WO2023190316A1 - Preventive or therapeutic agent for neurodegenerative disease - Google Patents

Preventive or therapeutic agent for neurodegenerative disease Download PDF

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WO2023190316A1
WO2023190316A1 PCT/JP2023/012161 JP2023012161W WO2023190316A1 WO 2023190316 A1 WO2023190316 A1 WO 2023190316A1 JP 2023012161 W JP2023012161 W JP 2023012161W WO 2023190316 A1 WO2023190316 A1 WO 2023190316A1
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klhl32
cells
nucleic acid
gene
disease
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治久 井上
孝之 近藤
拓也 山本
祐一郎 矢田
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国立大学法人京都大学
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Definitions

  • the present invention relates to a preventive or therapeutic drug for neurodegenerative diseases, which contains a drug that suppresses the expression of the KLHL32 gene.
  • AD Alzheimer's disease
  • FDA US Food and Drug Administration
  • Frontotemporal lobar degeneration is known as a disease that exhibits a progressive neurodegenerative disorder.
  • Frontotemporal lobar degeneration is the second or third most common early-onset neurodegenerative dementia after Alzheimer's disease, with symptoms of marked behavioral and personality changes, often accompanied by language impairment. This gradually develops into cognitive impairment and dementia. Also, although research is progressing in the same way as Alzheimer's disease, the full picture of the onset mechanism has not yet been clarified.
  • Parkinson's disease is a typical neurodegenerative disease along with Alzheimer's disease
  • Lewy bodies are known to appear in the substantia nigra of patients' brains.
  • Lewy bodies are aggregates of a protein called alpha-synuclein, which consists of 140 amino acid residues, and are associated with Lewy body diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. It is known that it also appears in the brains of patients with the disease, and diseases accompanied by the accumulation of ⁇ -synuclein in the brain are called ⁇ -synucleinopathy.
  • ⁇ -synuclein aggregation or ⁇ -synuclein fibril formation, plays an important role in the progression of ⁇ -synucleinopathy, and active research is being conducted in various fields on substances that inhibit ⁇ -synuclein fibril formation. It is progressing.
  • Non-Patent Document 1 A common pathological condition of many neurodegenerative diseases including Alzheimer's disease is neurodegeneration accompanied by activation of caspase 3 (for example, Non-Patent Document 1).
  • the present inventors previously developed a method for predicting the presence or absence and amount of neurodegeneration from phase contrast images using deep learning and induced pluripotent stem cell (iPS cell) technology (Patent Document 1).
  • iPS cell induced pluripotent stem cell
  • the objective of the present invention is to predict neurodegenerative death, which is most important at the time when neurological symptoms in neurodegenerative diseases are manifested and the disease progresses, and to identify molecular targets for treating neurodegenerative diseases.
  • Another object of the present invention is to provide a method for preventing or treating neurodegenerative diseases by suppressing the expression of the identified molecular target.
  • the present inventors constructed a model using deep learning, and used this model to identify cells with a high score (cells with a high probability of being caspase-3-activated cells) and cells with a low score (cells with a high probability of caspase-3 activation).
  • Single-cell RNA-seq analysis was performed on cells with a low probability of being expected to be activated.
  • KLHL32 was found to be a gene that is specifically expressed before caspase-3 activation in a group of cells with high scores.
  • the present invention is as follows.
  • a drug for preventing or treating neurodegenerative diseases which contains a drug that suppresses the expression of the KLHL32 gene.
  • the preventive or therapeutic agent according to [1], wherein the expression suppressing drug is selected from the group consisting of siRNA, heteroduplex nucleic acid, antisense nucleic acid, shRNA, miRNA, antigene nucleic acid, and CRISPR-Cas system.
  • the expression inhibitor is siRNA.
  • the preventive or therapeutic agent according to [2] wherein the CRISPR-Cas system is a CRISPR-dCas system.
  • Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, and progressive
  • [6] (1) A step of bringing a test substance into contact with cells expressing the KLHL32 gene; and (2) A step of selecting a test substance that reduces the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. , a screening method for preventive or therapeutic drugs for neurodegenerative diseases.
  • Neurodegenerative diseases including (1) a step of contacting cells with a test substance, and (2) a step of selecting a test substance that suppresses an increase in the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. screening methods for preventive or therapeutic drugs.
  • [8] The method according to [6] or [7], wherein the cells are model cells for neurodegenerative diseases.
  • the neurodegenerative disease model cell is a cell derived from a patient with a neurodegenerative disease or a cell induced to differentiate from a pluripotent stem cell having a mutation in a gene that causes the neurodegenerative disease.
  • the cell is a nerve cell.
  • the cells are human cells.
  • Biomarker for diagnosing neurodegenerative diseases consisting of KLHL32 protein or KLHL32 transcript.
  • a method for assisting in diagnosing whether a subject has a neurodegenerative disease comprising the step of detecting the biomarker according to [12] in the subject or a sample derived from the subject.
  • a kit for diagnosing a neurodegenerative disease comprising an antibody that specifically recognizes KLHL32 protein or a nucleic acid probe or primer that specifically recognizes KLHL32 transcript.
  • a method for preventing or treating a neurodegenerative disease in a mammal which comprises administering to the mammal an effective amount of a drug that suppresses the expression of the KLHL32 gene.
  • a drug that suppresses the expression of the KLHL32 gene for use in the prevention or treatment of neurodegenerative diseases.
  • [17] Use of a drug that suppresses KLHL32 gene expression for the production of a drug for preventing or treating neurodegenerative diseases.
  • a method for preventing or treating neurodegenerative diseases comprising the following steps (i) to (iii).
  • step (i) Detecting the biomarker according to [12] in a subject or a sample derived from a subject, (ii) diagnosing whether the subject has a neurodegenerative disease based on the results of step (i); (iii) A step of administering a preventive or therapeutic agent for a neurodegenerative disease to a subject diagnosed with a neurodegenerative disease according to (ii) [19] The method according to [18], wherein the preventive or therapeutic agent for neurodegenerative diseases is selected from the group consisting of KLHL32 gene expression inhibitors, aducanumab, donepezil, rivastigmine, galantamine, donepezil, and memantine.
  • the present invention makes it possible to predict neurodegeneration significantly in advance of neurodegeneration, and also provides preventive or therapeutic agents for neurodegenerative diseases based on a new molecular target (KLHL32 gene), methods to assist in diagnosis, and A method for screening the drug is provided.
  • KLHL32 gene new molecular target
  • FIG. 1 A Schematic diagram of the Deep-i model for predicting cells programmed for neurodegeneration.
  • FIG. 2 Schematic diagram of the transient NGN2-induced neuronal differentiation method and assay system for detecting activated caspase-3.
  • iPSCs Induced pluripotent stem cells
  • A Changes in activated caspase-3 signal counts were plotted when 0.1% DMSO was added to all 384 wells of a 384-well plate. The average counts for each row were plotted separately to assess the position effect of the signal at different locations and between different batches.
  • (A) Changes in signal counts of activated caspase-3 of experimental batch 2 were plotted upon addition of Z-VAD-FMK at different concentrations from 0 to 25 ⁇ M. Error bars indicate standard deviation (S.D.) of n 16.
  • (B) Changes in signal counts of activated caspase-3 of experimental batch 3 were plotted upon addition of Z-VAD-FMK at different concentrations from 0 to 25 ⁇ M. Error bars indicate standard deviation (S.D.) of n 16.
  • the DL-based prediction system showed reproducible results between different experimental batches at different time points and different lines, distinguishing genetic differences in the PSEN1 gene.
  • A Schematic diagram of image collection for deep learning (DL) training.
  • B Images from two plates, including plates A and B, were merged and processed as one batch for DL training, validation, and testing steps, respectively, with rotation.
  • C Schematic diagram for evaluating the error of DL-based prediction between images with different cropping sizes.
  • (E) The average DL prediction scores of two rows were plotted separately in a boxplot to assess the distribution of DL prediction scores between different locations and different batches. Boxplots show the median, 25% and 75% quartiles, minimum and maximum of DL prediction scores for each location or batch.
  • (F) Comparison between neurons with and without PSEN1 mutations. Correcting PSEN1 mutations in FAD neurons by CRISPR-Cas9 system to generate “corrected” neurons that retain the same genetic background except for the PSEN1 mutations that were corrected in two different batches (2 and 3). Got ready. The prediction scores by the Deep-i model were compared between FAD neurons and modified neurons. Plotted dots represent n 1,536 independent replicates.
  • KLHL32 showed a significant and unique increase only in cluster 12.
  • F Representative images of KLHL32 with and without 25 ⁇ M Z-VAD-FMK. **** indicates P ⁇ 0.0001 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test).
  • G Graph quantifying Figure 6F to show the difference in the number of KLHL32-positive cells between different Z-VAD-FMK concentrations. **** indicates P ⁇ 0.0001 (using Student's t-test). Single-cell transcriptome reveals KLHL32 as a marker of neurons programmed for neurodegeneration.
  • A Quality control of single cell RNA sequencing (scRNAseq).
  • scRNAseq UMAP shows the distribution of cluster 12 at different conditions of Z-VAD-FMK 0, 0.2, and 25 ⁇ M.
  • D Signal counts of activated caspase-3 were plotted 144 hours after the start of the assay. One-way analysis of variance revealed significant effects in different conditions.
  • KLHL32-positive cells accumulated in preferentially affected brain regions of Alzheimer model mice and postmortem Alzheimer's brain tissue.
  • Coronal brain sections from a representative 3-month-old non-transgenic littermate (left panel) and 5 ⁇ FAD mouse (right panel) to obtain images of the post-ampullar cortex or post-ampullar cortex. were stained with KLHL32 (top panel) or amyloid beta (A ⁇ ) (bottom panel) and photographed using a microscope. Scale bar 4 mm (for low magnification) (left panel), 400 ⁇ m (for high magnification).
  • KLHL32 was detected in senile plaques (left panel), neurites (middle panel, white arrowheads), neuropil aggregate structures (middle panel, black arrowheads), dot-like patterns (right panel, white arrowheads), and neuropils.
  • the intracellular space (right panel, black arrowhead) was stained.
  • Scale bar 100 ⁇ m.
  • Schematic diagram for counting KLHL32-positive populations Representative images for analyzing KLHL32-positive areas (gray) and labeling positive areas as a counting mask (right panel) in the retrosplenial cortex (RSP) or subiculum (SUB).
  • Preventive or therapeutic agents for neurodegenerative diseases As shown in the examples below, in a cell model of Alzheimer's disease (AD), knocking down the expression of the KLHL32 gene with siRNA suppresses the activation of caspase-3 and inhibits cell activation. It has been demonstrated by the inventors that death is suppressed. Neurodegeneration accompanied by activation of caspase-3 is considered to be a common pathology of all neurodegenerative diseases including AD. Therefore, a drug that suppresses the expression of the KLHL32 gene can be used as a prophylactic or therapeutic drug for neurodegenerative diseases in general, including AD.
  • AD Alzheimer's disease
  • the present invention provides a prophylactic or therapeutic drug for neurodegenerative diseases (hereinafter sometimes referred to as "the drug of the present invention”), which contains a drug that suppresses the expression of the KLHL32 gene. Furthermore, unless otherwise specified, the prophylactic or therapeutic agent (or method) for neurodegenerative diseases also includes a medicament (or method) that can prevent and treat the disease.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition containing the active ingredient, a drug that suppresses the expression of the KLHL32 gene, either alone or mixed with a pharmacologically acceptable carrier, excipient, diluent, etc. Alternatively, it can be administered orally or parenterally as a prophylactic or therapeutic agent. Furthermore, the medicament of the present invention can be administered to mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, dogs, cats, and monkeys). Therefore, there is also provided a method for preventing or treating neurodegenerative diseases in a mammal, which comprises administering to the mammal an effective amount of a drug that suppresses the expression of the KLHL32 gene.
  • mammals eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, dogs, cats, and monkeys. Therefore, there is also provided a method for preventing or treating neurodegenerative diseases
  • the neurodegenerative diseases to be prevented or treated in the present invention are not particularly limited, but include, for example: Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, progressive supranuclear disease Examples include sexual paralysis and corticobasal degeneration. Among them, AD is preferable.
  • AD to be prevented or treated may be either sporadic or familial AD.
  • the causative gene is not particularly limited, and any gene including amyloid precursor protein (APP), presenilin 1 (PSEN1), presenilin 2 (PSEN2), etc. It may be a known causative gene.
  • the APP gene mutations include dup APP mutation, APP KM670/671NL mutation, APP D678N mutation, APP E682K mutation, APP A692G mutation, APP E693K mutation, APP E693Q Changes, App E693G variant, App E693DEL (App E693 ⁇ ) variant, App D694N variant, App L705V variant, App A713T variant, App T714A Karizu, App T714I Campaign, App V715M Campaign, App i715A Detergent, App i716, App i716 V transformation, App i716F change, Examples include, but are not limited to, APP I716T mutation, APP V717I mutation, and the like.
  • APP E693 ⁇ means a deletion type mutation of E693 in APP.
  • ALS to be prevented or treated includes both sporadic and familial ALS.
  • the causative genes are not particularly restricted, and include SOD1, TDP-43, C9orf72, alsin, SETX, FUS/TLS, VAPB, ANG, FIG4, OPTN, ATXN2, DAO, UBQLN2, PFN1, DCTN1, CHPM2B, Examples include VCP.
  • the SOD1 gene mutation includes a mutation in which Leu at position 144 of the SOD1 protein is replaced with Phe-Val-Xaa (Xaa represents any amino acid).
  • SOD1-L144FVX a mutation in which Gly at position 93 is replaced with Ser
  • SOD1-G93S a mutation in which Leu at position 106 is replaced with Val
  • the present invention can also be suitably used for patients who have a mutation in the MAPT gene that has a mutation in exons 9-13.
  • mutations in exons 9-13 include K257T, I260V, G272V, N297K, K280 ⁇ , L284L, N296N, P301L, P301S, S305N, S305S, V337M, E342V, G389R, and R406W.
  • therapeutic drugs include not only drugs aimed at the radical treatment of neurodegenerative diseases, but also drugs aimed at suppressing the progression of these diseases, alleviation of symptoms (e.g., daily life, work This also includes medicines that aim to improve symptoms to minimal symptoms (MM) that do not interfere with health conditions or alleviate the after-effects.
  • MM minimal symptoms
  • neurodegenerative diseases are diseases that progress over a long period of time (usually on a yearly basis)
  • preventive drugs include not only drugs aimed at reducing the risk of developing neurodegenerative diseases in subjects who have not developed neurodegenerative diseases, but also drugs for neurodegenerative diseases.
  • the KLHL32 gene expression inhibitor is not limited as long as it is a substance that can suppress the expression of the gene at least in nerve cells, but the substance may be a nucleic acid, for example, an antisense nucleic acid (e.g. antisense oligonucleotide (ASO ), etc.) (including the nucleic acid encoding the aforementioned nucleic acid), siRNA (including the nucleic acid encoding the aforementioned siRNA), heteroduplex oligonucleotide (HDO), shRNA (including the nucleic acid encoding the aforementioned shRNA), Examples include miRNA (microRNA) (including nucleic acids encoding the miRNA), antigene nucleic acids, CRISPR-Cas systems, and the like.
  • an antisense nucleic acid e.g. antisense oligonucleotide (ASO ), etc.
  • ASO antisense oligonucleotide
  • siRNA including the nucleic acid en
  • the KLHL32 gene expression inhibitor when the KLHL32 gene expression inhibitor is a nucleic acid, such a nucleic acid may be referred to as "the nucleic acid of the present invention".
  • the KLHL32 gene expression inhibitor is a CRISPR-Cas system
  • the nucleic acid encoding the system i.e., the nucleic acid encoding Cas, the nucleic acid encoding guide RNA, and the nucleic acid encoding guide RNA and Cas
  • the medicament of the present invention may contain only one or two or more KLHL32 gene expression inhibitors. If two or more are included, they may be of the same type (e.g., multiple siRNAs with different target sequences) or different types (e.g., siRNA and siRNA). antisense nucleic acids, etc.).
  • the KLHL gene belongs to the KLHL (Kelch-like) gene family, which generally encodes proteins with a BTB/POZ domain, a BACK domain, and 5 to 6 Kelch motifs.
  • An example of the human KLHL32 protein is a 620 amino acid long protein containing a BTB/POZ domain, a BACK domain, and five Kelch motifs.
  • the KLHL family has known molecular types, KLHL1 to KLHL42, and some of these are predicted to be involved in tumorigenesis and the E3-mediated protein degradation system in the endoplasmic reticulum, but the KLHL32 protein The relationship between this and neurodegeneration has not been reported.
  • the expression suppressing drugs may be any as long as it can suppress the expression of at least the KLHL32 transcript encoding the full-length KLHL32 protein. Therefore, the expression inhibitor contains a base sequence complementary to a partial sequence of the KLHL32 transcript encoding the full-length KLHL32 protein (hereinafter referred to as target RNA sequence).
  • Such a nucleotide sequence is designed based on the cDNA nucleotide sequence (SEQ ID NO: 1 or 2) of the KLHL32 transcript encoding the full-length human KLHL32 protein, which is registered as Genbank Accession No. NM_001323252.2 or NM_052904.4, for example. can do.
  • the expression suppressing drug in the case of a CRISPR/Cas system, the guide RNA constituting the system
  • Such a base sequence can be designed, for example, based on the DNA base sequence of the human KLHL32 gene registered as Gene ID: 114792, location: NC_000006.12 (96898083..97145030) on NCBI Gene.
  • Specific examples of the target RNA sequence include, but are not limited to, the base sequences shown in any of SEQ ID NOs: 4 to 7.
  • the length of the target RNA sequence or target DNA sequence is not particularly limited as long as the KLHL32 gene expression inhibitor can specifically recognize and bind to the sequence, but it is preferably 12 nucleotides or more, more preferably 15 nucleotides.
  • the length is at least 17 nucleotides, more preferably 17 nucleotides or longer.
  • the upper limit of the length is also not particularly limited, but is, for example, 30 nucleotides or less, preferably 25 nucleotides or less, more preferably 22 nucleotides or less. Therefore, the length range of the target region includes, for example, 12 to 30 nucleotides, preferably 15 to 25 nucleotides, and more preferably 17 to 22 nucleotides.
  • expression of the KLHL32 gene is used to include at least "production of a KLHL32 transcript” unless otherwise specified, but preferably also includes “production of a functional KLHL32 protein”. used in meaning. Therefore, suppression of gene expression refers not only to a decrease in the amount of transcripts transcribed from the gene in cells due to contact with a drug that suppresses the expression of the KLHL32 gene, but also to a decrease in the amount of transcripts transcribed from the gene. The method may also include reducing the amount of protein present in the cell.
  • the KLHL32 transcript typically includes KLHL32 mRNA and KLHL pre-mRNA, but preferably KLHL32 mRNA.
  • nucleic acid may mean a monomeric nucleotide, but usually means a nucleotide consisting of multiple monomers (eg, oligonucleotide, polynucleotide, etc.). Therefore, when a monomeric nucleotide is intended, it may be written as a "nucleic acid nucleotide,” and such nucleic acids include, for example, ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid (PNA), and morpholinonucleic acid (PNA). Examples include nucleic acid).
  • nucleic acid when a nucleic acid is a nucleotide consisting of multiple monomers, each nucleotide residue (including nucleotides at the 5' and 3' ends) constituting the nucleic acid is simply referred to as a "nucleotide.” to be called.
  • nucleic acid strand or “strand” means a single-stranded nucleic acid unless otherwise specified. Therefore, “antisense strand” can be read as “single-stranded antisense nucleic acid.”
  • complementary means that nucleic acid bases are formed through hydrogen bonds, so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, fluctuations). A relationship that can form a base pair (such as a Wobble base pair). Therefore, a “complementary sequence” includes not only a sequence that is completely complementary (i.e., hybridizes without mismatch) to a target RNA sequence or target DNA sequence, but also a sequence that hybridizes under stringent conditions or under mammalian conditions. The term is used to include sequences containing one or several (eg, 2, 3, 4, 5, or more) mismatches, as long as they can hybridize with the target sequence under physiological conditions of the cell.
  • a sequence that is completely complementary to the target RNA sequence or target DNA sequence and 80% or more e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, most preferably 100%.
  • Stringent conditions may be low stringency conditions or high stringency conditions.
  • Low stringency conditions may be relatively low temperature and high salt concentration conditions, for example, 30° C., 2 ⁇ SSC, 0.1% SDS.
  • High stringency conditions may be relatively high temperature and low salt concentration conditions, for example, 65° C., 0.1 ⁇ SSC, 0.1% SDS.
  • the stringency of hybridization can be adjusted by varying conditions such as temperature and salt concentration.
  • 1x SSC contains 150mM sodium chloride and 15mM sodium citrate.
  • An antisense nucleic acid is a single-stranded nucleic acid that contains a sequence complementary to the target RNA sequence.
  • the antisense nucleic acid forms a double-stranded region with the target RNA sequence due to a complementary sequence to the target RNA sequence, and this double-stranded region is cleaved by ribonuclease H (RNase H), thereby converting the KLHL32 gene. Suppress expression.
  • the antisense nucleic acid is preferably a gapmer type nucleic acid from the viewpoint of in vivo stability and efficient cleavage of the transcript.
  • gapmer-type nucleic acid refers to a plurality of nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) that are recognized by RNase H.
  • Nucleotides (hereinafter referred to as "modified nucleotides”) whose internal regions (herein sometimes referred to as "gap regions”) have been subjected to various chemical modifications to confer resistance to ribonucleases.
  • an external region in this specification, The external region on the 3' side is sometimes referred to as the "3' wing region” and the external region on the 5' side is sometimes referred to as the "5' wing region.”
  • At least one nucleoside constituting each region of the 3' wing region and the 5' wing region is preferably a crosslinked nucleoside (specific examples will be described later).
  • the length of the antisense nucleic acid is not particularly limited, but is typically 10 to 50 nucleotides, preferably 10 to 30 nucleotides, more preferably 13 to 30 nucleotides, and even more preferably 15 to 30 nucleotides. ⁇ 20 nucleotides long.
  • the antisense nucleic acid is sequenced based on the sequence of the KLHL32 transcript, and a complementary sequence is synthesized using a commercially available automatic DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.) It can be prepared by:
  • siRNA is double-stranded RNA consisting of an RNA with a sequence complementary to the target RNA sequence (i.e., antisense strand) and its complementary strand.
  • it is a single-stranded RNA in which a sequence complementary to the target RNA sequence (first sequence) and its complementary sequence (second sequence) are linked via a hairpin loop portion, and the hairpin loop type RNA in which the first sequence forms a double-stranded structure with the second sequence (small hairpin RNA: shRNA) is also a preferred embodiment of siRNA.
  • shRNA may be in the form of a nucleic acid (eg, expression vector, etc.) encoding the shRNA.
  • HDO means a double-stranded nucleic acid composed of a main strand DNA (i.e., antisense strand) and an RNA (cRNA) strand complementary to the DNA.
