Epilepsy model

Abstract

To provide a mammalian epilepsy model.SOLUTION: A mammalian epilepsy model is obtained by a method comprising (i) injecting an RNA-interfering shRNA having a specific nucleic acid sequence of a nucleic acid encoding CREB-controlled transcriptional coactivator 1 into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNA-interfering shRNA having a specific nucleic acid sequence of a nucleic acid encoding CREB-controlled transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammal and injecting an RNA-interfering shRNA having a specific nucleic acid sequence of a nucleic acid encoding CREB-controlled transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an epilepsy model, and methods for producing and using said epilepsy model. [Background technology]

[0002] Epilepsy is a major neurological disorder affecting 0.4-1% of the world's population (Non-Patent Documents 1, 2 (https: / / www.who.int / en / news-room / fact-sheets / detail / epilepsy)). 20-30% of epilepsy patients suffer from poor seizure control or intolerable adverse events (Non-Patent Documents 3-8). Animal models, in combination with drug development, have significantly contributed to the development of effective drugs for treating these symptoms (Non-Patent Documents 3, 4, 9). Therefore, there is a need to develop a series of animal models targeting specific classes of epilepsy, which have traditionally been classified into 15 subtypes (Non-Patent Document 10) and have been more recently updated (Non-Patent Document 11). However, currently, there are only a limited number of suitable epilepsy models, and the development of new animal models is required.

[0003] In our previous study, we reported that after monocular injection of TTX (tetrodotoxin), marmosets were reared in the dark (dark rearing, or DR) for 1–2 days, and then light stimulation, several immediate early genes (IEGs) in the primary visual cortex (V1) showed expression patterns consistent with ocular dominance columns (ODCs) (Non-Patent Document 12). During this study (see, for example, Non-Patent Document 13), we noticed that phosphorylation of the transcription factor CREB (cAMP response element binding protein) was significantly elevated in the projection columns of healthy eyes under both dark rearing (DR) and normal rearing conditions, but transiently decreased after light stimulation. Based on this finding, we decided to investigate CRTC1 (or TORC1), which had previously been reported as a member of the cAMP response element binding protein (CREB) coactivator family (Non-Patent Document 14) and as a transducer regulating the activity of the transcription factor CREB (Non-Patent Document 15) through genome-wide functional analysis in mammalian cells. CRTC1 (TORC1) is downstream of the cascade triggered by salt-inducible kinases (SIK1, 2, 3) and is known to regulate CREB activity (Non-Patent Document 16). Furthermore, it has been reported that calcium influx due to synaptic activity transports dephosphorylated CRTC1 to the nucleus (Non-Patent Document 17), where it activates the CREB / CBP complex and promotes IEG expression (Non-Patent Document 18). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Watanabe, S. et al. Functional and molecular characterization of a non-human primate model of autism spectrum disorder shows similarity with the human disease. Nat Commun 12, 5388, doi:10.1038 / s41467-021-25487-6 (2021). [Non-patent document 2] Schmidt, D. & Sillanpaa, M. Evidence-based review on the natural history of the epilepsies. Curr Opin Neurol 25, 159-163, doi:10.1097 / WCO.0b013e3283507e73 (2012). [Non-patent document 3] Loscher, W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochem Res 42, 1873-1888, doi:10.1007 / s11064-017-2222-z (2017). [Non-patent document 4] Bialer, M. & White, HS Key factors in the discovery and development of new antiepileptic drugs. Nat Rev Drug Discov 9, 68-82, doi:10.1038 / nrd2997 (2010). [Non-Patent Document 5] White, HS Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia 44 Suppl 7, 2-8, doi:10.1046 / j.1528-1157.44.s7.10.x (2003). [Non-patent document 6] Depaulis, A., David, O. & Charpier, S. The genetic absence epilepsy rat from Strasbourg as a model to decipher the neural and network mechanisms of generalized idiopathic epilepsies. J Neurosci Methods 260, 159-174, doi:10.1016 / j.jneumeth.2015.05.022 (2016). [Non-Patent Document 7] van Luijtelaar, G. & Zobeiri, M. Progress and outlooks in a genetic absence epilepsy model (WAG / Rij). Curr Med Chem 21, 704-721, doi:10.2174 / 0929867320666131119152913 (2014). [Non-patent document 8] Pitkanen, A. et al. Epileptogenesis in experimental models. Epilepsia 48 Suppl 2, 13-20, doi:10.1111 / j.1528-1167.2007.01063.x (2007). [Non-Patent Document 9] Loscher , W. , Klitgaard , H. , Twyman , RE & Schmidt , D. New avenues for anti-epileptic drug discovery and development . Nat Rev Drug Discov 12, 757–776.

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[0005] To investigate the role of nuclear CREB-regulated transcriptional coactivator 1 (CRTC1) and phosphorylated CREB (pCREB) in IEG induction in marmoset V1, we knocked down CRTC1 RNA using short hairpin (sh) RNA interference. As a result, shCRTC1 injected via AAV vector induced sustained IEG induction not only at the local injection site but also throughout the V1 of both hemispheres. We hypothesized that shCRTC1 might induce abnormal neural activity throughout the entire hemisphere, and therefore performed a series of experiments ranging from histological analysis to electrocorticography (ECoG) and magnetic resonance imaging (MRI).

[0006] Our research has revealed a series of events induced by shCRTC1. First, the spread of neurodegeneration in infected cells (verified histologically) is prevented by the activation of surrounding glial cells, thereby limiting it to a localized state. Second, despite the first event, epileptic neuronal activity spreads throughout the cortex and eventually settles into a steady state. Therefore, this series of events is considered to represent the process of brain-wide functional recovery from abnormal EEG (epilepsy) caused by local injection of shCRTC1 in V1. Based on these findings, we have conceived the epilepsy model of the present invention. [Means for solving the problem]

[0007] The present invention includes, but is not limited to, the following aspects. [Aspect 1] 1. A mammalian epilepsy model comprising: (i) injecting an RNA interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 (CRTC1), or an RNA interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal; The epilepsy model is obtained by a method comprising: [Aspect 2] 2. The epilepsy model according to embodiment 1, wherein the epilepsy is (a) focal onset seizure type and / or (b) structural. [Aspect 3] 2. The epilepsy model according to embodiment 1, wherein the epilepsy is of the focal onset seizure type and is structural. [Aspect 4] An epilepsy model according to any one of aspects 1 to 3, wherein the RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. [Aspect 5] An epilepsy model according to any one of aspects 1 to 3, wherein the RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140. [Aspect 6] The RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has a base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. and The RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the nucleotide sequence of SEQ ID NO: 140 or a nucleotide sequence in which one or two nucleotides are substituted, added, deleted or inserted relative to SEQ ID NO: 140. 4. The epilepsy model according to any one of aspects 1-3. [Aspect 7] The epilepsy model according to any one of aspects 1-3, wherein the mammal is an animal belonging to the order Primates, Rodentia or Carnivora. [Aspect 8] The epilepsy model according to any one of aspects 1-3, wherein the mammal is an animal belonging to the order Primates. [Aspect 9] The epilepsy model according to any one of Aspects 1-3, wherein the mammal is an animal belonging to the infraorder Anthropoidea. [Aspect 10] A method for producing an epilepsy model in a mammal, comprising: (i) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1, or an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, of a nucleic acid encoding CREB-regulated transcriptional coactivator 1, into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal; The method comprising: [Aspect 11] 11. The method of embodiment 10, wherein the epilepsy is (a) focal onset seizure type and / or (b) structural. [Aspect 12] 11. The method of embodiment 10, wherein the epilepsy is focal onset seizure type and structural. [Aspect 13] A method according to any one of aspects 10 to 12, wherein the RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. [Aspect 14] A method according to any one of aspects 10 to 12, wherein the RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of the nucleic acid encoding CREB-controlled transcription coactivator 1, and has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140. [Aspect 15] The RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has a base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. and The RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the nucleotide sequence of SEQ ID NO: 140 or a nucleotide sequence in which one or two nucleotides are substituted, added, deleted or inserted relative to SEQ ID NO: 140. 13. The method of any one of embodiments 10-12. [Aspect 16] 13. The method of any one of aspects 10-12, wherein the mammal is an animal belonging to the order Primates, Rodentia or Carnivora. [Aspect 17] 13. The method of any one of aspects 10-12, wherein the mammal is an animal belonging to the order Primates. [Aspect 18] 13. The method of any one of aspects 10-12, wherein the mammal is an animal belonging to the infraorder Anthropoidea. [Aspect 19] an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1; and / or An RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1. Use of the method for producing an epilepsy model according to any one of Aspects 10 to 12. [Aspect 20] An shRNA that causes RNA interference, which has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. and / or An shRNA that causes RNA interference, which has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the base sequence of SEQ ID NO: 140 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140. Use of the method for producing an epilepsy model according to any one of Aspects 10 to 12. [Effects of the Invention]

[0008] The epilepsy model of the present invention makes it possible to elucidate how epilepsy occurs, develops and progresses, both in terms of development and seizures. [Brief explanation of the drawings]

