A GANGLIOGLIOMA-INDUCED ANIMAL MODEL AND A METHOD FOR DIAGNOSING AND TREATING GANGLIOGLIOMA AND RELATED DISEASES

Abstract

The present invention relates to a biomarker of epilepsy, a composition for diagnosing epilepsy, an epilepsy-induced animal, and a composition for preventing or treating epilepsy, and specifically, relates to a composition for diagnosing epilepsy comprising a BRAF mutant protein and a nucleic acid molecule, and an agent capable of detecting the protein or nucleic acid molecule, an epilepsy-induced animal transformed with the BRAF mutant nucleic acid molecule, and a composition for prevention or treatment of epilepsy comprising a BRAF mutant protein activity inhibitor.

Claims

1-34. (canceled)

35. A non-human animal with ganglioglioma or epilepsy caused by ganglioglioma, comprising a BRAF mutant protein consisting of an amino acid sequence comprising a mutation in which valine at position 600 is substituted to glutamic acid in an amino acid sequence of SEQ ID NO: 1, a BRAF mutant protein consisting of an amino acid sequence comprising a mutation in which valine at position 637 is substituted to glutamic acid in an amino acid sequence of SEQ ID NO: 3, or a mutant nucleic acid molecule encoding the BRAF mutant protein.

36. The non-human animal according to claim 35, wherein the animal is a mammal other than a human.

37. The non-human animal according to claim 35, wherein the animal has a spontaneous seizure accompanying an epilepsy wave.

38. The non-human animal according to the claim 35, wherein the mutant nucleic acid molecule comprises a mutation in which thymine at position 1799 is substituted to adenine in a nucleotide sequence of SEQ ID NO: 2, or thymine at position 1910 is substituted to adenine in a nucleotide sequence of SEQ ID NO: 4.

39. The non-human animal according to the claim 35, wherein the ganglioglioma is caused by a brain somatic mutation.

40. The non-human animal according to the claim 35, wherein the ganglioglioma or epilepsy caused by ganglioglioma is not caused by activation of the mTOR signaling pathway.

41. The non-human animal according to the claim 35, wherein the ganglioglioma or epilepsy caused by ganglioglioma is caused by expression of the mutant protein or the mutant nucleic acid molecule in a nerve cell.

42. The non-human animal according to the claim 35, wherein the ganglioglioma or epilepsy caused by ganglioglioma is caused by expression of the mutant protein or the mutant nucleic acid molecule in a nerve cell at an embryonic development stage.

43. A method for preparing a non-human animal with ganglioglioma or epilepsy caused by ganglioglioma, comprising a step of introducing a gene encoding a protein consisting of an amino acid sequence comprising a mutation in which valine at position 600 is substituted to glutamic acid in the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence comprising a mutation in which valine at position 637 is substituted to glutamic acid in the amino acid sequence of SEQ ID NO: 3 to the animal.

44. The method for preparing a non-human animal according to claim 43, wherein the gene is introduced by preparing a recombinant vector comprising a nucleic acid molecule comprising a mutation in which thymine at position 1799 is substituted to adenine, in the nucleotide sequence of SEQ ID NO: 2; and transforming the animal with the recombinant vector.

45. The method according to claim 44, wherein the recombinant vector gene is introduced to brain of an embryo during the formation of a cortical layer in the embryonic period.

46. A method for screening of therapeutic agents for ganglioglioma or epilepsy caused by ganglioglioma, comprising determining an alleviation of epilepsy after administering a candidate substance for epilepsy treatment to the non-human animal with ganglioglioma or epilepsy caused by ganglioglioma of claim 35.

47. A method for diagnosing and treating ganglioglioma or epilepsy caused by ganglioglioma, comprising diagnosing a subject by detecting a mutant protein consisting of an amino acid sequence comprising a mutation in which valine at position 600 is substituted to glutamic acid in an amino acid of SEQ ID NO: 1, or a mutant nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which thymine at position 1799 is substituted to adenine in a nucleotide sequence of SEQ ID NO: 2, as having ganglioglioma or epilepsy caused by ganglioglioma, and administering an inhibitor of the mutant protein or the mutant nucleic acid molecule to the subject at a therapeutically effective amount.

48. The method according to claim 47, wherein the ganglioglioma is caused by a brain somatic mutation.

49. The method according to claim 47, wherein the ganglioglioma or epilepsy caused by ganglioglioma is not caused by activation of the mTOR signaling pathway.

50. The method according to claim 47, wherein the ganglioglioma or epilepsy caused by ganglioglioma is caused by expression of the mutant protein or the mutant nucleic acid molecule in a nerve cell.

51. The method according to claim 47, wherein the ganglioglioma or epilepsy caused by ganglioglioma is caused by expression of the mutant protein or the mutant nucleic acid molecule in a nerve cell at an embryonic development stage.

52. The method according to claim 47, wherein the mutant nucleic acid molecule is detected by a primer, a probe or an antisense nucleic acid complementary to the nucleotide sequence, and the mutant protein is detected by an antibody or an aptamer specific to the protein.

53. The method according to claim 47, wherein the mutant protein is detected from a sample of a patient, and the sample is a brain tissue sample or a cerebrospinal sample of a patient.

54. The method according to claim 47, wherein the inhibitor is selected from the group consisting of Vemurafenib or its salt, and Dabrafenib or its salt.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0132] FIG. 1a is a picture of confirming that BRAF V600E is significantly present in pediatric low grade glioma with statistical significance.

[0133] FIG. 1b is a picture of confirming the magnetic resonance imaging opinion of the head of the ganglioglioma patient (GG221) (left) and the histopathological opinion confirming the corresponding lesion in the arrow during surgery (right).

[0134] FIG. 1c is a picture of confirming the magnetic resonance imaging opinion of the head before surgery of ganglioglioma patients (GG57, GG163, GG221, GG231, GG249, and GG381).

[0135] FIG. 2a is a schematized picture of the experimental method for presence or absence of BRAF V600E mutation in a nerve cell and glia using the laser capture microscope anatomical method.

[0136] FIG. 2b is a picture of confirming the presence or absence of BRAF V600E mutation in a nerve cell (left) and glia (right) using the laser capture microscope anatomical method.

[0137] FIG. 3a is a picture of confirming that the mouse produced according to one example of the present invention expresses a BRAF V600E mutant gene.

[0138] FIG. 3b is a picture of confirming that a spontaneous seizure accompanying epileptic spikes in the mouse in which the plasmid, in which the BRAF V600E mutant gene is inserted, is injected according to one example of the present invention.