  • cRNA RNA
  • the main chain DNA is typically a gapmer type nucleic acid
  • the wing region of the cRNA strand is typically composed of modified nucleotides such as 2'-O-methyl RNA (specific examples are given below). has been done.
  • typically all phosphodiester bonding portions of the main chain DNA and/or cRNA are sulfurized (ie, the internucleoside bonds are phosphorothioate bonds).
  • the main strand DNA which becomes a single-stranded antisense nucleic acid by cRNA strand cleavage, binds to the target RNA, and it is assumed that RNase H cleaves the target RNA again, thereby exerting its antisense effect.
  • the cRNA strand serves as a substrate for RNase H, the main chain DNA or cRNA strand may have modifications other than those described above.
  • a double-stranded nucleic acid may have an overhang (also referred to as an overhang) at the 5' or 3' end of one or both of the antisense strand and the complementary strand.
  • the overhang is typically one to several (eg, 1, 2, 3, 4, 5 or 6), preferably 1 to 3 bases at the end of the sense and/or antisense strand. It is formed by addition. Examples of the overhang include those made of dTdT or UU. Overhangs can be present only on the antisense strand, only on the sense strand, or on both the antisense and sense strands, but in the present invention, double-stranded nucleic acids having overhangs on both the antisense and sense strands is preferably used.
  • each nucleic acid strand of the double-stranded nucleic acid is not particularly limited as long as it can exhibit an antisense effect; be.
  • Double-stranded nucleic acids can be obtained by chemically synthesizing using conventionally known techniques, or by producing using genetic recombination technology. It is also possible to use appropriately commercially available nucleic acids.
  • double-stranded nucleic acids can be appropriately designed based on the target RNA sequence using commercially available software (eg, RNAiDesigner; Invitrogen).
  • the sense strand and antisense strand of the target sequence on RNA are synthesized using a commercially available automatic DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.), and the mixture is incubated at approximately 90°C to approximately 90°C in an appropriate annealing buffer.
  • HDO can also be produced, for example, by the method described in WO2013/089283.
  • miRNA refers to single-stranded RNA or double-stranded RNA that does not cleave the target RNA like siRNA, but rather recognizes the 3' untranslated region (UTR) of the target RNA and controls translation.
  • double-stranded RNA e.g. miRNA/miRNA * , etc.
  • miRNA refers to endogenous non-coding RNA (ncRNA) of approximately 20 to 25 bases encoded on the genome.
  • pri-miRNA is expressed from the miRNA gene, and then pre-miRNA is generated. After that, mature-miRNA is generated. Thereafter, mature-miRNA is taken up into RISC and single-stranded miRNA is generated.
  • the miRNA used in the present invention may be in the form of pri-miRNA, pre-miRNA, mature-miRNA (miRNA/miRNA * ), and even single-stranded miRNA. It may be in the form of RNA.
  • miRNAs used in the present invention include those that act in the nucleus and degrade transcripts in an RNase H-dependent manner due to their gapmer structure, which has RNA oligomers at both ends and DNA oligomers in the center. shall be taken as a thing.
  • miRNA may be in the form of a nucleic acid (eg, expression vector, etc.) encoding the miRNA.
  • miRNA may be synthesized in the same manner as the double-stranded nucleic acid synthesis method described above. Furthermore, miRNA can also be produced based on any endogenous miRNA by substituting the miRNA sequence according to the target RNA sequence. Such endogenous miRNAs include, but are not limited to, let-7, miR-15a, miR-143, miR-139 and their precursors.
  • the length of miRNA is not particularly limited as long as it can exhibit an antisense effect, but for example, it is 10 to 50 nucleotides, preferably 15 to 30 nucleotides. more preferably 20 to 27 nucleotides in length.
  • Antigene nucleic acid refers to a nucleic acid that can suppress target gene expression at the transcriptional level by forming a triplex with double-stranded DNA containing a target gene, and is also referred to as triplex forming oligonucleotide (TFO). .
  • TFO triplex forming oligonucleotide
  • the typical antigene method is based on the binding of a third nucleic acid to a DNA double strand, and the base of the third nucleic acid forms a hydrogen bond with the purine base of the purine-pyrimidine base pair, resulting in a flat surface.
  • Triple strand formation is possible by adopting a structure in which three bases are arranged consecutively within the chain.
  • TFO third nucleic acid
  • Hoogsteen binding type i.e., antigene nucleic acid
  • reverse Hoogsteen binding type the former has a parallel orientation (the same orientation as the base-pairing strand of the DNA duplex), and the latter has an antiparallel orientation (the opposite orientation to the base-pairing strand of the DNA duplex) It is.
  • Parallel orientation TFO has T (T:A-T base pair) and protonated C (C+:G-C base pair) for A and G of A-T base pair and G-C base pair in DNA duplex, respectively. Join.
  • TFO anti-parallel orientation
  • G G:G-C base pair
  • acidic conditions are required, which limits its use under physiological conditions, but methylcytidine, which can be protonated even under neutral conditions, and derivatives that do not require protonation ( This problem can be avoided by using eg 5-methyl-6-oxocytidine).
  • the nucleotide molecules constituting the antigene nucleic acid may be naturally occurring DNA or RNA, or may be treated with various chemicals to improve stability (chemical and/or anti-enzyme) and specific activity (affinity with DNA). It may also be a modified nucleotide (for example, a PNA nucleotide).
  • the length of the antigene nucleic acid is not particularly limited, but is typically 10 to 50 nucleotides, preferably 10 to 30 nucleotides, more preferably 13 to 30 nucleotides, and even more preferably 15 to 30 nucleotides. ⁇ 20 nucleotides long.
  • Antigene nucleic acids can be chemically synthesized, for example, by methods known per se described in WO 2005/021570, WO 03/068695, and WO 2001/007455, or methods analogous thereto. can.
  • the CRISPR/Cas system consists of a complex of short CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that forms a double strand with the target DNA sequence (i.e. double-stranded guide RNA), crRNA and tracrRNA.
  • the target DNA sequence is recognized by a single synthetic RNA (i.e., single-stranded guide RNA) or crRNA alone. Therefore, by designing the crRNA sequence based on the target DNA sequence, any sequence can be targeted.
  • the CRISPR/Cas system is provided as a complex of single-stranded or double-stranded guide RNA and Cas (also referred to as Cas nuclease).
  • the design of a complementary base sequence to the target DNA sequence contained in guide RNA can be done using, for example, the public guide RNA design website (CRISPR Design Tool , CRISPRdirect, etc.), it can be designed as appropriate.
  • CRISPR Design Tool CRISPRdirect, etc.
  • candidate sequences with a small number of off-target sites in the target host genome can be used as targeting sequences.
  • the guide RNA design software you use does not have the ability to search for off-target sites in the host genome, for example, 8 to 12 nucleotides on the 3' side of the candidate sequence (seed sequence with high discriminatory ability for the target nucleotide sequence) Off-target sites can be searched by performing a Blast search on the genome.
  • tracrRNA etc. can also be designed as appropriate depending on the type of Cas nuclease.
  • the Cas used in the present invention is not particularly limited, but is preferably Cas9 (also referred to as Cas9 nuclease) or Cpf1 (also referred to as Cpf1 nuclease).
  • Cas9 include Cas9 (SpCas9; PAM sequence (5' ⁇ 3' direction; same below) NGG (N is A, G, T or C; same below)) derived from Streptococcus pyogenes, yellow Cas9 from Staphylococcus aureus (SaCas9; PAM sequence NNGRR(T)), Cas9 from Streptococcus thermophilus (StCas9; PAM sequence NNAGAAW), Cas9 from Neisseria meningitidis (NmCas9) ; PAM sequence NNNNGATT), but are not limited to these.
  • Cpf1 examples include Cpf1 (FnCpf1; PAM sequence TTN) derived from Francisella novicida, Cpf1 (AsCpf1; PAM sequence TTTN) derived from Acidaminococcus sp., and Lachnospiraceae bacteria.
  • Cpf1 examples include, but are not limited to, Cpf1 (LbCpf1; PAM sequence TTTN) derived from (Lachnospiraceae bacterium).
  • the CRISPR-dCas system is preferable as the CRISPR-Cas system used in the present invention.
  • dCas for example, in the case of SpCas9, the 10th Asp residue is converted to an Ala residue, the D10A mutant lacks the ability to cleave the opposite strand of the strand that forms a complementary strand with the guide RNA, and the 840th His residue.
  • a double mutant of the H840A mutant which lacks the ability to cleave guide RNA and complementary strands, can be used.
  • mutants lacking the ability to cleave both chains, in which the Asp residue at position 917 was converted to an Ala residue (D917A) or the Glu residue at position 1006 was converted to an Ala residue (E1006A). Can be used. Other mutant Cas can be used as well.
  • a complex of guide RNA and Cas binds to the target DNA sequence, thereby inhibiting transcription of the target gene. Furthermore, by binding a transcriptional repressor to Cas, it is possible to further enhance the transcriptional repression of the target gene.
  • transcriptional repressors include, for example, KRAB, MBD2B, v-ErbA, SID (including SID concatemer (SID4X)), MBD2, MBD3, DNMT family (e.g. DNMT1, DNMT3A, DNMT3B, etc.), Rb, MeCP2, Examples include ROM2 and AtHD2A, preferably KRAB.
  • the protein from which it is derived is not particularly limited, but examples include KOX-1 (ZNF10), KOX8 (ZNF708), ZNF43, ZNF184, ZNF91, HPF4, HTF10, HTF34, etc. can be mentioned.
  • the CRISPR/Cas system can be used, for example, in the form of a complex between a guide RNA and Cas, a complex between a nucleic acid encoding Cas or an expression vector and guide RNA, or a complex between a nucleic acid encoding guide RNA or an expression vector and Cas. or in the form of a nucleic acid or expression vector (which may be a single nucleic acid or expression vector or a plurality of nucleic acids or expression vectors) encoding guide RNA and Cas. It can be done.
  • These nucleic acids may be RNA such as mRNA, but are preferably DNA.
  • Expression vectors used in the present invention include, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, immunodeficiency virus ( Viral vectors such as HIV) can be used.
  • adenovirus or adeno-associated virus vectors are used.
  • the expression vector typically has a promoter upstream of the DNA encoding the nucleic acid or protein of the present invention.
  • promoters include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter. etc. are used.
  • a promoter specific to a particular cell eg, nerve cell
  • nerve cell may be used.
  • the length of the nucleotide sequence complementary to the target DNA sequence contained in the guide RNA is not particularly limited as long as it can specifically bind to the target DNA sequence, but for example, 15 to 30 nucleotides, preferably 18 ⁇ 25 nucleotides.
  • CRISPR/Cas systems guide RNA-encoding nucleic acids, and Cas-encoding nucleic acids are produced by chemically synthesizing DNA strands or proteins, or by converting partially overlapping oligo DNA short strands into synthesized DNA strands using PCR methods or Gibson Assembly. It is also possible to construct DNA encoding the full length of a protein by connecting them using the method.
  • the constituent elements of the nucleotide constituting the nucleic acid of the present invention include a sugar moiety (eg, ribose, deoxyribose), a base, and a phosphoric acid.
  • modifiedifications include, for example, substitutions, additions and/or deletions in the constituent elements and/or internucleoside bonds, substitutions, additions and/or deletions of atoms and/or functional groups in the constituent elements and/or internucleoside bonds. or deletion.
  • Natural bases include adenine, cytosine, guanine, thymine and uracil.
  • modified bases in which the base is modified include 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine or N4-methylcytosine; N6-methyladenine or 8-bromoadenine ; and N2-methylguanine or 8-bromoguanine.
  • the modified base is preferably 5-methylcytosine.
  • modified internucleoside bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds, methylphosphonate bonds, methylthiophosphonate bonds, boranophosphate bonds, and phosphoroamidate bonds. , but not limited to.
  • Modifications of the sugar moiety include, for example, 2'-O-methoxyethyl modification of the sugar moiety, 2'-O-methyl modification of the sugar moiety, 2'fluoro modification of the sugar moiety, and 2' and 4' positions of the sugar moiety.
  • the nucleotide having the crosslinked structure is a crosslinked nucleotide.
  • Examples of bridged nucleotides include locked artificial nucleic acids (LNA), 2'-O, 4'-C-ethylene bridged nucleic acids (ENA), etc. can be mentioned. More specifically, examples of the bridged nucleotide include those having the following nucleoside structure.
  • R is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, or a heteroatom.
  • R represents an aryl group having 3 to 12 carbon atoms which may contain an atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom, or a protecting group for an amino group in nucleic acid synthesis.
  • R is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, or a heteroatom.
  • R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R is a hydrogen atom or a methyl group.
  • Base is a natural base or a modified base.
  • the modified nucleotide may be PNA, UNA (unlocked nucleic acid), HNA, morpholino nucleic acid, or the like.
  • the hydroxyl group of the hexopyranose moiety of the HNA may be deoxylated. Further, in the above HNA, the hydroxyl group of the hexopyranose moiety may be substituted with a fluorine atom.
  • the nucleic acids of the present invention may be modified at the 5' end, 3' end, and/or within the sequence of the nucleic acid strand (in the case of double-stranded nucleic acids, each nucleic acid strand) with one or more ligands or fluorophores. Nucleic acids modified with ligands or fluorophores are also often referred to as conjugated nucleic acids. By reacting a modifying agent that can react on the solid phase during the extension reaction on the solid phase, it is possible to modify the 5' end, 3' end, and/or the inside of the sequence.
  • a conjugate nucleic acid can also be obtained by synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on the nucleic acid.
  • the ligand may be any molecule that has affinity with biomolecules, such as cholesterol, fatty acids, tocopherol, lipids such as retinoids, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc.
  • saccharides full antibodies, antibodies such as Fab, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, peptides such as RGD, NGR, R9, CPP, low molecules such as folic acid, synthetic polyamino acids, etc.
  • LDL low density lipoprotein
  • peptides such as RGD, NGR, R9, CPP
  • low molecules such as folic acid, synthetic polyamino acids, etc.
  • Examples include synthetic polymers, nucleic acid aptamers, etc., and these can also be used in combination.
  • Fluorophores include the Cy3 series, Alexa series, and black hole quenchers.
  • the medicament of the present invention is administered to mammals orally or parenterally (e.g., subcutaneous injection, intramuscular injection, local injection (e.g., intraventricular administration, intrathecal administration), intraperitoneal administration, etc.). Although it is possible, it is preferable to administer the drug parenterally, particularly by intraventricular or intrathecal administration.
  • mammals orally or parenterally e.g., subcutaneous injection, intramuscular injection, local injection (e.g., intraventricular administration, intrathecal administration), intraperitoneal administration, etc.).
  • parenterally e.g., subcutaneous injection, intramuscular injection, local injection (e.g., intraventricular administration, intrathecal administration), intraperitoneal administration, etc.).
  • compositions for oral administration include solid or liquid dosage forms, in particular tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. formulations, emulsions, suspensions, etc.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may also be included. These formulations contain excipients (e.g.
  • sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminate metasilicate; phosphates such as calcium hydrogen phosphate; carbonic acid carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (such as stearic acid, metal salts of stearate such as calcium stearate, magnesium stearate; talc; Colloidal silica; waxes such as beeswax and gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid
  • cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, internally cross-linked sodium carboxymethylcellulose; carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone) emulsifiers (e.g. colloidal clays such as bentonite, vegum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; sodium lauryl sulfate, calcium stearate) anionic surfactants such as; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid ester.
  • colloidal clays such as bentonite, vegum
  • metal hydroxides such as magnesium hydroxide, aluminum hydroxide
  • sodium lauryl sulfate calcium stearate
  • anionic surfactants such as; cationic surfact
  • stabilizers paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; .
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.
  • the medicament of the present invention may further contain a nucleic acid introduction reagent in order to promote the introduction of the nucleic acid into target cells.
  • a nucleic acid introduction reagent include calcium chloride, Calcium enrichment reagent, atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, Alternatively, a cationic lipid such as poly(ethyleneimine) (PEI) can be used.
  • PEI poly(ethyleneimine)
  • the medicament of the present invention may be a pharmaceutical composition in which a KLHL32 gene expression inhibitor is encapsulated in a liposome.
  • Liposomes are microscopic closed vesicles that have an internal phase surrounded by one or more lipid bilayers, and can typically retain water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer.
  • encapsulation means that the KLHL32 gene expression inhibitor may be retained in the internal phase of the liposome or within the lipid bilayer.
  • the liposome used in the present invention may be a monolayer or a multilayer, and the particle size can be appropriately selected within the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering the delivery performance to the target tissue, the particle size is, for example, 200 nm or less, preferably 100 nm or less.
  • Methods for encapsulating water-soluble compounds such as nucleic acids in liposomes include the lipid film method (vortex method), reversed-phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method.
  • the method is not limited, and any known method can be selected as appropriate.
  • the dosage of the pharmaceutical of the present invention varies depending on the purpose of administration, the method of administration, the type and severity of the target disease, and the circumstances of the subject (sex, age, body weight, etc.).
  • the single dose of the expression inhibitor is usually 2 nmol/kg or more and 50 nmol/kg or less, and when locally administered, the dose is 1 pmol/kg or more and 10 nmol/kg or less. desirable. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times. The dose may be increased or decreased depending on the symptoms.
  • the drug of the present invention is used in combination with other preventive or therapeutic drugs (hereinafter sometimes referred to as "existing drugs") for neurodegenerative diseases (e.g., aducanumab, donepezil, rivastigmine, galantamine, donepezil, memantine, etc.). You can also do that. Therefore, in one embodiment of the present invention, a preventive or therapeutic drug for neurodegenerative diseases is provided, which contains a drug that suppresses the expression of the KLHL32 gene and one or more existing drugs.
  • existing drugs for neurodegenerative diseases
  • such a combination drug When used as a combination drug, such a combination drug can be formulated with a KLHL32 gene expression inhibitor and administered as a single formulation, or alternatively, it can be formulated separately from the KLHL32 gene expression inhibitor. It can also be administered (for example, as a kit) by the same or different route as the medicament of the invention, either simultaneously or at a staggered time. Further, the dosage of these combined drugs may be the amount normally used when the drugs are administered alone, or may be reduced from the amount normally used.
  • the present invention provides a method for screening for preventive or therapeutic agents for neurodegenerative diseases (hereinafter sometimes referred to as "Screening method 1 of the present invention"), which includes the following steps.
  • a step of bringing a test substance into contact with cells expressing the KLHL32 gene and (2) A step of selecting a test substance that reduces the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases.
  • the present invention provides a method for screening for preventive or therapeutic agents for neurodegenerative diseases (hereinafter sometimes referred to as "Screening method 2 of the present invention"), which includes the following steps. .
  • screening method of the present invention may be used to include screening method 1 of the present invention and screening method 2 of the present invention.
  • the cells to be contacted with the test substance are not particularly limited as long as they are cells that express the KLHL32 gene or cells that will express the KLHL32 gene by culturing, etc.; cultured cells may be used; , mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), or cells within a mammal.
  • the cells used in the present invention include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; (3) Nerve cells, lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain Examples include differentiated cells such as cells, lung cells, kidney cells, and adipocytes. Among these, nerve cells are preferred, and human nerve cells are more preferred.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells
  • tissue progenitor cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells
  • Nerve cells lymphocytes
  • the nerve cells used in the present invention are nerve cell-specific cells selected from the group consisting of ⁇ -III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2). These cells express at least one marker gene and have ⁇ -III tubulin-positive processes (ie, neurites).
  • the cells include model cells for neurodegenerative diseases.
  • Model cells for neurodegenerative diseases include, for example, cells derived from patients with neurodegenerative diseases, cells derived from model animals for neurodegenerative diseases, and cells induced to differentiate from pluripotent stem cells that have mutations in genes that cause neurodegenerative diseases. Examples include cells.
  • the above-mentioned pluripotent stem cells that have mutations in genes that cause neurodegenerative diseases are induced pluripotent stem cells (iPS cells) that are established by a method known per se using somatic cells collected from patients with neurodegenerative diseases. It is preferable that there be.
  • Pluripotent stem cells can be differentiated into any cells (preferably nerve cells) by a method known per se.
  • genes that cause neurodegenerative diseases, and mutants of the genes the contents described in the above "1.
  • Preventive or therapeutic agents for neurodegenerative diseases are cited.
  • iPS cells can be created by introducing specific reprogramming factors into somatic cells in the form of DNA or protein.
  • genes included in the reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, and ECAT15.
  • -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc., and these initialization factors may be used alone or in combination.
  • Combinations of reprogramming factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091 659, WO2009/101084, WO2009/ 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO2010/0339 06, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO201 0/
  • pluripotent stem cells such as iPS cells into specific cells
  • various known differentiation inducing methods can be selected and used as appropriate.
  • a method for differentiating into nerve cells (1) Hester As described in M.E. et al., Mol. Therapy, 19:1905-1912 (2011), three types of (motile) A method for differentiating into motor neurons by expressing neuronal cell lineage-specific transcription factors (Ngn2, Lhx3, and Isl1), (2) described in WO 2014/148646, in which the above three types are applied to pluripotent stem cells.
  • Examples of Alzheimer's disease model animals include animals in which mutations have been introduced in one or more genes selected from the group consisting of APP, PSEN1, and PSEN2, or animals in which mutants of these genes have been introduced externally. More specifically, (1) PSEN1 transgenic mice (mouse expressing exon 9 deletion (PSEN1dE9) under the control of mouse PrP promoter, Jankowsky J.L. et al., Hum Mol Genet. 13(2): 159-70 (2004)), (2) PSEN2 transgenic mouse (mice expressing human PSEN2 mutant (N141I) under the control of the ubiquitous CMV early enhancer and chicken ⁇ -actin promoter, Oyama F. et al., J. Neurochem.
  • mutant SOD1 e.g., one or more mutations selected from the group consisting of A4V, G37R, G41D, H46R, G85R, D90A, G93A, G93S, I112T, I113T, L114F and S134N is introduced.
  • Examples include transgenic mice obtained by Other examples include tauopathy model animals in which a mutation has been introduced into the MAPT gene, or a variant of this gene has been introduced externally. More specifically, examples include tauopathy model mice into which one or more mutant MAPT genes selected from the group consisting of G272V, N297K, P301L, P301S, V337M and R406W have been introduced.
  • the model animals mentioned here are just examples, and neurodegeneration models other than these can also be used in the screening method of the present invention.
  • the expression level of the KLHL32 gene (ie, the expression level of the KLHL32 transcript or the expression level of the KLHL32 protein) can be used as an indicator. It is known that these expression levels, which serve as indicators, are the expression levels in cells before contact with the test substance, the expression levels in cells that have not been contacted with the test substance, or that no preventive or therapeutic effect on neurodegenerative diseases has been observed. If the amount is low compared to the current amount in cells that have been contacted with a certain substance (control substance), the test substance has decreased the expression level of the KLHL32 gene, or the test substance has suppressed the increase in the expression level of the KLHL32 gene. It can be evaluated that
  • the expression level of the KLHL32 transcript can be detected or quantified by a known method using, for example, the transcript extracted from cells, and examples of such methods include RT-qPCR and digital PCR.