[0009] [Figure 1a]Figure 1 shows IEG induction in one hemisphere of marmoset V1 (Monkey 1) following three injections of shCRTC1#1 into the left hemisphere (V1). The left bottom panel of Figure 1a shows a lateral view of the left hemisphere of the marmoset, with the injection area (V1) indicated by a red circle. The right bottom panel of Figure 1a shows a schematic diagram of the injection sites in Monkey 1. The marmoset brain is viewed from the back, corresponding to the red circle in the left panel. The injection sites are indicated by shCRTC1 (blue circle) and scramble (shscr, scrambled shRNA) (red circle). The scale bar in the left bottom panel of Figure 1a is 1 cm. Abbreviations: A (anterior), P (posterior), L (lateral), M (medial), D (dorsal), V (ventral). The blue line D in the left bottom panel indicates the position of the coronal plane shown in Figure 1d. [Figure 1b] Figure 1 shows IEG induction in one hemisphere of marmoset V1 (Monkey 1) following three-site injection of shCRTC1#1. Figure 1b shows the experimental protocol from AAV injection to perfusion. The time course is from left to right. Abbreviations: NR (normal rearing with day and night), DR (dark rearing), LS (light stimulation). The time course is from left to right. [Figure 1c] Figure 1 shows IEG induction in one hemisphere of marmoset V1 (monkey 1) following three-site injection of shCRTC1#1. Figure 1c shows the expression of Sirius (top), CRTC1 (middle), and cFOS mRNA (bottom) at injection sites in the left (shCRTC1) and right (shscr) hemispheres. cFOS mRNA was stained by in situ hybridization, and CRTC1 and Sirius were stained by immunohistochemistry, and adjacent sections were analyzed. The scale bar in Figure 1c is 500 μm. [Figure 1d] Figure 1 shows IEG induction in one hemisphere of marmoset V1 (Monkey 1) following three-site infusion of shCRTC1#1. Figure 1d shows cFOS mRNA expression in a coronal section of V1 (section D) at both sides of the location shown in Figure 1a (lower left) of Monkey 1. The scale bar in Figure 1d is 1 mm. [Figure 1e]Figure 1 shows IEG induction in the unilateral hemisphere of marmoset V1 (monkey 1) following three-site injection of shCRTC1#1. Figure 1e shows a high-magnification image of the expression of five IEGs in the left and right V1. cFOS (IHC) shows the expression of cFOS protein by immunohistochemistry; the others show the mRNA expression pattern by ISH. [Figure 2a] Figure 2 shows cFOS expression after one or two injections of shCRTC1#1 in each hemisphere. Figure 2a (Monkey 2) shows normal feeding (NR), while Figure 2b (Monkey 3) shows normal feeding followed by darkness (41 hours) and 24 minutes of light stimulation (LS) without intraocular injection of TTX. Each monkey received one or two injections of shCRTC1#1 and scramble (shscr) into the left and right V1. As in Figure 1a, the shCRTC1#1 and scramble shRNA injection sites are indicated by blue and red circles, respectively. The right side of Figure 2a shows double-stained fluorescent ISH images of Sirius (green) and cFOS mRNA (red) at the AAV injection site. Induction of cFOS mRNA expression was not detected at the scramble injection site, but was strongly detected at and around the shCRTC1 injection site. Abbreviations: L1-6 (layers 1-6). Other abbreviations are the same as in Figure 1. [Figure 2b] Figure 2 shows cFOS expression after one or two injections of shCRTC1#1 in each hemisphere. Figure 2a (Monkey 2) shows normal feeding (NR), while Figure 2b (Monkey 3) shows normal feeding followed by darkness (41 hours) and 24 minutes of light stimulation (LS) without intraocular injection of TTX. Each monkey received one or two injections of shCRTC1#1 and scramble (shscr) into the left and right V1. As shown in Figure 1a, the shCRTC1#1 and scramble shRNA injection sites are indicated by blue and red circles, respectively. Figure 2b (top right) shows the experimental conditions for Monkey 3, and Figure 2b (bottom) shows Sirius (green) expression at the AAV injection sites (shCRTC1 and scr). Abbreviations: L1-6 (layers 1-6). Other abbreviations are the same as in Figure 1. The scale bar in Figure 2b is 500 μm. [Figure 2c] Figure 2 shows cFOS expression after one or two injections of shCRTC1#1 into each hemisphere. Figure 2a (Monkey 2) shows normal feeding (NR), while Figure 2b (Monkey 3) shows normal feeding followed by darkness (41 hours) and 24 minutes of light stimulation (LS) without TTX injection into the eyes. Each monkey received one or two injections of shCRTC1#1 and scramble (shscr) into the left and right V1. As in Figure 1a, the injection sites of shCRTC1#1 and scramble shRNA are indicated by blue and red circles, respectively. Figure 2c shows cFOS protein expression (IHC) (top) and cFOS mRNA expression (ISH) (bottom) at the shCRTC1 injection site under the conditions shown in Figure 2b. cFOS induction was much stronger at the shCRTC1#1 injection site and its surrounding area than the cFOS expression induced by ocular input. On the other hand, areas outside the injection site show the usual pattern of cFOS expression induced by light stimulation. Abbreviations: L1-6 (layers 1-6). Other abbreviations are the same as in Figure 1. The scale bars in Figure 2c (top) and (bottom) are 200 µm. [Figure 3a] Figure 3 shows the results of histological analysis of the area around the shRNA injection site in monkey 1 using Nissl staining and markers for neurons, glia, and cell death. Neurons were detected by antibody staining for NeuN, microglia for IBA1, and activated astrocytes for GFAP. TUNEL staining was performed as a marker for cell death. Sirius (green) was used to detect the injection site. The upper panel of Figure 3a shows the injection site and the location of the tissues used for analysis (dashed lines), and the lower panel shows the results of Nissl staining around the injection site. The scale bar in the lower panel of Figure 3a is 500 μm. [Figure 3b]Figure 3 shows the results of histological analysis of the area around the shRNA injection site in monkey 1 using Nissl staining and markers for neurons, glia, and cell death. Neurons were detected by antibody staining for NeuN, microglia for IBA1, and activated astrocytes for GFAP. TUNEL staining was performed as a marker for cell death. Sirius (green) was used to detect the injection site. The left panel in the upper row of Figure 3b shows the IBA1 signal in red, and the right panel shows the GFAP signal in red. The Sirius signal is shown in green (G). The bottom row of Figure 3b shows an image where the red and green signals are superimposed. [Figure 3c] Figure 3 shows the results of histological analysis of the area around the shRNA injection site in monkey 1 using Nissl staining and markers for neurons, glia, and cell death. Neurons were detected by antibody staining for NeuN, microglia for IBA1, and activated astrocytes for GFAP. TUNEL staining was performed as a marker for cell death. Sirius (green) was used to detect the injection site. Figure 3c shows the results of double antibody staining for NeuN (magenta) and Sirius (green) at the shCRTC1#1 injection site. The right panel of Figure 3c is a superimposed image of magenta and green. The scale bar in Figure 3c is 100 μm. [Figure 3d] Figure 3 shows the results of histological analysis of the area around the shRNA injection site in monkey 1 using Nissl staining and markers for neurons, glia, and cell death. Neurons were detected by antibody staining for NeuN, microglia for IBA1, and activated astrocytes for GFAP. TUNEL staining was performed as a marker for cell death. Sirius (green) was used to detect the injection site. Figure 3d shows the results of TUNEL staining of the injection site. Left (Sirius), middle (TUNEL), and right (enlarged TUNEL). The scale bar in Figure 3d is 500 μm. [Figure 3e]Figure 3 shows the results of histological analysis of the area around the shRNA injection site in monkey 1 using Nissl staining and markers for neurons, glia, and cell death. Neurons were detected by antibody staining for NeuN, microglia for IBA1, and activated astrocytes for GFAP. TUNEL staining was performed as a marker for cell death. Sirius (green) was used to detect the injection site. Figure 3e shows the results of antibody staining for Sirius (left), phosphorylated CREB (middle), and CRTC1 (right) at the shCRTC1#1 injection site. Adjacent sections were analyzed. Phosphorylated CREB signals (middle) were detected at the injection site (Sirius, left). The scale bar in Figure 3e is 500 μm. [Figure 4a] Figure 4 shows that local injection of shCRTC1 induced local and cortical-wide HFOs. Figure 4a shows the time course of the experiment (in vivo MRI, in vivo CT, ECoG recording, postmortem MRI (T2 / DTI), histochemical analysis). [Figure 4b] Figure 4 shows that local injection of shCRTC1 induced local and cortical-wide HFOs. Figure 4b shows the locations of injection sites (open circles) and ECoG electrodes (colored dots) in monkeys 5 and 6. The dot colors indicate the grouped cortical regions: V1 (primary visual cortex, blue), Vis (higher-order visual cortex, dark orange), Aud (auditory cortex, ochre), TC (temporal cortex, purple), SM (somatosensory cortex, yellow-green), PC (parietal cortex, cyan), and FC (frontal cortex, magenta). The color code is the same as in Figure 4f. [Figure 4c] Figure 4 shows that local injection of shCRTC1 induced local and cortical-wide HFOs. Figure 4c shows an example of HFA (80-200 Hz) in monkey 5. The left panel of Figure 4c shows the results 22 days after injection of shCRTC1, and the right panel shows the results 39 days after injection of shCRTC1. The vertical bar at the bottom of Figure 4c indicates the onset of cwHFOs. The black triangle corresponds to the onset of cwHFOs, which is shown enlarged in Figure 4d. [Figure 4d]Figure 4 shows that local injection of shCRTC1 induced local and cortical-wide HFOs. Figure 4d shows examples of cwHFOs following HFOs in V1 (left panel) or TC (right panel). [Figure 4e] Figure 4 shows that local injection of shCRTC1 induced local and cortical-wide HFOs. Figure 4e shows 15-min average HFA recordings in V1 (blue) and PC (cyan) on each experimental day. Open triangles indicate results 28 days after injection of shCRTC1. [Figure 4f] Figure 4 shows that local injection of shCRTC1 induced localized and cortical-wide HFOs. Figure 4f shows the number of cwHFOs for each experimental day. The color of each bar indicates the cortical region with the highest HFA between -250 and 0 ms, before the onset of cwHFOs. [Figure 5a] Figure 5 shows that the altered functional network recovered 5 weeks after shCRTC1 injection. Figure 5a shows that shCRTC1 injection induced changes in the functional network. The left column shows the average correlation matrices of ECoG signals from monkeys 5 (top) and 6 (bottom). The center and right columns show the correlation matrices 3 weeks (22 days) and 8 weeks (56 days) after injection, respectively. The color scale is the same as in Figure 5d. [Figure 5b] Figure 5 shows that the altered functional networks were restored 5 weeks after shCRTC1 injection. Figure 5b shows the deviation from the mean functional connections. Each functional connection is plotted on the x-axis for each experimental day and on the y-axis for the mean network values ​​in Figure 5a. [Figure 5c] Figure 5 shows that the altered functional network recovered 5 weeks after shCRTC1 injection. Figure 5c shows the stability of functional connectivity. The left and right panels show the correlation matrices of functional connectivity for monkeys 5 and 6 on each experimental day. The open triangles on the horizontal axis indicate 28 days after shCRTC1 injection. [Figure 5d]Figure 5 shows that the altered functional network recovered 5 weeks after shCRTC1 injection. Figure 5d shows intra- and inter-regional functional connectivity. The left and right panels show the results for monkeys 5 and 6, respectively. [Figure 5e] Figure 5 shows that the altered functional network recovered 5 weeks after shCRTC1 injection. Figure 5e shows a transient decrease in functional connectivity within V1. The horizontal line indicates the 5% significance level (z = -1.645). [Figure 6a] Figure 6 shows the results of histological analysis 63 and 148 days after shCRTC1 injection. Marmosets injected with shCRTC1#1 at three sites in the right hemisphere of the brain (V1) were fixed and histologically analyzed 63 and 148 days after injection (Monkeys 5 and 6 in Table 3). Figure 6a shows histological images of two injection sites in Monkey 5 (#1-1, upper panel, and #1-2, lower panel), analyzed by (IBA1, NeuN, Sirius), (GFAP) only, (Nissle and TUNEL), and (TUNEL) only, from left to right in four panels. The scale bar in Figure 6a is 500 μm. [Figure 6b] Figure 6 shows the results of histological analysis 63 and 148 