Composition for delaying or treating huntington's disease through NNAT expression activity

Abstract

The present invention relates to a composition comprising an NNAT expression promoter or activity promoter as an active ingredient for preventing, alleviating, or treating Huntington's disease. The present disclosure also relates to various substances and a screening method therefor that can provide preventive or therapeutic effects on Huntington's disease by promoting the expression or activity of NNAT, which is reduced in expression and activity in patients with Huntington's disease.

Description

Composition for delaying or treating Huntington's disease through NNAT expression activation

[0001] The present invention relates to a pharmaceutical composition that has a delaying or therapeutic effect on Huntington's disease by activating NNAT (neuronatin) gene expression.

[0002] Huntington's disease (HD) is a genetic disorder caused by an excessive increase in CAG repeats in the huntingtin (HTT) gene on chromosome 4. Common symptoms of HD, which usually develop in adulthood, include involuntary movements, abnormal gait, slurred speech, dysphagia, cognitive impairment, and personality disorders. Huntington's disease is one of the representative neurodegenerative brain diseases and is accompanied by movement disorders and cognitive impairment. As the disease progresses, abnormal movement disorders become more severe, making it difficult for patients to perform daily activities. Most patients with HD survive 15 to 20 years after the onset of symptoms.

[0003] Huntington's disease is known to be caused by a mutation in the huntingtin protein, which increases the number of glutamine repeats in the polyglutamine domain by more than 40, leading to the loss of neurons in the striatum, a brain region. While there is currently no treatment that can alleviate the symptoms of Huntington's disease, medication can alleviate chorea, slowed movement, and personality traits.

[0004] The growing elderly population is leading to a higher prevalence of age-related degenerative diseases. Various treatments are currently being developed for Huntington's disease, and gene therapies, including gene editing, are being studied for effective treatment. There is a pressing need to identify and correct genes that serve as treatment targets for Huntington's disease, thereby developing highly effective treatment methods.

[0005] Accordingly, the present inventors confirmed that NNAT (neuronatin) gene expression is suppressed in Huntington's disease patients, and discovered that this can suppress the expression of nerve cells, particularly the expression of regenerative GABAergic neurons, thereby completing the present invention for preventing and treating Huntington's disease by delaying the onset of the disease through promotion of NNAT gene expression.

[0006] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Huntington's disease, which comprises an NNAT (neuronatin) gene expression promoting agent or an activity promoting agent.

[0007] Another object of the present invention is to provide a method for screening a candidate substance for the prevention or treatment of Huntington's disease, comprising the steps of: treating a test substance to be analyzed to a cell containing the NNAT (neuronatin) gene; measuring the activity or expression level of NNAT in the cell; and selecting the test substance as a candidate substance for the prevention or treatment of Huntington's disease if the activity or expression of NNAT is increased.

[0008]

[0009] Another object of the present invention is to provide a diagnostic composition for Huntington's disease, comprising a preparation for measuring the mRNA or protein expression level of the NNAT (neuronatin) gene.

[0010] Another object of the present invention is to provide a method for providing information for diagnosing Huntington's disease, comprising the steps of measuring the mRNA or protein expression level of the NNAT (neuronatin) gene from a biological sample of a patient; and comparing the mRNA or protein expression level with that of a control sample.

[0011]

[0012] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating Huntington's disease, which comprises an agent for promoting the expression or activity of the NNAT (neuronatin) gene.

[0013] In order to achieve another object of the present invention, the present invention provides a method for screening a candidate substance for the prevention or treatment of Huntington's disease, comprising the steps of: treating a test substance to be analyzed to a cell containing an NNAT (neuronatin) gene; measuring the activity or expression level of NNAT in the cell; and selecting the test substance as a candidate substance for the prevention or treatment of Huntington's disease when the activity or expression of NNAT is increased.

[0014] In order to achieve another object of the present invention, the present invention provides a diagnostic composition for Huntington's disease, comprising a preparation for measuring the mRNA or protein expression level of the NNAT (neuronatin) gene.