  • RT-qPCR can be performed using a well-known method.
  • cDNA is synthesized using reverse transcriptase using total RNA as a template, and a set of primers specific to the target gene, DNA polymerase, Furthermore, it can be carried out by performing PCR in the presence of a dye or probe (eg, TaqMan® probe, etc.) that can function as a DNA intercalator for quantifying the expression level.
  • a dye or probe eg, TaqMan® probe, etc.
  • the primers used for RT-qPCR can be a set of primers that are present on the synthesized cDNA and are specific to the gene to be amplified.
  • the primers have various characteristics such as amplification size (e.g., preferably 80-150 bp), primer size (e.g., 17-25 bases), GC content (e.g., 40-60%), 3'-end sequence (e.g., , use G or C as the base at the 3' end, avoid primers with too much GC content near the 3' end), sequence gaps (e.g., avoid sequence repeats), sequence complementarity.
  • Tm value (For example, make sure that there are no more than 3 bases complementary within the upstream primer or between primers), Tm value (For example, make sure that the Tm values of the upstream and downstream primers are the same.
  • the Tm value is 2(A+T)+4(G +C)) etc., and can be designed using primer design software well known to those skilled in the art. Design of PCR primers can also be requested from Applied Biosystems Inc., etc.
  • the expression level of KLHL32 protein can be detected or quantified by known methods. For example, using an antibody that specifically recognizes the KLHL32 protein, it can be detected or quantified by Western blotting, immunostaining, enzyme immunoassay (e.g., EIA, ELISA), etc. It is not limited to these methods as long as it can be detected or quantified.
  • an antibody that specifically recognizes the KLHL32 protein it can be detected or quantified by Western blotting, immunostaining, enzyme immunoassay (e.g., EIA, ELISA), etc. It is not limited to these methods as long as it can be detected or quantified.
  • Test substances used in the screening method of the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, Examples include synthetic low-molecular compounds and natural compounds.
  • the test substance can also be used in (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound” library methods, and (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography selection.
  • Contact between the test substance and cells can be achieved by adding the test substance to the culture medium or buffer containing the cells in the case of cultured cells, or by adding the test substance to cells in mammals, such as by adding the test substance to non-human mammals. It can be carried out by administering to animals orally or parenterally.
  • the amount added to a medium etc. and the amount administered to mammals can be appropriately selected depending on the test substance.
  • the contact between the test substance and the cells is not particularly limited, but examples include, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more.
  • the concentration of the added test substance can be adjusted as appropriate depending on the type of compound (solubility, toxicity, etc.).
  • the cell culture medium used when bringing the test substance into contact with cells is not particularly limited as long as it is a medium that can culture the cells, and may be a basic medium, but preferably (hereinafter sometimes referred to as "neural differentiation induction medium").
  • a neuronal differentiation induction medium can be prepared by adding neurotrophic factors to a basal medium supplemented with neurotrophic factors.
  • Neurotrophic factors are membrane receptor ligands that play an important role in maintaining the survival and function of nerve cells, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT -3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) and LIF.
  • preferred neurotrophic factors in the present invention are GDNF, BDNF, and/or NT-3.
  • Examples of the basic medium include Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 (F12).
  • Media Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium medium (Lifetechnologies), and mixed media thereof are included.
  • the basal medium may contain serum or may be serum-free.
  • the medium may contain, for example, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, May contain one or more serum replacements such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential It may also contain one or more substances such as amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine.
  • KSR Knockout Serum Replacement
  • the culture temperature at which the test substance and cells are brought into contact is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed in an atmosphere containing CO2 , with a CO2 concentration of: Preferably it is about 2-5%.
  • Biomarkers for diagnosing neurodegenerative diseases and their uses As shown in the examples below, when autopsy brains of AD patients were analyzed, expression of KLHL32 was found to be specific to areas where ⁇ -amyloid accumulates. In a mouse model of Alzheimer's disease, it was confirmed that it is expressed in neurons prior to caspase-3 activation. Therefore, KLHL32 protein and KLHL32 transcript can be used as a biomarker for diagnosing neurodegenerative diseases (hereinafter sometimes referred to as "biomarker of the present invention").
  • KLHL32 protein is known to have multiple isoforms (i.e., isoforms a to o, X1 to X3), but unless otherwise specified, the term "KLHL32 protein” will be used to include all isoforms. Use terminology. Among the isoforms of KLHL32 protein, full-length KLHL32 protein (isoform a in the case of human KLHL32 protein) is preferred as a biomarker for diagnosing neurodegenerative diseases. Therefore, regarding the KLHL32 transcript, unless otherwise specified, the term “KLHL32 transcript” is used to include all isoforms. Among the isoforms of KLHL32 transcripts, KLHL32 mRNA, which encodes the full-length KLHL32 protein, is preferred as a biomarker for diagnosing neurodegenerative diseases.
  • the KLHL32 protein used as a biomarker of the present invention is a known protein, and in the case of humans, NCBI Accession No.: NP_443136.2 or NP_443136.2 (isoform a; SEQ ID NO: 3), NP_001273179.1 (isoform b).
  • the KLHL32 protein may be an amino acid sequence represented by any of the NCBI Accession Nos. It may be a protein containing an amino acid sequence substantially the same as these, and the KLHL32 protein of an animal other than human It may also be a protein encoded by an ortholog of a gene.
  • amino acid sequences that are substantially identical to the amino acid sequences represented by any of the above NCBI Accession Nos. (for example, the sequence shown in SEQ ID NO: 3) include, for example, 60% or more, preferably 70%, of these amino acid sequences.
  • similarity herein refers to the optimal alignment of two amino acid sequences using a mathematical algorithm known in the art (preferably, the algorithm refers to the ratio (%) of identical and similar amino acid residues to all overlapping amino acid residues (in which the introduction of a gap in one or both may be considered).
  • Similar amino acids means amino acids similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn ), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is predicted that such a substitution with a similar amino acid will not result in a change in the protein phenotype (ie, it is a conservative amino acid substitution).
  • KLHL32 protein can be produced according to known protein synthesis methods, such as solid phase synthesis and liquid phase synthesis.
  • the obtained protein can be purified and isolated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and a combination thereof.
  • it may be isolated and purified from a biological sample by a method known per se.
  • the KLHL32 protein can also be produced by culturing a transformant containing a nucleic acid encoding it, and separating and purifying the protein from the resulting culture.
  • a nucleic acid may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA.
  • the nucleic acid may be double-stranded or single-stranded.
  • amino acid sequence that is substantially the same as the amino acid sequence represented by any of the above NCBI Accession No. is one or two or more (preferably about 1 to 100, preferably 1 or more) of these amino acid sequences.
  • the KLHL32 transcription product is a known transcription product, and includes, for example, RNA encoding the above-mentioned KLHL32 protein.
  • RNA encoding isoform a shown in SEQ ID NO: 1 or 2, but also NM_001286250.2 (RNA encoding isoform b), NM_001286251.2 (isoform c NM_001286252.2 (RNA encoding isoform d), NM_001286254.3 (RNA encoding isoform e), NM_001323253.2 (RNA encoding isoform RNA encoding form g), NM_001323255.2 (RNA encoding isoform h), NM_001323256.2 (RNA encoding isoform i), NM_001323257.2 (RNA encoding isoform j), NM_001323258.2 (RNA encoding isoform k), NM_001323260.2 (RNA encoding isoform a shown
  • the KLHL32 transcript may be any nucleic acid having the above nucleotide sequence (T should be read as U), and any nucleotide sequence that is the same or substantially the same as these nucleotide sequences. It may be a nucleic acid containing. Alternatively, it may be a transcription product transcribed from an ortholog of the human KLHL32 gene of an animal other than humans.
  • Nucleic acids containing base sequences that are substantially identical to the above base sequences include, for example, 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and even more Examples include nucleic acids that contain a base sequence that preferably has an identity of 95% or more, most preferably 98% or more, and encode a protein that has substantially the same activity as the KLHL32 protein.
  • the KLHL32 transcription product can be obtained, for example, by isolating and purifying a biological sample containing the transcription product by a method known per se.
  • the present invention provides a method for diagnosing whether a subject has a neurodegenerative disease (e.g., diagnosis, , judgment, differentiation, testing, etc.), methods to assist in the diagnosis, etc., or methods to analyze the possibility or probability of neurodegenerative diseases (e.g., analysis, evaluation, calculation, etc.) may be collectively referred to as "the methods of the present invention”).
  • the number of biomarkers to be detected may be one or two or more (for example, both KLHL32 protein and KLHL32 mRNA may be detected).
  • "assisting diagnosis, etc.” refers to providing information that serves as an indicator for determining whether a subject has a neurodegenerative disease, and is a medical procedure.
  • detecting a biomarker refers to the expression of the KLHL32 gene observed in at least a part of the subject's brain (e.g., senile plaques, degenerated neurites, etc.) or in a sample derived from the subject ( That is, it includes not only determining whether a protein or transcription product is present in an amount exceeding the detection limit of the detection method, but also measuring (quantifying) its expression level.
  • Subjects that can be tested in the method of the present invention may be animals other than humans.
  • animals suspected of having neurodegenerative diseases are mentioned.
  • types of animals include mammals (e.g., humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, rats, etc.), birds (e.g., chickens, etc.) Examples include.
  • it is a mammal, more preferably a human.
  • Samples derived from the subject include, for example, blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal discharge, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, lymph fluid, etc. From a clinical point of view, blood, serum, plasma and cerebrospinal fluid are preferred, and serum and plasma are more preferred.
  • serum and plasma can be prepared by collecting blood from a subject according to a conventional method and separating the humoral components
  • cerebrospinal fluid can be prepared by It can be collected by known means such as puncture.
  • RNA e.g., total RNA, mRNA
  • the method of the invention comprises detecting a KLHL32 transcript using a nucleic acid probe or a nucleic acid primer, each of which is capable of specifically recognizing the KLHL32 transcript.
  • RNA fractions can be performed using known methods such as guanidine-CsCl ultracentrifugation and AGPC methods. Highly pure total RNA can also be rapidly and easily prepared from a specimen. Examples of methods for detecting KLHL32 transcripts in RNA fractions include methods using hybridization (Northern blot, dot blot, etc.), or methods using PCR (RT-PCR, competitive PCR, real-time PCR, etc.). can be mentioned. Quantitative PCR methods such as competitive PCR and real-time PCR are preferred because expression can be detected quickly and easily from a trace amount of sample.
  • KLHL32 transcripts can be performed, for example, using a nucleic acid probe that can specifically recognize KLHL32 transcripts.
  • the nucleic acid probe may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA.
  • the nucleic acid used as a probe may be double-stranded or single-stranded. If it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single strand, one containing an antisense strand sequence can be used.
  • the above nucleic acid probe can be obtained by chemically synthesizing it using a commercially available automatic DNA/RNA synthesizer or the like. Furthermore, the nucleic acid probe is preferably labeled with a labeling agent to enable detection of the target nucleic acid.
  • Detection of KLHL32 protein in a sample derived from a subject can be investigated by preparing a protein fraction from the sample and detecting or quantifying the translation product of the gene (i.e., KLHL32 protein) contained in the fraction. . Detection or quantification of these proteins can be performed by immunoassays (e.g. ELISA, FIA, RIA, Western blotting, etc.) using antibodies that specifically recognize each protein, but preferably immunoassays A method using a chemical measurement method, particularly ELISA, is more preferred.
  • immunoassays e.g. ELISA, FIA, RIA, Western blotting, etc.
  • Antibodies that can specifically recognize KLHL32 protein can be produced by existing general production methods using these proteins or partial peptides having epitopes as immunogens.
  • antibodies include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), chimeric antibodies that can be produced using genetic recombination technology, humanized antibodies, and single chain antibodies, and their binding properties. This includes, but is not limited to, fragments.
  • the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof.
  • the binding fragment refers to a partial region of the above-mentioned antibody that has specific binding activity, and specifically includes F(ab') 2 , Fab', Fab, Fv, sFv, dsFv, sdAb, etc. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).
  • the class of antibodies is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. Preferably, it is IgG or IgM, and considering ease of purification, etc., IgG is more preferable. In the present invention, it is also preferable to use commercially available antibodies or kits, arrays, etc. containing the antibodies as antibodies that can specifically recognize the KLHL32 protein.
  • Detection of a biomarker in a subject can also be performed using, for example, an antibody labeled directly or indirectly with a radionuclide.
  • radionuclides include positron-emitting nuclides (e.g. 68 Ga, 64 Cu, 86 Y, 89 Zr, etc.) that can be used in PET (Positron Emission Tomography) examinations, and SPECT (Single Photon Emission Computed Tomography) examinations.
  • Examples include nuclides that emit gamma rays (e.g.
  • 111 In, etc. that can be used for Preparation of an antibody labeled with a radionuclide and detection of a biomarker using the antibody can be performed by a method known per se (for example, the method described in WO2013/157102, WO2021/075544, etc.).
  • the antibodies are encapsulated in microcapsules (microcapsules of hydroxymethyl cellulose, gelatin, polymethyl methacrylate, etc.) and colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, etc.). ) ("Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.).
  • a functional peptide such as a cell membrane-penetrating peptide (eg, TAT peptide, a modified version of the peptide, etc.) may be added to the antibody.
  • the amount of the biomarker of the present invention in a healthy person or a sample derived from a healthy person (hereinafter sometimes referred to as a "control sample") and a subject targeted by the present invention or a sample derived from a subject is determined. This can be done by quantifying and comparing the amounts of both. Alternatively, it may be performed by comparing the amount of the biomarker with a reference value. As the "reference value" used in the present invention, the amount of the biomarker of the present invention in a control sample may be used.
  • a value preset from the quantitative value of the biomarker in the control sample may be used.
  • the reference value may be, for example, the average value or the mode of the measured values of a plurality of individuals, with the plurality of individuals as a control group.
  • a "healthy person” means a person who has not been definitively diagnosed with a neurodegenerative disease.
  • the above reference value may be a cutoff value.
  • the "cutoff value” is a value that satisfies both high diagnostic sensitivity (prevalence rate) and high diagnostic specificity (predictability rate) when a disease is determined based on the value. For example, a value that shows a high positive rate in a neurodegenerative disease patient group and a high negative rate in a healthy person group can be set as a cutoff value.
  • Methods for calculating cutoff values are well known in the field. For example, the amount of the biomarker of the present invention in serum collected from neurodegenerative disease patients and healthy subjects is quantified, the diagnostic sensitivity and diagnostic specificity of the quantified values are determined, and based on these values, commercially available Create a ROC (Receiver Operating Characteristic) curve using analysis software. Then, the value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can be determined, and this value can be used as the cutoff value.
  • ROC Receiveiver Operating Characteristic
  • the biomarker of the present invention is higher in the subject or sample derived from the subject than in a healthy person or a control sample, or if it is higher than the above reference value.
  • the subject can be diagnosed with a neurodegenerative disease, and the biomarker of the present invention is at the same level or lower than that of a healthy person or a control sample, or the above standard value If it is below, it can be diagnosed that the subject does not have a neurodegenerative disease.
  • the biomarker of the present invention is detected in the subject or a sample derived from the subject, or if the level is higher than that of a healthy person or a control sample, or if the above-mentioned If the value is above the standard value, it can be analyzed that there is a high possibility or probability that the subject has a neurodegenerative disease, and if the value is the same or lower than that of healthy subjects or control samples, or If it is less than the above reference value, it can be analyzed that the possibility or probability that the subject has a neurodegenerative disease is low.
  • a preventive or therapeutic drug for the neurodegenerative disease to be administered to the test subject is selected or determined based on the results of the diagnosis, etc.
  • Neurodegenerative diseases can be prevented or treated by administering a therapeutically effective amount of a prophylactic or therapeutic agent to the subject. That is, a method for preventing or treating neurodegenerative diseases is also provided, which includes the following steps (i) to (iii).
  • step (i) detecting the biomarker of the present invention in a subject or a sample derived from a subject; (ii) diagnosing whether the subject has a neurodegenerative disease based on the results of step (i); (iii) A step of administering a preventive or therapeutic agent for a neurodegenerative disease to a subject diagnosed as having a neurodegenerative disease according to (ii).
  • the preventive or therapeutic agents for the above neurodegenerative diseases include the pharmaceutical of the present invention, 1.
  • Examples include existing drugs described in , and combinations thereof.
  • compositions for oral administration include solid or liquid dosage forms, in particular tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. formulations, emulsions, suspensions, etc.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may also be included.
  • the dosage of the prophylactic or therapeutic drug can be appropriately determined depending on various conditions such as the type of compound, the symptoms of the subject, age, body weight, and drug acceptability.
  • the present invention further provides a kit for diagnosing neurodegenerative diseases (hereinafter referred to as "the present invention (sometimes referred to as “diagnostic kit”).
  • the present invention sometimes referred to as “diagnostic kit”
  • the present invention for diagnosing neurodegenerative diseases
  • the present invention for definitions, specific examples, detection methods, etc. of the KLHL32 protein, antibodies that specifically recognize the protein, KLHL32 transcripts, and nucleic acid probes or primers that specifically recognize the transcripts, please refer to "3. Neurodegenerative Diseases" above. The contents described in “Diagnostic Biomarkers and Their Uses” are incorporated.
  • the diagnostic kit of the present invention is a kit that can detect the presence or absence of the biomarker of the present invention in a sample by simply bringing the sample into contact with a substrate using the ELISA method (hereinafter referred to as "the diagnostic kit of the present invention”).
  • the simple kit of the present invention includes a substrate on which an antibody that specifically recognizes the KLHL32 protein (hereinafter also referred to as "first antibody”) is immobilized.
  • the simple kit of the present invention also includes an antibody (hereinafter also referred to as "second antibody”) that specifically recognizes the KLHL32 protein, which is different from the above-mentioned antibody (typically, the antibody is labeled. It is preferable that the antibody is an antibody that has been used in the present invention.
  • the biomarker of the present invention can be detected by a sandwich ELISA method.
  • nucleic acid probe or nucleic acid primer also simply referred to as "nucleic acid”
  • these nucleic acids can also be provided as a solid in a dry state or in an alcohol-precipitated state. However, it can also be provided in a state dissolved in water or an appropriate buffer (eg, TE buffer, etc.).
  • the nucleic acid can be provided in a state that has been labeled in advance with any of the above-mentioned labeling substances, or it can be provided separately with each labeling substance and labeled before use.
  • the nucleic acid can also be provided in a state immobilized (also referred to as supported or immobilized) on a suitable substrate.
  • the substrate include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like.
  • an immobilization means a functional group such as an amino group, an aldehyde group, an SH group, or a biotin is introduced into the nucleic acid in advance, and a functional group (e.g., an aldehyde group) that can react with the nucleic acid is also placed on the substrate.
  • electrostatic bonding can be achieved by coating the substrate with polycations. Examples include, but are not limited to, methods such as immobilizing nucleic acids using a method of immobilizing nucleic acids.
  • the diagnostic kit of the present invention may contain other substances necessary for the reaction for detecting the expression of the biomarker of the present invention. These other substances may be provided in coexistence with the nucleic acid, antibody, etc., or may be provided together with separate reagents, as long as they do not adversely affect the reaction.
  • the reaction for detecting the expression of the biomarker of the present invention is PCR
  • examples of the other substances include reaction buffer, dNTPs, thermostable DNA polymerase, and the like.
  • a competitor nucleic acid, a fluorescent reagent the above-mentioned intercalator, fluorescent probe, etc. can be further included.
  • the reaction for detecting the expression of the biomarker of the present invention is an antigen-antibody reaction
  • the other substances include, for example, a reaction buffer, a competitor antibody, a labeled secondary antibody (for example, if the primary antibody is In the case of rabbit antibodies, examples include mouse anti-rabbit IgG labeled with peroxidase or alkaline phosphatase, etc.), blocking solutions, and ELISA plates.
  • the determination kit of the present invention may include an instruction manual that describes how to use the kit and reagents, disease determination criteria, and the like.
  • the above-mentioned determination kit may contain one or more types of biomarkers of the present invention, for example, for use as a positive control.
  • iPSC iPSCs
  • PBMCs peripheral blood mononuclear cells
  • human cDNA for reprogramming factors is an episomal vector (SOX2, KLF4, OCT4, L-MYC, LIN28, p53 dominant negative).
  • SOX2, KLF4, OCT4, L-MYC, LIN28, p53 dominant negative was used to transduce human PBMC.
  • PBMCs were collected and replated onto iMatrix-coated dishes. The next day, the medium was replaced with StemFit AK03 or AK02N. Thereafter, the medium was replaced every other day. Twenty days after transduction, iPSC colonies were picked.
  • the established PBMC-derived iPSCs were expanded and cultured (Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)).
  • iN-iPSCs Preparation of iN-iPSCs
  • NNN2 Human neurogenin 2
  • tetO tetracycline-inducible promoter
  • Thermo Fisher Scientific Inc., Waltham, MA Thermo Fisher Scientific Inc., Waltham, MA.
  • the piggyBac vector containing tetO::NGN2 was used (Kim et al., 2016).
  • Activated caspase-3 in apoptotic neurons was detected using a fluorogenic substrate consisting of the 4-amino acid peptide DEVD conjugated with a nucleic acid-binding dye (CellEvent Caspase-3/7 Green Detection Reagent, Thermo Fisher) did.
  • the reagent is non-fluorescent in nature because the DEVD peptide inhibits the dye's ability to bind to DNA.
  • the fluorescent dye can bind to the DNA, resulting in a fluorogenic response.
  • I resized the cropped image to 256 x 256.
  • a training image of size 256 x 256 was randomly flipped horizontally with a probability of 50%, and a region of 224 x 224 pixels was randomly cropped from the image.
  • the 224 x 224 cropped region was finally used as input to the network.
  • 224 x 224 images were cropped without flipping.
  • the logarithmic scale concentration of Z-VAD-FMK on the micromolar scale was defined as the target.
  • Three independent batches of experiments were performed, and two 384-well plates were used in each batch. Images from 0 to 12 hours after DMSO/Z-VAD-FMK processing from two experimental batches (768 images for each class) were used for training and validation (training dataset), and after processing from the remaining experimental batches. Images at specific times were used for testing (128 images for each class; test dataset). The same cropping size as determined in the cell fate classification analysis was also employed. For evaluation of images of cells treated with other compounds and CRISPR-modified cells, all wells of the plate were used as the test data set.
  • the network was pre-trained on the ImageNet dataset, which has 1.2 million labeled images of objects in 1,000 categories.
  • the number of input nodes was adjusted according to the image size.
  • the weights of all layers were reoptimized. Mean squared error (regression analysis) was used as the loss function. Errors were backpropagated through the network and weights were optimized by stochastic gradient descent (SGD) using mini-batches.
  • SGD stochastic gradient descent
  • the network parameters at the epoch when the network showed the highest classification accuracy on the training dataset were used for testing. We rotated the training and testing experimental batches and calculated the average of the results of the three trials.