days after shCRTC1 injection. Marmosets in which shCRTC1#1 was injected into three locations in the right brain V1 were fixed and histologically analyzed 63 and 148 days after injection (Monkeys 5 and 6 in Table 3). Figure 6b shows histological images of monkey 6 analyzed in the same way as monkey 5 in Figure 6a. [Figure 6c] Figure 6 shows the results of histological analysis 63 and 148 days after shCRTC1 injection. Marmosets injected with shCRTC1#1 at three sites in the right hemisphere of the brain (V1) were fixed and histologically analyzed 63 and 148 days after injection (Monkeys 5 and 6 in Table 3). Figure 6c shows in vivo MRI (left) and ex vivo MRI (right). The insets in the upper right corner of each image show a magnified view of the lesion in the temporal cortex (presumably TE3). [Figure 6d]Figure 6 shows the results of histological analysis 63 and 148 days after shCRTC1 injection. Marmosets injected with shCRTC1#1 at three sites in the right hemisphere V1 were fixed and histologically analyzed 63 and 148 days after injection (Monkeys 5 and 6 in Table 3). Figure 6d shows Nissl-stained images (left panel) and "backlit" images (right panel) from sections of Monkey 5 corresponding to the locations shown in Figure 6c. The scale bar in Figure 6d is 500 μm. [Figure 6e] Figure 6 shows the results of histological analysis 63 and 148 days after shCRTC1 injection. Marmosets injected with shCRTC1#1 at three sites in the right hemisphere V1 were fixed and histologically analyzed 63 and 148 days after injection (Monkeys 5 and 6 in Table 3). Figure 6e shows images of antibody staining for GFAP (top two rows) and IBA1 (bottom two rows) in the temporal lobe of Monkey 5. These images were obtained from adjacent sections to the Nissl staining in Figure 6d. The small squares below the larger images of GFAP and IBA1 are enlarged images of the respective red squares on the right, and low-magnification images of the entire cross section on the left. [Figure 7a] Figure 7 shows that diffusion tensor imaging (DTI) after shCRTC1 injection demonstrated cortical asymmetry. Figure 7a shows the correlation between left and right profiles of DTI connectivity in each supervoxel. Red areas indicate low correlation values, implying asymmetric connectivity between the left and right cortex. [Figure 7b]Figure 7 shows that diffusion tensor imaging (DTI) after shCRTC1 injection demonstrated cortical asymmetry. Figure 7b shows the left-right asymmetry (AF) of 31 cortical regions of interest. Box plots show ex vivo DTI AF from 36 normal marmosets. Post-injection AF of monkey 5, post-injection AF of monkey 6, and pre-injection AF of monkey 6 are shown as solid blue, solid red, and open red circles, respectively. Abbreviations: HiF=hippocampal formation;V6=visual cortex V6;V2=secondary visual cortex;V1=primary visual cortex;TPO=temporal pole;STP=superior temporal polysensory cortex;PHG=parahippocampal gyrus;IT=inferior temporal area;S2=secondary somatosensory cortex;PA=anterior Striatal cortical area; PC = piriform cortex; Pcu = precuneus; PPC = posterior parietal area; S = subiculum; STR = superior temporal rostral area; PPCv = ventral posterior parietal area; GC = gustatory area; MT = middle temporal area; EC = entorhinal cortex; Aud = auditory cortex ;PFCvl = ventrolateral frontal cortex;PR = perirhinal cortex;RSC = retrosplenial cortex;ACC = anterior cingulate cortex;PCC = posterior cingulate cortex;V3 = visual area 3;PFCmv = ventromedial prefrontal cortex;OFC = orbitofrontal cortex;FP = frontal pole;PFCm = medial prefrontal cortex;PFCdl = dorsolateral prefrontal cortex;PM = premotor cortex;M1 = primary motor cortex;S1 = primary somatosensory cortex;IPS = intraparietal sulcus;INS = insular cortex;ON = olfactory nucleus;OB = olfactory bulb. [Figure 8a] Figure 8 shows the DTI bundle analysis by Hata et al. Figure 8a shows the results of the DTI bundle analysis. The left image in Figure 8a shows the DTI bundle with V1 and the frontal cortex as the regions of interest projected in green onto an MRI cross section passing through V1 and the frontal cortex. The right image shows the DTI bundle with V1 and the temporal lobe as the regions of interest projected in blue onto an MRI cross section passing through V1 and the temporal lobe. [Figure 8b] Figure 8 shows the DTI bundle analysis by Hata et al. Figure 8b shows the results of the DTI bundle analysis. Figure 8b shows the asymmetry of fiber bundles between the left and right hemispheres. The crosses (X) and circles on the figure correspond to the proportion of right DTI bundles relative to the total of left and right fiber bundles, respectively. The proportion of right DTI bundles after injection in monkey 5, after injection in monkey 6, and before injection in monkey 6 are shown as solid blue, solid red, and open red circles, respectively. As control data, the proportion of right DTI bundles in normal marmosets is shown as black crosses. [Figure 9a] Figure 9 shows the effect and specificity of shCRTC1. Figure 9a shows the specificity of shCRTC1#1 and #2. 293T cells were transfected with each shCRTC1 plasmid and a marmoset CRTC1 expression vector, and the expression level of CRTC1 was assessed by Western blotting. The left panel shows the marmoset CRTC1 expression vector construct, and the right panel shows the results of Western blot analysis. Each column in the Western blot shows the results from transfection with no shRNA, shscr, shCRTC1#1, and shCRTC1#2. From top to bottom, each row shows detection with CRTC1 antibody, HA antibody, and β-actin antibody. [Figure 9b] Figure 9 shows the effect and specificity of shCRTC1. Figure 9b shows antibody staining results for Sirius (upper left panel) and CRTC1 (lower left panel) at the shCRTC#1 and shscr(scramble) injection sites in marmoset V1. These were stained in adjacent sections. Right panels a) and b) show high-magnification images of the white-boxed area of ​​CRTC1 (lower left panel). From top to bottom, images of Sirius (naive fluorescence, no antibody staining), CRTC1 (IHC), and a superimposition of the two are shown. The scale bar in Figure 9b is 1 mm. [Figure 10] Figure 10 shows that in Monkey 1, in which shCRTC1#1 was injected into three sites on one side, shCRTC1#1 injection significantly reduced mRNA expression of Nur77, an IEG, except in layer 2. ISH analysis shows (from left) Sirius expression, Nur77 expression, and an overlay of the two at the shCRTC1#1 injection site. [Figure 11]Figure 11 shows the results of double ISH analysis of the expression patterns of four IEGs around the shCRTC1 and shscr injection sites in Monkey 2, which received one or two unilateral injections of shCRTC1#1. Left panel: (from top to bottom) mRNA expression of cFOS, ARC, ZIF268, and NURR1. Middle panel: Sirius mRNA expression. Right panel: Overlay of the images in the left and middle panels. The scale bar in Figure 11 is 500 μm. [Figure 12a] Figure 12, related to Figures 3 and 11, shows the results of analysis of glial and apoptotic markers at the shCRTC1 and shscre injection sites in monkey 2. Figure 12a shows the shRNA injection sites in monkey 2 and the locations of the sections analyzed in Figure 12 with dashed lines. [Figure 12b] Figure 12, related to Figures 3 and 11, shows the results of analysis using glial and apoptotic markers at the shCRTC1 and shscre injection sites in monkey 2. Figure 12b shows the upper image of IBA1 (left) and GFAP (right) expression (magenta) at the shRNA injection site superimposed with Sirius expression (green). The lower image shows only the IBA1 signal and GFAP signal. These stainings were performed on adjacent sections. [Figure 12c] Figure 12, related to Figures 3 and 11, shows the results of analysis of glial and apoptotic markers at the shCRTC1 and shscre injection sites in monkey 2. Figure 12c shows that TUNEL signals were detected only at the shCRTC1#1 injection site. The squared area in the left panel is enlarged in the image on the right. [Figure 12d] Figure 12, related to Figures 3 and 11, shows the results of glial and apoptotic marker analysis at the shCRTC1 and shscr injection sites in monkey 2. Figure 12d shows NeuN expression at the shCRTC1#1 and shscr injection sites. The left panel shows the NeuN signal (magenta), and the right panel shows the overlay with Sirius (green). The scale bar in Figure 12d is 500 μm. [Figure 13a1]Figure 13a shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#1 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13a2] Figure 13a shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#1 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13b1]Figure 13b shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#2 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13b2] Figure 13b shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#2 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13c1]Figure 13c shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#3 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13c2] Figure 13c shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#3 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13d1]Figure 13d shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#4 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13d2] Figure 13d shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#4 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13e1]Figure 13e shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#5 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 13e2] Figure 13e shows the results of a BLAST (Basic Local Alignment Search Tool) homology analysis between shCRTC1#5 and the common marmoset genome (19 base pairs) on the U.S. National Library of Medicine website. The following information is included: Scientific Name (species name), Total Score (evaluation score; the higher the score, the higher the homology), Query Coverage (what percentage of the 19 bases matched?), E Value (statistical probability of homology with the searched bases; the lower the E Value, the more likely the sequence is identical), and Accession (gene ID number). The BLAST analysis revealed that shCRTC1#1-5 all share extremely high homology with the CRTC1 gene sequence compared to other gene sequences in the marmoset genome (100% match of the 19 bases, P value of 0.01 or less). [Figure 14]Figure 14 shows ECoG signals from monkey 5 22 days (left panel) and 39 days (right panel) after injection. (a) An example of a 2-minute ECoG signal. (b) An example of HFA (80–200 Hz) from monkey 5. The x-axis represents the same time range as in (a), and the y-axis represents each electrode, with the electrodes sorted in the same order as in (a). (c) The percentage of electrodes showing HFO (HFA > 3 SD) at each time point. The horizontal line indicates the 50% criterion for cortical-wide HFO (cwHFO). The x-axis in (c) represents the same time range as in (a), and the vertical bar below the figure indicates the onset of cwHFO. [Figure 15] Figure 15 shows ECoG signals from monkey 6 at 18 days (left panel) and 38 days (right panel) after injection, similar to monkey 5 in Figure 14. (a) Example of a 2-minute ECoG signal. (b) Example of HFA (80-200 Hz) from monkey 6. (c) Percentage of electrodes showing HFO (HFA > 3 SD) at each time point. The vertical bars in the bottom row indicate the onset of cwHFO, as in the previous figure. [Figure 16] Figure 16 shows that cwHFOs appeared following HFOs in V1 (left panel) or TC (right panel) of monkey 6. (Top) Example of HFA (80-200 Hz) in monkey 6. The vertical bar at the bottom of the figure indicates the onset of cwHFOs. The black triangle corresponds to the onset of the enlarged cwHFO in the bottom inset. (Bottom) Example of cwHFOs appeared following HFOs in V1 (left) or TC (right). (a) 18 days after injection, (b) 38 days after injection. [Figure 17] Figure 17 shows ECoG signals from Monkey 6 6 days after knockdown (day 145). (a) An example of a 2-minute (left panel) and a 10-second (right panel) ECoG signal. The shaded area on the x-axis in the left panel corresponds to the time range shown in the right panel. (b) An example of HFA (80–200 Hz). The x-axis represents the same time range as in (a), and the y-axis represents electrodes. The electrodes were sorted in the same order as in (a). (c) The percentage of electrodes showing HFO (HFA > 3 SD) at each time point. The horizontal line indicates the 50% criterion for cortical-wide HFO (cwHFO). The x-axis represents the same time range as in (a), and the vertical bar below the panel indicates the onset of cwHFO. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention includes, but is not limited to, the following embodiments. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in this specification, it means that the embodiment is not limited, i.e., is not limited.