[0139] FIGS. 3c is a picture of confirming pathological features specific to epilepsy in the brain tissue obtained from the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0140] FIG. 4 is a picture of confirming that synchronized burst firing is shown in which spontaneous activity waves and temporally short period of high amplitude energy are simultaneously emitted from several channels, in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0141] FIG. 5 is a picture of confirming that dysplasia of the nerve cell characteristically shown in ganglioglioma is accompanied, in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0142] FIG. 6a is picture showing a process of mating conditional floxed tdTomato mice for tracing daughter cells of neural progenitor cells having BRAF V600E mutation.

[0143] FIG. 6b is a picture of confirming that glia-related astrocytes or oligodendrocytes are significantly increased compared to normal brain tissue in the neural progenitor cells having BRAF V600E mutation produced according to one example of the present invention.

[0144] FIG. 6c is a picture of magnifying the photograph of tissue of FIG. 6b at high magnification and retaking it.

[0145] FIG. 6d is a picture of confirming that CD34 expression is increased by immunohistochemical staining in the actual patient tissue.

[0146] FIG. 7 is a picture of confirming that the expression of CD34 is increased in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0147] FIG. 8 is a picture showing the immunofluorescent staining opinion (left) and immunohistochemical staining opinion (right) which show the increase of the CD34 marker in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0148] FIG. 9 is a picture of observing abnormal lamination of the cortex in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0149] FIG. 10 is a picture of confirming that when a mouse model is produced by the method of one example of the present invention, the brain tissue having BRAF V600E mutation has the changed distribution in the upper and lower parts different from normal brain tissue and shows cortical dysplasia. In the picture at the bottom of FIG. 10, this observation was magnified and it was confirmed that there was no change in the amount of the total nerve cells.

[0150] FIG. 11 is a picture of confirming that patterns of behavior of epilepsy are not shown, but the number of glia-based cells is increased, in the mouse expressing BRAF V600E mutation produced according to one example of the present invention only in glia.

[0151] FIG. 12 is a picture showing the method for producing a BRAF V600E mutant mouse which is inducible using tamoxifen in an adult mouse.

[0152] FIG. 13 is a picture showing the method for producing a BRAF V600E mutant mouse which is inducible using a virus in an adult mouse.

[0153] FIG. 14 is a picture of confirming the abnormal cytological aspect in the case of inducing BRAF V600E mutation in an adult mouse.

[0154] FIG. 15 is a picture of confirming reduction of seizures through chronic intracerebroventricular injection (cICV) of the BRAF V600E-specific inhibitor in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention using measurement of interictal spikes through video monitoring brainwave analysis. POD (post operation day) means days after surgery, and ictal seizure means an epileptic seizure.

[0155] FIG. 16 is a picture of confirming reduction of seizures through chronic intracerebroventricular injection of the BRAF V600E-specific inhibitor in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention using measurement of interictal spikes and electrographic seizure spikes through video monitoring brainwave analysis.

[0156] FIG. 17 is a picture of confirming that there was no reduction of seizures in the video monitoring brainwave analysis through chronic intracerebroventricular injection (cICV) of the BRAF V600E-specific inhibitor and oral administration (PO, per oral) in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

[0157] FIG. 18 is a picture of confirming the activation of the mTOR signaling pathway in the mouse expressing the BRAF V600E mutant protein produced according to one example of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0158] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

EXAMPLE 1

Genome Range Profiling Through Genome Public Data Analysis

[0159] Pediatric low grade glioma has much different clinical aspects from brain tumor of general adults, and particularly, it is known that the ratio of accompanying epilepsy is high in this patient group. To confirm why epilepsy is caused with high frequency in pediatric low grade glioma, by analyzing genome public data such as TCGA (The Cancer Genome Atlas) and PeCan (Pediatric Cancer Genomic Data Portal), mutant gene types distinguished in adult and pediatric brain tumors were classified.

[0160] As a result, as shown in FIG. 1, mutations of genes such as IDH1 and TP53 were present remarkably many in the brain tumor of adults, but BRAF V600E mutation was present significantly many in pediatric low grade glioma with statistical significance.

TABLE-US-00001 TABLE 1 Adult brain Pediatric Pediatric low tumor brain tumor grade glioma Number of patients (n) 283 274 73 Gene type Number of mutations (percentage %) IDH1 215(75.97) 3(1.09) 1(1.37) TP53 128(45.23) 38(13.87) 0(0) H3F3A 0(0).sup. 47(17.15) 3(4.11) CIC 26(9.19) 1(0.36) 1(1.37) BRAF 2(0.71) 5(1.82) 19(26.03) ATRX 16(5.65) 3(1.09) 1(1.37) PIK3CA 2(0.71) 16(5.84) 0(0) SMARCA4 8(2.83) 9(3.28) 0(0) ACVR1 0(0).sup. 16(5.84) 0(0) NOTCH1 14(4.95) 2(0.73) 0(0) Total 411 140 25

EXAMPLE 2

Confirmation of BRAF V600E Mutation Using Whole Exome Sequencing

2-1. Confirmation of BARF V600E Candidates by Whole Exome Sequencing in 5 Patients

[0161] To accurately reflect a tumor-derived epilepsy patient model to an animal model, ganglioglioma known as the biggest cause causing epilepsy-related tumor in children was selected, and to sort gene mutations present in the ganglioglioma, brain tissue and blood samples of 5 ganglioglioma patients (named GG29, GG30, GG221, GG231, and GG249) were collected.

[0162] In the brain tissue sample of ganglioglioma patients, the whole exome sequencing (deep whole exome sequencing, read depth 630-672) was performed, and the candidate mutation found both algorithms of Strelka and Mutect of the whole exome sequencing simultaneously, BRAF V600E was sorted.

[0163] Specifically, a sequencing library was constructed using Agilent library preparation protocols (Agilent Human All Exon 50 Mb kit) provided by the manufacturer for acquisition of data of whole exome sequencing. Sequencing was conducted for the constructed sequencing library using Hiseq2000 (Illumina), and to increase the accuracy of analysis, sequencing was performed with 5 times depth (500) of the general sequencing depth.

[0164] The data after the sequencing were converted into a file in a form to be analyzed using Broad Institute best practice pipeline (https://www.broadinstitute.org/gatk/) (barn file) and were stored.

TABLE-US-00002 TABLE 2 Total Total depth of Average depth Sample yield (bp) target site (X) of target site (X) GG221-Blood 44,917,301,962 630.4 293.35 GG221-Brain 39,837,168,006 559.1 296.33 GG29-Blood 47,943,121,572 579.8 327.14 GG29-Brain 43,394,449,010 596.3 328.11 GG30-Blood 43,443,678,228 609.0 329.51 GG30-Brain 41,317,104,442 609.7 341.22 GG231-Blood 45,607,318,610 630.7 351.96 GG231-Brain 42,488,301,048 640.0 352.19 GG249-Blood 44,944,663,064 669.9 360.64 GG249-Brain 47,736,544,858 672.8 361.99

[0165] As a result, as shown in Table 2, it could be confirmed that the BRAF V600E mutation was present commonly in 3 patients of 5 ganglioglioma patients, and mutations of other genes except for BRAF V600E mutation were not observed. The BRAF V600E mutation confirmed in ganglioglioma is a mutation in which the 1799 thymine of human gene BRAF gene of SEQ ID NO: 2 is substituted to adenine, and this is a mutation in which valine (V) at position 600 of human BRAF protein of SEQ ID NO: 1 is substituted to glutamic acid (E).