  • GradCAM provides gradient-based class activation maps that allow identification of localized regions where CNNs are utilized to make decisions (Selvaraju, RR et al., arXiv:1610.02391v1 (2016)). They applied this method to a trained CNN to obtain a map that visualizes the CNN's focus on an image when the network determines that the image has a high prediction score. For a more detailed interpretation, we also applied Guided GradCAM to the CNN, which is a combination of guided error backpropagation and GradCAM (Springenberg et al., 2014).
  • Example 1 In vitro prediction of degenerating neurons using a deep learning-based iPSC model
  • the purpose of this example was to predict iPSC-derived neurons using only phase contrast images, as shown in the figure.
  • the aim was to develop a deep learning-based iPSC model (Deep-i model) that predicts the cell fate of cells (Figure 1A).
  • Neuronal cell death is the most characteristic histopathological feature of neurodegenerative diseases, including AD, and activated caspase-3 is a molecular marker of neuronal cell death in the brains of AD patients (Christie LA et al., Neurobiol. Dis. 26, 165-173 (2007); Louneva N. et al., Am. J. Pathol.
  • the performance of the trained network was evaluated on the test dataset.
  • the network was trained using the main part of the training dataset and the loss was evaluated on the remaining training dataset at each epoch. The best learning state of the network across training epochs was saved.
  • the tests used images from independent batches of the two batches used in the training step.
  • the test data set was images collected 24 hours after the start of tracking, and caspase-3 signals were not significantly different between DMSO and Z-VAD-FMK conditions ( Figure 1E).
  • the trained network produced DL prediction scores from test images.
  • the DL prediction score showed a linear correlation with the actual Z-VAD-FMK concentration 24 hours after the start of the assay (Fig. 2D).
  • BSI IV a ⁇ -secretase inhibitor
  • the DL prediction score increased in a dose-dependent manner of BSI IV, which shows a similar trend with mild inhibition of cell death compared to the results of Z-VAD-FMK (Fig. 2H).
  • necrostatin-1 which is a potent inhibitor of RIP1 kinase and suppresses necrotic cell death.
  • necrostatin-1 partially rescued neurodegeneration and showed higher DL prediction scores at intermediate to high concentration conditions ( Figures 2I and 2J).
  • necrostatin-1 analogs that can suppress neurodegeneration only under high concentration conditions (Takahashi N. et al., Cell Death Dis.
  • Example 2 Identification of molecular markers of cells programmed for neurodegeneration by single-cell RNA-sequencing analysis
  • the Deep-i model can detect hidden neurodegeneration during the process leading up to neuronal cell death. was completed. However, while the Deep-i model can detect which cells begin to degenerate, it cannot detect the molecular signature of the cells.
  • To find the molecular signature of neurons programmed for neurodegeneration in vitro we simultaneously performed single-cell RNA-sequencing analysis. Twelve hours after the start of the assay (Time 0), an increase in caspase-3 activation was not yet detected even in the negative control group without the addition of Z-VAD-FMK.
  • Cluster 12 contains 1.42% cells in the DMSO control group, 1.22% cells in the Z-VAD-FMK 1 ⁇ M treated group, and 0.75% cells in the Z-VAD-FMK 25 ⁇ M treated group, respectively ( Figures 6C and 2B). This result indicates that the population of neurons in cluster 12 is one that is destined to undergo neurodegeneration after a few hours.
  • Table 1 shows the target sequences of the siRNAs used. A mixture of four types of siRNA was used for each target gene (including non-targeting control).
  • Z-VAD-FMK Z-Val-Ala-Asp(OMe)-CH 2 F
  • FIGGS. 6F and 6G The cell bodies of KLHL32-positive cells were enlarged and had a rounded shape, similar to that of dying neurons.
  • the positive rate for KLHL32 was approximately a few percent ( Figure 6G), which was consistent with the abundance of cluster 12 in single-cell RNA-seq analysis. Based on these results and analysis at the single cell level, KLHL32 was identified as an early marker before cell death fate.
  • Example 3 Identification of preferential accumulation sites of neurodegeneratively programmed cells in a mouse model of Alzheimer's disease (AD) or postmortem AD patient brain tissue. Provide marker information to know what is happening, when and where. Next, we investigated whether the identified marker, KLHL32, is found in the process of neurodegeneration in the brain of an AD mouse model or in the brain tissue of postmortem AD patients.
  • AD Alzheimer's disease
  • KLHL32 the identified marker
  • KLHL-32-positive cells A quantified area of KLHL-32-positive cells was observed predominantly only in 5 ⁇ FAD mice compared to control littermates (non-transgenic mice) ( Figures 9B and 10). The distribution of KLHL32-positive cells was similar to A ⁇ pathology ( Figures 9A and 9B). On the other hand, no KLHL32-positive cells were observed in hippocampal CA1 (CA1) and entorhinal cortex (EN), where A ⁇ pathology was not observed at 3 months of age, except for subependymal astrocytes ( Figures 9A and 9B). .
  • CA1 hippocampal CA1
  • EN entorhinal cortex
  • KLHL32 is observed to be associated with AD pathology before the onset of neurodegeneration, and may be useful for early identification of cells programmed for neurodegeneration, causing cell death and subsequent brain atrophy. It became clear that it could be used as a marker.
  • KLHL32 is also distributed in the brains of AD patients.
  • Postmortem brain samples from neuropathologically diagnosed AD and healthy elderly controls were used.
  • immunopositivity for KLHL32 we found that KLHL32 was positively stained in neurons and astrocytes in the temporal cortex (Figure 9C).
  • KLHL32 is distributed in senile plaques, a neuropathological hallmark of AD, and in degenerating neurites, indicating a pre-stage of neurodegeneration. (Figure 9D).
  • Drugs that suppress KLHL32 gene expression are useful for preventing or treating neurodegenerative diseases. Furthermore, a drug that suppresses the expression of the KLHL32 gene is useful as a combination drug for the treatment of neurodegenerative diseases.

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Abstract

The present invention provides a preventive or therapeutic agent for neurodegenerative disease, the agent containing an agent for suppressing KLHL32 gene expression. The present invention also provides a screening method for a preventive or therapeutic agent for neurodegenerative disease, the method comprising: (1) a step for bringing a test substance into contact with cells that express the KLHL32 gene; and (2) a step for selecting, as a candidate for the preventive or therapeutic agent for neurodegenerative disease, a test substance which has caused a reduction in KLHL32 gene expression.

Description

神経変性疾患の予防または治療薬Drugs for preventing or treating neurodegenerative diseases
　本発明は、KLHL32遺伝子の発現抑制薬を含有してなる、神経変性疾患の予防または治療薬に関する。 The present invention relates to a preventive or therapeutic drug for neurodegenerative diseases, which contains a drug that suppresses the expression of the KLHL32 gene.
　アルツハイマー病（Alzheimer’s disease; AD）は、認知症の代表とされる脳の変性疾患であり、その患者数は増加の一途をたどっている。アルツハイマー病患者の脳神経機能低下に伴う生活の質（Quality of Life）の低下は、患者本人に加え家族など周囲に多大な影響を与え、現代社会の抱える深刻な問題となっている。2021年6月米食品医薬品局（FDA）は、アルツハイマー病治療薬の候補である「アデュカヌマブ」について、条件付きで承認を認めた。一方で、FDAは、アデュカヌマブがアルツハイマー病の症状を治療に役立つかどうかを確認するための追跡調査を行うことを、開発者の１社であるバイオジェン社に要求している。 Alzheimer's disease (AD) is a degenerative brain disease that is a typical form of dementia, and the number of patients is steadily increasing. The decline in the quality of life of Alzheimer's disease patients due to decline in cranial nerve function has a significant impact not only on the patients themselves but also on their families and other people around them, making it a serious problem facing modern society. In June 2021, the US Food and Drug Administration (FDA) granted conditional approval for aducanumab, a candidate drug for the treatment of Alzheimer's disease. Meanwhile, the FDA is requiring one of its developers, Biogen, to conduct follow-up studies to see if aducanumab can help treat symptoms of Alzheimer's disease.
　アルツハイマー病同様に、進行性の神経変性障害を示す疾患として、前頭側頭葉変性症（FTLD）が知られている。前頭側頭葉変性症は、アルツハイマー病に次いで２番目若しくは３番目に頻度の高い早期発症型神経変性認知症であり、顕著な挙動および人格変化の症状を示し、しばしば言語機能障害が付随して起こり、これが徐々に認知障害および認知症へと発展していくこととなる。また、アルツハイマー病同様に研究が進められているが、発症機構の全貌は未だ明らかになっていない。 Like Alzheimer's disease, frontotemporal lobar degeneration (FTLD) is known as a disease that exhibits a progressive neurodegenerative disorder. Frontotemporal lobar degeneration is the second or third most common early-onset neurodegenerative dementia after Alzheimer's disease, with symptoms of marked behavioral and personality changes, often accompanied by language impairment. This gradually develops into cognitive impairment and dementia. Also, although research is progressing in the same way as Alzheimer's disease, the full picture of the onset mechanism has not yet been clarified.
　また、パーキンソン病はアルツハイマー病と並ぶ神経変性疾患の代表的な疾患であり、患者の脳には黒質にレビー小体（Lewy body）が出現することが知られている。レビー小体とは、αシヌクレインと呼ばれる140アミノ酸残基からなるタンパク質の凝集体であり、パーキンソン病の他にレビー小体型認知症、多系統萎縮症等のようなレビー小体病と称される疾患の患者の脳にも出現することが知られており、脳内におけるαシヌクレインの蓄積を伴う疾患は、αシヌクレイノパチーと呼ばれている。αシヌクレイノパチーの進行には、αシヌクレインの凝集、すなわちαシヌクレイン線維形成が重大な役割を果たしていると考えられており、αシヌクレイン線維形成を抑制等する物質について各方面で活発に研究が進められている。 Furthermore, Parkinson's disease is a typical neurodegenerative disease along with Alzheimer's disease, and Lewy bodies are known to appear in the substantia nigra of patients' brains. Lewy bodies are aggregates of a protein called alpha-synuclein, which consists of 140 amino acid residues, and are associated with Lewy body diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. It is known that it also appears in the brains of patients with the disease, and diseases accompanied by the accumulation of α-synuclein in the brain are called α-synucleinopathy. It is believed that α-synuclein aggregation, or α-synuclein fibril formation, plays an important role in the progression of α-synucleinopathy, and active research is being conducted in various fields on substances that inhibit α-synuclein fibril formation. It is progressing.
　しかしながら、これらの神経変性症の研究は精力的に進められているが、その発症機構の全貌は明らかではなく、その根治薬はいまだ開発されていないのが現状である。アルツハイマー病を始めとする、多くの神経変性疾患の共通病態として、caspase 3の活性化を伴う神経変性が挙げられる（例えば、非特許文献１）。本発明者らは以前、深層学習と人工多能性幹細胞（iPS細胞）技術を用いて、神経変性の有無および多寡を位相差画像から予測する方法を開発した（特許文献１）。しかしながら、この予測方法のメカニズムや、深層学習を施したモデルが、神経変性を予測する際に何を指標としたかについては依然として不明なままであった。 However, although research into these neurodegenerative diseases is being vigorously pursued, the full picture of their onset mechanisms is not clear, and no cure has yet been developed. A common pathological condition of many neurodegenerative diseases including Alzheimer's disease is neurodegeneration accompanied by activation of caspase 3 (for example, Non-Patent Document 1). The present inventors previously developed a method for predicting the presence or absence and amount of neurodegeneration from phase contrast images using deep learning and induced pluripotent stem cell (iPS cell) technology (Patent Document 1). However, the mechanism behind this prediction method and the indicators used by deep learning models to predict neurodegeneration remain unclear.
国際公開第２０２０／２４１７７２号International Publication No. 2020/241772
　従って、本発明の課題は、神経変性疾患における神経症状が発露し病状が進行する時期において最も重要な神経変性死を予測し、神経変性疾患を治療するための分子標的を特定することである。そして、該特定した分子標的の発現を抑制することで、神経変性疾患を予防または治療する方法を提供することである。 Therefore, the objective of the present invention is to predict neurodegenerative death, which is most important at the time when neurological symptoms in neurodegenerative diseases are manifested and the disease progresses, and to identify molecular targets for treating neurodegenerative diseases. Another object of the present invention is to provide a method for preventing or treating neurodegenerative diseases by suppressing the expression of the identified molecular target.
　本発明者らは、深層学習を施したモデルを構築し、該モデルにより、スコアが高い細胞（カスパーゼ-3が活性化する予定の細胞である確率が高い細胞）と、スコアが低い細胞（カスパーゼ-3が活性化する予定の細胞である確率が低い細胞）について、単一細胞RNAシーケンス（single cell RNA-seq）解析を行った。その結果、スコアが高い細胞群において、カスパーゼ-3の活性化前に特異的に発現する遺伝子としてKLHL32を見出した。次に、アルツハイマー病患者の剖検脳を解析したところ、βアミロイドが蓄積している部位特異的にKLHL32の発現が認められること、アルツハイマー病マウスモデルでは、カスパーゼ-3の活性化に先行して神経細胞で発現することを確認した。さらに、アルツハイマー病の細胞モデルにおいて、siRNAによりKLHL32遺伝子の発現をノックダウンすると、カスパーゼ-3の活性化が抑制されて細胞死が抑制されることが明らかとなった。本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。 The present inventors constructed a model using deep learning, and used this model to identify cells with a high score (cells with a high probability of being caspase-3-activated cells) and cells with a low score (cells with a high probability of caspase-3 activation). Single-cell RNA-seq analysis was performed on cells with a low probability of being expected to be activated. As a result, KLHL32 was found to be a gene that is specifically expressed before caspase-3 activation in a group of cells with high scores. Next, when we analyzed the autopsy brains of Alzheimer's disease patients, we found that KLHL32 is expressed specifically in areas where β-amyloid accumulates, and that in Alzheimer's disease mouse models, the activation of caspase-3 is preceded by activation of caspase-3. It was confirmed that it was expressed in cells. Furthermore, in a cell model of Alzheimer's disease, knocking down the expression of the KLHL32 gene with siRNA suppressed caspase-3 activation and cell death. As a result of further research based on these findings, the present inventors have completed the present invention.
　すなわち、本発明は以下の通りのものである。
［１］
　KLHL32遺伝子の発現抑制薬を含有してなる、神経変性疾患の予防または治療薬。
［２］
　前記発現抑制薬が、siRNA、ヘテロ二本鎖核酸、アンチセンス核酸、shRNA、miRNA、アンチジーン核酸およびCRISPR-Casシステムからなる群から選択される、［１］に記載の予防または治療薬。
［３－１］
　前記発現抑制薬がsiRNAである、［２］に記載の予防または治療薬。
［３－２］
　前記CRISPR-CasシステムがCRISPR-dCasシステムである、［２］に記載の予防または治療薬。
［４］
　神経変性疾患が、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症（ＡＬＳ）、脊髄小脳変性症、前頭側頭葉変性症、レビー小体型認知症、多系統萎縮症、ハンチントン病、進行性核上性麻痺および大脳皮質基底核変性症からなる群から選択される、［１］～［３－２］のいずれか１つに記載の予防または治療薬。
［５］
　神経変性疾患がアルツハイマー病である、［１］～［４］のいずれか１つに記載の予防または治療薬。
［６］
　（１）KLHL32遺伝子を発現する細胞に、被験物質を接触させる工程、および
　（２）KLHL32遺伝子の発現量を減少させた被験物質を神経変性疾患の予防または治療薬の候補として選択する工程
を含む、神経変性疾患の予防または治療薬のスクリーニング方法。
［７］
　（１）細胞に、被験物質を接触させる工程、および
　（２）KLHL32遺伝子の発現量の増加を抑制した被験物質を神経変性疾患の予防または治療薬の候補として選択する工程
を含む、神経変性疾患の予防または治療薬のスクリーニング方法。
［８］
　前記細胞が神経変性疾患のモデル細胞である、［６］または［７］に記載の方法。
［９］
　前記神経変性疾患のモデル細胞が、神経変性疾患の患者由来の細胞または神経変性疾患の原因である遺伝子に変異を有する多能性幹細胞から分化誘導した細胞である、［８］に記載の方法。
［１０］
　前記細胞が神経細胞である、［６］～［９］のいずれか１つに記載の方法。
［１１］
　前記細胞がヒト細胞である、［６］～［１０］のいずれか１つに記載の方法。
［１２］
　KLHL32タンパク質またはKLHL32転写産物からなる、神経変性疾患診断用バイオマーカー。
［１３］
　被験者または該被験者由来の試料において、［１２］に記載のバイオマーカーを検出する工程を含む、該被験者が神経変性疾患であるかの診断を補助する方法。
［１４］
　KLHL32タンパク質を特異的に認識する抗体またはKLHL32転写産物を特異的に認識する核酸プローブ若しくは核酸プライマーを含む、神経変性疾患診断用キット。
［１５］
　哺乳動物に対して、KLHL32遺伝子の発現抑制薬の有効量を投与することを特徴とする、該哺乳動物における神経変性疾患の予防または治療方法。
［１６］
　神経変性疾患の予防または治療における使用のための、KLHL32遺伝子の発現抑制薬。
［１７］
　神経変性疾患の予防または治療薬の製造のための、KLHL32遺伝子の発現抑制薬の使用。
［１８］
　以下の(i)～(iii)の工程を含む、神経変性疾患の予防または治療方法。
　（i）被験者または被験者由来の試料における、［１２］に記載のバイオマーカーを検出する工程、
　（ii）前記工程(i)の結果に基づき、該被験者が神経変性疾患であるかの診断を行う工程、
　（iii）（ii）により神経変性疾患であると診断された被験者に、神経変性疾患の予防または治療薬を投与する工程
［１９］
　前記神経変性疾患の予防または治療薬が、KLHL32遺伝子の発現抑制薬、アデュカヌマブ、ドネペジル、リバスチグミン、ガランタミン、ドネペジルおよびメマンチンからなる群から選択される、［１８］に記載の方法。 That is, the present invention is as follows.
[1]
A drug for preventing or treating neurodegenerative diseases, which contains a drug that suppresses the expression of the KLHL32 gene.
[2]
The preventive or therapeutic agent according to [1], wherein the expression suppressing drug is selected from the group consisting of siRNA, heteroduplex nucleic acid, antisense nucleic acid, shRNA, miRNA, antigene nucleic acid, and CRISPR-Cas system.
[3-1]
The prophylactic or therapeutic agent according to [2], wherein the expression inhibitor is siRNA.
[3-2]
The preventive or therapeutic agent according to [2], wherein the CRISPR-Cas system is a CRISPR-dCas system.
[4]
Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, and progressive The prophylactic or therapeutic agent according to any one of [1] to [3-2], which is selected from the group consisting of supranuclear palsy and corticobasal degeneration.
[5]
The preventive or therapeutic agent according to any one of [1] to [4], wherein the neurodegenerative disease is Alzheimer's disease.
[6]
(1) A step of bringing a test substance into contact with cells expressing the KLHL32 gene; and (2) A step of selecting a test substance that reduces the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. , a screening method for preventive or therapeutic drugs for neurodegenerative diseases.
[7]
Neurodegenerative diseases, including (1) a step of contacting cells with a test substance, and (2) a step of selecting a test substance that suppresses an increase in the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. screening methods for preventive or therapeutic drugs.
[8]
The method according to [6] or [7], wherein the cells are model cells for neurodegenerative diseases.
[9]
The method according to [8], wherein the neurodegenerative disease model cell is a cell derived from a patient with a neurodegenerative disease or a cell induced to differentiate from a pluripotent stem cell having a mutation in a gene that causes the neurodegenerative disease.
[10]
The method according to any one of [6] to [9], wherein the cell is a nerve cell.
[11]
The method according to any one of [6] to [10], wherein the cells are human cells.
[12]
Biomarker for diagnosing neurodegenerative diseases consisting of KLHL32 protein or KLHL32 transcript.
[13]
A method for assisting in diagnosing whether a subject has a neurodegenerative disease, the method comprising the step of detecting the biomarker according to [12] in the subject or a sample derived from the subject.
[14]
A kit for diagnosing a neurodegenerative disease, comprising an antibody that specifically recognizes KLHL32 protein or a nucleic acid probe or primer that specifically recognizes KLHL32 transcript.
[15]
A method for preventing or treating a neurodegenerative disease in a mammal, which comprises administering to the mammal an effective amount of a drug that suppresses the expression of the KLHL32 gene.
[16]
A drug that suppresses the expression of the KLHL32 gene for use in the prevention or treatment of neurodegenerative diseases.
[17]
Use of a drug that suppresses KLHL32 gene expression for the production of a drug for preventing or treating neurodegenerative diseases.
[18]
A method for preventing or treating neurodegenerative diseases, comprising the following steps (i) to (iii).
(i) Detecting the biomarker according to [12] in a subject or a sample derived from a subject,
(ii) diagnosing whether the subject has a neurodegenerative disease based on the results of step (i);
(iii) A step of administering a preventive or therapeutic agent for a neurodegenerative disease to a subject diagnosed with a neurodegenerative disease according to (ii) [19]
The method according to [18], wherein the preventive or therapeutic agent for neurodegenerative diseases is selected from the group consisting of KLHL32 gene expression inhibitors, aducanumab, donepezil, rivastigmine, galantamine, donepezil, and memantine.
　本発明により、神経変性に大幅に先んじて神経変性を予測することが可能となり、さらには、新たな分子標的（KLHL32遺伝子）に基づく神経変性疾患の予防または治療薬、診断を補助する方法、および前記薬のスクリーニング方法が提供される。 The present invention makes it possible to predict neurodegeneration significantly in advance of neurodegeneration, and also provides preventive or therapeutic agents for neurodegenerative diseases based on a new molecular target (KLHL32 gene), methods to assist in diagnosis, and A method for screening the drug is provided.