[0015] In order to achieve another object of the present invention, the present invention provides a method for providing information for diagnosing Huntington's disease, comprising the steps of measuring the mRNA or protein expression level of the NNAT (neuronatin) gene from a biological sample of a patient; and comparing the mRNA or protein expression level with that of a control sample.

[0016] Hereinafter, the present invention will be described in detail.

[0017] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating Huntington's disease, comprising an NNAT (neuronatin) gene expression promoting agent or an activator.

[0018] In the present invention, the NNAT (neuronatin) is an RNA-binding protein that regulates the expression of proteolipid proteins that may be involved in the regulation of ion channels during brain development. This protein may contribute to the formation and maintenance of the nervous system's structure and is specifically expressed primarily in neural tissues, including the brain. This gene exists within an intron of the bladder cancer associated protein gene, but is located on the opposite strand of the gene. Furthermore, this gene is known to be an imprinted gene, expressed only from the paternal allele.

[0019] In the present invention, the NNAT expression or activity promoter refers to a substance that acts directly or indirectly on NNAT to improve, induce, stimulate, or increase the expression or activity of NNAT. Such substances include single compounds such as organic or inorganic compounds, biopolymers such as peptides, proteins, nucleic acids, carbohydrates, and lipids, and complexes of multiple compounds. The mechanism by which the substance promotes the expression or activity of NNAT is not particularly limited. For example, the substance may act as a mechanism to increase gene expression such as transcription or translation, or to convert an inactive form to an active form. Preferably, the substance that promotes the expression or activity of NNAT is a biopolymer such as peptides, proteins, nucleic acids, carbohydrates, and lipids. For NNAT, whose nucleic acid and protein sequences are already known, those skilled in the art can prepare or screen single compounds such as organic or inorganic compounds that act as promoters, biopolymers such as peptides, proteins, nucleic acids, carbohydrates, and lipids, and complexes of multiple compounds, using techniques in the art.

[0020] The NNAT expression or activity promoter of the present invention may be provided in the form of a vector capable of expressing NNAT in cells for use in gene therapy, etc. Accordingly, the present invention relates to a pharmaceutical composition for the prevention or treatment of Huntington's disease, comprising as an active ingredient a nucleic acid encoding NNAT, preferably a recombinant vector containing the nucleic acid.

[0021] In the present invention, the sequence encoding the NNAT may include a sequence having substantial sequence identity or substantial sequence homology to the above sequences. Here, the terms "substantial sequence identity" or "substantial sequence homology" indicate substantial structural or functional identity of the sequence with another sequence, and may be mutated by substitution, deletion, insertion, or a combination thereof at one or more nucleic acid bases, as long as the sequence encodes a protein with equivalent activity. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0022] The vector of the present invention includes, but is not limited to, liposomes, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. In the present invention, examples of preferred viral vectors include adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, alpha virus, and the like. The recombinant vector of the present invention may include a nucleic acid encoding NNAT and a regulatory sequence for its transcription or translation. Particularly important regulatory sequences are those that regulate transcription initiation, such as a promoter or enhancer. In addition, the vector may include a regulatory sequence consisting of a start codon, a stop codon, a polyadenylation signal, a Kozak, an enhancer, a signal sequence for membrane targeting and secretion, an IRES (Internal Ribosome Entry Site), and the like. These regulatory sequences and the nucleic acid encoding NNAT should be operably linked.

[0023] The term "operably linked" above means that the binding between nucleic acid sequences is functionally related. Any nucleic acid sequence is operably linked when any nucleic acid sequence is positioned so as to have a functional relationship with another nucleic acid sequence. In the present invention, if any transcriptional regulatory sequence affects the transcription of a nucleic acid molecule encoding NNAT, the transcriptional regulatory sequence is said to be operably linked to the nucleic acid molecule. In the present invention, treatment includes inhibiting or preventing Huntington's disease, or reducing, alleviating, or reversing symptoms associated with Huntington's disease, and inhibiting the progression of Huntington's disease.

[0024] The expression promoter of the above NNAT may be any one selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the gene encoding NNAT, and the activity promoter of the NNAT protein may be, but is not limited to, a compound, peptide, peptide mimetic, aptamer, antibody, and natural product that specifically bind to the NNAT protein.