2-2. Confirmation of BRAF V600E Mutation in Expanded Patient Group

[0166] To confirm whether BRAF V600E confirmed as present in 5 ganglioglioma patients in Example 2-1 was present in other patient group, by expanding the patient group, gene mutations present in 12 patients in total (including 5 patients confirmed in Example 2-1 (GG29, GG30, GG221, GG231, and GG249)) were investigated (Table 3).

TABLE-US-00003 TABLE 3 BRAF V600E Period of mutation Age at occurrence allele Patient surgery Main of epilepsy Pathological Radiologic Sequencing percentage code (year) opinion (year) Gender opinion opinion method (%) GG29 14 Recurrent 3 Male Ganglioglioma In the Whole seizure in white matter preoperative exome image, a 1 cm sequencing round lesion accompanying edema was located in supramarginal gyrus GG30 15 Tonic 4 Male Low grade In the Whole intermittent glioma preoperative exome spasmodic accompanying image, cystic sequencing seizure multiple lesion with calcification increased size in close to thalamus ganglioglioma GG54 16 Recurrent 10 Female Low grade Low grade Target seizure glioma close cortex tumor panel to close to sequencing ganglioglioma ganglioglioma in left parahippocampal gyrus, fusiform gyrus and onsil GG57 14 Long-term 1 Male Low grade 1.4 cm lump ii Target 18.50 intractable glioma close mesial temporal panel epilepsy to lobe sequencing ganglioglioma GG163 6 Long-term 0.5 Male Low grade Cystic lesion Target 28.87 intractable glioma close increased at T2 panel epilepsy to in posteromedial sequencing ganglioglioma temporal lobe GG221 6 Long-term 0.5 Female Low grade 1 cm Cystic Whole 15.44 intractable glioma close lesion in left exome epilepsy to temporal lobe sequencing ganglioglioma GG231 7 Long-term 2 Male Low grade 1.5 cm T2 Whole 17.86 intractable glioma close increased lesion exome epilepsy to in right uncus sequencing ganglioglioma GG249 5 Long-term 1 Female Low grade T2 increased Whole 19.05 intractable glioma close lesion in right exome epilepsy to temporalis sequencing ganglioglioma posterior GG263 2 Recurrent 1 Male Low grade T2 increased Target seizure glioma close signal in left panel to frontal lobe sequencing ganglioglioma GG351 5 Seizure 1 Male Tumor-related Target intractable panel epilepsy close sequencing to ganglioglioma GG356 9 Seizure 2 Male Tumor-related Target intractable panel epilepsy close sequencing to ganglioglioma GG381 7 Long-term 1 Male Tumor-related 2.8 cm cystic Target 7.41 intractable intractable lesion in regions panel epilepsy epilepsy close including uncus, sequencing to parahippocampal ganglioglioma gyrus and inferotemporal lobe gyrus

[0167] The BRAF V600E mutation was not found in blood of patients, but was found specifically in the brain tissue sample.

[0168] As a result, as could be seen in Table 3, it could be confirmed that the BRAF V600E mutation was present in 6 patients in 12 ganglioglioma patients in total (Genetic variation ratio was 50%), and it could be seen that the ratio of the BRAF V600E mutation allele to the BRAF normal allele present in each patient was about 7 to 30%.

2-3. Patient Sample Collection and Genome DNA Extraction

[0169] For 12 patients of intractable epilepsy caused by ganglioglioma (GG) used in Example 2-2, with the consent of all the patients, their brain tissue (1-2g), saliva (1-2 mL), blood (about 5 mL), frozen tissue and formalin fixed paraffin-embedded brain tissue were obtained (Severance Hospital Pediatric Neurosurgery and Pediatric Neurology). Using the following kits with the manufacturers' protocols, the genome DNA of brain tissue, blood, saliva, frozen and formalin fixed paraffin-embedded brain tissue of patients was separated:

[0170] brain tissue: Qiamp mini DNA kit (Qiagen, USA), blood: Flexigene DNA kit (Qiagen, USA), saliva: prepIT2P purification kit (DNAgenotek, USA), frozen tissue and formalin fixed paraffin-embedded brain tissue: Qiamp mini FFPE DNA kit (Qiagen, USA).

2-4. Ganglioglioma-Specific Gene Mutation Sequencing

[0171] To further confirm whether the BRAF V600E mutation was present in ganglioglioma patients, for the rest 7 patients except for 5 patients confirmed in Example 2-1 among 12 patients confirmed in Example 2-2, hybrid capture sequencing was performed by the following method so that the lead depth was 100-17,700. For the hybrid capture sequencing, a BRAF gene-specific probe was produced using SureDesign online tools (Agilent Technologies). Using Agilent library preparation protocols provided by the manufacturer, a sequencing library was constructed. For the constructed sequencing library, using Hiseq2500 (illumina), the sequencing was performed so that the central lead depth was 500. The data after the sequencing were made into a file in a form that could be analyzed using Broad Institute best practice pipeline (https://www.broadinstitute.org/gatk/).

[0172] To find a brain tissue-specific de novo somatic mutation, the somatic mutation found in both algorithms of Strelka and Mutect among gene sequencing results of blood and brain tissue simultaneously was selected. In addition, only genetic mutations satisfying screening criteria of 100 or more depth and 3 or more mutated calls (30 or more mapping quality) among genetic mutations all found in the hybrid capture sequencing result were selected as a disease-related gene candidate.

[0173] To definitely eliminate errors caused in the sequencing process, only the cases of 1% or more genetic variation rate were considered as positive, and only the cases that all variations were shown when the hybrid capture exome sequencing was performed in the genome DNA of blood and brain tissue were considered as positive, to select these variations as genuine mutations.

[0174] As a result, as shown in the following Table 4, the BRAF V600E mutation was not found in the saliva and blood (control group) of genetic variation positive patients (1% or more genetic variation rate) (negative). However, the BRAF V600E mutation was repeatedly detected in 3 patients (GG221, GG231, GG249), and the percentage of BRAF V600E mutation allele to BRAF normal allele was 7% to 30%.