アルツハイマー病の神経細胞は、活性化（activated）カスパーゼ-3による細胞死に向けて徐々に変性した。（A）神経変性がプログラムされた細胞を予測するためのDeep-iモデルの概略図。（B）一過性NGN2誘導による神経細胞の分化法および活性化カスパーゼ-3を検出するためのアッセイシステムの概略図。変異PSEN1（G384A）を有するアルツハイマー病の患者から樹立された人工多能性幹細胞（iPSC）を、多能性マーカーであるNANOGおよびTRA1-60により陽性に染色した（スケールバー＝100μm）。ヒトNGN2の一過性発現により分化した神経細胞は、皮質神経細胞であるMAP2およびSATB2を示した（スケールバー＝50μm）。（C）時間依存的にAD神経細胞の段階的な神経細胞死を分析するための実験計画。代表的なタイムラプス画像は、活性化カスパーゼ-3の段階的な増加と一致する、時間依存的な神経細胞の収縮を示した。スケールバー＝20μm。（D）活性化カスパーゼ-3の経時的増加は、全カスパーゼ阻害剤であるZ-VAD-FMKを用いた条件で抑制された。スケールバー＝50μm。（E）活性化カスパーゼ-3のシグナルカウント（signal count）の変化（時間=0からの変化）は、アッセイ開始（時間=0（Time 0））後30時間以上、Z-VAD-FMKありの状態（平均±標準偏差）とZ-VAD-FMKなしの状態（平均±標準偏差）の間に有意差を示した（散布図）。二元配置反復測定分散分析（Two-way repeated-measures ANOVA）は、時間の有意な効果を明らかにした（F（20、940） = 581.4、P <0.0001）。2つの異なる条件の違いは、シダックの多重比較補正（Sidak's multiple comparisons correction）を使用した事後ペアワイズ比較（post-hoc pairwise comparisons）によって分析した（* P <0.001）。略語：Z-VAD-FMK = Z-Val-Ala-Asp（OMe）-CH2F、DMSO =ジメチルスルホキシド。Neurons in Alzheimer's disease gradually degenerate toward cell death due to activated caspase-3. (A) Schematic diagram of the Deep-i model for predicting cells programmed for neurodegeneration. (B) Schematic diagram of the transient NGN2-induced neuronal differentiation method and assay system for detecting activated caspase-3. Induced pluripotent stem cells (iPSCs) established from an Alzheimer's disease patient with mutant PSEN1 (G384A) were positively stained with pluripotency markers NANOG and TRA1-60 (scale bar = 100 μm). Neurons differentiated by transient expression of human NGN2 exhibited cortical neurons MAP2 and SATB2 (scale bar = 50 μm). (C) Experimental design to analyze gradual neuronal death of AD neurons in a time-dependent manner. Representative time-lapse images showed time-dependent neuronal contraction consistent with a gradual increase in activated caspase-3. Scale bar = 20 μm. (D) The increase in activated caspase-3 over time was suppressed using the pan-caspase inhibitor Z-VAD-FMK. Scale bar = 50 μm. (E) Change in signal count of activated caspase-3 (change from time = 0) with Z-VAD-FMK over 30 hours after assay initiation (Time 0). Significant differences were shown between the condition (mean ± standard deviation) and the condition without Z-VAD-FMK (mean ± standard deviation) (scatter plot). Two-way repeated-measures ANOVA revealed a significant effect of time (F (20, 940) = 581.4, P < 0.0001). Differences between the two different conditions were analyzed by post-hoc pairwise comparisons using Sidak's multiple comparisons correction (*P<0.001). Abbreviations: Z-VAD-FMK = Z-Val-Ala-Asp(OMe) -CH2F , DMSO = dimethyl sulfoxide. Deep-iモデルは、遺伝子型の違いや化合物処理に依存して神経変性を予測した。（A）0～25μMの異なる濃度でZ-VAD-FMKを添加した場合の、活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）である。（B）予測方法の概略図。DL分析用の画像は、アッセイ開始後24時間以内に取得した。（C）トレーニング用の256 × 256、512 × 512、768 × 768、1,024 × 1,024ピクセルを含む異なるサイズ間の誤差の比較。エラーバーは、N = 3の独立した実験からの平均および標準偏差（S.D.）を表す。*は、P <0.05（一元配置分散分析（one-way ANOVA）とそれに続くテューキーの多重比較事後検定（Tukey’s multiple comparison post hoc test）を使用）を示す。（D）皮質神経細胞にゼロから25μMまでの8点の濃度でZ-VAD-FMKを投与した。CNNを、画像からZ-VAD-FMK濃度を予測するようにトレーニングした。予測濃度は、Deep-iモデルによる予測スコアとして定義した。エラーバーは、n = 128の独立したレプリケイト（replicate）からの平均および標準偏差（S.D.）を表す。Deep-iモデルによる予測スコアは、実際のZ-VAD-FMK濃度と相関していた。R二乗（決定係数）値（R-squared value）は0.987であった。（E）トレーニングの24～72時間の異なる時点での平均二乗誤差の比較。エラーバーは、N = 3の独立した実験からの平均および標準偏差（S.D.）を表す。*は、P <0.05（一元配置分散分析とそれに続くテューキーの多重比較事後検定を使用）を示す。（F）トレーニングの24～72時間の異なる時点でのR二乗の比較。エラーバーは、N = 3の独立した実験からの平均および標準偏差（S.D.）を表す。*は、P <0.05（一元配置分散分析とそれに続くテューキーの多重比較事後検定を使用）を示す。（G）PSEN1に変異を有する神経細胞と変異を有さない神経細胞の比較。CRISPR-Cas9システムによりFAD神経細胞のPSEN1の変異を修正して、修正された(corrected)PSEN1変異を除いて同じ遺伝的背景を保持する「修正された」神経細胞を準備した。Deep-iモデルによる予測スコアを、FAD神経細胞と修正された神経細胞の間で比較した。プロットされたドットは、n = 1,536の独立したレプリケイトを表す。****は、P <0.0001（一元配置分散分析とそれに続くテューキーの多重比較事後検定を使用）を示す。（H）用量依存的にAβ産生を抑制するβ-セクレターゼ阻害剤であるBSI IVの効果。Deep-iモデルによる予測スコアを、各濃度について計算した。エラーバーは、N = 3の独立した実験バッチからの平均および標準偏差（S.D.）を表す。DLスコア（Y軸）と実際のBSI IV濃度（X軸）との間の相関は有意性が高かった：線形回帰分析で示されるように、P = 0.0136（バッチ1）、0.0025（バッチ2）、および0.0045（バッチ3）であった（R二乗（r2）値は0.665（バッチ1）、0.8054（バッチ2）、および0.7643（バッチ3）であった）。（I）DL予測スコア（DL-predicted score）に対するネクロスタチン-1の効果を各濃度について計算した。エラーバーは、N = 3の独立した実験バッチからの平均および標準偏差（S.D.）を表す。（J）0～25μMの異なる濃度でネクロスタチン-1を添加した場合の、活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）である。（K）DL予測スコアに対するネクロスタチン-1アナログの効果を各濃度で計算した。エラーバーは、N = 3の独立した実験バッチからの平均および標準偏差（S.D.）を表す。（L）0～25μMの異なる濃度でネクロスタチン-1アナログを添加した場合の活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）である。（M）DL予測スコアに対するドコサヘキサエン酸（DHA）の効果を各濃度で計算した。エラーバーは、N = 3の独立した実験バッチからの平均および標準偏差（S.D.）を表す。（N）0～25μMの異なる濃度でDHAを添加した場合の、活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）である。The Deep-i model predicted neurodegeneration depending on genotype differences and compound treatment. (A) Changes in signal counts of activated caspase-3 were plotted when Z-VAD-FMK was added at different concentrations from 0 to 25 μM. Error bars are standard deviations (SD) of n = 16. (B) Schematic diagram of the prediction method. Images for DL analysis were acquired within 24 hours after starting the assay. (C) Comparison of error between different sizes including 256 × 256, 512 × 512, 768 × 768, and 1,024 × 1,024 pixels for training. Error bars represent the mean and standard deviation (SD) from N = 3 independent experiments. * indicates P < 0.05 using one-way ANOVA followed by Tukey's multiple comparison post hoc test. (D) Z-VAD-FMK was administered to cortical neurons at 8 concentrations ranging from zero to 25 μM. A CNN was trained to predict Z-VAD-FMK concentration from images. Predicted concentration was defined as the predicted score by the Deep-i model. Error bars represent mean and standard deviation (SD) from n = 128 independent replicates. The predicted score by the Deep-i model was correlated with the actual Z-VAD-FMK concentration. The R-squared value was 0.987. (E) Comparison of mean squared errors at different time points from 24 to 72 hours of training. Error bars represent the mean and standard deviation (SD) from N = 3 independent experiments. * indicates P < 0.05 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test). (F) Comparison of R-squared at different time points from 24 to 72 hours of training. Error bars represent the mean and standard deviation (SD) from N = 3 independent experiments. * indicates P < 0.05 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test). (G) Comparison of neurons with and without mutations in PSEN1. By correcting the PSEN1 mutation in FAD neurons using the CRISPR-Cas9 system, we prepared "corrected" neurons that retain the same genetic background except for the corrected PSEN1 mutation. The prediction scores by the Deep-i model were compared between FAD neurons and modified neurons. Plotted dots represent n = 1,536 independent replicates. **** indicates P < 0.0001 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test). (H) Effect of BSI IV, a β-secretase inhibitor that suppresses Aβ production in a dose-dependent manner. A predicted score by the Deep-i model was calculated for each concentration. Error bars represent the mean and standard deviation (SD) from N = 3 independent experimental batches. The correlation between DL score (Y-axis) and actual BSI IV concentration (X-axis) was highly significant: P = 0.0136 (batch 1), 0.0025 (batch 2) as shown by linear regression analysis. , and 0.0045 (batch 3) (R-squared ( r2 ) values were 0.665 (batch 1), 0.8054 (batch 2), and 0.7643 (batch 3)). (I) The effect of Necrostatin-1 on DL-predicted score was calculated for each concentration. Error bars represent the mean and standard deviation (SD) from N = 3 independent experimental batches. (J) Changes in signal counts of activated caspase-3 upon addition of necrostatin-1 at different concentrations from 0 to 25 μM were plotted. Error bars are standard deviations (SD) of n = 16. (K) The effect of necrostatin-1 analog on DL prediction score was calculated at each concentration. Error bars represent the mean and standard deviation (SD) from N = 3 independent experimental batches. (L) Changes in signal counts of activated caspase-3 upon addition of necrostatin-1 analogs at different concentrations from 0 to 25 μM were plotted. Error bars are standard deviations (SD) of n = 16. (M) The effect of docosahexaenoic acid (DHA) on DL prediction score was calculated at each concentration. Error bars represent the mean and standard deviation (SD) from N = 3 independent experimental batches. (N) Changes in signal counts of activated caspase-3 were plotted when DHA was added at different concentrations from 0 to 25 μM. Error bars are standard deviations (SD) of n = 16. 神経変性の経時変化のモニタリングシステムは、384ウェルプレートの異なるウェル間での位置効果（positional effect）、および異なるバッチ間の変動性を示さなかった。（A）384ウェルプレートの384ウェルすべてに0.1％DMSOを添加した場合の、活性化カスパーゼ-3のシグナルカウントの変化をプロットした。各行の平均カウントを個別にプロットして、さまざまな位置およびさまざまなバッチ間のシグナルの位置効果を評価した。エラーバーは、各バッチの行ごとにn = 24の標準偏差（S.D.）を示す。The neurodegeneration time course monitoring system showed no positional effects between different wells of the 384-well plate and no variability between different batches. (A) Changes in activated caspase-3 signal counts were plotted when 0.1% DMSO was added to all 384 wells of a 384-well plate. The average counts for each row were plotted separately to assess the position effect of the signal at different locations and between different batches. Error bars indicate standard deviation (S.D.) of n = 24 per row for each batch. 2つの追加バッチにおける活性化カスパーゼ-3のシグナルカウントの変化。（A）0～25μMの異なる濃度でZ-VAD-FMKを添加した場合の、実験バッチ2の活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）を示す。（B）0～25μMの異なる濃度でZ-VAD-FMKを添加した場合の、実験バッチ3の活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 16の標準偏差（S.D.）を示す。Changes in signal counts of activated caspase-3 in two additional batches. (A) Changes in signal counts of activated caspase-3 of experimental batch 2 were plotted upon addition of Z-VAD-FMK at different concentrations from 0 to 25 μM. Error bars indicate standard deviation (S.D.) of n = 16. (B) Changes in signal counts of activated caspase-3 of experimental batch 3 were plotted upon addition of Z-VAD-FMK at different concentrations from 0 to 25 μM. Error bars indicate standard deviation (S.D.) of n = 16. DLベースの予測システムは、さまざまな時点およびさまざまな行でのさまざまな実験バッチ間で再現性のある結果を示し、PSEN1遺伝子における遺伝的差異を区別した。（A）深層学習（DL）トレーニング用の画像収集の概要図。（B）プレートAおよびBを含む2つのプレートからの画像を統合し、回転させながらそれぞれDLトレーニング、検証、およびテストステップのために1つのバッチとして処理した。（C）異なるクロッピングサイズの画像間でのDLベースの予測の誤差を評価するための概略図。（D）Deep-iモデルによる予測スコアを、各濃度で計算した。予測スコアとZ-VAD-FMKの実際の濃度との相関関係をプロットして、異なる時点間、および3つの異なるバッチ間でそれらを比較した。エラーバーは、n = 64の独立した実験画像からの平均および標準偏差（S.D.）を表す。（E）2つの行の平均DL予測スコアを箱ひげ図に個別にプロットして、異なる位置および異なるバッチ間のDL予測スコアの分布を評価した。箱ひげ図は、各位置またはバッチのDL予測スコアの中央値、25％四分位数および75％四分位数、最小および最大を示す。（F）PSEN1変異を有する場合と有さない場合の神経細胞間の比較。CRISPR-Cas9システムによりFAD神経細胞のPSEN1の変異を修正して、2つの異なるバッチ（2と3）で修正されたPSEN1変異を除いて同じ遺伝的背景を保持する「修正された」神経細胞を準備した。Deep-iモデルによる予測スコアをFAD神経細胞と修正された神経細胞間で比較した。プロットされたドットは、n = 1,536の独立したレプリケイトを表す。****は、P <0.0001（一元配置分散分析とそれに続くテューキーの多重比較事後検定を使用）を示す。The DL-based prediction system showed reproducible results between different experimental batches at different time points and different lines, distinguishing genetic differences in the PSEN1 gene. (A) Schematic diagram of image collection for deep learning (DL) training. (B) Images from two plates, including plates A and B, were merged and processed as one batch for DL training, validation, and testing steps, respectively, with rotation. (C) Schematic diagram for evaluating the error of DL-based prediction between images with different cropping sizes. (D) Prediction scores by the Deep-i model were calculated at each concentration. The correlation between the predicted score and the actual concentration of Z-VAD-FMK was plotted to compare them between different time points and between three different batches. Error bars represent the mean and standard deviation (S.D.) from n = 64 independent experimental images. (E) The average DL prediction scores of two rows were plotted separately in a boxplot to assess the distribution of DL prediction scores between different locations and different batches. Boxplots show the median, 25% and 75% quartiles, minimum and maximum of DL prediction scores for each location or batch. (F) Comparison between neurons with and without PSEN1 mutations. Correcting PSEN1 mutations in FAD neurons by CRISPR-Cas9 system to generate “corrected” neurons that retain the same genetic background except for the PSEN1 mutations that were corrected in two different batches (2 and 3). Got ready. The prediction scores by the Deep-i model were compared between FAD neurons and modified neurons. Plotted dots represent n = 1,536 independent replicates. **** indicates P < 0.0001 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test). Deep-iモデルと単一細胞RNAシーケンス解析との組み合わせにより、カスパーゼ-3の活性化前に神経変性がプログラムされた細胞のクラスターが同定された。（A）神経変性がプログラムされた細胞のクラスター同定の概略図。（B）単一細胞RNAシーケンス解析のUMAPは、異なる条件間で同様のパターンを示した。（C）クラスターのサイズ（分析したすべての細胞の％）を、異なる条件でプロットした。クラスター12は、Z-VAD-FNKの濃度に反比例してクラスターのサイズの増加を示した。（D）異なるZ-VAD-FMK濃度に従ってクラスター12で変化した遺伝子セットに関連する経路のリスト。（E）KLHL32は、クラスター12でのみ顕著で独特な増加を示した。（F）25μMのZ-VAD-FMKを用いた場合と用いていない場合のKLHL32の代表的な画像。****は、P <0.0001（一元配置分散分析とそれに続くテューキーの多重比較事後検定を使用）を示す。（G）異なるZ-VAD-FMK濃度間のKLHL32陽性細胞数の違いを示すための、図6Fを定量化したグラフ。****は、P <0.0001（スチューデントのt検定を使用）を示す。Combining the Deep-i model with single-cell RNA-seq analysis identified clusters of cells programmed for neurodegeneration prior to caspase-3 activation. (A) Schematic representation of cluster identification of cells programmed for neurodegeneration. (B) Single-cell RNA-seq analysis of UMAP showed similar patterns between different conditions. (C) Cluster size (% of all cells analyzed) was plotted at different conditions. Cluster 12 showed an increase in cluster size inversely proportional to the concentration of Z-VAD-FNK. (D) List of pathways associated with gene sets that changed in cluster 12 according to different Z-VAD-FMK concentrations. (E) KLHL32 showed a significant and unique increase only in cluster 12. (F) Representative images of KLHL32 with and without 25 μM Z-VAD-FMK. **** indicates P < 0.0001 (using one-way ANOVA followed by Tukey's multiple comparisons post hoc test). (G) Graph quantifying Figure 6F to show the difference in the number of KLHL32-positive cells between different Z-VAD-FMK concentrations. **** indicates P < 0.0001 (using Student's t-test). 単一細胞トランスクリプトームにより、神経変性がプログラムされた神経細胞のマーカーとしてのKLHL32が明らかとなった。（A）単一細胞RNAシーケンス（scRNAseq）の品質管理。各データセットのnFeature_RNA、nCount_RNA、およびPercent_mitoのバイオリン図。（B）scRNAseqのUMAPは、Z-VAD-FMK 0、0.2、25 μMの異なる条件でのクラスター12の分布を示す。（C）異なる非標的対照またはKLHL32、CASP3、CASP8もしくはCASP9を標的とするsiRNAを添加した場合の、活性化カスパーゼ-3のシグナルカウントの変化をプロットした。エラーバーは、n = 12の標準偏差（S.D.）を示す。（D）アッセイ開始から144時間後の活性化カスパーゼ-3のシグナルカウントをプロットした。一元配置分散分析により、異なる条件での有意な効果が明らかとなった。2つの条件の違いを、シダックの多重比較補正を使用した事後ペアワイズ比較によって分析した（****； P <0.001）。Single-cell transcriptome reveals KLHL32 as a marker of neurons programmed for neurodegeneration. (A) Quality control of single cell RNA sequencing (scRNAseq). Violin diagram of nFeature_RNA, nCount_RNA, and Percent_mito for each dataset. (B) scRNAseq UMAP shows the distribution of cluster 12 at different conditions of Z-VAD-FMK 0, 0.2, and 25 μM. (C) Changes in activated caspase-3 signal counts upon addition of different non-targeting controls or siRNA targeting KLHL32, CASP3, CASP8 or CASP9 were plotted. Error bars indicate standard deviation (S.D.) with n = 12. (D) Signal counts of activated caspase-3 were plotted 144 hours after the start of the assay. One-way analysis of variance revealed significant effects in different conditions. Differences between the two conditions were analyzed by post hoc pairwise comparisons using Sidak's multiple comparisons correction (****; P < 0.001). Deep-iが焦点を合わせる場所を規定するためのGradCAM（Gradient-weighted Class Activation Mapping）。（A）GradCAM分析の概略図。（B）KLHL32陽性細胞（Mark）、細胞体マスク（Cell）、およびGradCAM顕著性マップ（Grad）のマージされた領域を分析するためのベン図の概略図。（C）Dense Block 3における位相、細胞体マスク、KLHL32染色神経細胞、およびGradCAM顕著性マップの代表的な画像。（D）細胞体マスクとGradCAM顕著性マップの間の重複率をプロットした。重複領域（％）は、すべての画像で60～80％を示した。GradCAM (Gradient-weighted Class Activation Mapping) to define where Deep-i focuses. (A) Schematic diagram of GradCAM analysis. (B) Schematic representation of a Venn diagram for analyzing the merged regions of KLHL32-positive cells (Mark), cell body masks (Cell), and GradCAM saliency maps (Grad). (C) Representative images of phase, cell body mask, KLHL32-stained neurons, and GradCAM saliency map in Dense Block 3. (D) The overlap rate between the cell body mask and GradCAM saliency map is plotted. The overlapping area (%) was 60-80% for all images. KLHL32陽性細胞は、アルツハイマーモデルマウスの優先的に影響を受けた脳領域および死後のアルツハイマーの脳組織に蓄積された。（A）脳梁膨大後皮質または脳梁膨大後部の画像を取得するため、代表的な生後3ヶ月の非トランスジェニック同腹仔（左パネル）および5×FADマウス（右パネル）からの冠状脳切片をKLHL32（上パネル）またはアミロイドベータ（Aβ）（下パネル）で染色し、顕微鏡で写真を撮った。スケールバー＝4 mm（低倍率の場合）（左パネル）、400 μm（高倍率の場合）。（B）脳梁膨大後部皮質（RSP）、背側海馬台（SUB）、海馬CA1領域（CA1）、および嗅内野（ENT）におけるKLHL32陽性細胞数の定量化。（C）認知機能障害のない健康な高齢者対照（左パネル）およびアルツハイマー病患者（右パネル）からの脳切片をKLHL32で染色した。白い矢じりは、小さな神経細胞における陽性染色を示し、黒い矢じりは、星状細胞で陽性染色を示す。スケールバー＝1 mm（x4対物レンズの場合）、400 μm（x40対物レンズの場合）。（D）KLHL32を、老人斑（左パネル）、神経突起（中央パネル、白い矢じり）、神経網の凝集構造（中央パネル、黒い矢じり）、ドット状のパターン（右パネル、白い矢じり）、および神経細胞内のスペース（右パネル、黒い矢じり）で染色した。スケールバー＝100 μm。（E）側頭皮質のKLHL32陽性領域をプロットして、対照の脳と、ADと診断された剖検例とを比較した。エラーバーは、n = 4の標準偏差（S.D.）である。スチューデントのt検定を用いたp値が0.05未満を有意であるとみなした。KLHL32-positive cells accumulated in preferentially affected brain regions of Alzheimer model mice and postmortem Alzheimer's brain tissue. (A) Coronal brain sections from a representative 3-month-old non-transgenic littermate (left panel) and 5× FAD mouse (right panel) to obtain images of the post-ampullar cortex or post-ampullar cortex. were stained with KLHL32 (top panel) or amyloid beta (Aβ) (bottom panel) and photographed using a microscope. Scale bar = 4 mm (for low magnification) (left panel), 400 μm (for high magnification). (B) Quantification of the number of KLHL32-positive cells in the retrosplenial cortex (RSP), dorsal subiculum (SUB), hippocampal CA1 area (CA1), and entorhinal cortex (ENT). (C) Brain sections from healthy elderly controls without cognitive impairment (left panel) and Alzheimer's disease patients (right panel) were stained with KLHL32. White arrowheads indicate positive staining in small neurons and black arrowheads indicate positive staining in astrocytes. Scale bar = 1 mm (for x4 objective), 400 μm (for x40 objective). (D) KLHL32 was detected in senile plaques (left panel), neurites (middle panel, white arrowheads), neuropil aggregate structures (middle panel, black arrowheads), dot-like patterns (right panel, white arrowheads), and neuropils. The intracellular space (right panel, black arrowhead) was stained. Scale bar = 100 μm. (E) KLHL32-positive regions in the temporal cortex were plotted to compare control brains and autopsy cases diagnosed with AD. Error bars are standard deviations (S.D.) of n = 4. A p-value of less than 0.05 using Student's t-test was considered significant. KLHL32陽性集団を数える概略図。KLHL32陽性領域（灰色）を分析し、脳梁膨大後部皮質（RSP）または海馬台（SUB）におけるcounting mask（右パネル）として陽性領域にラベルを付すための代表的な画像。Schematic diagram for counting KLHL32-positive populations. Representative images for analyzing KLHL32-positive areas (gray) and labeling positive areas as a counting mask (right panel) in the retrosplenial cortex (RSP) or subiculum (SUB).