[0011] 1. Epilepsy Model In one aspect, the present invention relates to a mammalian epilepsy model. The epilepsy model of the present invention includes, but is not limited to: (i) injecting an RNA interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 (CRTC1), or an RNA interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into two or more different locations in a mammalian animal, and injecting an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into one or more different brain or cranial nerve regions of the mammalian animal; The method is obtained by a method including:

[0012] "shRNA" is short hairpin RNA. shRNA is generally used in a vector-based approach to deliver short interfering RNA (siRNA). When shRNA is introduced into cells, it reduces the gene expression of the complementary sequence by RNA interference (RNAi). In this specification, "shRNA" may be used to include the siRNA delivered therefrom.

[0013] In one embodiment, the epilepsy model is obtained by injecting each shRNA into different brain or cranial nerve regions of the cerebral cortex of a mammal. In one aspect, the epilepsy model comprises: (i) comprising an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1, or an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, in three or more different brain or cranial nerve regions of a mammal; or (ii) Injecting an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal.

[0014] In one embodiment, the epilepsy model comprises each shRNA in a different region of the brain or cranial nerve of the mammal's cerebral cortex. "Cranial nerve regions" refers to peripheral nerve regions that enter and exit the brain. "Different brain or cranial nerve regions" preferably refer to different but adjacent brain or cranial nerve regions in the brain (e.g., cerebral cortex). "Adjacent brain or cranial nerve regions" refer to brain or cranial nerve regions that are close enough together that shRNAs injected into different locations can interact with each other to cause RNA interference. "Different brain or cranial nerve regions" are, for example, within 5 mm, more preferably within 2 mm, in the case of a macaque brain.

[0015] "Epilepsy" refers, without limitation, to a neurological disease or condition in which abnormal neural activity occurs in cells in the brain, resulting in seizures. The International League Against Epilepsy (ILAE) classification of epilepsy has three levels. The first level is the "seizure type" diagnosis, which is divided into three types: focal onset seizures, generalized onset seizures, and seizures of unknown onset. The next level after the seizure type diagnosis is the "epileptic disease type" diagnosis, which is divided into focal epilepsy, generalized epilepsy, generalized focal combined epilepsy, and epilepsy of unknown type. The third level is the "epileptic syndrome" diagnosis, which allows for the diagnosis of a specific syndrome. The ILAE classification further incorporates an "etiology" diagnosis at each level. Etiology is divided into six subgroups (structural, diagnoses, infectious, metabolic, immune, and unknown etiology) selected based on their potential impact on treatment.

[0016] In the epilepsy model, the type of epilepsy is not particularly limited. In one embodiment, the epilepsy is focal onset seizure type. In one embodiment, the epilepsy is structural. In one embodiment, the epilepsy is focal onset seizure type and structural.

[0017] An "epilepsy model" is an animal model that exhibits the human disease epilepsy or symptoms similar to epilepsy. Animal models are used to study the development and progression of the disease and to test new treatments before administering them to humans.

[0018] CREB is a transcription factor and a central nuclear protein involved in transcriptional activation via transcriptional regulation of the cAMP response element (CRE) present in gene transcription factors on the genome. CREB transcriptional activity is controlled via various second messenger pathways, and in neurons, in addition to cAMP-dependent activity regulation, increases in intracellular calcium concentration due to synaptic activation and receptor activation by neurotrophic factors function as major factors for CREB activation.

[0019] CREB-controlled transcriptional coactivators (CRTCs) are coactivators that bind to the bZIP domain involved in CREB dimerization and DNA binding. In mammals, the CRTC gene group forms a gene family consisting of three species (CRTC1, CRTC2, and CRTC3), but CRTC1 and CRTC2 are mainly expressed in the nervous system. The subject of this invention is CREB-controlled transcriptional coactivator 1. Under basal conditions, the CRTC gene family is phosphorylated and exists in the cytoplasm in a complex with 14-3-3 protein. When cAMP or Ca is added to this complex, 2+ Upon stimulation by an increase in concentration, the CRTC gene family is dephosphorylated by calcineurin, dissociates from 14-3-3, and rapidly translocates into the nucleus where it binds to CREB.

[0020] "An shRNA that causes RNA interference, having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1 (CRTC1)," and "an shRNA that causes RNA interference, having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1," can be easily prepared by a person skilled in the art based on the sequence of SEQ ID NO: 1 using known methods for preparing shRNA for RNA interference. For example, the method described in Non-Patent Document 37 can be referenced.

[0021] In one aspect, the "RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1" and the "RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1" are shCRTC1#1 and shCRTC1#2, respectively, as described in the examples of this specification.

[0022] In one aspect, an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO: 139.

[0023] Depending on the animal species or mutant, the sequence corresponding to bases 194-212 of SEQ ID NO: 1 may be slightly different from the base sequence of SEQ ID NO: 139. In such cases, the shRNA that induces RNA interference may have a base sequence that corresponds to the sequence of the animal species or mutant. In such cases, the shRNA that induces RNA interference may have a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO: 139.

[0024] In one aspect, an RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1 has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140.

[0025] Depending on the animal species or mutant, the sequence corresponding to bases 403-421 of SEQ ID NO: 1 may be slightly different from the base sequence of SEQ ID NO: 139. In such cases, the shRNA that induces RNA interference may have a base sequence that corresponds to the sequence of the animal species or mutant. In this case, the shRNA that induces RNA interference may have a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO: 140.

[0026] In one embodiment, the RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of the nucleic acid encoding CREB-regulated transcription coactivator 1, and has a base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO: 139. and An RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcription coactivator 1 has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140.

[0027] In one embodiment, the "substitution" is a substitution in which the amino acid residue encoded by the substitution of the base sequence does not change ("silent substitution"); Alternatively, in one embodiment, the "substitution" is a substitution in which the substitution of the encoded amino acid residue resulting from the substitution of the base sequence is a "conservative substitution." A conservative substitution is the replacement of a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution is acceptable as long as it does not substantially change the structural characteristics of the original sequence. For example, any substitution is acceptable as long as the substituted amino acid does not disrupt the helix present in the original sequence or other types of secondary structure that characterize the original sequence. Below, examples of conservative substitutions of amino acid residues are listed, categorized by substitutable residue, but the substitutable amino acid residues are not limited to those listed below.

[0028] Group A: leucine, isoleucine, valine, alanine, methionine, glycine, cysteine, proline Group B: aspartic acid, glutamic acid Group C: asparagine, glutamine D group: lysine, arginine Group E: serine, threonine Group F: phenylalanine, tyrosine, tryptophan, histidine In the case of non-conservative substitutions, one member of the above-mentioned group can be exchanged for a member of another group. For example, amino acids in the above-mentioned groups B, D, and E can be substituted with amino acids from other groups to eliminate inadvertent glycosylation. Alternatively, cysteines can be deleted or substituted with other amino acids to prevent folding into a protein in a tertiary structure. Alternatively, amino acids can be substituted taking into account the hydropathic index of amino acids, which is an index of hydrophobicity / hydrophilicity for amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, pp. 105-132, 1982), to maintain a balance between hydrophilicity and hydrophobicity or to increase hydrophilicity for easier synthesis.

[0029] As shown below, the amino acid and nucleotide sequences of CREB-regulated transcriptional coactivator 1 from various mammals are known. SEQ ID NO: 1 is the nucleic acid sequence encoding CREB-regulated transcriptional coactivator 1 from the common marmoset, and SEQ ID NO: 2 is the encoded amino acid sequence.

[0030] The "corresponding sequences" of "a sequence corresponding to bases 194-212 of SEQ ID NO: 1 in a nucleic acid encoding CREB-controlled transcriptional coactivator 1" and "a sequence corresponding to bases 403-421 of SEQ ID NO: 1 in a nucleic acid encoding CREB-controlled transcriptional coactivator 1" can be easily determined by comparing the nucleic acid and amino acid sequences of each mammal. Those skilled in the art can select an appropriate shRNA depending on each sequence. For example, bases 194-212 of SEQ ID NOs: 3, 5, 79, 11, 57, 59, 61, 63, and 65 correspond to bases 194-212 of SEQ ID NO: 1. For example, bases 403-421 of SEQ ID NOs: 3, 5, 7, 57, and 59, and bases 355-373 of SEQ ID NOs: 9 and 11 correspond to bases 403-421 of SEQ ID NO: 1. Within each of the base sequences of SEQ ID NOs: 3-137 (including odd-numbered SEQ ID NOs: 1), there are also sequences that are slightly different from bases 194-212 of SEQ ID NO: 1 and bases 403-421 of SEQ ID NO: 1, for example, with one or two bases substituted, added, deleted, or inserted, but which may correspond to shRNA that interferes with RNA.

[0031] Examples of nucleic acids encoding CREB-regulated transcriptional coactivator 1 from various mammals are shown in Table 1.

[0032] [Table 1-1]

[0033] [Table 1-2]

[0034] [Table 1-3]

[0035] [Table 1-4]

[0036] [Table 1-5]

[0037] [Table 1-6]

[0038] [Table 1-7]

[0039] [Table 1-8]

[0040] [Table 1-9]

[0041] [Table 1-10]

[0042] [Table 1-11]

[0043] [Table 1-12]

[0044] [Table 1-13]

[0045] [Table 1-14]

[0046] Those skilled in the art can appropriately use shRNAs corresponding to the CRTC1 sequence depending on the type of mammal. Non-limiting examples include an "RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcription coactivator 1" and an "RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1" injected into a total of three or more different brain or cranial nerve regions. Preferably, the injection sites are three or more, four or more, or five or more different sites. There is no particular limit to the number of injection sites. In one embodiment, the injection sites are 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer.

[0047] Only "an shRNA that causes RNA interference, having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcription coactivator 1" may be injected, or only "an shRNA that causes RNA interference, having a sequence corresponding to bases 403-421 of SEQ ID NO: 1" may be injected, or both may be injected.

[0048] In addition to "shRNA that causes RNA interference, having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1" and "shRNA that causes RNA interference, having a sequence corresponding to bases 403-421 of SEQ ID NO: 1," other shRNA that causes RNA interference with a nucleic acid encoding CREB-controlled transcriptional coactivator 1 may be injected. For example, in addition to shCRTC1#1 and shCRTC1#2 described in the Examples herein, shCRTC1#3 may be injected.

[0049] The method for injecting shRNA into mammals is not particularly limited, and known methods for injecting shRNA into mammals can be used. The type of "mammal" is not particularly limited. In one embodiment, the mammal is a northern eutherian, including the superorder Euthromynia and the superorder Laurasia. In one embodiment, the mammal is an animal belonging to the order Primates, Rodentia, or Carnivora. In one embodiment, the mammal is an animal belonging to the order Primates. In one embodiment, the mammal is an animal belonging to the infraorder Anthropoidea. In one embodiment, the mammal is an animal belonging to the order Platyrrhine. In one embodiment, the mammal is selected from the group consisting of common marmosets, capuchin monkeys, macaques, rhesus monkeys, tufted capuchin monkeys, southern pigtailed monkeys, green monkeys, cynomolgus monkeys, and greater galagos.