TABLE-US-00004 TABLE 4 Saliva (Presence or absence Blood (Presence or absence of BRAF V600E mutation allele Patient of BRAF V600E mutation) BRAF V600E mutation) percentage in brain tissue (%) GG221 15.44 GG231 17.86 GG249 19.05

EXAMPLE 3

Confirmation of Cell-Specific Presence of BRAF V600E Mutation Using Laser Capture Cell Lamination

[0175] A prototype of an appropriate model living organism should be prepared to use for patient treatment, but before preparing the prototype of the model organism, it was required to confirm whether the BRAF V600E mutation was present accurately in a nerve cell, or whether it was present in a glia-based cell line, and therefore the following experiment was conducted.

[0176] To confirm whether the BRAF V600E mutation was present accurately in a nerve cell, or whether it was present in a glia-based cell line, after staining tissue of GG221 and GG231 patients in which the BRAF V600E mutation was confirmed with a marker specific to a nerve cell and a marker specific to glia, respectively, using an immunofluorescent staining method, only a specific tissue type of cells were separated using a method called laser capture microscope anatomy to confirm presence or absence of BRAF V600E.

[0177] The surgical tissue block in which BRAF V600E mutation was confirmed among ganglioglioma patient tissue used in Example 2 (GG221, GG231 patient tissue) was cultured in newly prepared 4% (w/v) paraformaldehyde under phosphate-buffered (PB) (fixation), 20% (w/v) buffered sucrose (cryoprotect), and 7.5% (w/v) gelatin under 10% (w/v) sucrose/PB overnight to make a gelatin-embedded tissue block, and it was stored at 80 C.

[0178] The prepared gelatin-embedded tissue block was rapidly soaked in 2-methylbutanes at 50 C. and the gelatin embedded tissue block was rapidly frozen, and a cryostat-cut section having a thickness of 10 um was made using a cryostat microtome (Leica) under the circumstance of 20 C. or below, and the cryostat-cut section was put on a glass slide, and it was blocked with PBS-GT (0.2% (w/v) gelatin and 0.2% (v/v) Triton X-100 in PBS) at a room temperature for one hour, and it was stained with the following antibodies: mouse monoclonal anti-NeuN (1:200, #MAB377, Millipore) and rabbit polyclonal anti-oligodendrocyte Lineage Transcription Factor 2 (Olig2) (1:500, AB9610, Millipore). After 3 times of PBS cleaning, the tissue slide was stained with the following secondary antibodies: Alexa Fluor 555-conjugated goat antibody to mouse (1:200 dilution; A21422, Invitrogen) or Alexa Fluor 488-conjugated goat antibody to rabbit (1:200 dilution; A11008, Invitrogen). DAPI comprised in a mounting solution (P36931, Life technology) was used for nuclear staining. A fluorescent image was obtained using Leica DMI3000 B inverted microscope. Approximately 50 cells only that were NeuN positive and Olig2 negative, or in contrast, NeuN negative and Olig2 positive were detached using PALM MicroBeam (Carl Zeiss) microscope among stained tissue, and only the genomic DNA was extracted with QlAamp DNA Micro Kit, and then PCR was performed using primers of Table 5, and then the amplified PCR products were purified using MEGAquick spin total fragment purification kit (Intron, Korea), and then Sanger sequencing was performed using BioDye Terminator and automatic sequencer system (Applied Biosystems).

TABLE-US-00005 TABLE5 SEQ Name Primer IDNO BRAF_LCM_F 5-TGCTTGCTCTGATAGGAAAATG-3 5 BRAF_LCM_R 5-AGCCTCAATTCTTACCATCCAC-3 6

[0179] As a result, as could be confirmed in FIG. 2b, it could be confirmed that the BRAF V600E mutation (BRAF Chr7: 140453136 for c.1799T>A mutation) was present in two different kinds of cells that were a nerve cell (left in FIG. 2b) and glia (right in FIG. 2b).

[0180] As shown in FIG. 2b, the presence of the same mutation in two different cells, a nerve cell and glia, suggested that the mutation occurred in the common ancestral cells of both nerve cell and glia.

EXAMPLE 4

Confirmation of Prototypes of Epilepsy Patients in Mice Expressing BRAF V600E Mutation

4-1. Confirmation of Spontaneous Seizures in Animal Model Mice

[0181] Conditional floxed BRAF V600E mice capable of expressing BRAF V600E mutation shown in epilepsy patients confirmed in Examples 1 to 3 were produced, and the mice were mated with conditional floxed tdTomato mice to produce a timed pregnant of 14 days of embryo, and then a plasmid gene having a Cre recombinase was introduced in uterus by electroporation, and thereby an animal model similar to conditions of ganglioglioma patients.

[0182] Specifically, in one example of the present invention, using the known site-directed mutagenesis method, a gene in which the 1910th thymine was substituted to adenine in the sequence disclosed in SEQ ID NO: 4 was amplified, and a product in which this was added to a Cre-dependent LoxP sequence was injected into a mouse embryo, thereby producing a transformed mouse which was substituted by a Cre-dependent condition mutation, and at the 14th day of pregnancy of a mouse obtained by mating the produced mouse and tdTomato mouse (E14), the uterine horn was exposed, and 2 ug/ml of Fast Green (F7252, Sigma, USA) combined to a 2 to 3 ug of plasmid having a Cre recombinase was injected to lateral ventricles of each embryo using a pulled glass capillary. The plasmid having a Cre recombinase (pCAG-Cre-IRES2-GFP, addgene, #26646) was subjected to electroporation by discharging 50V to the head of embryo with an ECM830 eletroporator (BTX-harvard apparatus) which was an electric pulse 5 times of 100 ms at an interval of 900 ms.

[0183] Genotyping PCR was conducted using primers of Table 6 and i-Taq TM DNA Polymerase kit (Intron, #25021), by detaching the brain tissue of the mouse embryo electroporated with the plasmid having a Cre recombinase.

TABLE-US-00006 TABLE6 SEQ Name Primer IDNO LSL-Braf.sup.V600EFwd 5-CCCAGGCTCTTTATGAGAA-3 7 LSL-Braf.sup.WTRev 5-AGTCAATCATCCACAGAGACCT-3 8 LSL-Braf.sup.V600ERev 5-GCTTGGCTGGACGTAAACTC-3 9

[0184] As a result, as shown in FIG. 3a, it could be confirmed that the produced mice expressed the BRAF V637E mutant gene (It is mouse BRAF V637E mutation, but it is commonly referred to as BRAF V600E as same as human BRAF V600E, so hereinafter it is described as BRAF V600E).