１．神経変性疾患の予防または治療薬
　下述の実施例で示される通り、アルツハイマー病（AD）の細胞モデルにおいて、siRNAによりKLHL32遺伝子の発現をノックダウンすると、カスパーゼ-3の活性化が抑制されて細胞死が抑制されることが、本発明者らにより実証された。カスパーゼ-3の活性化を伴う神経変性は、ADを始めとするあらゆる神経変性疾患の共通病態であると考えられている。よって、KLHL32遺伝子の発現抑制薬は、ADを含む神経変性疾患一般の予防または治療薬として用いることができる。即ち、本発明は、KLHL32遺伝子の発現抑制薬を含有してなる、神経変性疾患の予防または治療薬（以下、「本発明の医薬」と称することがある。）を提供する。また、特に断らない限り、神経変性疾患の予防または治療薬（または方法）には、該疾患を予防でき、かつ治療できる医薬（または方法）も包含される。 1. Preventive or therapeutic agents for neurodegenerative diseases As shown in the examples below, in a cell model of Alzheimer's disease (AD), knocking down the expression of the KLHL32 gene with siRNA suppresses the activation of caspase-3 and inhibits cell activation. It has been demonstrated by the inventors that death is suppressed. Neurodegeneration accompanied by activation of caspase-3 is considered to be a common pathology of all neurodegenerative diseases including AD. Therefore, a drug that suppresses the expression of the KLHL32 gene can be used as a prophylactic or therapeutic drug for neurodegenerative diseases in general, including AD. That is, the present invention provides a prophylactic or therapeutic drug for neurodegenerative diseases (hereinafter sometimes referred to as "the drug of the present invention"), which contains a drug that suppresses the expression of the KLHL32 gene. Furthermore, unless otherwise specified, the prophylactic or therapeutic agent (or method) for neurodegenerative diseases also includes a medicament (or method) that can prevent and treat the disease.
　本発明の医薬は、有効成分であるKLHL32遺伝子の発現抑制薬をそのまま単独で、または薬理学的に許容される担体、賦形剤、希釈剤等と混合し、適当な剤型の医薬組成物または予防もしくは治療剤として経口的または非経口的に投与することができる。また、本発明の医薬は、哺乳動物（例：ヒト、ラット、マウス、モルモット、ウサギ、ヒツジ、ウマ、ブタ、ウシ、イヌ、ネコ、サル）に対して投与することが可能である。よって、哺乳動物に対し、KLHL32遺伝子の発現抑制薬の有効量を投与することを特徴とする、該哺乳動物における神経変性疾患の予防または治療方法も提供される。 The pharmaceutical composition of the present invention is a pharmaceutical composition containing the active ingredient, a drug that suppresses the expression of the KLHL32 gene, either alone or mixed with a pharmacologically acceptable carrier, excipient, diluent, etc. Alternatively, it can be administered orally or parenterally as a prophylactic or therapeutic agent. Furthermore, the medicament of the present invention can be administered to mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, dogs, cats, and monkeys). Therefore, there is also provided a method for preventing or treating neurodegenerative diseases in a mammal, which comprises administering to the mammal an effective amount of a drug that suppresses the expression of the KLHL32 gene.
　カスパーゼ-3の活性化を伴う神経変性は、あらゆる神経変性疾患の共通病態と考えられているため、本発明において、予防または治療の対象となる神経変性疾患としては、特に制限されないが、例えば、アルツハイマー病（AD）、パーキンソン病、筋萎縮性側索硬化症(ALS)、脊髄小脳変性症、前頭側頭葉変性症、レビー小体型認知症、多系統萎縮症、ハンチントン病、進行性核上性麻痺、大脳皮質基底核変性症などが挙げられる。中でも、ADが好ましい。 Since neurodegeneration accompanied by activation of caspase-3 is considered to be a common pathological condition of all neurodegenerative diseases, the neurodegenerative diseases to be prevented or treated in the present invention are not particularly limited, but include, for example: Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, progressive supranuclear disease Examples include sexual paralysis and corticobasal degeneration. Among them, AD is preferable.
　本発明において、予防または治療の対象となるADは、孤発性および家族性のいずれのADであってもよい。家族性ADの場合、原因遺伝子は特に制限されず、アミロイド前駆体タンパク質（Amyloid Precursor Protein; APP）、プレセニリン１(Presenilin 1; PSEN1)、プレセニリン2（Presenilin 2; PSEN2）等をはじめとする任意の既知原因遺伝子であり得る。一実施態様において、APP変異を有する家族性ADの場合、当該APP遺伝子変異としては、dup APP 変異、APP KM670/671NL 変異、APP D678N 変異、APP E682K 変異、APP A692G 変異、APP E693K 変異、APP E693Q 変異、APP E693G 変異、APP E693del(APP E693Δ) 変異、APP D694N 変異、APP L705V 変異、APP A713T 変異、APP T714A 変異、APP T714I 変異、APP V715M 変異、APP V715A 変異、APP I716V 変異、APP I716F 変異、APP I716T 変異、APP V717I 変異などが挙げられるが、これらに限定されない。ここで、例えばAPP E693Δとは、APP内にE693の欠損型変異を意味する。 In the present invention, AD to be prevented or treated may be either sporadic or familial AD. In the case of familial AD, the causative gene is not particularly limited, and any gene including amyloid precursor protein (APP), presenilin 1 (PSEN1), presenilin 2 (PSEN2), etc. It may be a known causative gene. In one embodiment, for familial AD with APP mutations, the APP gene mutations include dup APP mutation, APP KM670/671NL mutation, APP D678N mutation, APP E682K mutation, APP A692G mutation, APP E693K mutation, APP E693Q Changes, App E693G variant, App E693DEL (App E693}) variant, App D694N variant, App L705V variant, App A713T variant, App T714A Karizu, App T714I Campaign, App V715M Campaign, App i715A Detergent, App i716, App i716 V transformation, App i716F change, Examples include, but are not limited to, APP I716T mutation, APP V717I mutation, and the like. Here, for example, APP E693Δ means a deletion type mutation of E693 in APP.
　本発明において、予防または治療の対象となるALSは、孤発性および家族性のいずれのALSも包含される。家族性ALSの場合、原因遺伝子は特に制限されず、SOD1、TDP-43、C9orf72、alsin、SETX、FUS/TLS、VAPB、ANG、FIG4、OPTN、ATXN2、DAO、UBQLN2、PFN1、DCTN1、CHPM2B、VCPなどが挙げられる。一実施態様において、SOD1変異を有する家族性ALSの場合、当該SOD1遺伝子変異としては、SOD1タンパク質の144番目のLeuがPhe-Val-Xaa（Xaaは任意のアミノ酸を示す）に置換される変異（SOD1-L144FVX）、93番目のGlyがSerに置換される変異（SOD1-G93S）、106番目のLeuがValに置換される変異（SOD1-L106V）等が挙げられるが、これらに限定されない。また、本発明には、エクソン9-13に変異を有するMAPT遺伝子に変異を有する患者に対しても好適に用いることができる。エクソン9-13の変異としては、例えば、K257T、I260V、G272V、N297K、K280Δ、L284L、N296N、P301L、P301S、S305N、S305S、V337M、E342V、G389R、R406Wなどが挙げられる。 In the present invention, ALS to be prevented or treated includes both sporadic and familial ALS. In the case of familial ALS, the causative genes are not particularly restricted, and include SOD1, TDP-43, C9orf72, alsin, SETX, FUS/TLS, VAPB, ANG, FIG4, OPTN, ATXN2, DAO, UBQLN2, PFN1, DCTN1, CHPM2B, Examples include VCP. In one embodiment, in the case of familial ALS having an SOD1 mutation, the SOD1 gene mutation includes a mutation in which Leu at position 144 of the SOD1 protein is replaced with Phe-Val-Xaa (Xaa represents any amino acid). SOD1-L144FVX), a mutation in which Gly at position 93 is replaced with Ser (SOD1-G93S), a mutation in which Leu at position 106 is replaced with Val (SOD1-L106V), etc., but is not limited to these. Furthermore, the present invention can also be suitably used for patients who have a mutation in the MAPT gene that has a mutation in exons 9-13. Examples of mutations in exons 9-13 include K257T, I260V, G272V, N297K, K280Δ, L284L, N296N, P301L, P301S, S305N, S305S, V337M, E342V, G389R, and R406W.
　本明細書において、「治療薬」には、神経変性疾患の根治治療を目的とする医薬だけでなく、例えば、これらの疾患の進行抑制を目的とする医薬、症状の軽減（例えば、生活、仕事の支障がない症状軽微(minimal manifestations MM)への改善）を目的する、または後遺症を軽減する医薬も含まれるものとする。例えば、神経変性疾患は、長期間（通常年単位）にわたり進行する病気であることから、早期に治療を開始することで、症状の進行を予防することができる。また、本明細書において、「予防薬」には、神経変性疾患を発症していない対象に対して、神経変性疾患を発症するリスクを低減させることを目的とする医薬だけでなく、神経変性疾患を発症した対象に対して、神経変性疾患が再発するリスクを低減させることを目的とする医薬も包含されるものとする。例えば、潜在的に、神経変性疾患に罹患しやすい遺伝的バックグラウンドを有するような患者に対して、神経変性疾患の症状を呈する前に本発明の医薬を投与することにより、神経変性疾患の発症を予防することもできる。「治療方法」および「予防方法」についても同様である。 As used herein, "therapeutic drugs" include not only drugs aimed at the radical treatment of neurodegenerative diseases, but also drugs aimed at suppressing the progression of these diseases, alleviation of symptoms (e.g., daily life, work This also includes medicines that aim to improve symptoms to minimal symptoms (MM) that do not interfere with health conditions or alleviate the after-effects. For example, since neurodegenerative diseases are diseases that progress over a long period of time (usually on a yearly basis), the progression of symptoms can be prevented by starting treatment early. In addition, as used herein, "preventive drugs" include not only drugs aimed at reducing the risk of developing neurodegenerative diseases in subjects who have not developed neurodegenerative diseases, but also drugs for neurodegenerative diseases. It also includes medicines aimed at reducing the risk of recurrence of neurodegenerative diseases in subjects who have developed neurodegenerative diseases. For example, by administering the medicament of the present invention to patients who have a genetic background that makes them potentially susceptible to neurodegenerative diseases, the onset of neurodegenerative diseases can be can also be prevented. The same applies to "treatment methods" and "prevention methods."
　KLHL32遺伝子の発現抑制薬として、少なくとも神経細胞において該遺伝子の発現を抑制できる物質であれば限定されないが、前記物質は核酸であってよく、例えば、アンチセンス核酸（例：アンチセンスオリゴヌクレオチド（ASO）等）（該核酸をコードする核酸も含む）、siRNA（該siRNAをコードする核酸も含む）、ヘテロ二本鎖核酸（heteroduplex oligonucleotide; HDO）、shRNA（該shRNAをコードする核酸も含む）、miRNA（microRNA）（該miRNAをコードする核酸も含む）、アンチジーン核酸、CRISPR-Casシステムなどが挙げられる。以下では、KLHL32遺伝子の発現抑制薬が核酸である場合、かかる核酸を「本発明の核酸」と称することがある。KLHL32遺伝子の発現抑制薬がCRISPR-Casシステムの場合には、該システムをコードする核酸（即ち、Casをコードする核酸、ガイドRNAをコードする核酸、ならびにガイドRNAおよびCasをコードする核酸）およびガイドRNAも、「本発明の核酸」に包含されるものとする。本発明の医薬には、KLHL32遺伝子の発現抑制薬が、１つのみ含まれていてもよく、２つ以上含まれていてもよい。２つ以上含まれる場合には、同種のものが含まれていてもよく（例えば、標的配列が異なる複数のsiRNAが含まれる等）、異種のものが含まれていてもよい（例えば、siRNAとアンチセンス核酸とが含まれる等）。 The KLHL32 gene expression inhibitor is not limited as long as it is a substance that can suppress the expression of the gene at least in nerve cells, but the substance may be a nucleic acid, for example, an antisense nucleic acid (e.g. antisense oligonucleotide (ASO ), etc.) (including the nucleic acid encoding the aforementioned nucleic acid), siRNA (including the nucleic acid encoding the aforementioned siRNA), heteroduplex oligonucleotide (HDO), shRNA (including the nucleic acid encoding the aforementioned shRNA), Examples include miRNA (microRNA) (including nucleic acids encoding the miRNA), antigene nucleic acids, CRISPR-Cas systems, and the like. In the following, when the KLHL32 gene expression inhibitor is a nucleic acid, such a nucleic acid may be referred to as "the nucleic acid of the present invention". When the KLHL32 gene expression inhibitor is a CRISPR-Cas system, the nucleic acid encoding the system (i.e., the nucleic acid encoding Cas, the nucleic acid encoding guide RNA, and the nucleic acid encoding guide RNA and Cas) and the guide RNA is also included in the "nucleic acid of the present invention". The medicament of the present invention may contain only one or two or more KLHL32 gene expression inhibitors. If two or more are included, they may be of the same type (e.g., multiple siRNAs with different target sequences) or different types (e.g., siRNA and siRNA). antisense nucleic acids, etc.).
　KLHL遺伝子は、一般にBTB/POZドメイン、BACKドメイン、および5～6個のケルチモチーフ（Kelch motif）を有するタンパク質をコードするKLHL（Kelch-like）遺伝子ファミリーに属している。ヒトのKLHL32タンパク質としては、BTB/POZドメイン、BACKドメイン、および5個のケルチモチーフを含む620アミノ酸長のタンパク質が例示される。KLHLファミリーには、KLHL1～KLHL42の分子タイプが知られており、この一部は腫瘍原生との関わり、小胞体におけるE3を介したタンパク質分解系に関与することが予測されているが、KLHL32タンパク質と神経変性との関係性については報告されていない。 The KLHL gene belongs to the KLHL (Kelch-like) gene family, which generally encodes proteins with a BTB/POZ domain, a BACK domain, and 5 to 6 Kelch motifs. An example of the human KLHL32 protein is a 620 amino acid long protein containing a BTB/POZ domain, a BACK domain, and five Kelch motifs. The KLHL family has known molecular types, KLHL1 to KLHL42, and some of these are predicted to be involved in tumorigenesis and the E3-mediated protein degradation system in the endoplasmic reticulum, but the KLHL32 protein The relationship between this and neurodegeneration has not been reported.
　KLHL32タンパク質には、複数のアイソフォームが存在する。KLHL32遺伝子の発現抑制薬が、アンチセンス核酸、siRNA、ヘテロ二本鎖核酸、shRNAまたはmiRNAなどのKLHL32転写産物を標的とする場合、予防または治療の対象がヒトの場合には、該発現抑制薬は、少なくとも完全長KLHL32タンパク質をコードするKLHL32転写産物の発現を抑制できるものであればよい。よって、前記発現抑制薬は、完全長KLHL32タンパク質をコードするKLHL32転写産物の部分配列（以下、標的RNA配列）に対して相補的な塩基配列を含む。かかる塩基配列は、例えば、Genbank Accession No. NM_001323252.2またはNM_052904.4として登録されている、完全長ヒトKLHL32タンパク質をコードするKLHL32転写産物のcDNA塩基配列（配列番号１または２）に基づいて設計することができる。また、KLHL32遺伝子の発現抑制薬が、アンチジーン核酸、CRISPR/CasシステムなどのKLHL32遺伝子を標的とする場合、該発現抑制薬（CRISPR/Casシステムの場合には、該システムを構成するガイドRNA）は、KLHL32遺伝子の部分配列（以下、標的DNA配列）に対して相補的な塩基配列を含む。かかる塩基配列は、例えば、NCBI GeneのGene ID: 114792, location: NC_000006.12 (96898083..97145030)として登録されているヒトKLHL32遺伝子のDNA塩基配列に基づいて設計することができる。標的RNA配列として、具体例には、配列番号４～７のいずれかで示される塩基配列が挙げられるが、これらに限定されない。 There are multiple isoforms of the KLHL32 protein. When the KLHL32 gene expression suppressing drug targets KLHL32 transcripts such as antisense nucleic acids, siRNAs, heteroduplex nucleic acids, shRNAs, or miRNAs, when the target of prevention or treatment is humans, the expression suppressing drugs may be any as long as it can suppress the expression of at least the KLHL32 transcript encoding the full-length KLHL32 protein. Therefore, the expression inhibitor contains a base sequence complementary to a partial sequence of the KLHL32 transcript encoding the full-length KLHL32 protein (hereinafter referred to as target RNA sequence). Such a nucleotide sequence is designed based on the cDNA nucleotide sequence (SEQ ID NO: 1 or 2) of the KLHL32 transcript encoding the full-length human KLHL32 protein, which is registered as Genbank Accession No. NM_001323252.2 or NM_052904.4, for example. can do. In addition, when the KLHL32 gene expression suppressing drug targets the KLHL32 gene using an antigene nucleic acid, a CRISPR/Cas system, etc., the expression suppressing drug (in the case of a CRISPR/Cas system, the guide RNA constituting the system) contains a base sequence complementary to a partial sequence of the KLHL32 gene (hereinafter referred to as target DNA sequence). Such a base sequence can be designed, for example, based on the DNA base sequence of the human KLHL32 gene registered as Gene ID: 114792, location: NC_000006.12 (96898083..97145030) on NCBI Gene. Specific examples of the target RNA sequence include, but are not limited to, the base sequences shown in any of SEQ ID NOs: 4 to 7.
　標的RNA配列または標的DNA配列の長さは、KLHL32遺伝子の発現抑制薬が該配列を特異的に認識して結合し得る限り、特に制限されないが、好ましくは12ヌクレオチド長以上、より好ましくは15ヌクレオチド長以上、さらに好ましくは17ヌクレオチド長以上である。長さの上限も特に制限されないが、例えば30ヌクレオチド以下、好ましくは25ヌクレオチド以下、より好ましくは22ヌクレオチド以下である。従って、該標的領域の長さの範囲としては、例えば12～30ヌクレオチド、好ましくは15～25ヌクレオチド、より好ましくは17～22ヌクレオチドが挙げられる。 The length of the target RNA sequence or target DNA sequence is not particularly limited as long as the KLHL32 gene expression inhibitor can specifically recognize and bind to the sequence, but it is preferably 12 nucleotides or more, more preferably 15 nucleotides. The length is at least 17 nucleotides, more preferably 17 nucleotides or longer. The upper limit of the length is also not particularly limited, but is, for example, 30 nucleotides or less, preferably 25 nucleotides or less, more preferably 22 nucleotides or less. Therefore, the length range of the target region includes, for example, 12 to 30 nucleotides, preferably 15 to 25 nucleotides, and more preferably 17 to 22 nucleotides.
　本明細書において、「KLHL32遺伝子の発現」とは、特に断らない限り、少なくとも「KLHL32転写産物の産生」を含む意味で用いられるが、好ましくは、さらに「機能的なKLHL32タンパク質の産生」を含む意味で用いられる。従って、遺伝子の発現抑制とは、KLHL32遺伝子の発現抑制薬との接触により、該遺伝子から転写される転写産物の細胞中での存在量が減少することだけでなく、該遺伝子にコードされる機能的なタンパク質の細胞中での存在量が減少することも含んでいてもよい。また、本明細書において、KLHL32転写産物には、典型的には、KLHL32 mRNAおよびKLHL pre-mRNAが含まれるが、好ましくはKLHL32 mRNAである。 As used herein, "expression of the KLHL32 gene" is used to include at least "production of a KLHL32 transcript" unless otherwise specified, but preferably also includes "production of a functional KLHL32 protein". used in meaning. Therefore, suppression of gene expression refers not only to a decrease in the amount of transcripts transcribed from the gene in cells due to contact with a drug that suppresses the expression of the KLHL32 gene, but also to a decrease in the amount of transcripts transcribed from the gene. The method may also include reducing the amount of protein present in the cell. Further, in this specification, the KLHL32 transcript typically includes KLHL32 mRNA and KLHL pre-mRNA, but preferably KLHL32 mRNA.
　本明細書において、「核酸」は、モノマーのヌクレオチドを意味してもよいが、通常は複数のモノマーからなるヌクレオチド（例：オリゴヌクレオチド、ポリヌクレオチド等）を意味する。従って、モノマーのヌクレオチドを意図する場合には、「核酸ヌクレオチド」と表記することがあり、かかる核酸としては、例えば、リボ核酸、デオキシリボ核酸、ペプチド核酸(peptide nucleic acid; PNA)、モルフォリノ核酸（morpholino nucleic acid）などが挙げられる。また、特に断らない限り、核酸が複数のモノマーからなるヌクレオチドの場合には、該核酸を構成する各ヌクレオチド残基（5’末端および3’末端のヌクレオチドも含まれる)を、単に「ヌクレオチド」と称する。さらに、本明細書において、「核酸鎖」または「鎖」は、特に断らない限り、いずれも一本鎖核酸を意味する。従って、「アンチセンス鎖」は「一本鎖アンチセンス核酸」と読み替えることができる。 As used herein, "nucleic acid" may mean a monomeric nucleotide, but usually means a nucleotide consisting of multiple monomers (eg, oligonucleotide, polynucleotide, etc.). Therefore, when a monomeric nucleotide is intended, it may be written as a "nucleic acid nucleotide," and such nucleic acids include, for example, ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid (PNA), and morpholinonucleic acid (PNA). Examples include nucleic acid). In addition, unless otherwise specified, when a nucleic acid is a nucleotide consisting of multiple monomers, each nucleotide residue (including nucleotides at the 5' and 3' ends) constituting the nucleic acid is simply referred to as a "nucleotide." to be called. Furthermore, in this specification, "nucleic acid strand" or "strand" means a single-stranded nucleic acid unless otherwise specified. Therefore, "antisense strand" can be read as "single-stranded antisense nucleic acid."
　本明細書において、「相補的」とは、核酸塩基同士が水素結合を介して、いわゆるワトソン-クリック塩基対(天然型塩基対)または非ワトソン-クリック塩基対(フーグスティーン型塩基対、ゆらぎ塩基対（Wobble base pair）など)を形成し得る関係を意味する。従って、「相補的な配列」には、標的RNA配列または標的DNA配列に対して、完全相補的な（即ち、ミスマッチなくハイブリダイズする）配列のみならず、ストリンジェントな条件下で、あるいは哺乳動物細胞の生理的条件下で標的配列とハブリダイズし得る限り、1ないし数個（例：2、3、4、5個またはそれ以上）のミスマッチを含む配列をも含む意味で用いられる。例えば、標的RNA配列または標的DNA杯配列に対して完全相補的な配列と、80%以上（例：85%以上、90%以上、95%以上、96%以上、97%以上、98%以上、99%以上）、最も好ましくは100%の同一性を有する配列が挙げられる。 In this specification, "complementary" means that nucleic acid bases are formed through hydrogen bonds, so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, fluctuations). A relationship that can form a base pair (such as a Wobble base pair). Therefore, a "complementary sequence" includes not only a sequence that is completely complementary (i.e., hybridizes without mismatch) to a target RNA sequence or target DNA sequence, but also a sequence that hybridizes under stringent conditions or under mammalian conditions. The term is used to include sequences containing one or several (eg, 2, 3, 4, 5, or more) mismatches, as long as they can hybridize with the target sequence under physiological conditions of the cell. For example, a sequence that is completely complementary to the target RNA sequence or target DNA sequence and 80% or more (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more), most preferably 100%.