[0025] The term “prevention” in this specification means any action that can suppress symptoms of Huntington’s disease or delay the onset of the disease by administering the pharmaceutical composition according to the present invention.

[0026] The term “treatment” as used herein refers to any action that improves or benefits symptoms by administering the pharmaceutical composition according to the present invention.

[0027] The pharmaceutical composition of the present invention can be formulated and used in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and in parenteral dosage forms such as external preparations, suppositories, and sterile injection solutions, according to conventional methods, and may additionally include carriers or excipients necessary for the formulation.

[0028] Pharmaceutically acceptable carriers, excipients and diluents that may be additionally included in the above active ingredients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil. When formulated, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants.

[0029] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0030] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0031] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. However, the present invention is not limited thereto, and pharmaceutical compositions according to one aspect may be formulated according to the intended purpose using any method known in the art without limitation.

[0032] The effective dosage of the pharmaceutical composition may vary depending on the patient's age, sex, and weight, but may be administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0033] In another aspect, the present invention relates to a method for screening a candidate substance for preventing or treating Huntington's disease, the method comprising the following steps.

[0034] 1) A step of treating a test substance to be analyzed into a cell containing the NNAT (neuronatin) gene;

[0035] 2) a step of measuring the activity of NNAT protein or the expression level of the gene in the cell of 1); and

[0036] 3) A step of selecting the test substance as a candidate substance for preventing or treating Huntington's disease, when the activity of the NNAT protein or the expression of the gene is increased in the above 2).

[0037] The above test substance may be any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, miRNA, ribozymes, DNAzymes, PNAs, antibodies, aptamers, natural extracts, and synthetic compounds.

[0038] In addition, the method for measuring the activity of the NNAT protein or the expression level of the gene is preferably performed by any one method selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, immunoprecipitation, immunohistochemical analysis, and fluorescence-activated cell sorting (FACS), but any method for measuring the level of gene expression or protein expression known to those skilled in the art may be applied without limitation.

[0039] In addition, the method for measuring the activity level of the NNAT protein is preferably performed by any one method selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and protein chip, but any method for measuring the level of protein activity known to those skilled in the art can be applied without limitation.

[0040] In another aspect, the present invention relates to a composition for diagnosing Huntington's disease, comprising a preparation for measuring the mRNA or protein expression level of the NNAT (neuronatin) gene.

[0041] The term "diagnosis" as used in the present invention means confirming the presence or characteristics of a pathological condition, and in the present invention, diagnosis means confirming whether Huntington's disease has occurred.

[0042] The diagnostic composition of the present invention may be a biomarker, and includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which show a significant increase or decrease in a specific gene expression level or protein expression level in individuals with advanced Huntington's disease compared to a normal control group, and preferably refers to the NNAT (neuronatin) gene and its mRNA, and proteins encoded therefrom.

[0043] In one example, the mRNA expression level of NNAT in neural progenitor cells (HD iPSC derived NPC) derived from fibroblast-induced pluripotent stem cells of a Huntington's disease patient and the mRNA expression level of NNAT in neural cells differentiated from the neural progenitor cells were confirmed to be significantly reduced compared to neural progenitor cells or neural cells of a normal person.

[0044] The term "mRNA expression level measurement" used in the present invention refers to the process of determining the presence and expression level of mRNA of a biomarker for diagnosing chronic liver disease in a biological sample, thereby measuring the amount of mRNA. Analytical methods for this include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0045] The preparation for measuring the mRNA level of the above NNAT is characterized by being a primer pair, probe or antisense nucleotide that specifically binds to the gene of the above NNAT, and preferably includes the base sequence of SEQ ID NO: 1, and these primer pairs, probes or antisense nucleotides can be designed based on the above sequence.

[0046] Additionally, the agent for measuring the protein level of the NNAT may be characterized as an antibody, interacting protein, ligand, nanoparticle or aptamer that specifically binds to the protein or peptide fragment.

[0047] The present invention also relates to a method for providing information for diagnosing Huntington's disease, comprising the steps of measuring the mRNA or protein expression level of the NNAT (neuronatin) gene from a biological sample of a patient; and comparing the mRNA or protein expression level with that of a control sample.