[0050] In one embodiment, the mammal is a rodent. In one embodiment, the mammal is a carnivore. In one embodiment, the mammal is selected from the group consisting of a mouse, a rat, a guinea pig, a dog, a cat, and a ferret.

[0051] 2. Method for producing epilepsy models The present invention also relates to a method for producing the mammalian epilepsy model of the present invention, including, but not limited to, the method comprising: (i) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1, or an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, of a nucleic acid encoding CREB-regulated transcriptional coactivator 1, into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNA interference shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal; This includes:

[0052] The meaning of each term is as described in "1. Epilepsy model." All aspects described in "1. Epilepsy model" are also included in the aspects of the method for producing the epilepsy model.

[0053] In one embodiment, in the above method, the epilepsy is (a) focal onset seizure type and / or (b) structural. In one embodiment, in the above method, the epilepsy is focal onset seizure type and structural.

[0054] In one aspect, in the above method, the RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of the nucleic acid encoding CREB-controlled transcription coactivator 1 has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139.

[0055] In one aspect, in the above method, the RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of the nucleic acid encoding CREB-controlled transcription coactivator 1 has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140.

[0056] In one embodiment, in the above method, the RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of the nucleic acid encoding CREB-controlled transcription coactivator 1 has a base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. and An RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcription coactivator 1 has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140.

[0057] In one embodiment, in the above method, the mammal is an animal belonging to the order Primates, Rodentia, or Carnivora. In one embodiment, in the above method, the mammal is an animal belonging to the order Primates.

[0058] In one embodiment, in the above method, the mammal is an animal belonging to the infraorder Anthropoidea. 3.Use In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1; and / or An RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcriptional coactivator 1. and the use of the above in the method for producing an epilepsy model of the present invention.

[0059] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: An shRNA that causes RNA interference, which has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 139. and / or An shRNA that causes RNA interference, which has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-regulated transcription coactivator 1, and has the base sequence of SEQ ID NO: 140 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO: 140. and the use of the above in the method for producing an epilepsy model of the present invention.

[0060] The meaning of each term is as described in "1. Epilepsy model." All of the aspects described in "1. Epilepsy model" are also included in the above-mentioned aspects of use. [Example]

[0061] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily make modifications and variations to the present invention based on the description in this specification, and such modifications and variations are within the technical scope of the present invention.

[0062] 1. Materials and Methods Unless otherwise stated, the examples described herein followed the following materials and methods. Experimental procedures, including tissue preparation, in situ hybridization (ISH), and immunohistochemistry (IHC), were essentially performed as described or cited in Non-Patent Document 34, unless otherwise noted below. Experiments in the examples herein were conducted in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and were approved by the Animal Experimentation Committee of the National Institutes of Natural Sciences. All other experimental procedures were approved by the RIKEN Laboratory Animal Committee.

[0063] shRNA preparation / injection The shCRTC1 RNAi sequence was prepared as follows. #1: TGCCCAATGTGAACCAGAT (SEQ ID NO: 139) (194-212 of SEQ ID NO: 1); #2: CTGTCAGTGGACAAACATG (SEQ ID NO: 140) (403-421 of SEQ ID NO: 1); #3: GTGGCATCCCCAATATCAT (SEQ ID NO: 141) (1715-1733 of SEQ ID NO: 1); #4: TCGCCAGTCTCCAATCAAG (SEQ ID NO: 142) (1429-1447 of SEQ ID NO: 1); #5: GGAGCAATTCAACATGATG (SEQ ID NO: 143) (1563-1581 of SEQ ID NO: 1; scr: TCGCTAGATCGAGACAGAT (designed based on #1) (SEQ ID NO: 144) BLAST analysis of authentic genes for the marmoset CRTC1 gene [CREB-regulated transcriptional coactivator 1 {Callithrix jacchus (white-tufted-ear marmoset)} Gene ID: 100385682 (updated May 29, 2021; Ensembl: ENSCJAG0000007421)] yielded 100% query coverage and an E-value of 0.006 for shCRTC1#1, #2, #3, #4, and #5. However, the lowest E-value among other marmoset genes was 0.05 or higher. We used 30-well plates for injecting adeno-associated viruses (AAV1) encoding shRNA into the marmoset V1 cortex. o The Nanoject needle was placed shallowly in the cortex, waited 2 minutes, and injected 0.1 μl / 2.5 minutes. The needle was then advanced deeper, waited 2 minutes, and injected 0.1 μl / 2.5 minutes. The needle was then withdrawn after another 2 minutes. After the injection, the marmosets were given the same anesthesia and care as on Day 1.

[0064] Experimental marmosets and procedure In the examples, a total of nine adult common marmosets (Callithrix jacchus, 14-69 months, both sexes) were used (see Table 3 below).

[0065] Marmosets were reared under 12-hour light and 12-hour dark conditions (normal rearing, NR). Three marmosets (monkeys 3, 5, 6, 7, and 8) were not injected with TTX and were reared under normal conditions (NR). After the animals had been reared under light conditions for at least 6 hours, they were perfusion-fixed. For light-induced experiments using monkeys 1 and 4, TTX was injected into one eye, and the marmosets were reared in the dark for 24–43 hours (DR), followed by 24 minutes of light stimulation and immediate perfusion fixation. Monkeys 2 and 9 were reared in the dark for 24–43 hours, followed by 24 minutes of light stimulation and perfusion fixation. As shown in Table 3, monkeys 5 and 6 were injected with shCRTC1#1 at three sites in the right hemisphere V1. After a one-week recovery period, ECoG recordings were performed.

[0066] Intravitreal injection of TTX to block retinal impulse activity Under anesthesia with a mixture of ketamine and xylazine (25 and 2.0 mg / kg, respectively), 1.4–1.6 μl of TTX (4.7 mM; Wako Pure Chemical Industries, Osaka, Japan) was manually injected into the vitreous cavity of one eye of a marmoset using a Hamilton syringe. After TTX injection, the loss of pupillary reflex in the injected eye was confirmed. After waking from anesthesia, marmosets were housed under dark conditions for 24–43 h before stimulation to allow the transcripts already induced by normal rearing to completely decrease to basal levels.

[0067] The shRNA injection sites for monkey 1 (monocular TTX injection, DR and LS (24 min of light stimulation)) are shown in Figures 1a and 3a, while the injection sites for monkey 2 (NR) and monkey 3 (NR 4 weeks, DR 41 h, 24 min of light stimulation) are shown in Figures 2a, 2b, and 12a, respectively. The injection sites for each shRNA were spaced approximately 2-5 mm apart.

[0068] In situ hybridization (ISH) In situ hybridization using the floating method was performed as described in Non-Patent Document 12. Briefly, brain sections sliced ​​to 25 μm were pretreated with 0.1 M phosphate buffer, 0.75% glycine / 0.1 M phosphate buffer, or 0.3% Triton X-100 / 0.1 M phosphate buffer, followed by proteinase K treatment (5 μg / mL, 37°C for 30 minutes), acetylation (room temperature for 10 minutes), prehybridization (60-72°C for 1 hour), and hybridization with each probe (60-72°C overnight).

[0069] The probes used are as shown in the table below.

[0070] [Table 2]

[0071] *1: The species of cDNA clone used as a template for each probe *2: Sequence referenced when designing PCR primers *3: Probes used in previous papers (a: Non-patent document 12, b: Non-patent document 38) The next day, sections were washed with 2x SSC / 50% formamide / 0.1% NLS and treated with RNase (20 μg / mL). Then, sections were washed with 2x SSC / 0.1% NLS and 0.2x SSC / 0.1% NLS, and blocked with 1% blocking solution (room temperature for 60 minutes). For single-color ISH, antibodies were then reacted with Anti-Digoxigenin-AP, Fab fragment (Merck) and developed with NBT / BCIP. For double-color ISH, the DIG probe was detected with an HNPP Fluorescence Detection Set (11758888001, Merck). The FITC probe was detected by antibody reaction using Peroxidase-IgG Fraction Monoclonal Mouse Anti-FITC (#200-032-037, Jackson ImmunoResearch laboratory), signal amplification using the TSA PLUS system (NEL747A001KT, Akoya), and then labeling with Alexa Fluor 488-conjugated anti-DNP antibody (Molecular Probe). The composition of each reaction solution was based on Non-Patent Document 12.

[0072] Immunohistochemistry (IHC) Immunohistochemistry (antibody staining) using the floating method was also performed as described in Non-Patent Document 12. Briefly, brain sections were rinsed with Tris-buffered saline (TBS) and immersed in blocking buffer (5% bovine serum albumin, 0.1% Triton X-100, 4% normal goat serum in TBS) for 1 hour at room temperature for blocking, followed by antibody incubation with primary antibodies for 16–72 hours at 4°C.

[0073] The primary antibodies used were as follows: TORC1 / CRTC1(C71D11)Rabbit mAb (2587, Cell Signaling Technology, Inc.), Anti-phospho-CREB (Ser133) Antibody, clone 10E9 (05-667, Merck), Anti-GFAP antibody(ab7260,abcam), Anti Iba1,Rabbit(019-19741,FUJIFILM Wako), Anti-c-Fos polyclonal rabbit IgG (sc-52, SantaCruz Biotechnology).

[0074] After washing with TNT buffer, the cells were incubated with a secondary antibody (2 hours at room temperature). The secondary antibodies were biotinylated antibodies (Jackson ImmunoResearch Laboratories), fluorescently labeled antibodies [Cy TM 5. Cy TM 3. Cy TM 2 (Jackson ImmunoResearch Laboratories), Alexa Fluor 488 (Molecular Probes)] were used.

[0075] After washing with TNT buffer, sections were attached to gelatin-coated slides and mounted for fluorescent multi-staining. For single staining, sections were labeled with avidin-biotin-labeled enzyme complex using the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature, developed with DAB, and mounted on slides.

[0076] Cell death assay (TUNEL staining) TUNEL staining was performed using the IN Situ Apoptosis Detection Kit (TAKARA) according to the kit's protocol with minor modifications. Briefly, free-floating brain sections were post-fixed in PB buffer / 4% PFA for 2 hours at room temperature or overnight at 4°C. The sections were then treated sequentially with 0.75% glycine / 0.1M phosphate buffer, 0.3% Triton X-100, 5 μg / ml proteinase K (incubated at 37°C for 30 minutes), and 1% H2O2 / PBS (incubated at room temperature for 30 minutes). Subsequent procedures were performed according to the kit's protocol. After staining with DAB, sections were mounted on gelatin-coated slides and, if necessary, counterstained with Nissl stain.