[0185] In order to confirm that the epilepsy wave actually occurred electrophysiologically in the brain of the produced model mice, after a mouse embryo which was electroporated with the plasmid having a Cre recombinase was born, a video-electroencephalography (video-EEG) test was performed from the 3rd week after birth. To analyze the seizure wave with a signal, video electroencephalography surveillance was conducted from the 3rd week after birth. After separating the embryo with its mother, whether a tonic-clonic seizure was started was confirmed by video surveillance for 12 hours a day. Then, the video-electroencephalography for the mice exhibiting a seizure was conducted for 6 hours a day for 2 days or more, thereby investigating the spontaneous seizure exhibiting a seizure wave.

[0186] To measure the frequency of the interictal spike and nonspastic brainwave seizure, video electroencephalography data filmed for about 10 to 12 hours were used, and from these data, data of 1 minute were extracted at an interval of 1 hour and were analyzed.

[0187] The frequency of the interictal spike and nonspastic brainwave seizure was measured by an observer who did not know the genotype of mice. The interictal spike showed a wave in an epilepsy shape of 200 ms or less at a certain interval and it was defined as the case having an amplitude of 2 times or more than the background brainwave, and the nonspastic brainwave seizure showed at least 2 or more of connected spike-wave (14 Hz) with an amplitude of 2 times or more than the background brainwave and it was defined as the case observed in all 4 electrodes.

[0188] Specifically, after the mice were weaned (>3 weeks), occurrence of a seizure was confirmed only by video monitoring, and then surgery to implant an electrode to measure electroencephalography was progressed. The electrodes were located on an epidural layer, and A total of 5 electrodes were implanted by implanting 2 in the frontal region (AP+2.8 mm, ML1.5 mm), 2 in the temporal region (AP-2.4mm, ML2.4 mm), and 1 in the cerebellum region, based on the bregma. After the recovery period of 4 days, measurement of electroencephalography for 2-5 days (6 hours a day) per mouse was performed at 6 PM to 2 AM. The electroencephalography signal was amplified by an amplifier (GRASS model 9 EEG/Polysomnograph, GRASS technologies, USA), and the signal was analyzed using pCLAMP program (Molecular Devices, USA) or RHD2000 amplifier, board (Intan technoloties, USA) and MATLAB EEGLAB (http://sccn.ucsd.edu/eeglab).

[0189] As a result, as shown in FIG. 3b, 90% or more of mice expressing BRAF V600E mutation showed the spontaneous seizure with the epilepsy wave, and the epilepsy wave showed a high frequency of high amplitude, a spike-wave of high amplitude, and a high frequency of low amplitude. It could be confirmed that the interictal spike was also shown in mice expressing the BRAF V600E mutant gene. The mice showing such a spontaneous seizure showed a systemic tonic-clonic seizure consisting of a tonic phase, a clonic phase and a post-ictal phase, and this was similar to symptoms occurred in ganglioglioma patients. In addition, it was confirmed that the tonic brainwave of the mice showed low-voltage and high-frequency synchronized multi-frequency, and the brainwave of the clonic phase showed a constant form of high-voltage, and the post-ictal phase showed the synchronized damped amplitude. On the other hand, the mice expressing the wildtype BRAF protein did not show a spontaneous seizure or an epilepsy wave.

[0190] Thus, based on the above result, it could be seen that a spontaneous seizure with an epilepsy wave occurred in mice in which a plasmid, in which the BRAF V600E mutant gene was inserted, was injected.

4-2. Confirmation of Hyperactivity of Nerve Cells in Animal Model Mice

[0191] In order to analyze the epilepsy wave shown in the model mice produced in Example 4-1 electrophysiologically, the experiment was performed by the following method.

[0192] Specifically, for the model mice produced in Example 4-1, mouse brain cortex slices were obtained by vibratome and then the spontaneous action potential was measured in artificial cerebrospinal fluid. The composition of the artificial cerebrospinal fluid was as follows: in mM, 124 NaCl, 26 NaHCO3, 3 KC1, 1.25 KH2PO4, 2 CaC12, 1 MgSO4, and 10 D-glucose. The mouse brain cortex slices were put on a brain multichannel electrode recorder (MED64 probe, #P515A, Panasonic Alpha-Med Sciences), and were supported using a slice anchor kit (SHD-22CKIT, Warner Instruments), and then the artificial cerebrospinal fluid was flowed at a rate of 2 mL/minute for 15 minutes and the epilepsy wave occurring under the condition of 37 C./5% CO2 was measured. Spikes were detected using Mobius software (Alpha Med Scientific).

[0193] The result was shown in FIG. 3c and FIG. 4.

[0194] According to the left in FIG. 3c, in case of inducing BRAF V600E mutation, an epilepsy seizure occurred in mice close to 90%, and according to the middle in FIG. 3c, in case of mice causing a seizure, they showed an epilepsy prototype from 6 weeks to 7 weeks on average in FIG. 3c.

[0195] According to the right in FIG. 3c, the timing of individual seizures of BRAF V600E mutation type of mice could be confirmed.

[0196] According to FIG. 4, it was confirmed that synchronized burst firing was shown, in which a spontaneous activity wave and a short period of high amplitude energy were simultaneously emitted on several channels, which was distinctive of epilepsy in brain tissue having BRAF V600E mutation, different from the control group.

[0197] Thus, through the above result, it could be confirmed that synchronized burst firing was shown, in which a spontaneous activity wave and a short period of high amplitude energy were simultaneously emitted on several channels, which was distinctive of epilepsy in brain tissue obtained from mice expressing the BRAF V600E mutant protein, different from the brain tissue obtained in mice expressing a wildtype BRAF protein that was the control group. Through this, it could be seen that nerve cells were hyperactivated in the model mice produced in Example 4-1.

EXAMPLE 5

Confirmation of Epilepsy-Related Tumor Specificity of Mice Expressing BRAF V600E Mutation

5-1 Immunofluorescent Staining of BRAF V600E Mutation Animal Model Mice

[0198] For the brain tissue of the animal model mice produced in Example 4-1, the experiment was performed by the method as Example 3 to prepare gelatin-embedded tissue block, and it was stored at 80 C.