[0048] In one embodiment, the method for providing information for diagnosing Huntington's disease may further include a step of providing information that Huntington's disease is present if the level of mRNA expression of the NNAT gene or the level of protein activity of NNAT is reduced compared to a control group.

[0049] The present invention relates to a pharmaceutical composition for preventing or treating Huntington's disease, which comprises an NNAT (neuronatin) gene expression promoting agent or an NNAT (neuronatin) gene activity promoting agent. Since the NNAT gene exhibits abnormally low expression in Huntington's disease patients, the composition has the effect of improving symptoms of Huntington's disease by enhancing its expression and increasing its activity.

[0050] Figure 1 shows the results of RNA-sequence analysis between neural progenitor cells derived from fibroblasts of normal individuals and Huntington's disease patients and neural cells differentiated from the neural progenitor cells.

[0051] Figure 2 shows the results of RNA-sequence analysis between neural progenitor cells derived from fibroblasts of normal individuals and Huntington's disease patients and neural cells differentiated from the neural progenitor cells.

[0052] Figure 3 shows the results of RNA-sequence analysis between neural progenitor cells derived from induced pluripotent stem cells of normal individuals and Huntington's disease patients and neural cells differentiated from the neural progenitor cells.

[0053] Figure 4 shows the results of confirming the mRNA expression levels of six genes, NNAT, MEG3, SVIL-AS1, GSTM1, HOXD12, and TBX5, in neural progenitor cells derived from fibroblast-induced pluripotent stem cells of normal individuals and Huntington's disease patients using qPCR (real-time polymerase chain reaction).

[0054] Figure 5 shows the results of confirming the mRNA expression levels of six genes, NNAT, MEG3, SVIL-AS1, GSTM1, HOXD12, and TBX5, in neural cells differentiated from the corresponding neural precursor cells using qPCR (real-time polymerase chain reaction).

[0055] Figure 6 shows the results of confirming the NNAT mRNA expression levels of neural progenitor cells derived from fibroblast-induced pluripotent stem cells of Huntington's disease patients (HD iPSC derived NPC) and neural progenitor cells derived from fibroblast-induced pluripotent stem cells of normal individuals (WT iPSC derived NPC), as well as the NNAT mRNA expression levels of neural cells differentiated from the corresponding neural progenitor cells.

[0056] Figure 7 shows a plasmid map for knocking down NNAT in induced pluripotent stem cells or neural progenitor cells of Huntington's disease patients.

[0057] Figure 8 is a plasmid map for overexpressing NNAT in normal human induced pluripotent stem cells or neural progenitor cells.

[0058] Figure 9 shows the results of confirming the level of NNAT mRNA expression after lentiviral packaging of the plasmid of Figure 7 and viral transduction into Huntington's disease neural progenitor cells.

[0059] Figure 10 shows the results of confirming the level of NNAT mRNA expression after lentiviral packaging of the plasmid of Figure 8 and viral transduction into normal human neural progenitor cells.

[0060] Figure 11 is an optical micrograph showing the progression of differentiation into neural cells after increasing the expression of NNAT in Huntington's disease neural progenitor cells.

[0061] Figure 12 is an immunofluorescence staining photograph of TUJ1, an immature neuronal marker, in each group on day 7 during the differentiation process of Figure 11.

[0062] Figure 13 is an immunofluorescence staining photograph of MAP2, a mature neuronal marker, and GABA, a GABAergic neuron marker, in each group on day 21 of the differentiation process of Figure 11.

[0063] Figure 14 shows the ratio of each marker in the immunofluorescence staining photographs of Figures 12 and 13.

[0064] Figure 15 shows the mRNA expression levels of TUJ1, an immature neuronal marker, in a sample on the 7th day of differentiation of Figure 11, MAP2, a mature neuronal marker, and GABA, a GABAergic neuronal marker, in a sample on the 21st day of differentiation.