[0077] ECoG recordings from CHRTC1 KD animals Pan-cortical 64-channel and 96-channel ECoG arrays (Cir-Tech Inc., Japan) were epidurally implanted into the right hemisphere of two monkeys (Monkeys 5 and 6). Ten electrodes in Monkey 5 were amputated during implantation. The implantation procedure is described in detail in non-patent literature 24. At the same time (Monkey 5) or 1 week before implantation (Monkey 6), the CRTC1 KD AAV vector was injected into three sites in V1 of the right hemisphere of the monkeys (Figure 4a). Longitudinal ECoG recordings and behavioral monitoring were performed for 2 and 5 months after AAV injection in Monkeys 5 and 6, respectively. During daily 1-2 h of monitoring, the monkeys were seated in a primate chair in a dimly lit room. To examine auditory evoked responses, the tone stimuli used in non-patent literature 25 were presented for the first 20 min of recording. ECoG signals were recorded using a Cerebus system (Blackrock Microsystems, USA) at a sampling rate of 1 kHz per channel.

[0078] (1) Anatomical localization of electrodes The location of each electrode was identified based on postoperative computed tomography and pre-obtained T2-weighted anatomical magnetic resonance images. The localization procedure is detailed in

[24] or the Brain / MINDS data portal. Based on the estimated cortical area, the electrodes were divided into seven groups: primary visual (V1), visual (Vis), auditory (Au), temporal (TC), sensorimotor (SM), parietal (PC), and frontal (FC). Vis was defined as the visual cortex excluding V1, the site of KD. In all monkeys, the electrodes covered the frontal, parietal, occipital, and temporal cortices (Figure 4a).

[0079] (2) Data analysis (i) Pretreatment For quantitative analysis, ECoG signals were clipped to the first 15 minutes of recording on each experimental day. The signals were then re-referenced using a common mean reference (CMR) montage (Figures 13a and 14a). In this process, signals from each channel were re-referenced to a common mean signal across all channels, excluding channels with a standard deviation greater than 250 µV. Functional connectivity was calculated using the signals. To extract high-frequency activity (HFA), a band-pass filter (80-200 Hz) was applied to the signals, and then the envelope was calculated using the Hilbert transform (Figures 13b and 14b).

[0080] (ii) Detection of whole-cortex high-frequency oscillations (cwHFOs) Here, we defined an HFO as one whose HFA was greater than twice the mean + standard deviation of the HFA across all channels. Next, we counted the number of electrodes that showed an HFO at each time point (Figures 14a, 14b, and 14c). If the number of electrodes showed an HFO exceeded 50%, the event was marked as a cwHFO. We then merged the marked events into a single cwHFO with an inter-event interval of less than 1 second. During some putative cwHFOs, Monkey 5 occasionally bit the chair, making it difficult to distinguish this noise from neural activity. Therefore, we used a supervised learning algorithm to discard these events from the estimated cwHFOs.

[0081] We recorded videos of marmosets 7 days after knockdown (22, 35, 42, 51, 55, 57, and 62 days) and used them to manually distinguish chewing events from putative cwHFOs. We identified 39 chewing events from a total of 296 cwHFOs. We then used a linear support vector machine (SVM) method to classify these cwHFOs into chewing events and other events.

[0082] We focused on the time-averaged HFA of 33 electrodes, which was stably observed across all experimental days. SVM was applied to the time-averaged signal for the 0-400 ms time window of cwHFO onset. Note that the size of this time window was optimized using 10-fold cross-validation. We found that 94% of cwHFOs were correctly detected by SVM. Finally, we applied this SVM to cwHFOs on days when no movies were recorded, and events labeled as mastication events were removed from the following analysis. To investigate which cortical region was the origin of the cwHFO, we calculated the average HFA of seven cortical groups in the -250-0 ms time window for each cwHFO (V1, Vis, Au, TC, SM, PC, and FC).

[0083] (iii) functional linkage; Functional connectivity matrices were obtained for each recording using the correlation coefficients of the ECoG signals from each electrode over a 15-minute period. All matrices were then averaged and compared with the daily connectivity matrix to assess deviations from the daily average. To examine the day-to-day stability of functional connectivity, we calculated (1) the correlation of the connectivity matrices across experimental days and (2) the average functional connectivity within and between regions. Finally, functional connectivity was z-scored for each experimental day to visualize changes in functional connectivity within the knockdown site in both monkeys.

[0084] (iv) spatial interpolation of signals We constructed left and right surfaces of a standard marmoset brain using iso2Mesh (35), which spatially interpolates HFA onto the surface of the standard marmoset brain using the BMA 2019 Ex vivo (see NanoZoomer Artificial Intelligence Connectomics Pipeline for Tracer Injection Studies of the Marmoset Brain). Here, the number of vertices per hemisphere is 1672, and the average vertex distance is 1 mm. Gaussian process regression (36) with a 3D isotropic Gaussian kernel (sigma = 4 mm) was used to calculate the interpolated HFA for each vertex time point. Finally, a movie of the interpolated HFA on the surface was obtained to apply the spatial interpolation to HFA at all time points.

[0085] DTI analysis Small animal imaging was performed using a Bruker's system. Diffusion-weighted (DW) imaging was performed using a spin-echo DW echo-planar imaging sequence with a repetition time (TR) of 3000 ms and an echo time (TE) of 24.8 ms. The b-value was 3000 s / mm. 2 The native isotropic image resolution was 0.2 mm. The diffusion sampling protocol included 128 unique diffusion directions. The structural connectivity of 36 individual ex vivo diffusion MRIs was obtained using a global tractography method (doi:10.1016 / j.neuroimage.2010.09.016). A standard brain MRI was used in the Brain / MINDS portal, and its left and right medial surfaces were defined based on 167,082 vertices.

[0086] We randomly sampled 12,428 vertices from the original surface and defined corresponding supervoxels on a standard brain. We obtained a connectivity matrix between supervoxels by counting the number of streamlines passing through two supervoxels. We focused on the asymmetry of the convex patterns in the left and right cortices. We set two symmetrical points on the left and right cortices and calculated the correlation between the connectivity profiles of the two points in each cortex. A low correlation indicates an asymmetric connection pattern at this point (red area in Figure 7a). To assess asymmetry in the epileptic marmoset, we calculated the z-score of the asymmetric connectivity pattern of the normal marmoset averaged across brain regions, which we refer to as the asymmetry feature (AF) in Figure 7b.

[0087] 2. Experimental Results (1) IEG expression throughout the cerebral hemisphere was achieved by injecting shCRTC1 into three or more sites per side.

[0088] In our previous study, we found that ser133-phosphorylated CREB increased in the nucleus in the 4Cβ layer of marmoset V1 under dark conditions and transiently decreased upon light exposure. We also found that nuclear translocation of CRTC1 protein transiently increased upon light exposure, leading us to investigate the relationship between CRTC1 nuclear transport and CREB phosphorylation.

[0089] We designed a CRTC1 knockdown sequence and analyzed the induction of IEG expression in marmoset V1 using known ISH and IHC methods (Non-Patent Document 12). Among the shCRTC1s we constructed (Table 1), shCRTC1#1 demonstrated the highest knockdown efficacy by Western blot and in situ hybridization (ISH) (Figure 9). We injected shCRTC1#1 into three sites and a scrambled sequence (TCGCTAGATCGAGACAGAT (designed based on shCRTC1#1) (SEQ ID NO: 144) into two sites in the left hemisphere of marmosets. We also injected only a single scrambled sequence into the right hemisphere of V1. Four weeks after shRNA injection, one eye was injected with TTX and kept in the dark for 48 hours. After 24 minutes of light exposure, the eye was immediately perfusion-fixed (Figures 1a and 1b).

[0090] We first investigated cFOS expression by ISH. In the right hemisphere of V1, where shscr was injected only once, cFOS mRNA expression was detected in an ODC-like pattern (Non-Patent Document 12) following light stimulation from the normal eye, as shown in Figure 1d (right). Surprisingly, however, the entire left V1, including the CRTC1 shRNA injection site, showed an abnormally strong IEG expression pattern that was completely different from the normal pattern (Figure 1c, bottom left, left 1d).

[0091] Next, we examined several other IEGs other than cFOS (Fig. 1e). In left V1, where shCRTC1#1 was injected at three sites, the overall expression pattern of ARC and ZIF268 mRNA was strongest in layer 2, and the ODC in layer 4 was less distinct than in right V1. This was similar to that of cFOS. However, in right V1, where shCRTC1#1 was not injected, the expression of cFOS was similar to that observed in healthy marmosets following visual stimulation. Like the other three IEGs, NUR77 showed abnormal expression throughout the hemisphere (Fig. 1e). However, at the shCRTC1#1 injection site, expression was significantly reduced in all areas except layer 2 (Fig. 10).

[0092] These results indicated that shCRTC1#1 KD affected the expression of specific IEGs downstream of CREB. On the other hand, NURR1 expression was slightly more pronounced than normal in layer 2 of the left V1, but little abnormal expression was observed in other layers (Fig. 1e). Normally, in animals perfusion-fixed 24 min after light stimulation, light stimulation-induced cFOS protein expression is not yet observed. In contrast, immunohistochemistry (IHC) showed that cFOS protein expression was already strong in layer 2 of the left V1 after shCRTC1#1 injection. In the hemisphere not injected with shCRTC1#1 (right V1, Fig. 1e), cFOS protein expression was very low, similar to that in the normal V1. In contrast to the hemisphere not injected with shCRTC1#1, abnormal IEG expression in the left V1 was induced regardless of retinal light stimulation.

[0093] (2) When shCRTC1 was injected into only one or two sites per hemisphere, it induced localized IEG expression. The AAV infections performed in the present study spread approximately 1 mm around the injection site. The marmoset brain is approximately 3 cm and 2 cm long in the AP and ML axes, respectively (Non-Patent Document 19), and the infection site is only a small portion of the brain (see Figure 1a). To understand why local injections cause such widespread cortical activation, we further performed local knockdown by injecting ShCRTC1#1 into only one or two sites in one hemisphere of monkeys 2 and 3. Monkey 2 was perfusion-fixed after 4 weeks of normal feeding and IEG expression was analyzed. With this limited number of local injections, strong expression of IEGs, including cFOS, was observed only around the shCRTC1 injection site. No abnormal IEG expression was observed around the shscr injection site (Figures 2a and 11). cFOS, ARC, and ZIF268 (EGR1 or NGFIA) were strongly expressed in neurons in all layers of the brain at each injection site, and also showed strong abnormal expression around infected cells in layers 2 and 3 (Fig. 2a, Fig. 11). This suggests that the strong excitatory activation of shCRTC1-injected neurons propagates to nearby neurons. NURR1 did not exhibit any abnormal gene expression patterns, except in layer 6, where it showed decreased expression (Fig. 11). When shCRTC1 was injected into one or two sites per hemisphere, IEG expression was observed only near the injection site. Furthermore, monkey 3 was dark-housed for 41 hours, followed by a 24-minute light exposure and immediate perfusion fixation 4 weeks after AAV injection. The cFOS mRNA expression pattern was similar to that of control animals without shCRTC1, except at the injection site under the same light exposure. Expression of cFOS(ISH) near the injection site was much higher than in other areas (Fig. 2c), and induction of cFOS protein was also detected by IHC (Fig. 2b), indicating that this abnormal expression was induced under these experimental conditions. Similar to monkey 1, these results demonstrated that CRTC1 knockdown with shCRTC1#1 induced strong excitatory responses that propagated to surrounding neurons, particularly in layers 2 and 3, regardless of rearing, TTX monocular suppression, or light exposure. Taken together with the results in Fig. 1, it is likely that injection of shCRTC1#1 into a wider area (three or more adjacent sites) in ipsilateral V1 induced even stronger neural activity that propagated throughout the ipsilateral V1.