[0199] By the method as Example 3, the gelatin-embedded tissue block was produced as cryostat-cut sections, and they were put on a glass slide, and were stained with the following antibodies: mouse monoclonal anti-NeuN (1:200, #MAB377, Millipore), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:500, #z0334, DAKO), rabbit polyclonal anti-oligodendrocyte Lineage Transcription Factor 2 (Olig2) (1:500, AB9610, Millipore), rabbit monoclonal anti-S100 beta (1:500, ab52642, Abcam), rabbit monoclonal anti-CD34 (1:500, ab81289, Abcam), mouse monoclonal anti-glutamate decarboxylase 67 (GAD67) (1:500, #MAB5406, Millipore), mouse monoclonal anti-parvalbumin (PV) (1:500, #MAB1572, Millipore), rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (1:500, #135 303, Synaptic Systems), rabbit polyclonal anti-vesicular GABA transporter (VGAT) (1:500, #135 002, Synaptic Systems), mouse monoclonal anti-GFP (1:500, #Ab1218, Abcam), rabbit polyclonal anti-GFP (1:500, #Ab290, Abcam), rabbit polyclonal anti-REST (1:200, IHC-00141, Bethyl Laboratories) and rabbit polyclonal anti-CUX1 (1:400, SC13024, Santa cruz). After three times of PBS washing, the tissue slide was stained with secondary antibodies as follows: Alexa 488-conjugated goat anti-rabbit IgG (1:500, #A11008, Invitrogen) or Alexa 555-conjugated goat anti-rabbit IgG (1:500, #21428, Invitrogen). DAPI comprised in a mounting solution (P36931, Life technology) was used for nuclear staining. Images were obtained using Leica DMI3000 B inverted microscope or Zeiss LSM780 confocal microscope. The number of cells which were positive to NeuN was measured using 10 objective lens; 4 to 5 fields were obtained per one subject in the neuron-rich region, and 100 or more cells were recorded per region. The number of DAPI-positive cells represents the total number of cells. The size of neuron cells was measured using an automated counting protocol of ImageJ software (http://rsbweb.nih.gov/ij/) in NeuN positive cells. The circularity and aspect ratio of nerve cells and the angle to the cortex side of dendrites were referenced to values calculated automatically by ImageJ.

5-2. Confirmation of Nerve Cell Dysplasia Through Quantitative Analysis of Immunofluorescent Stained Tissue

[0200] The result of immunofluorescent staining by the method as Example 5-1 was shown in FIG. 5.

[0201] As a result, as could be seen in FIG. 5, it could be confirmed that in case of GFP positive cells of cerebral region in which BRAF V600E mutation was induced by electroporation by the method as Example 4, dysplasia of nerve cells distinctively shown in ganglioglioma was accompanied (A in FIG. 5), and it could be confirmed that in case of the GFP positive cells, the size of cells became bigger and the shape of cells were dented (B in FIG. 5), and the shape of cells and arrangement of branches of nerve cells were arranged in any direction different to normal nerve cells (C in FIG. 5).

5-3. Confirmation of Increases of Glia Proliferation Through Quantitative Analysis of Immunofluorescent Stained Tissue

[0202] In order to trace daughter cells differentiated from neural progenitor cells having BRAF V600E mutation in animal model mice produced using the method as Example 4-1, mice were obtained by mating transformed mice substituted with a gene expressing BRAD V600E protein dependent to a Cre recombinase with mice capable of producing tdTomato fluorescent protein using the method as Example 4-1, and a Cre recombinase was introduced by electroporation in uterus for the mice, and daughter cells differentiated from neural progenitor cells having BRAF V600E mutation derived from the mice were traced by tdTomato fluorescent staining

[0203] As a result, as could be seen in FIG. 6b, in the daughter cells differentiated from neural progenitor cells having BRAF V600E mutation, glia-based related astroglia cells (left picture in FIG. 6b) or oligodendroglia (right picture in FIG. 6b) were significantly increased compared to the brain tissue of mice expressing a BRAF normal gene.

[0204] To sum up the results of Examples 5-2 and 5-3, it could be seen that the BRAF V600E mutation produced in Example 4 was an animal model which reflected a ganglioglioma disease accompanied with dysplasia of nerve cells and numerical increment of glia well.

5-4. Confirmation of CD34 Marker Through Quantitative Analysis of Immunofluorescent Stained Tissue

[0205] It was known that ganglioglioma had a characteristic of increased expression of CD34 pathologically in tumor tissue in the past, and therefore the expression of CD34 marker was confirmed by performing immunofluorescent staining by the method as Example 5-1 in the animal model mouse tissue produced by the method as Example 4-1.

[0206] As a result, as shown in FIG. 7, it could be confirmed that the expression of CD34 increased 2.2 times even in animal model mouse tissue produced by the method as Example 4-1, different from the control group.

[0207] In addition, in order to further confirm characteristics of ganglioglioma, the animal model mouse tissue produced by the method as Example 4-1 was stained using hematoxylin-eosin and DAB immunohistochemical staining used when diagnosing ganglioglioma pathologically.

[0208] Specifically, formalin (Sigma, # HT501128) was injected through heart of the animal model mice produced by the method as Example 4-1 and was fixed, and then paraffin was penetrated into a paraffin embedding device (Leica TP1020) over one day to obtain formalin-fixed paraffin-embedded mouse brain tissue. The formalin-fixed paraffin-embedded mouse brain tissue was cut in a thickness of 4 um using microtome (Leica RM 2135), and then general hematoxylin-eosin staining was conducted. For immunohistochemical staining of CD34, at first, antigen collection for slides was performed using sodium citrate, and they were hydrated using a gradual concentration of alcohol, and then the intrinsic peroxidase activity was removed using 3% (w/w) hydrogen peroxide solution (H202) (Sigma aldrich, H6520). Then, the tissue was blocked using a blocking solution, PBS-GT solution (0.2% (w/v) gelatin and 0.2% (v/v) Triton X-100 under PBS), and then it was stored at a room temperature using an anti-CD34 primary antibody (1:500, ab81289, Abcam). After three times of PBS washing and staining with a secondary antibody, antigen-binding sites in tissue were visualized using 3,3-Diaminobenzidine substrate solution (DAB, Vector Laboratories). To exclude a bias by the image intensity, normal and BRAF V600E mutant mouse tissue was reacted for the same time, and background staining was performed with hematoxylin.

[0209] As a result, as shown in the right picture of FIG. 8, it could be confirmed that the expression of CD34 which was not stained in the normal tissue was remarkable in the area where the mutation occurred in the BRAF V600E mutant mouse brain tissue.

[0210] To sum up the results of FIG. 7 to FIG. 9, it could be seen that the expression of CD34 was significantly increased in the mouse brain tissue having BRAF V600E somatic genome mutation, compared to the mouse brain tissue having a normal gene, and such a pattern was distinctively shown along with nerve cells and tissue in which the mutation was induced by electroporation in uterus particularly.

5-5. Confirmation of Cortical Dyslamination Through Quantitative Analysis of Immunofluorescent Stained Tissue

[0211] Since ganglioglioma has been known to accompany local cortical dysplasia or have many cases of observed cortical dyslamination as a part of malformation of cortical development in tumor tissue, in order to confirm whether the similar aspect was shown in the animal model mice produced by the method as Example 4-1, the animal model mouse tissue produced by the method as Example 401 was fixed and prepared by the method as Example 5-4, and then the layer of cortex was classified by the method as Example 5-1, and thereby staining was performed by Cux1 marker which could be confirmed.