[0065] Figure 16 shows the results of measuring the mRNA expression levels of TUJ1, an immature neuronal marker, MAP2, a mature neuronal marker, and GAD65 and GAD67, GABAergic neuronal markers, in a sample on day 21 of the differentiation process of Figure 11.

[0066] Figure 17 shows the results of measuring calcium activity using a calcium fluorescent indicator (fluo-4-AM) from neural progenitor cells derived from normal human induced pluripotent stem cells.

[0067] Figure 18 shows the measurement of calcium activity using a calcium fluorescent indicator (fluo-4-AM) from neural progenitor cells derived from induced pluripotent stem cells of a Huntington's disease patient.

[0068] Hereinafter, examples will be described in detail to specifically explain this specification. However, the embodiments according to this specification may be modified in various different forms, and the scope of this specification is not limited to the embodiments described below. The embodiments of this specification are provided to more fully explain this specification to those of average skill in the art.

[0069]

[0070] Example 1. Gene expression analysis of cells derived from Huntington's disease patients.

[0071] RNA sequencing (RNA-seq) was performed on neural stem cells (HDciNSC) derived from fibroblasts of Huntington's disease patients and neural stem cells (CRI3_6) in which the number of CAG repeats in fibroblasts of Huntington's disease patients was corrected using the CRISPR-cas9 system. Genes with differences in mRNA expression levels between the two groups were selected (Fig. 1). As confirmed in Fig. 2, the mRNA of approximately 30 genes was overexpressed compared to normal subjects, and the mNRA of approximately 25 genes was decreased compared to normal subjects, confirming differences in expression levels.

[0072] Additionally, RNA-sequence analysis was performed between neural progenitor cells derived from induced pluripotent stem cells of normal individuals and Huntington's disease patients, and neural cells differentiated from these neural progenitor cells. Two normal cell lines and two Huntington's disease patient cell lines were compared, and genes exhibiting similar trends were selected. Twenty-two genes were highly expressed and eight genes were downregulated in Huntington's disease patients compared to normal individuals (Fig. 3).

[0073] Among these, when selecting genes with a difference in mRNA expression level of 4 times or more and a p-value of 0.05 or less between each group, it was confirmed that six genes, NNAT, MEG3, SVIL-AS1, GSTM1, HOXD12, and TBX5, had abnormal expression levels in Huntington's disease patient cells compared to normal cells. As shown in Figure 4, the NNAT gene is abnormally expressed at a lower level in Huntington's disease cells than in normal cells, and the MEG3 GSTM1, HOXD12, and TBX5 genes are expressed at a higher level in Huntington's disease cells than in normal cells.

[0074] In addition, the results of confirming the expression levels of the above genes using qPCR confirmed that the NNAT and SVIL-AS1 genes were abnormally expressed at lower levels in Huntington's disease cells compared to normal cells, and the MEG3, HOXD12, and TBX5 genes were expressed at higher levels in Huntington's disease cells compared to normal cells (Fig. 5).

[0075]

[0076] Example 2. Changes in NNAT expression levels during stem cell differentiation.

[0077] To confirm the function of NNAT related to the onset and progression of Huntington's disease, the mRNA expression levels of NNAT in neural stem cells (iPSC-derived NPCs) derived from fibroblasts of Huntington's disease patients and neural stem cells (WT iPSC-derived NPCs) derived from fibroblasts of normal individuals, as well as the mRNA expression levels of NNAT during the differentiation process into neurons, were determined.

[0078] As a result, as shown in Fig. 6, it was confirmed that the difference in the expression level of NNAT mRNA between neural stem cells derived from Huntington's disease patients and normal cells increased during the process of differentiating from neural stem cells (NPCs) into neural cells, and it was found that the expression level of NNAT mRNA in neural stem cells derived from Huntington's disease patients was reduced compared to normal cells.

[0079]

[0080] Example 3. Effects of NNAT overexpression and knockdown

[0081] 3-1. Construction and transduction of plasmids for NNAT overexpression or knockdown

[0082] We conducted experiments to determine whether the expression of various factors involved in neurogenesis in neural stem cells increases or decreases when NNAT is knocked down or overexpressed.