[0094] (3) Tissue damage caused by CRTC1 KD in monkey 1 Tissue damage (neurodegeneration) in the V1 of monkey 1 was examined histologically using Nissl staining (Figure 3a) and immunohistochemistry for the neuronal marker NeuN, the microglial marker IBA1, and the activated astrocyte marker GFAP (Figure 3b). NeuN signals were reduced in AAV-infected cells at the shCRTC1#1 injection site (Figure 3c). GFAP signals were observed at and near the injection site, suggesting astrocytic activation in these areas (Figure 3b, right panel). Furthermore, IBA1 signals, indicating the accumulation of microglial cells, were observed (Figure 3b, left panel). On the other hand, monkeys 2 and 3, which received shCRTC1#1 injections at one or two sites on one side of the V1, showed less neuronal loss or tissue damage. The immunohistochemistry for IBA1 and GFAP signals were less intense than those in monkey 1 (Figure 1b). TUNEL staining confirmed that the neuronal loss in monkey 1 was due to cell death caused by shCRTC1#1 injection. TUNEL signals were observed in the central region of the injection site (Fig. 3d). In contrast, the decrease in NeuN signals was barely observed after shscr injection. Furthermore, TUNEL signals were absent at the shscr injection site in monkey 2 (Fig. 12) and monkey 3 (data not shown).

[0095] (4) Tissue damage correlates with knockdown efficiency Four other shCRTC1 sequences were constructed and injected into marmosets V1 (monkeys 4, 7-9; Table 3). Antibody staining confirmed a significant reduction in CRTC1 signaling in these marmosets. NeuN expression was reduced by shCRTC1 (shCRTC1#1 and #2) but not by shscr. Furthermore, shCRTC1 (shCRTC1#3 and #5) had little effect on reducing NeuN expression. IBA1 and GFAP signals tended to increase with increasing KD efficacy. TUNEL staining signals were only detected around the injection sites of shCRTC1#1 and #2. Injection of the five shCRTC1 sequences, particularly shCRTC1#1 and #2, consistently induced localized or hemisphere-wide IEG expression after brain fixation in all monkeys except monkey 4 (Table 3). In monkey 4, no abnormal IEG expression was observed despite injection of each of the five shCRTC1 sequences into each hemisphere. On the other hand, decreased IEG expression was observed at the injection site (Table 3). This may be because this monkey (Monkey 4) had received a monocular injection of TTX before dark rearing (DR), and neuronal propagation during light exposure was suppressed in the ocular dominance column corresponding to the TTX-injected eye, making it less likely that abnormal IEG expression would occur.

[0096] [Table 3]

[0097] Table 3: List of monkeys injected with five types of shCRTC1. Each row in Table 3 indicates a marmoset (sometimes referred to as "monkey" in this specification) used in the examples. Each column describes the characteristics of the experiment and the animal. From left to right, (i) monkey ID; (ii) gender; (iii) age in months; (iv) experimental duration: number of days from injection to perfusion fixation; (v) Hemisphere (R: right hemisphere, L: left hemisphere); (vi) IEG expression pattern. "Normal" means a pattern similar to that in the uninjected hemisphere. "Diffusible" means abnormal expression of IEGs throughout the injected hemisphere, as in Figure 1d. "Partially diffusible" means expression beyond the injection area but not throughout V1. "Local" means IEGs expressed only around the injection site, as in Figure 2; (vii) Number of shCRTC1: The number of injection sites of shCRTC1 types (shscr, shCRTC1#1–#5) is shown under the title of each column in the two hemispheres; (viii) Notes: Experimental condition for each monkey.

[0098] The transcription factor CREB is a key regulator of activity-dependent transcriptional regulation and is also involved in memory (Non-Patent Documents 20, 21). Nuclear transport via dephosphorylation of CRTC1 and Ser133 phosphorylation of CREB in the nucleus occur as parallel, independent processes (Non-Patent Documents 15, 18, 21). We examined CREB phosphorylation at Ser133 by immunohistochemistry (IHC) in shCRTC1-injected monkeys. Regardless of the injection site, phosphorylated CREB signals were detected only at the injection sites of shCRTC1#1 and #2 (Fig. 3e). When shCRTC1#1 was injected into only one or two sites on one side of V1, little tissue damage was observed (Fig. 12). In contrast, localized abnormal expression of IEGs and tissue damage were observed around two or more injection sites on one side of V1 injected with shCRTC1#2 (Table 3).

[0099] (5) Abnormal high-frequency activity in shCRTC1 KD marmosets The abnormal IEG expression in layer 2 of monkey 1 closely resembled the IEG pattern reported in human epileptic tissue (Non-Patent Document 22). The above-mentioned histological observations, such as the decrease in NeuN-positive cells, the strong GFAP-positive signal suggestive of gliosis, and neuronal loss, also closely matched those in human epilepsy (Non-Patent Document 23). Therefore, we considered that cortical-wide epilepsy may have occurred in marmosets injected with shCRTC1 into V1.

[0100] To demonstrate this possibility, we performed longitudinal recordings of neural activity in the cortex of two shCRTC1 KD marmosets (Monkeys 5 and 6). shCRTC1#1 was injected into three sites in one side of V1, and ECoG electrodes (NPL 24) covering the lateral hemisphere on the same day and one week after injection were implanted in Monkeys 5 and 6, respectively (Figure 4a). After recovery, Monkeys 5 and 6 underwent ECoG recordings and behavioral monitoring for 1–2 hours per day for 2 and 5 months, respectively. During recording, the monkeys sat in a primate chair without performing any tasks, and for the first 20 minutes of recording, they listened to repeated pure tones used in previous studies (NPL 25). Auditory responses were confirmed to be normal in the shCRTC1#1 KD marmosets. In addition to electrophysiological monitoring, preoperative in vivo MRI (T2) and postoperative ex vivo MRI (T2 and DTI images) were obtained to examine structural changes (Figure 4a). Cortical synchronized activity was observed for 2 months in Monkey 5 and for 5 months in Monkey 6 (Figures 4, 14-17). To quantitatively analyze synchronized activity, we focused on the high-frequency (80-200 Hz) ECoG signals obtained from the first 15 minutes of daily recordings, because high-frequency oscillations (HFOs) are considered biomarkers of epileptic foci (26, 27, 28). High-frequency activity (HFA) (80-200 Hz; >3 std) was examined at all electrodes, and we confirmed that defined HFAs, known as cortical-wide HFOs (cwHFOs), extended to more than 50% of the electrodes (Figures 4b, 14, and 15). In both shCRTC1 KD marmosets, HFAs in V1, including the injection site, were repeatedly observed and decreased 5 weeks after injection (Figures 4c, 4d). To determine the site of cwHFO onset, we calculated the average HFA 250 ms immediately preceding the cwHFO. We observed cwHFOs following cwHFAs in V1 during the early stages of ShCRTC1 injection, but 4 weeks after injection, these cwHFOs decreased, and HFAs in the temporal cortex tended to precede cwHFOs (Fig. 4c, 4d, 16). Both marmosets showed a tendency to look around and conspicuously increase their attention when strong HFA appeared in the occipital cortex (Fig. 4e), but this tendency was maintained under high HFA in the temporal cortex (Fig. 4f).Additionally, cheek hair twitching was observed approximately one month after injection.

[0101] (6) Functional network reorganization during epilepsy To understand the significance of changes in HFA patterns, we examined the daily functional network by calculating the correlation between each electrode on each recording day and obtained the average correlation across all days (Fig. 5a). Functional connectivity (correlation values) for early recordings from the time of injection tended to dissociate from the averaged functional connectivity, whereas functional connectivity for later recordings was close to the average (Fig. 5b). Comparison of functional connectivity between days revealed that connectivity was not stable immediately after shCRTC1 injection and became stable approximately 5 weeks after injection in both marmosets (Fig. 5c). To examine this variability more precisely, we compared the average and daily values ​​of functional connectivity between cortical areas. We found that some functional connectivity within 5 weeks after injection tended to significantly increase or decrease from the average (Fig. 5d). Among the changes in functional connectivity, values ​​for putative V1-V1 (intra-V1) connectivity significantly decreased in both monkeys (Fig. 5e). These results suggest that local functional connectivity in visual areas decreased immediately after shCRTC1 injection and recovered to its stable level 5 weeks after injection.

[0102] (7) Progressive cell death at the shCRTC1 injection site accompanied the development of epilepsy Histological analysis was performed after ECoG recording. We fixed the brains of monkeys 1, 2, 3, and 4 one month after injection, but monkey 5 was fixed two months after injection. Sirius-positive neurons were widely lost (Fig. 6a), and TUNEL-positive neurons significantly increased (Fig. 6a). Strong IBA1 signals in the neuronal loss area suggest that dead neurons were phagocytosed (Fig. 6a). Immunostaining with GFAP antibodies indicated that activated astrocytes forming a reticular structure were in a state of gliosis (Fig. 6a). In monkey 6, fixed 5 months later, most shCRTC1-positive neurons were lost, leaving only cells roughly around the injection site border. Furthermore, GFPA signals disappeared in the injection center, forming a glial scar (nonspecifically, Ref. 29) (Fig. 6b). Although HFOs were observed by ECoG on the same day as fixation in monkeys 5 and 6, ISH analysis did not reveal widespread cFOS expression in either hemisphere. Instead, monkey 5 showed slight cFOS expression around the injection site and irregular BDNF mRNA expression in layer 2 (30). No clear differences in the cFOS expression pattern outside the injection site were observed between these two monkeys and normally housed control marmosets.

[0103] Furthermore, we observed signal loss in a part of the right temporal cortex in postmortem MRI T2 images of monkey 5, but not in in vivo MRI T2 images (Fig. 6c). Nissl staining also showed columnar cell death in the same part of the right temporal cortex of monkey 5 (Fig. 6d). We examined the expression of IBA1, GFAP, and TUNEL in the corresponding area and found that although there were faint IBA1 and GFAP signals, no TUNEL-positive neurons were observed (Fig. 6e).