[0212] As a result, as shown in FIG. 9, it could be confirmed that the Cux1 marker was intensively observed in the bottom part as well as the top part of cortex in the mouse brain tissue having BRAF V600E somatic genome mutation, different from the mouse brain tissue having a normal gene. In particular, it was confirmed that the distribution of nerve cells which were positive to Cuxl had negative correlation statistically significantly in mice having BRAF V600E mutation different from the normal group, and thereby it could be seen that cortical dyslamination of mouse brain tissue having BRAF V600E somatic genome mutation was remarkably shown.

[0213] In addition, when the animal model mouse tissue produced by the method as Example 4-1 was under immunohistofluorescent analysis to confirm the distribution of cells which were positive to NeuN and tdTomato concurrently, as could be confirmed in the top of FIG. 10, it could be confirmed that nerve cells which were positive to NeuN and positive to tdTomato were significantly distributed in the layer of the bottom part of cortex in which cells derived from the mouse brain tissue having BRAF V600E mutation were not normally distributed (r=0.1122, p<0.0001).

[0214] However, by the bottom of FIG. 10, it could be seen that hyperplasia in glia shown in Example 5-3 was not simultaneously transmitted in nerve cells. In other words, only the malposition in cortex was shown without proliferation of cells in nerve cells.

5-6. Result Arrangement G Summary

[0215] Through the results of Examples 5-2, 5-3, 5-4 and 5-5, it could be confirmed that the animal model mouse tissue produced by the method as Example 4-1 reflected opinions of tumors with high frequency of accompanying epilepsy among pediatric low grade glioma including ganglioglioma. The above opinions include dysplasia of nerve cells, glia hyperplasia, abnormality of arrangement of dendrites of nerve cells, CD34-positive opinion and transmission of cortical dyslamination, by BRAF V600E mutation, and the like.

EXAMPLE 6

Phenotypes According to Spatial Acquisition of BRAF V600E Mutation

[0216] In case of pediatric epilepsy-related tumor and pediatric low grade glioma and the animal model mice suggested in the present invention examined in Examples 3 and 5-2, the BRAF V600E mutation was present in glia, and therefore it is required to confirm which mutation plays an important role in inducing epilepsy among mutations derived from nerve cells or glia.

[0217] For this, a mouse capable of expressing the BRAF V600E mutation found in ganglioglioma patients in a Cre-dependent manner was produced by a similar method to Example 4-1, and a plasmid having a Cre recombinase at the 1th day after the mouse birth was electroporated, and then behavior was monitored for about 90 days.

[0218] Specifically, in the differentiation of cortical nervous system, the differentiation of nerve cells and glia were actively proceeded at E14 and after birth, respectively, and therefore, when a Cre plasmid was injected at the 14th day of pregnancy of the mother mouse who was in pregnancy of an embryo capable of expressing a Cre-dependent gene, the mutation occurred in nerve cells and glia, and when a Cre plasmid was injected at the first day after birth of the mouse, the mutation occurred in glia, so in order to generate BRAF V600E mutation only in glia using the above fact, mice were produced as Example 4-1, but a Cre plasmid was injected at the first day after birth of the mouse capable of expressing a Cre-dependent gene.

[0219] As a result, as shown in B of FIG. 11, it could be confirmed that a seizure did not occur at all in the mouse produced so as to generate BRAF V600E mutation only in glia (GFAP, and OLIG2 were glia-confirming markers).

[0220] In addition, in order to confirm whether glia hyperplasia was caused in the mouse produced so as to generate BRAF V600E mutation only in glia, the experiment was performed as Example 5-3, and as a result, as shown in C of FIG. 11, it could be confirmed that glia hyperplasia was significantly shown continuously.

[0221] Thus, to sum up the results, it could be seen that the development of epilepsy by BRAF V600E somatic genome mutation was independent of the mutation originated from glia and BRAF V600E mutant generated in nerve cells played an important role in an epilepsy pathogenic mechanism, and in addition, it could be seen that proliferation of a benign tumor in which BRAF V600E mutation occurred only in glia increased.

EXAMPLE 7

Phenotypes According to Temporal Acquisition of BRAF V600E Mutation

[0222] Since epilepsy-related tumor was found with a high ratio in children, whether there was a phenotypic difference between acquisition of BRAF V600E mutation of childhood and acquisition of the mutation in adult mice in the mouse model of the present invention such as epidemiological information of patients was confirmed by the following method.

7-1. BRAF V600E Mutation Inducible Using Tamoxifen in Adult Mice

[0223] In order to change time of occurrence of BRAF V600E mutation in the animal model mouse, the following method was used.

[0224] Timed pregnant of conditional floxed BRAF V600E mice was made by the method as Example 4-1, and a plasmid gene having a tamoxifen inducible Cre recombinase was introduced in uterus, and then from the 30th day after birth, tamoxifen was intraperitoneally injected to induce the BRAF V600E somatic genome mutation in model mouse brain appropriately. Tamoxifen was treated from the 30th day after birth, and behavior of mice was monitored for about 30 days.

[0225] Specifically, for introduction of genome in uterus, the uterine horn was exposed of timed pregnant 14th day (E14), and 2 ug/ml of Fast Green (F7252, Sigma, USA) combined to a 2 to 3 ug of plasmid having a Cre recombinase was injected to lateral ventricles of each embryo using a pulled glass capillary. The plasmid was subjected to electroporation by discharging 50V to the head of embryo with an ECM830 eletroporator (BTX-harvard apparatus) which was an electric pulse 5 times of 100 ms at an interval of 900 ms. In addition, tamoxifen (Sigma, T5648) was shaded in corn oil (Sigma, C8267) at a concentration of 10 mg/ml at 37 C. for 1 day to store, and 100 ug/g tamoxifen was intraperitoneally injected for both normal mice and mutant mice, and it was treated once a day for 5 consecutive days, and after the withdrawal period for immediately next one week, it was treated again for 5 consecutive days. After treatment of tamoxifen, behavior observation for mice was started, and behavior was compared using a mouse treated with only corn oil without tamoxifen or a normal mouse treated with tamoxifen as a control group. A schematic flow of the above experimental procedure was shown in FIG. 12.

[0226] As a result, as could be confirmed in the right graph in FIG. 12, in case that the BRAF V600E mutation was induced in nerve cells of adult mice of after 30 days after birth, mice did not exhibit icta seizures.

7-2. BRAF V600E Mutation Adult Mice Using a Virus

[0227] In order to change time of occurrence of BRAF V600E mutation in the animal model mice, BRAF V600E mutation adult mice were produced using a virus by the following method.