　ストリンジェントな条件は、低ストリンジェントな条件であっても高ストリンジェントな条件であってもよい。低ストリンジェントな条件は、比較的低温で、かつ高塩濃度の条件、例えば、30℃、2×SSC、0.1％SDSであってよい。高ストリンジェントな条件は、比較的高温で、かつ低塩濃度の条件、例えば、65℃、0.1×SSC、0.1％SDSであってよい。温度および塩濃度などの条件を変えることによって、ハイブリダイゼーションのストリンジェンシーを調整できる。ここで、1×SSCは、150mM塩化ナトリウムおよび15mMクエン酸ナトリウムを含む。 Stringent conditions may be low stringency conditions or high stringency conditions. Low stringency conditions may be relatively low temperature and high salt concentration conditions, for example, 30° C., 2×SSC, 0.1% SDS. High stringency conditions may be relatively high temperature and low salt concentration conditions, for example, 65° C., 0.1×SSC, 0.1% SDS. The stringency of hybridization can be adjusted by varying conditions such as temperature and salt concentration. Here, 1x SSC contains 150mM sodium chloride and 15mM sodium citrate.
　アンチセンス核酸は、標的RNA配列と相補的な配列を含む一本鎖核酸である。アンチセンス核酸は、該標的RNA配列と相補的な配列により、標的RNA配列と二本鎖領域を形成し、該二本鎖領域がリボヌクレアーゼH（RNase H）により切断されることで、KLHL32遺伝子の発現を抑制する。 An antisense nucleic acid is a single-stranded nucleic acid that contains a sequence complementary to the target RNA sequence. The antisense nucleic acid forms a double-stranded region with the target RNA sequence due to a complementary sequence to the target RNA sequence, and this double-stranded region is cleaved by ribonuclease H (RNase H), thereby converting the KLHL32 gene. Suppress expression.
　アンチセンス核酸は、生体内での安定性および効率的な転写産物の切断の観点から、ギャップマー（Gapmer）型核酸であることが好ましい。本発明において「ギャップマー型核酸」とは、RNase Hにより認識される複数(例：5、6、7、8、9、10、11、12、13、14、15またはそれ以上）のヌクレオチドを有する内部領域（本明細書において、「ギャップ領域」と称することがある。）が、リボヌクレアーゼに対する耐性を付与するように種々の化学修飾が施されたヌクレオチド（以下、「修飾ヌクレオチド」と称することがある。また、具体例は後述する。）を１つ以上（例：1、2、3、4、5、6、7、8、9、10またはそれ以上）有する外部領域（本明細書において、３’側の外部領域を「３’ウイング領域」と、５’側の外部領域を「５’ウイング領域」と称することがある。）間に配置されるキメラアンチセンス核酸を意味する。３’ウイング領域と５’ウイング領域の各領域を構成する少なくとも一つのヌクレオシドは、架橋型ヌクレオシド（具体例は後述する。）であることが好ましい。 The antisense nucleic acid is preferably a gapmer type nucleic acid from the viewpoint of in vivo stability and efficient cleavage of the transcript. In the present invention, "gapmer-type nucleic acid" refers to a plurality of nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) that are recognized by RNase H. Nucleotides (hereinafter referred to as "modified nucleotides") whose internal regions (herein sometimes referred to as "gap regions") have been subjected to various chemical modifications to confer resistance to ribonucleases. In addition, an external region (in this specification, The external region on the 3' side is sometimes referred to as the "3' wing region" and the external region on the 5' side is sometimes referred to as the "5' wing region." At least one nucleoside constituting each region of the 3' wing region and the 5' wing region is preferably a crosslinked nucleoside (specific examples will be described later).
　アンチセンス核酸の長さは、特に制限されないが、典型的には10～50ヌクレオチド長であり、好ましくは10～30ヌクレオチド長であり、より好ましくは13～30ヌクレオチド長であり、さらに好ましくは15～20ヌクレオチド長である。 The length of the antisense nucleic acid is not particularly limited, but is typically 10 to 50 nucleotides, preferably 10 to 30 nucleotides, more preferably 13 to 30 nucleotides, and even more preferably 15 to 30 nucleotides. ~20 nucleotides long.
　アンチセンス核酸は、KLHL32転写産物の配列に基づいて配列を決定し、市販のDNA/RNA自動合成機（アプライド・バイオシステムズ社、ベックマン社等）を用いて、これに相補的な配列を合成することにより調製することができる。 The antisense nucleic acid is sequenced based on the sequence of the KLHL32 transcript, and a complementary sequence is synthesized using a commercially available automatic DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.) It can be prepared by:
　siRNAとは、標的RNA配列と相補的な配列を有するRNA（即ち、アンチセンス鎖）とその相補鎖から構成される二本鎖RNAである。また、ヘアピンループ部分を介して、標的RNA配列に相補的な配列（第１の配列）と、その相補配列（第２の配列）とが連結された一本鎖RNAであって、ヘアピンループ型の構造をとることにより、第１の配列が第２の配列と二本鎖構造を形成するRNA（smallhairpin RNA：shRNA）もsiRNAの好ましい態様の１つである。また、shRNAは、該shRNAをコードする核酸（例：発現ベクター等）の形態であってもよい。 siRNA is double-stranded RNA consisting of an RNA with a sequence complementary to the target RNA sequence (i.e., antisense strand) and its complementary strand. In addition, it is a single-stranded RNA in which a sequence complementary to the target RNA sequence (first sequence) and its complementary sequence (second sequence) are linked via a hairpin loop portion, and the hairpin loop type RNA in which the first sequence forms a double-stranded structure with the second sequence (small hairpin RNA: shRNA) is also a preferred embodiment of siRNA. Furthermore, shRNA may be in the form of a nucleic acid (eg, expression vector, etc.) encoding the shRNA.
　HDOは、主鎖DNA（即ち、アンチセンス鎖）と、該DNAに相補的なRNA（cRNA）鎖から構成される二本鎖核酸を意味する。HDOは、細胞内に取り込まれると、細胞内でRNase HによりcRNA鎖が切断される。主鎖DNAは、典型的には、ギャップマー型核酸であり、cRNA鎖のウイング領域は、典型的には、2’-O-methyl RNAなどの修飾ヌクレオチド（具体例は後述する。）で構成されている。また、典型的には、主鎖DNAおよび／またはcRNAのすべてのリン酸ジエステル結合部分が硫黄化されている（即ち、ヌクレオシド間結合がホスホロチオエート結合となっている）。HDOは、cRNA鎖の切断によって1本鎖のアンチセンス核酸となった主鎖DNAは標的RNAに結合し、再びRNase Hが標的RNAを切断することでアンチセンス効果を発揮すると推測されている。cRNA鎖がRNase Hの基質となる限り、主鎖DNAやcRNA鎖には、上記以外の修飾が付されていてもよい。 HDO means a double-stranded nucleic acid composed of a main strand DNA (i.e., antisense strand) and an RNA (cRNA) strand complementary to the DNA. When HDO is taken into cells, the cRNA strand is cleaved by RNase H within the cells. The main chain DNA is typically a gapmer type nucleic acid, and the wing region of the cRNA strand is typically composed of modified nucleotides such as 2'-O-methyl RNA (specific examples are given below). has been done. Additionally, typically all phosphodiester bonding portions of the main chain DNA and/or cRNA are sulfurized (ie, the internucleoside bonds are phosphorothioate bonds). In HDO, the main strand DNA, which becomes a single-stranded antisense nucleic acid by cRNA strand cleavage, binds to the target RNA, and it is assumed that RNase H cleaves the target RNA again, thereby exerting its antisense effect. As long as the cRNA strand serves as a substrate for RNase H, the main chain DNA or cRNA strand may have modifications other than those described above.
　二本鎖核酸は、アンチセンス鎖もしくは相補鎖の一方または双方の５’末端または３’末端においてオーバーハング（overhang）（突出部ともいう）を有していてもよい。オーバーハングは、典型的には、センス鎖および／またはアンチセンス鎖の末端における1～数個（例えば、1、2、3、4、5または6個）、好ましくは1～3個の塩基の付加により形成されるものである。オーバーハングとして、例えばdTdTまたはUUからなるものが挙げられる。オーバーハングは、アンチセンス鎖のみ、センス鎖のみ、およびアンチセンス鎖とセンス鎖の両方に有することができるが、本発明において、アンチセンス鎖とセンス鎖の両方に突出部を有する二本鎖核酸が好ましく用いられる。 A double-stranded nucleic acid may have an overhang (also referred to as an overhang) at the 5' or 3' end of one or both of the antisense strand and the complementary strand. The overhang is typically one to several (eg, 1, 2, 3, 4, 5 or 6), preferably 1 to 3 bases at the end of the sense and/or antisense strand. It is formed by addition. Examples of the overhang include those made of dTdT or UU. Overhangs can be present only on the antisense strand, only on the sense strand, or on both the antisense and sense strands, but in the present invention, double-stranded nucleic acids having overhangs on both the antisense and sense strands is preferably used.
　二本鎖核酸の各核酸鎖の長さは、アンチセンス効果を発揮できる限り特に制限されないが、例えば、10～50ヌクレオチド長、好ましくは15～30ヌクレオチド長、より好ましくは20～27ヌクレオチド長である。 The length of each nucleic acid strand of the double-stranded nucleic acid is not particularly limited as long as it can exhibit an antisense effect; be.
　二本鎖核酸は、従来公知の手法を用いて、化学的に合成することにより、または遺伝子組み換え技術を用いて産生することにより得ることができる。また、適宜市販されている核酸を用いることも可能である。例えば、二本鎖核酸は、標的RNA配列に基づいて、市販のソフトウエア（例：RNAiDesigner； Invitrogen）を用いて適宜設計することができる。RNA上の標的配列のセンス鎖およびアンチセンス鎖を市販のDNA/RNA自動合成機（アプライド・バイオシステムズ社、ベックマン社等）でそれぞれ合成し、適当なアニーリング緩衝液中、約90℃～約95℃で約1分程度変性させた後、約30℃～約70℃で約1～約8時間アニーリングさせることにより調製することができる。また、HDOは、例えば、WO2013/089283に記載の方法により作製することもできる。 Double-stranded nucleic acids can be obtained by chemically synthesizing using conventionally known techniques, or by producing using genetic recombination technology. It is also possible to use appropriately commercially available nucleic acids. For example, double-stranded nucleic acids can be appropriately designed based on the target RNA sequence using commercially available software (eg, RNAiDesigner; Invitrogen). The sense strand and antisense strand of the target sequence on RNA are synthesized using a commercially available automatic DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.), and the mixture is incubated at approximately 90°C to approximately 90°C in an appropriate annealing buffer. It can be prepared by denaturing it at ℃ for about 1 minute and then annealing it at about 30 ℃ to about 70 ℃ for about 1 to about 8 hours. Further, HDO can also be produced, for example, by the method described in WO2013/089283.
　本明細書において、「miRNA」とは、siRNAのように標的RNAを切断するのではなく、標的RNAの３’非翻訳領域（UTR）を認識して、翻訳を制御する一本鎖RNAまたは二本鎖RNA（例：miRNA/miRNA*等）を意味する。miRNAは、本来ゲノム上にコードされた内在性の20～25塩基程度の非コードRNA（non-codingRNA：ncRNA）を意味し、miRNA遺伝子からpri-miRNAが発現し、その後pre-miRNAの生成を経て、mature-miRNAが生成される。その後、mature-miRNAがRISCに取り込まれて一本鎖miRNAが生成される。本発明で用いるmiRNAは、pri-miRNAの形態であってもよく、pre-miRNAの形態であってもよく、mature-miRNA（miRNA/miRNA*）の形態であってよく、さらには一本鎖RNAの形態であってもよい。また、本発明で用いるmiRNAには、両端部にRNAオリゴマー、中央部にDNAオリゴマーを配したギャップマー構造により、核で作用してRNase H依存的に転写産物を分解するものも包含されるものものとする。また、miRNAは、該miRNAをコードする核酸（例：発現ベクター等）の形態であってもよい。 As used herein, "miRNA" refers to single-stranded RNA or double-stranded RNA that does not cleave the target RNA like siRNA, but rather recognizes the 3' untranslated region (UTR) of the target RNA and controls translation. Refers to double-stranded RNA (e.g. miRNA/miRNA * , etc.). miRNA refers to endogenous non-coding RNA (ncRNA) of approximately 20 to 25 bases encoded on the genome. pri-miRNA is expressed from the miRNA gene, and then pre-miRNA is generated. After that, mature-miRNA is generated. Thereafter, mature-miRNA is taken up into RISC and single-stranded miRNA is generated. The miRNA used in the present invention may be in the form of pri-miRNA, pre-miRNA, mature-miRNA (miRNA/miRNA * ), and even single-stranded miRNA. It may be in the form of RNA. Furthermore, miRNAs used in the present invention include those that act in the nucleus and degrade transcripts in an RNase H-dependent manner due to their gapmer structure, which has RNA oligomers at both ends and DNA oligomers in the center. shall be taken as a thing. Furthermore, miRNA may be in the form of a nucleic acid (eg, expression vector, etc.) encoding the miRNA.
　miRNAは、前述の二本鎖核酸の合成方法と同様に合成してもよい。また、miRNAは、任意の内在性のmiRNAを基礎にして、標的RNA配列に応じて該miRNAの配列を置換することで作製することもできる。かかる内在性のmiRNAとしては、let-7、miR-15a、miR-143、miR-139およびそれらの前駆体などが挙げられるが、これらに限定されない。 miRNA may be synthesized in the same manner as the double-stranded nucleic acid synthesis method described above. Furthermore, miRNA can also be produced based on any endogenous miRNA by substituting the miRNA sequence according to the target RNA sequence. Such endogenous miRNAs include, but are not limited to, let-7, miR-15a, miR-143, miR-139 and their precursors.
　miRNAの長さ（miRNAが二本鎖の場合には、各核酸鎖の長さ）は、アンチセンス効果を発揮できる限り特に制限されないが、例えば、10～50ヌクレオチド長、好ましくは15～30ヌクレオチド長、より好ましくは20～27ヌクレオチド長である。 The length of miRNA (if the miRNA is double-stranded, the length of each nucleic acid strand) is not particularly limited as long as it can exhibit an antisense effect, but for example, it is 10 to 50 nucleotides, preferably 15 to 30 nucleotides. more preferably 20 to 27 nucleotides in length.
　アンチジーン核酸は、標的遺伝子を有する二本鎖DNAと三重鎖を形成することで標的遺伝子発現を転写レベルで抑制できる核酸を意味し、三重鎖形成核酸（Triplex Forming Oligonucletide; TFO）とも称される。典型的なアンチジーン法は、DNA二重鎖に対して３本目の核酸が結合することを基盤としており、３本目核酸の塩基がプリン－ピリミジン塩基対のプリン塩基と水素結合することで、平面内に３つの塩基が連続して並ぶ構造をとることによって三重鎖形成が可能となる。三本目の核酸（TFO）（即ち、アンチジーン核酸）の塩基の１’位の炭素の位置が外向きのもの（Hoogsteen結合型）とその反対のもの（逆Hoogsteeen結合型）の２種類があり、前者がパラレル配向性（DNA二本鎖の塩基対形成する側の鎖と同じ配向性）、後者がアンチパラレル配向性（DNA二本鎖の塩基対形成する側の鎖と逆の配向性）である。パラレル配向性TFOは、DNA二本鎖中のA-T塩基対およびG-C塩基対のAとGに対して、それぞれT（T:A-T塩基対）とプロトン化されたC（C+：G-C塩基対）が結合する。一方、アンチパラレル配向性TFOは、DNA二本鎖中のA-T塩基対およびG-C塩基対のAとGに対して、それぞれT（T:A-T塩基対）とG（G:G-C塩基対）が結合する。Cがプロトン化されるためには酸性条件である必要があるため、生理的条件下での利用が制限されるが、中性でもプロトン化されるメチルシチジンや、プロトン化の必要のない誘導体（例、５－メチル－６－オキソシチジン）を用いることで、この問題は回避できる。 Antigene nucleic acid refers to a nucleic acid that can suppress target gene expression at the transcriptional level by forming a triplex with double-stranded DNA containing a target gene, and is also referred to as triplex forming oligonucleotide (TFO). . The typical antigene method is based on the binding of a third nucleic acid to a DNA double strand, and the base of the third nucleic acid forms a hydrogen bond with the purine base of the purine-pyrimidine base pair, resulting in a flat surface. Triple strand formation is possible by adopting a structure in which three bases are arranged consecutively within the chain. There are two types of third nucleic acid (TFO) (i.e., antigene nucleic acid) with the 1' carbon of the base facing outward (Hoogsteen binding type) and the opposite (reverse Hoogsteen binding type). , the former has a parallel orientation (the same orientation as the base-pairing strand of the DNA duplex), and the latter has an antiparallel orientation (the opposite orientation to the base-pairing strand of the DNA duplex) It is. Parallel orientation TFO has T (T:A-T base pair) and protonated C (C+:G-C base pair) for A and G of A-T base pair and G-C base pair in DNA duplex, respectively. Join. On the other hand, in anti-parallel orientation TFO, T (T:A-T base pair) and G (G:G-C base pair) bind to A and G of A-T base pair and G-C base pair in DNA duplex, respectively. do. In order for C to be protonated, acidic conditions are required, which limits its use under physiological conditions, but methylcytidine, which can be protonated even under neutral conditions, and derivatives that do not require protonation ( This problem can be avoided by using eg 5-methyl-6-oxocytidine).
　アンチジーン核酸を構成するヌクレオチド分子は、天然型のDNA若しくはRNAでもよいし、安定性（化学的および／または対酵素）や比活性（DNAとの親和性）を向上させるために、種々の化学修飾が施されたヌクレオチド（例えば、PNAヌクレオチドなど）であってもよい。 The nucleotide molecules constituting the antigene nucleic acid may be naturally occurring DNA or RNA, or may be treated with various chemicals to improve stability (chemical and/or anti-enzyme) and specific activity (affinity with DNA). It may also be a modified nucleotide (for example, a PNA nucleotide).
　アンチジーン核酸の長さは、特に制限されないが、典型的には10～50ヌクレオチド長であり、好ましくは10～30ヌクレオチド長であり、より好ましくは13～30ヌクレオチド長であり、さらに好ましくは15～20ヌクレオチド長である。 The length of the antigene nucleic acid is not particularly limited, but is typically 10 to 50 nucleotides, preferably 10 to 30 nucleotides, more preferably 13 to 30 nucleotides, and even more preferably 15 to 30 nucleotides. ~20 nucleotides long.
　アンチジーン核酸は、例えば、国際公開第2005/021570号、国際公開第03/068695号、国際公開第2001/007455号に記載の自体公知の手法或いはそれに準じる方法により、化学的に合成することができる。 Antigene nucleic acids can be chemically synthesized, for example, by methods known per se described in WO 2005/021570, WO 03/068695, and WO 2001/007455, or methods analogous thereto. can.
　CRISPR/Casシステムは、標的DNA配列と二本鎖を形成する短鎖CRISPR RNA（crRNA）とトランス活性化型crRNA（tracrRNA）との複合体（即ち、二本鎖ガイドRNA）、crRNAとtracrRNAとを組合せた単一の合成RNA（即ち、一本鎖ガイドRNA）、またはcrRNA単体により標的DNAの配列を認識する。従って、標的DNAの配列に基づきcrRNAの配列を設計することで、任意の配列を標的化することができる。CRISPR/Casシステムは、一本鎖もしくは二本鎖ガイドRNAとCas（Casヌクレアーゼとも称する。）との複合体として提供される。 The CRISPR/Cas system consists of a complex of short CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that forms a double strand with the target DNA sequence (i.e. double-stranded guide RNA), crRNA and tracrRNA. The target DNA sequence is recognized by a single synthetic RNA (i.e., single-stranded guide RNA) or crRNA alone. Therefore, by designing the crRNA sequence based on the target DNA sequence, any sequence can be targeted. The CRISPR/Cas system is provided as a complex of single-stranded or double-stranded guide RNA and Cas (also referred to as Cas nuclease).
　ガイドRNA（crRNA単体もガイドRNAに含まれるものとする。以下同様。）に含まれる、標的DNA配列に対して相補的な塩基配列の設計は、例えば公開のガイドRNA設計ウェブサイト（CRISPR Design Tool、CRISPRdirect等）を用いることなどにより、適宜設計できる。これらの候補の中から、目的の宿主ゲノム中のオフターゲットサイト数が少ない候補配列をターゲッティング配列として用いることができる。使用するガイドRNA設計ソフトウエアに宿主ゲノムのオフターゲットサイトを検索する機能がない場合、例えば、候補配列の3’側の8～12ヌクレオチド（標的ヌクレオチド配列の識別能の高いseed配列）について、宿主ゲノムに対してBlast検索をかけることにより、オフターゲットサイトを検索することができる。tracrRNAなども、Casヌクレアーゼの種類に応じて適宜設計することができる。 The design of a complementary base sequence to the target DNA sequence contained in guide RNA (crRNA alone is also included in guide RNA; the same shall apply hereinafter) can be done using, for example, the public guide RNA design website (CRISPR Design Tool , CRISPRdirect, etc.), it can be designed as appropriate. Among these candidates, candidate sequences with a small number of off-target sites in the target host genome can be used as targeting sequences. If the guide RNA design software you use does not have the ability to search for off-target sites in the host genome, for example, 8 to 12 nucleotides on the 3' side of the candidate sequence (seed sequence with high discriminatory ability for the target nucleotide sequence) Off-target sites can be searched by performing a Blast search on the genome. tracrRNA etc. can also be designed as appropriate depending on the type of Cas nuclease.