[0083] First, a plasmid for knocking down NNAT in neural stem cells derived from Huntington's disease patients was constructed as shown in Figure 7. The target sequence for knocking down NNAT is as shown in SEQ ID NO: 1.

[0084] In addition, in order to overexpress NNAT in normal human induced pluripotent stem cells or neural progenitor cells, a plasmid was constructed as shown in Fig. 8, and the target sequence for overexpressing NNAT is as shown in sequence number 2.

[0085] After infecting Huntington's disease stem cells with the plasmids of Figs. 7 and 8 produced above using lentivirus, the expression level of NNAT mRNA was confirmed (Figs. 9 and 10).

[0086] When a plasmid containing the overexpression target sequence of Fig. 8 was packaged with lentivirus and transduced into iPSC-derived Huntington's disease neural progenitor cells, the expression level of NNAT was confirmed to be increased to the level of normal neural progenitor cells (Fig. 9). This result indicates that the expression of NNAT was effectively suppressed in neural stem cells derived from Huntington's disease patients.

[0087] In addition, after packaging the plasmid containing the knockdown target sequence of FIG. 7 with lentivirus, the amount of NNAT mRNA expression was confirmed after viral transduction into normal human neural progenitor cells, and it was confirmed that NNAT expression was effectively suppressed in normal human neural progenitor cells, as shown in FIG. 10.

[0088] 3-2. Effect of cell death by regulating NNAT expression level

[0089] In the above 3-1, apoptosis during differentiation of NNAT-overexpressing neural stem cells into neurons was observed using an optical microscope. As shown in Fig. 11, optical micrographs were observed during differentiation into neurons after overexpressing NNAT in Huntington's disease neural stem cells. As a result of observing the optical microscope photographs, it was confirmed that when NNAT was knocked down in the normal group, the number of apoptotic cells increased and the number of neurites decreased. In addition, it was confirmed that when NNAT was overexpressed in the Huntington's disease group, the number of apoptotic cells decreased and the number of neurites increased.

[0090] Example 4 Changes in the process of neural cell differentiation

[0091] 4-1. Confirmation of expression of immature or mature neural markers

[0092] As in Example 3 above, the differentiation process of neural stem cells in which the expression of NNAT was regulated through transduction was observed, and the expression of immature or mature neural markers was confirmed through immunofluorescence staining as follows.

[0093] Samples were obtained on days 7, 14, and 21 of differentiation of neural stem cells, and the expression levels of immature neuronal markers, mature neuronal markers, and GABAergic neuronal markers were confirmed from these samples, respectively. First, on day 7 of differentiation, the immunofluorescence staining images of TUJ1, an immature neuronal marker, in each group were confirmed as in Figure 12, and on day 21 of the differentiation process, the immunofluorescence staining images of MAP2, a mature neuronal marker, and GABAergic neuronal marker, GABA, in each group were confirmed as in Figure 13. As a result of confirming the relative mRNA expression levels from the immunofluorescence staining images as described above, it was confirmed that the expression levels of immature neuronal markers were normalized by the overexpression of NNAT. In addition, after 21 days of differentiation, immunofluorescence staining images and relative mRNA expression levels of MAP2, a mature neuronal marker, and GABA, a GABAergic neuronal marker, in each group were confirmed, and it was observed that the expression levels of mature neuronal markers were increased by overexpression of NNAT and approached the normal range. The same results were confirmed in the graphs showing their ratios. (Figs. 14 to 16)

[0094] 4-2. Measurement of calcium activity

[0095] NNAT is known to be involved in the regulation of intracellular calcium homeostasis. Therefore, to determine the expression level of NNAT, an experiment was conducted to determine the presence of intracellular calcium activity. As described in Example 3, calcium activity was measured using a calcium fluorescent marker (fluo-4-AM) in neural progenitor cells (NPCs) derived from induced pluripotent stem cells of normal individuals and Huntington's disease patients, in which NNAT expression was regulated. The differences between normal individuals and Huntington's disease patients were confirmed.

[0096] Figure 17 shows the calcium activity of neural progenitor cells derived from induced pluripotent stem cells of a normal individual, and Figure 18 shows the calcium activity of neural progenitor cells derived from induced pluripotent stem cells of a Huntington's disease patient. Compared to normal individuals, the fluorescence activity of neural progenitor cells derived from Huntington's disease patients was significantly reduced.

