which is a symptom of cranial nerve disease.
6. The composition of claim 1 or 2, wherein the composition restores a decrease in motor function, which is a symptom of a cranial nerve disease.
7. The composition of claim 1 or 2, wherein the composition induces angiogenesis in brain tissue or induces proliferation of astrocytes or neuroprogenitor cells.
8. The composition according to claim 1 or 2, wherein the composition is administered to brain tissue and four pluripotent factors are expressed in brain tissue.
9. The composition according to claim 1 or 2, wherein the nucleic acid molecule is contained in a gene carrier or naked DNA.
10. 10. The composition of claim 9, wherein said gene carrier is a viral vector, plasmid, liposome, niosome, Lipid-DNA complex or Polymer-DNA complex.
11. Encoding four pluripotent factors: Octamer-binding transcription factor 4, Sox2 (SRY-box containing gene 2), c-Myc (c-myelocytomatosis oncogene) and Klf4 (Kruppel-like factor 4) A method of treating neurological disease through in vivo gene therapy comprising administering to a subject in need thereof a pharmaceutical composition comprising a single nucleic acid molecule.
12. Separately encodes four pluripotent factors: Octamer-binding transcription factor 4, Sox2 (SRY-box containing gene 2), c-Myc (c-myelocytomatosis oncogene) and Klf4 (Kruppel-like factor 4) A method of treating neurological disease through in vivo gene therapy comprising administering to a subject in need thereof a pharmaceutical composition comprising a plurality of nucleic acid molecules.

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