[0104] (8) Ex vivo DTI revealed cortical asymmetry in shCRTC1-injected monkeys. To examine structural changes in the global cortical network, we analyzed ex vivo DTI data from monkeys 5 and 6. We calculated global cortical DTI connectivity in both monkeys 5 and 6 and compared it with that of 31 healthy subjects. We found increased lateral asymmetry in connectivity in both ex vivo DTI data from monkeys 5 and 6, but most connectivity tended to be symmetric in normal marmosets and in vivo DTI data from monkey 6 obtained before shCRTC1 injection (Fig. 7a). We then calculated z-scores of asymmetry features in 39 cortical regions of interest (ROIs). The ROIs that showed significant asymmetry (z-score > 1.96) in both monkeys were V1, V3, and S1 (Fig. 7b).

[0105] Furthermore, DTI analysis of the white matter bundles between V1 and PFC (prefrontal cortex) and between V1 and TE was performed to quantitatively measure the fiber bundles of the white matter and compare them between the injected and contralateral sides. The results showed that the difference in V1-TE between the left and right sides was not significant in the two control animals, but was significant in the two shCRTC131-injected animals (Figure 8). These results indicated that the animals injected with shCRTC1#1 at three sites had a decrease in V1-TE white matter fiber bundles.

[0106] 3. Summary of Examples In this study, we found that abnormal neuronal activity induced by shCRTC1 spread to surrounding neurons in two ways: through electrical activity throughout the cortex, and through cell death in the surrounding injection site and the temporal cortex (Figures 6a-d). The period of repeated HFOs coincided well with the time period of fixation approximately 1 month after injection used for histological analysis. Neuronal loss, astrocytic activation, and microglial accumulation, indicative of tissue damage, occurred in monkeys injected with efficient shCRTC1 at three or more sites (Table 3). On the other hand, although IEG expression was observed around the injection site (Monkeys 2 and 3), tissue damage was minimal when only one or two sites were injected. Monkey 6, fixed 5 months after shCRTC1 injection and exposed to HFOs for 3 months longer than monkey 5, showed neuronal death and associated tissue damage localized within the primary shCRTC1-expressing cells and the secondary damaged temporal cortex.

[0107] Injection of a certain shCRTC1 into three or more sites induced cell death, resulting in a decrease in NeuN and positive TUNEL signals at the injection sites (Fig. 6b-c), as well as abnormal neuronal activity that spread throughout the cortex (Figs. 4 and 5). We found that the five shCRTC1 varieties differed in their extent and effects on IEG induction and cell death (Table 3).

[0108] (i) IEG expression throughout the entire V1 region was induced by injection of shCRTC1#1 at three or more adjacent sites, and significant TUNEL1, IBA1, and GFPA expression was observed. In contrast, injection of shCRTC1#1 at one or two sites induced relatively strong IEG expression, but weak IBA1 and GFPA expression, and little or no TUNEL expression.

[0109] (ii) We investigated five different shCRTC1 injections into bilateral V1 (Monkey 4). NeuN expression at each injection site was reduced depending on the efficacy of the shCRTC1s (strong efficacy with shCRTC1#1 and #2). Three or more injection sites of shCRTC1#1 in unilateral V1 resulted in abnormal IEG expression and electrical activity measured by ECoG in Monkey 1, Monkey 5 (plus #2 in both hemispheres), and Monkey 6.

[0110] (iii) shCRTC1#2 showed a slightly weaker knockdown effect than shCRTC1#1 as assessed by Western blot analysis, but showed strong neuronal damage and associated signals of GFAP, IBA1, and TUNEL at the V1 injection site. shCRTC1#2 also induced IEG expression when injected into three or more sites, but this did not extend to the entire V1 due to preceding tissue damage.

[0111] Injections of one or two shCRTC1#2, #3, or #5 did not result in abnormal IEG expression (Monkey 8). In Monkey 7, injections of shCRTC1#1 into one site each in bilateral V1 and co-injections of CRTC1#2 and #3 into three or more sites each in unilateral V1 induced diffuse IEG expression. Here, the combination of shCRTC1#1 with #2 and #3 appears to be important for diffuse (abnormal) IEG expression, with all three or more sites compared to the total of 10 shCRTC1 injection sites in Monkey 4, which did not show diffuse IEG expression (Table 3).

[0112] These results suggest that cell death and abnormal neural activity are synergistically linked, and that both the reduction of CRTC1 protein and excitatory neural activity are required to trigger cell death, at least initially. While the precise mechanism of shCRTC1-induced neuronal death is currently difficult to determine, given the existence of over a dozen different forms of neuronal death (Non-Patent Document 31), the occurrence of abnormal electrical neural activity was strongly supported by in vivo ECoG analysis or diffuse IEG expression in monkeys 1, 5, and 6. Once abnormal neural activity is induced, it may trigger glutamate-induced cell death, further promoting neuronal death (non-specifically, Reference 31) and promoting the spread of abnormal neural activity beyond the injection site.

[0113] We observed GFAP- and IBA1-positive signals around the injection site. GFAP-positive signals suggest the involvement of activated astrocytes (Non-Patent Document 29). IBA1-positive signals suggest the involvement of microglia (Non-Patent Document 32). Activated astrocytes mediate pro-inflammatory responses, and microglia play an important role in phagocyte phagocytosis. In the early stages of shCRTC1#1 injection, inflammation is promoted, astrocyte accumulation, and phagocytosis progress. In the latter stages, activated astrocytes act as anti-inflammatories through severe astrogliosis accompanied by compact glial scar formation, preventing further tissue damage.

[0114] Wide-area ECoG recordings suggest that HFOs were elicited at three injection sites in the V1 of shCRTC1#1 mice, spreading to surrounding areas and ultimately throughout the cortex. cwHFOs were also elicited from electrodes in the temporal cortex (Figure 6d), not only around the injection site but also from electrodes in the temporal cortex (Figure 6d). MRI and Nissl staining confirmed histological lesions in the temporal cortex in monkey 5 (Figure 6d). In rodent seizure models, caspase-8 has been implicated in the extrinsic / death pathway via apoptosis (Non-Patent Documents 33, 34, 35, 36). Therefore, tissue loss may be caused by abnormal neural activity spreading throughout the temporal cortex. Our functional connectivity analysis showed that whole-brain networks transitioned to instability and then stabilized (steady state) 5 weeks after injection. Following the recovery of stable cortical neuronal activity, the neuronal death and glial scar barrier blocked further generation of excessive activity in the temporal cortex, as well as in the surrounding area of ​​V1 where shCRTC1#1 was injected. A 5-month observation of Monkey 6 revealed that cwHFOs rarely occurred after 3 months (Figures 4f and 17), but scattered localized HFOs and low-frequency oscillations were observed over a wide cortical area (Figure 17, left).

[0115] Analysis of the correlation between left and right profiles of ex vivo DTI connectivity showed lower levels in CRTC1-injected marmosets 5 and 6 compared with normal marmosets. Three ROIs, V1, V3, and S1, showed significantly higher z-scores (>1.96) in both Mokeny 5 and 6 compared with normal marmosets (Figure 7). Structural changes after CRTC1 injection were not only observed in the temporal lobe at 5 months (Figure 6d), but also in both shCRTC1-injected marmosets. Laterality of white matter tracts (Figure 7a) was detected by ex vivo DTI. [Industrial Applicability]

[0116] The present invention provides an epilepsy model that enables elucidation of the onset, development, and progression of epilepsy from both the developmental and seizure perspectives. The epilepsy model is also useful for testing new pharmacological treatments for epileptogenesis, inflammation, and subsequent brain function recovery after brain injury (Non-Patent Document 4).

Claims

1. 1. A mammalian epilepsy model comprising: (i) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1, or an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, of a nucleic acid encoding CREB-regulated transcriptional coactivator 1 (CRTC1) into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding a CREB-controlled transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding a CREB-controlled transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal; The epilepsy model is obtained by a method comprising:

2. 2. The epilepsy model of claim 1, wherein the epilepsy is (a) focal onset seizure type and / or (b) structural.

3. 2. The epilepsy model of claim 1, wherein the epilepsy is of focal onset seizure type and structural.

4. The epilepsy model according to any one of claims 1 to 3, wherein the RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO:

139.

5. The epilepsy model according to any one of claims 1 to 3, wherein the RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of the nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO:

140.

6. The shRNA that causes RNA interference has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

139. and The shRNA that causes RNA interference has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 140 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

140. The epilepsy model according to any one of claims 1 to 3.

7. The epilepsy model according to any one of claims 1 to 3, wherein the mammal is an animal belonging to the order Primates, Rodents or Carnivora.

8. The epilepsy model according to any one of claims 1 to 3, wherein the mammal is an animal belonging to the order Primates.

9. The epilepsy model according to any one of claims 1 to 3, wherein the mammal is an animal belonging to the infraorder Anthropoidea.

10. A method for producing an epilepsy model in a mammal, comprising: (i) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1, or an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1, of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, into three or more different brain or cranial nerve regions of a mammal; or (ii) injecting an RNAi-interfering shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding a CREB-controlled transcriptional coactivator 1 into two or more different brain or cranial nerve regions of a mammalian animal, and injecting an RNAi-interfering shRNA having a sequence corresponding to bases 403-421 of SEQ ID NO: 1 of a nucleic acid encoding a CREB-controlled transcriptional coactivator 1 into one or more different brain or cranial nerve regions of a mammalian animal; The method comprising:

11. 11. The method of claim 10, wherein the epilepsy is (a) focal onset seizure type and / or (b) structural.

12. 11. The method of claim 10, wherein the epilepsy is focal onset seizure type and structural.

13. The method of any one of claims 10-12, wherein the RNA interference shRNA has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139, or a base sequence in which one or two bases are substituted, added, deleted, or inserted relative to SEQ ID NO:

139.

14. The method of any one of claims 10 to 12, wherein the RNA interference shRNA has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 140, or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

140.

15. The shRNA that causes RNA interference has a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

139. and The shRNA that causes RNA interference has a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and has the base sequence of SEQ ID NO: 140 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

140. The method according to any one of claims 10 to 12.

16. The method according to any one of claims 10 to 12, wherein the mammal is an animal belonging to the order Primates, Rodentia or Carnivora.

17. The method according to any one of claims 10 to 12, wherein the mammal is an animal belonging to the order Primates.

18. The method according to any one of claims 10 to 12, wherein the mammal is an animal belonging to the infraorder Anthropoidea.

19. an RNA interference shRNA having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1; and / or An RNA interference shRNA having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1. Use of the method for producing an epilepsy model according to any one of claims 10 to 12.

20. An shRNA that causes RNA interference, having a sequence corresponding to bases 194-212 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and having the base sequence of SEQ ID NO: 139 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

139. and / or An shRNA that causes RNA interference, having a sequence corresponding to 403-421 of SEQ ID NO: 1 of a nucleic acid encoding CREB-controlled transcriptional coactivator 1, and having the base sequence of SEQ ID NO: 140 or a base sequence in which one or two bases are substituted, added, deleted or inserted relative to SEQ ID NO:

140. Use of the method for producing an epilepsy model according to any one of claims 10 to 12.

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