[0228] Specifically, mice after 30 days after birth were anesthetized, and heads were fixed in a stereotactic surgery device (Stoelting Co., Wood Dale, Ill.) and a hole having a diameter of about 1 mm was made in a brain bone with a dental drill. 0.5 uL AAV9.CamKII.HI.eGFP-Cre.WPRE.SV40 (Penn Vector Core Philadelphia, Pa.; titer 6.541013 GCml-1) was injected into somatosensory cortex (AP: 0.5; ML: 2.0-2.5; DV: -0.5) using a glass micropipette at a slow rate (1 nL per sec). In 2 weeks, behavior of mice in which a virus was injected was observed and measurement of brainwaves was started. After observation was finished, frozen tissue slides of brain were prepared in a thickness of 20 um with paraformaldehyde by the method as Example 5-1, and they were observed with a microscope, and whether fluorescent reporter protein injected through the virus expressed well was re-confirmed.

[0229] As a result, as could be confirmed in FIG. 13, as same as inducement of BRAF V600E mutation in adult mice using tamoxifen in Example 7-1, a seizure was not shown also in mice in which the mutation was induced using a virus.

7-3. Confirmation of a Cytologically Abnormal Aspect by BRAF V600E Mutation Induction in Adult Mice

[0230] After inducing the BRAF V600E somatic genome mutation in adult mice as the method of Examples 7-2 and 7-3, dysplasia of nerve cells was confirmed by the method as Example 5-2.

[0231] As a result, as could be confirmed in FIG. 14, in case of inducing (BRAFWT/LSL-V600E) the BRAF V600E mutation in adult mice, as same as the BRAF V600E mutant mouse produced by the method as Example 4-1, compared to the mouse having a normal BRAF gene (BRAFWT/WT), there was no big difference in the size of cells (A in FIG. 14), circularity and aspect ratio of cells (B in FIG. 14) and the angle to the cortical surface of dendrites (C in FIG. 14) and the like from the mouse having a normal BRAF gene (absence of dysplasia of nerve cells).

[0232] In addition, in case of inducing the BRAF V600E mutation in adult mice using tamoxifen, compared to the mouse having a normal BRAF gene, there was no big difference in the size of cells, the circularity and aspect ratio of cells, and the angle to the cortical surface of dendrites and the like from the mouse having a normal BRAF gene (D in FIG. 14) (absence of dysplasia of nerve cells).

[0233] Through the above result, it could be seen that when the BRAF V600E mutation occurred only in nerve cells at an embryonic development stage, dysplasia of nerve cells was induced and thereby epilepsy was caused.

EXAMPLE 8

Confirmation of Reduction of Seizures by Injection of a BRAF V600E Specific Inhibitor

[0234] In order to reduce the frequency of seizures generated in BRAF V600E mutant mice produced by the method as Example 4-1, a BRAF V600E specific inhibitor was treated and its effect was confirmed.

[0235] Specifically, a BRAF V600E mutant protein-specific activity inhibitor (Vemurafenib) was injected into brain tissue of BRAF V600E mutant mice produced by the method as Example 4-1 for a long period. They were generally anesthetized by peritoneal injection of a solution in which Zoletil (0.01 mg/kg) and rompun (0.2 mg/kg) were mixed, and vehicle (50% (v/v) DMSO in DW) or 500 um PLX4032 (Vemurafenib) (V-2800, LC laboratories) was under chronic intracranioventricular injection (cICV) into right ventricle space (AP: -0.6; ML: -1.0; DV: -2.0) in an osmotic pump (ALZET pumps 2004 or 2006, ALZET Osmotic Pumps, Cupertino, Calif.) with infusion cannula (ALZET brain infusion kit 3). In order to trace brainwaves, after inserting 4 EEG probes (left/right frontal lobes and left temporal lobes, cerebellum as a reference) into the animal model mouse head by the method as Example 4-1, brainwave signals were obtained, and 5-6 weeks later, mice were sacrificed and brain was removed to use for the further research, and then whether the semipermeable membrane in the osmotic pump was reduced was re-confirmed. Drug injection was conducted by 4 weeks 6 weeks, and at that time, the brainwaves of mice were monitored and recorded.

[0236] The result was shown in FIG. 15 to FIG. 17.

[0237] According to FIG. 15, it could be confirmed that icta seizures were reduced about 4 times in case of mice in which the BRAF V600E specific inhibitor was injected (FIG. 15 B), and it could be electrophysiologically confirmed that seizures were continuously reduced when treating the BRAF V600E specific inhibitor for 6 weeks (FIG. 15 C).

[0238] According to FIG. 16, it could be confirmed that the electrographic seizures were reduced 2 times to 3 times (middle in FIG. 16), and the interictal spikes were reduced 1.3 times to 1.5 times.

[0239] In addition, according to FIG. 17, it could be confirmed that the seizure frequency was not reduced in case of acute intracerebroventricular injection (3 times/day, 5 uL injection for 8 minutes) (left in FIG. 17) or oral administration (PO, per oral) (right in FIG. 17) of the BRAF V600E specific inhibitor.

EXAMPLE 9

Seizure-Related Mechanism by BRAF V600E Mutation

[0240] The abnormal activity of the mTOR signaling pathway has been known to induce an epileptic seizure, and the BRAF V600E mutation has been known to activate the MARK signaling pathway of Ras-Raf-MEK-ERK. On the other hand, the MAPK signaling pathway of Ras-Raf-MEK-ERK known to be activated by the BRAF V600E mutation has been known to activate the mTOR signaling pathway by crosstalk with the mTOR signaling pathway, and it has been reported that the expression of mTOR signaling pathway-related proteins was increased in tissue of ganglioglioma patients. Thus, in order to confirm whether an epilepsy seizure was caused by activation of the mTOR signaling pathway by the BRAF V600E mutation, the following experiment was performed.

[0241] Specifically, for the animal model mice produced by the method as Example 4-1, rapamycin which was an mTOR-specific inhibitor was intraperitoneally injected, and then the change was confirmed. Rapamycin (LC Labs, USA) was diluted by 20 mg/ml in 100% ethanol to prepare a stock solution, and then was stored at 20 C. Before injecting rapamycin to the animal model mice, the rapamycin stock solution was diluted in 5% (w/v) polyethylene glycol 400 and 5% (v/v) Tween80 to prepare a solution of 1 mg/mL rapamycin and 4% (v/v) ethanol. The prepared solution was administered by intraperitoneal injection at a concentration of 1 to 10 mg/kg for 2 weeks (10 mg/kg/d intraperitoneal injection, for 2 weeks).

[0242] As a result, as shown in FIG. 18, despite of administration of rapamycin, the frequency of epilepsy seizures of BRAF V600E animal model mice did not decrease, and the BRAF V600E mutation animal model mouse tissue did not show a big difference compared to the normal mouse tissue in phosphorylation of S6 protein in the mTOR subpath.