[0097] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0098] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating Huntington's disease, comprising an NNAT (neuronatin) gene expression promoting agent or an activator.

[0099] Specifically, the NNAT expression promoter may be selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to mRNA of a gene encoding the NNAT protein.

[0100] Additionally, the activity promoter of the NNAT protein may be selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NNAT protein.

[0101] As an example, the expression promoter of the NNAT may include an expression vector comprising an oligonucleotide consisting of the base sequence of SEQ ID NO: 2.

[0102] In another aspect, the present invention relates to a method for screening a candidate substance for the prevention or treatment of Huntington's disease, comprising the steps of: 1) treating a test substance to be analyzed to a cell containing an NNAT (neuronatin) gene; 2) measuring the activity of the NNAT protein or the expression level of the NNAT gene in the cell of 1); and 3) selecting the test substance as a candidate substance for the prevention or treatment of Huntington's disease when the activity of the NNAT protein or the expression of the NNAT gene is increased in 2).

[0103] As an example, the test substance may be selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, miRNA, ribozymes, DNAzymes, PNAs, antibodies, aptamers, natural extracts, and synthetic compounds.

[0104] As another example, a method for measuring the NNAT protein activity or the expression level of the NNAT gene may be selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, immunoprecipitation, immunohistochemical analysis, and fluorescence-activated cell sorting (FACS).

[0105] As another example, the method for measuring the NNAT protein activity level may be performed by any one method selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and protein chip.

[0106] In another aspect, the present invention relates to a composition for diagnosing Huntington's disease, comprising a preparation for measuring the mRNA or protein expression level of the NNAT (neuronatin) gene.

[0107] In another aspect, the present invention relates to a method for providing information for diagnosing Huntington's disease, comprising the steps of measuring the mRNA or protein expression level of the NNAT (neuronatin) gene from a biological sample of a patient; and comparing the mRNA or protein expression level with that of a control sample.

Claims

1. A pharmaceutical composition for preventing or treating Huntington's disease, comprising an agent that promotes the expression or activity of the NNAT (neuronatin) gene.

2. In paragraph 1, A pharmaceutical composition for preventing or treating Huntington's disease, wherein the NNAT expression promoter is any one selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of a gene encoding the NNAT protein.

3. In paragraph 1, A pharmaceutical composition for preventing or treating Huntington's disease, wherein the NNAT protein activity promoter is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NNAT protein.

4. In paragraph 1, A pharmaceutical composition for preventing or treating Huntington's disease, wherein the expression promoter of the above NNAT comprises an expression vector containing an oligonucleotide consisting of the base sequence of sequence number 2. 5.1) A step of treating a test substance to be analyzed into a cell containing the NNAT (neuronatin) gene; 2) a step of measuring the activity of NNAT protein or the expression level of NNAT gene in the cells of 1); and 3) A method for screening a candidate substance for the prevention or treatment of Huntington's disease, comprising a step of selecting the test substance as a candidate substance for the prevention or treatment of Huntington's disease when the activity of the NNAT protein or the expression of the NNAT gene is increased in the above 2).

6. In paragraph 5, The method wherein the test substance is any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA, antibodies, aptamers, natural extracts, and synthetic compounds.

7. In paragraph 5, A method for measuring the level of expression of the NNAT protein activity or the NNAT gene is performed by any one method selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, immunoprecipitation, immunohistochemical analysis, and fluorescence-activated cell sorting (FACS).

8. In paragraph 5, A method for measuring the level of NNAT protein activity is performed by any one method selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and protein chip.

9. A composition for diagnosing Huntington's disease, comprising a preparation for measuring the mRNA or protein expression level of the NNAT (neuronatin) gene.

10. A step of measuring the mRNA or protein expression level of the NNAT (neuronatin) gene from a biological sample of a patient; and A method for providing information for diagnosing Huntington's disease, comprising a step of comparing the mRNA or protein expression level with a control sample.

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