　本発明で用いるCasは、特に制限はないが、好ましくはCas9（Cas9ヌクレアーゼとも称する。）またはCpf1（Cpf1ヌクレアーゼとも称する。）である。Cas9としては、例えばストレプトコッカス・ピオゲネス（Streptococcus pyogenes）由来のCas9（SpCas9; PAM配列（5’→3’方向；以下同じ）NGG(NはA、G、TまたはC。以下同じ。)）、黄色ブドウ球菌（Staphylococcus aureus）由来のCas9（SaCas9; PAM配列NNGRR(T)）、ストレプトコッカス・サーモフィラス（Streptococcus thermophilus）由来のCas9（StCas9; PAM配列NNAGAAW）、ナイセリア・メニンギチジス（Neisseria meningitidis）由来のCas9（NmCas9; PAM配列NNNNGATT）等が挙げられるが、それらに限定されない。好ましくはSpCas9である。また、Cpf1としては、例えば、フランシセラ・ノヴィシダ（Francisella novicida）由来のCpf1（FnCpf1; PAM配列TTN）、アシダミノコッカス sp.（Acidaminococcus sp.）由来のCpf1（AsCpf1;PAM配列TTTN）、ラクノスピラ科細菌（Lachnospiraceae bacterium）由来のCpf1（LbCpf1; PAM配列TTTN）等が挙げられるが、それらに限定されない。 The Cas used in the present invention is not particularly limited, but is preferably Cas9 (also referred to as Cas9 nuclease) or Cpf1 (also referred to as Cpf1 nuclease). Examples of Cas9 include Cas9 (SpCas9; PAM sequence (5'→3' direction; same below) NGG (N is A, G, T or C; same below)) derived from Streptococcus pyogenes, yellow Cas9 from Staphylococcus aureus (SaCas9; PAM sequence NNGRR(T)), Cas9 from Streptococcus thermophilus (StCas9; PAM sequence NNAGAAW), Cas9 from Neisseria meningitidis (NmCas9) ; PAM sequence NNNNGATT), but are not limited to these. Preferably it is SpCas9. Examples of Cpf1 include Cpf1 (FnCpf1; PAM sequence TTN) derived from Francisella novicida, Cpf1 (AsCpf1; PAM sequence TTTN) derived from Acidaminococcus sp., and Lachnospiraceae bacteria. Examples include, but are not limited to, Cpf1 (LbCpf1; PAM sequence TTTN) derived from (Lachnospiraceae bacterium).
　ヒト臨床に利用することを考慮すれば、DNA二本鎖切断（DSB）を生じるのは好ましくないので、Casとしては、DNA切断活性を失活したもの（dCas）が好ましい。よって、本発明に用いるCRISPR-Casシステムとして、CRISPR-dCasシステムが好ましい。dCasとして、例えば、SpCas9の場合、10番目のAsp残基がAla残基に変換した、ガイドRNAと相補鎖を形成する鎖の反対鎖の切断能を欠くD10A変異体、840番目のHis残基がAla残基で変換した、ガイドRNAと相補鎖の切断能を欠くH840A変異体の二重変異体を用いることができる。また、FnCpf1の場合、917番目のAsp残基がAla残基（D917A）に、あるいは1006番目のGlu残基がAla残基（E1006A）に変換した、両方の鎖の切断能を欠く変異体を用いることができる。他の変異Casも同様に用いることができる。 Considering clinical use in humans, it is undesirable to generate DNA double-strand breaks (DSB), so it is preferable to use Cas with inactivated DNA cleaving activity (dCas). Therefore, the CRISPR-dCas system is preferable as the CRISPR-Cas system used in the present invention. As dCas, for example, in the case of SpCas9, the 10th Asp residue is converted to an Ala residue, the D10A mutant lacks the ability to cleave the opposite strand of the strand that forms a complementary strand with the guide RNA, and the 840th His residue. A double mutant of the H840A mutant, which lacks the ability to cleave guide RNA and complementary strands, can be used. In the case of FnCpf1, we created mutants lacking the ability to cleave both chains, in which the Asp residue at position 917 was converted to an Ala residue (D917A) or the Glu residue at position 1006 was converted to an Ala residue (E1006A). Can be used. Other mutant Cas can be used as well.
　CRISPR/Casシステムを用いると、標的DNA配列でガイドRNAとCasの複合体が結合し、それにより標的遺伝子の転写が阻害される。また、Casに転写抑制因子を結合させることで、標的遺伝子の転写抑制をより高めることも可能である。かかる転写抑制因子としては、例えば、KRAB、MBD2B、v-ErbA、SID（SIDのコンカテマー（SID4X）を含む）、MBD2、MBD3、DNMTファミリー（例：DNMT1、DNMT3A、DNMT3B等）、Rb、MeCP2、ROM2およびAtHD2Aなどが挙げられ、好ましくは、KRABである。本発明の転写抑制因子としてKRABを用いる場合に、その由来とするタンパク質は特に制限されないが、例えば、KOX-1（ZNF10）、KOX8（ZNF708）、ZNF43、ZNF184、ZNF91、HPF4、HTF10、HTF34などが挙げられる。 When using the CRISPR/Cas system, a complex of guide RNA and Cas binds to the target DNA sequence, thereby inhibiting transcription of the target gene. Furthermore, by binding a transcriptional repressor to Cas, it is possible to further enhance the transcriptional repression of the target gene. Such transcriptional repressors include, for example, KRAB, MBD2B, v-ErbA, SID (including SID concatemer (SID4X)), MBD2, MBD3, DNMT family (e.g. DNMT1, DNMT3A, DNMT3B, etc.), Rb, MeCP2, Examples include ROM2 and AtHD2A, preferably KRAB. When using KRAB as the transcription repressor of the present invention, the protein from which it is derived is not particularly limited, but examples include KOX-1 (ZNF10), KOX8 (ZNF708), ZNF43, ZNF184, ZNF91, HPF4, HTF10, HTF34, etc. can be mentioned.
　CRISPR/Casシステムは、例えば、ガイドRNAとCasとの複合体の形態、Casをコードする核酸または発現ベクターとガイドRNAの複合体の形態、ガイドRNAをコードする核酸または発現ベクターとCasの複合体の形態、あるいはガイドRNAおよびCasをコードする核酸または発現ベクター（単一の核酸または発現ベクターであってもよく、複数の核酸または発現ベクターであってもよい）の形態で本発明の医薬に含まれ得る。これらの核酸は、mRNAなどのRNAであってもよいが、好ましくはDNAである。 The CRISPR/Cas system can be used, for example, in the form of a complex between a guide RNA and Cas, a complex between a nucleic acid encoding Cas or an expression vector and guide RNA, or a complex between a nucleic acid encoding guide RNA or an expression vector and Cas. or in the form of a nucleic acid or expression vector (which may be a single nucleic acid or expression vector or a plurality of nucleic acids or expression vectors) encoding guide RNA and Cas. It can be done. These nucleic acids may be RNA such as mRNA, but are preferably DNA.
　本発明で用いる発現ベクターとしては、例えば、無毒化したレトロウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンドビスウイルス、センダイウイルス、SV40、免疫不全症ウイルス（HIV）等のウイルスベクターを用いることができる。好ましくはアデノウイルスまたはアデノ随伴ウイルスベクターが挙げられる。 Expression vectors used in the present invention include, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, immunodeficiency virus ( Viral vectors such as HIV) can be used. Preferably, adenovirus or adeno-associated virus vectors are used.
　発現ベクターは、典型的には、本発明の核酸またはタンパク質をコードするDNAの上流にプロモーターを有する。かかるプロモーターとしては、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが用いられる。また、特定の細胞（例えば神経細胞）特異的なプロモーターを用いてもよい。 The expression vector typically has a promoter upstream of the DNA encoding the nucleic acid or protein of the present invention. Such promoters include SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter. etc. are used. Furthermore, a promoter specific to a particular cell (eg, nerve cell) may be used.
　ガイドRNAに含まれる、標的DNA配列に対して相補的な塩基配列の長さは、標的DNA配列に対して特異的に結合し得る限り特に制限はないが、例えば15～30ヌクレオチド、好ましくは18～25ヌクレオチドである。 The length of the nucleotide sequence complementary to the target DNA sequence contained in the guide RNA is not particularly limited as long as it can specifically bind to the target DNA sequence, but for example, 15 to 30 nucleotides, preferably 18 ~25 nucleotides.
　CRISPR/Casシステム、ガイドRNAをコードする核酸およびCasをコードする核酸は、化学的にDNA鎖またはタンパク質を合成するか、もしくは合成した一部オーバーラップするオリゴDNA短鎖を、PCR法やGibson Assembly法を利用して接続することにより、タンパク質全長をコードするDNAを構築することも可能である。 CRISPR/Cas systems, guide RNA-encoding nucleic acids, and Cas-encoding nucleic acids are produced by chemically synthesizing DNA strands or proteins, or by converting partially overlapping oligo DNA short strands into synthesized DNA strands using PCR methods or Gibson Assembly. It is also possible to construct DNA encoding the full length of a protein by connecting them using the method.
　本発明の核酸を構成するヌクレオチドの構成要素として、糖部（例：リボース、デオキシリボース）、塩基およびリン酸を含む。また、「修飾」として、例えば、該構成要素および／またはヌクレオシド間結合における置換、付加および／または欠失、前記構成要素および／またはヌクレオシド間結合における原子および／または官能基の置換、付加および／または欠失が挙げられる。 The constituent elements of the nucleotide constituting the nucleic acid of the present invention include a sugar moiety (eg, ribose, deoxyribose), a base, and a phosphoric acid. Furthermore, "modifications" include, for example, substitutions, additions and/or deletions in the constituent elements and/or internucleoside bonds, substitutions, additions and/or deletions of atoms and/or functional groups in the constituent elements and/or internucleoside bonds. or deletion.
　天然塩基として、アデニン、シトシン、グアニン、チミンおよびウラシルが挙げられる。また、該塩基に修飾が施された修飾塩基として、例えば、5-メチルシトシン、5-フルオロシトシン、5-ブロモシトシン、5-ヨードシトシンまたはN4-メチルシトシン； N6-メチルアデニンまたは8-ブロモアデニン；ならびにN2-メチルグアニンまたは8-ブロモグアニンが挙げられるが、これらに限定されない。修飾塩基は、好ましくは、5-メチルシトシンである。 Natural bases include adenine, cytosine, guanine, thymine and uracil. In addition, examples of modified bases in which the base is modified include 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine or N4-methylcytosine; N6-methyladenine or 8-bromoadenine ; and N2-methylguanine or 8-bromoguanine. The modified base is preferably 5-methylcytosine.
　修飾が施されたヌクレオシド間結合としては、例えば、ホスホロチオエート結合、ホスホロジチオエート結合、ホスホトリエステル結合、メチルホスホネート結合、メチルチオホスホネート結合、ボラノホスフェート結合、ホスホロアミデート結合などが挙げられるが、これらに限定されない。 Examples of modified internucleoside bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds, methylphosphonate bonds, methylthiophosphonate bonds, boranophosphate bonds, and phosphoroamidate bonds. , but not limited to.
　糖部の修飾としては、例えば、糖部の2’-O-メトキシエチル修飾、糖部の2’-O-メチル修飾、糖部の2’フルオロ修飾、糖部の2’位と4’位との架橋（該架橋構造を有するヌクレオチドが、架橋型ヌクレオチドである）などが挙げられる。架橋型ヌクレオチドとしては、例えば、ロックト人工核酸（LNA：Locked Nucleic Acid）、2’－O，4’-C-エチレン架橋核酸（ENA：2’-O，4’-C-Ethylenebridged Nucleic Acid）などが挙げられる。架橋型ヌクレオチドとして、より具体的には、下記ヌクレオシド構造を有するものが挙げられる。 Modifications of the sugar moiety include, for example, 2'-O-methoxyethyl modification of the sugar moiety, 2'-O-methyl modification of the sugar moiety, 2'fluoro modification of the sugar moiety, and 2' and 4' positions of the sugar moiety. (The nucleotide having the crosslinked structure is a crosslinked nucleotide). Examples of bridged nucleotides include locked artificial nucleic acids (LNA), 2'-O, 4'-C-ethylene bridged nucleic acids (ENA), etc. can be mentioned. More specifically, examples of the bridged nucleotide include those having the following nucleoside structure.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　（式中、Rは、水素原子、分岐または環を形成していてもよい炭素数1から7のアルキル基、分岐または環を形成していてもよい炭素数2から7のアルケニル基、ヘテロ原子を含んでいてもよい炭素数3から12のアリール基、ヘテロ原子を含んでいてもよい炭素数3から12のアリール部分を有するアラルキル基、または核酸合成のアミノ基の保護基を表す。好ましくは、Rは、水素原子、メチル基、エチル基、n-プロピル基、イソプロピル基、フェニル基、またはベンジル基であり、より好ましくは、Rは、水素原子またはメチル基である。Baseは、天然塩基または修飾塩基である。） (In the formula, R is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, or a heteroatom. represents an aryl group having 3 to 12 carbon atoms which may contain an atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom, or a protecting group for an amino group in nucleic acid synthesis. Preferably. , R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R is a hydrogen atom or a methyl group.Base is a natural base or a modified base.)
　また、修飾ヌクレオチドは、PNA、UNA(unlocked nucleic acid)、HNA、モルフォリノ核酸などであってもよい。上記HNAは、そのヘキソピラノース部分の水酸基がデオキシ化されていてもよい。また、上記HNAは、そのヘキソピラノース部分の水酸基がフッ素原子に置換されていてもよい。 Additionally, the modified nucleotide may be PNA, UNA (unlocked nucleic acid), HNA, morpholino nucleic acid, or the like. The hydroxyl group of the hexopyranose moiety of the HNA may be deoxylated. Further, in the above HNA, the hydroxyl group of the hexopyranose moiety may be substituted with a fluorine atom.
　本発明の核酸は、核酸鎖（二本鎖核酸の場合には、各核酸鎖）の5'末端、3'末端および／または配列内部が１つ以上のリガンドや蛍光団により修飾されていてもよく、リガンドや蛍光団により修飾された核酸をコンジュゲート核酸とも呼ぶ。固相上での伸張反応時に、固相上で反応可能な修飾剤を反応させることで、5'末端、3'末端および／または配列内部に修飾を施すことができる。また、アミノ基、メルカプト基、アジド基または３重結合などの官能基を導入した核酸をあらかじめ合成および精製しておき、それらに修飾化剤を作用させることでコンジュゲート核酸を得ることもできる。リガンドとしては、生体分子と親和性のある分子であれば良いが、例えば、コレステロール、脂肪酸、トコフェロール、レチノイドなどの脂質類、N-アセチルガラクトサミン(GalNAc)、ガラクトース(Gal)、マンノース(Man)などの糖類、フル抗体、Fab、VHHなどの抗体、低密度リポタンパク質（LDL）、ヒト血清アルブミンなどのタンパク質、RGD、NGR、R9、CPPなどのペプチド類、葉酸などの低分子、合成ポリアミノ酸などの合成ポリマー、あるいは核酸アプタマーなどがあげられ、これらを組み合わせて用いることもできる。蛍光団としてはCy3シリーズ、Alexaシリーズ、ブラックホールクエンチャーなどが挙げられる。 The nucleic acids of the present invention may be modified at the 5' end, 3' end, and/or within the sequence of the nucleic acid strand (in the case of double-stranded nucleic acids, each nucleic acid strand) with one or more ligands or fluorophores. Nucleic acids modified with ligands or fluorophores are also often referred to as conjugated nucleic acids. By reacting a modifying agent that can react on the solid phase during the extension reaction on the solid phase, it is possible to modify the 5' end, 3' end, and/or the inside of the sequence. Alternatively, a conjugate nucleic acid can also be obtained by synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on the nucleic acid. The ligand may be any molecule that has affinity with biomolecules, such as cholesterol, fatty acids, tocopherol, lipids such as retinoids, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc. saccharides, full antibodies, antibodies such as Fab, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, peptides such as RGD, NGR, R9, CPP, low molecules such as folic acid, synthetic polyamino acids, etc. Examples include synthetic polymers, nucleic acid aptamers, etc., and these can also be used in combination. Fluorophores include the Cy3 series, Alexa series, and black hole quenchers.
　哺乳動物への本発明の医薬の投与は、経口的にまたは非経口的（例えば、皮下注射、筋肉注射、局所注入（例：脳室内投与、髄腔内投与）、腹腔内投与など）に行うことが可能であるが、非経口的に（特に、脳室内投与または髄腔内投与により）投与するのが望ましい。 The medicament of the present invention is administered to mammals orally or parenterally (e.g., subcutaneous injection, intramuscular injection, local injection (e.g., intraventricular administration, intrathecal administration), intraperitoneal administration, etc.). Although it is possible, it is preferable to administer the drug parenterally, particularly by intraventricular or intrathecal administration.
　経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。一方、非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。これらの製剤は、賦形剤（例えば、乳糖、白糖、葡萄糖、マンニトール、ソルビトールのような糖誘導体；トウモロコシデンプン、バレイショデンプン、α澱粉、デキストリンのような澱粉誘導体；結晶セルロースのようなセルロース誘導体；アラビアゴム；デキストラン；プルランのような有機系賦形剤；および、軽質無水珪酸、合成珪酸アルミニウム、珪酸カルシウム、メタ珪酸アルミン酸マグネシウムのような珪酸塩誘導体；燐酸水素カルシウムのような燐酸塩；炭酸カルシウムのような炭酸塩；硫酸カルシウムのような硫酸塩等の無機系賦形剤である）、滑沢剤（例えば、ステアリン酸、ステアリン酸カルシウム、ステアリン酸マグネシウムのようなステアリン酸金属塩；タルク；コロイドシリカ；ビーズワックス、ゲイ蝋のようなワックス類；硼酸；アジピン酸；硫酸ナトリウムのような硫酸塩；グリコール；フマル酸；安息香酸ナトリウム；DLロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウムのようなラウリル硫酸塩；無水珪酸、珪酸水和物のような珪酸類；および、上記澱粉誘導体である）、結合剤（例えば、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、マクロゴール、および、前記賦形剤と同様の化合物である）、崩壊剤（例えば、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、内部架橋カルボキシメチルセルロースナトリウムのようなセルロース誘導体；カルボキシメチルスターチ、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドンのような化学修飾されたデンプン・セルロース類である）、乳化剤（例えば、ベントナイト、ビーガムのようなコロイド性粘土；水酸化マグネシウム、水酸化アルミニウムのような金属水酸化物；ラウリル硫酸ナトリウム、ステアリン酸カルシウムのような陰イオン界面活性剤；塩化ベンザルコニウムのような陽イオン界面活性剤；および、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンソルビタン脂肪酸エステル、ショ糖脂肪酸エステルのような非イオン界面活性剤である）、安定剤（メチルパラベン、プロピルパラベンのようなパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコールのようなアルコール類；塩化ベンザルコニウム；フェノール、クレゾールのようなフェノール類；チメロサール；デヒドロ酢酸；および、ソルビン酸である）、矯味矯臭剤（例えば、通常使用される、甘味料、酸味料、香料等である）、希釈剤等の添加剤を用いて周知の方法で製造される。 Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. formulations, emulsions, suspensions, etc. On the other hand, as compositions for parenteral administration, for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may also be included. These formulations contain excipients (e.g. sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminate metasilicate; phosphates such as calcium hydrogen phosphate; carbonic acid carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (such as stearic acid, metal salts of stearate such as calcium stearate, magnesium stearate; talc; Colloidal silica; waxes such as beeswax and gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl such as sodium lauryl sulfate and magnesium lauryl sulfate sulfates; silicic acids such as silicic anhydride and silicic acid hydrate; and the above-mentioned starch derivatives), binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the above-mentioned excipients) disintegrants (e.g. cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, internally cross-linked sodium carboxymethylcellulose; carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone) emulsifiers (e.g. colloidal clays such as bentonite, vegum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; sodium lauryl sulfate, calcium stearate) anionic surfactants such as; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid ester. ), stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; .
　医薬上許容される担体としては、例えば、ショ糖、デンプン等の賦形剤、セルロース、メチルセルロース等の結合剤、デンプン、カルボキシメチルセルロース等の崩壊剤、ステアリン酸マグネシウム、エアロジル等の滑剤、クエン酸、メントール等の芳香剤、安息香酸ナトリウム、亜硫酸水素ナトリウム等の保存剤、クエン酸、クエン酸ナトリウム等の安定剤、メチルセルロース、ポリビニルピロリド等の懸濁剤、界面活性剤等の分散剤、水、生理食塩水等の希釈剤、ベースワックス等が挙げられるが、それらに限定されるものではない。 Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.
　KLHL32遺伝子の発現抑制薬が核酸の形態である場合に、該核酸の標的細胞内への導入を促進するために、本発明の医薬は更に核酸導入用試薬を含んでいてもよい。該核酸導入用試薬としては、例えば、塩化カルシウム、Calcium enrichment試薬、アテロコラーゲン；リポソーム；ナノパーティクル；リポフェクチン、リプフェクタミン（lipofectamine）、DOGS（トランスフェクタム）、DOPE、DOTAP、DDAB、DHDEAB、HDEAB、ポリブレン、あるいはポリ（エチレンイミン）（PEI）等の陽イオン性脂質等を用いることができる。 When the KLHL32 gene expression inhibitor is in the form of a nucleic acid, the medicament of the present invention may further contain a nucleic acid introduction reagent in order to promote the introduction of the nucleic acid into target cells. Examples of the nucleic acid introduction reagent include calcium chloride, Calcium enrichment reagent, atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, Alternatively, a cationic lipid such as poly(ethyleneimine) (PEI) can be used.
　また、本発明の医薬は、KLHL32遺伝子の発現抑制薬がリポソームに封入されてなる医薬組成物であってもよい。リポソームは、１以上の脂質二重層により包囲された内相を有する微細閉鎖小胞であり、通常は水溶性物質を内相に、脂溶性物質を脂質二重層内に保持することができる。本明細書において「封入」という場合には、KLHL32遺伝子の発現抑制薬はリポソーム内相に保持されてもよいし、脂質二重層内に保持されてもよい。本発明に用いられるリポソームは単層膜であっても多層膜であってもよく、また、粒子径は、例えば10～1000nm、好ましくは50～300nmの範囲で適宜選択できる。標的組織への送達性を考慮すると、粒子径は、例えば200nm以下、好ましくは100nm以下である。 Furthermore, the medicament of the present invention may be a pharmaceutical composition in which a KLHL32 gene expression inhibitor is encapsulated in a liposome. Liposomes are microscopic closed vesicles that have an internal phase surrounded by one or more lipid bilayers, and can typically retain water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer. As used herein, "encapsulation" means that the KLHL32 gene expression inhibitor may be retained in the internal phase of the liposome or within the lipid bilayer. The liposome used in the present invention may be a monolayer or a multilayer, and the particle size can be appropriately selected within the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering the delivery performance to the target tissue, the particle size is, for example, 200 nm or less, preferably 100 nm or less.
　核酸のような水溶性化合物のリポソームへの封入法としては、リピドフィルム法（ボルテックス法）、逆相蒸発法、界面活性剤除去法、凍結融解法、リモートローディング法等が挙げられるが、これらに限定されず、任意の公知の方法を適宜選択することができる。 Methods for encapsulating water-soluble compounds such as nucleic acids in liposomes include the lipid film method (vortex method), reversed-phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method. The method is not limited, and any known method can be selected as appropriate.
　本発明の医薬の投与量は、投与の目的、投与方法、対象疾患の種類、重篤度、投与対象の状況（性別、年齢、体重など）によって異なるが、例えば、KLHL32遺伝子の発現抑制薬を核酸の形態で成人に全身投与する場合、通常、該発現抑制薬の一回投与量として2 nmol/kg以上50 nmol/kg以下、局所投与する場合、1 pmol/kg以上10 nmol/kg以下が望ましい。かかる投与量を1～10回、より好ましくは5～10回投与することが望ましい。症状に応じて増量若しくは減量してもよい。 The dosage of the pharmaceutical of the present invention varies depending on the purpose of administration, the method of administration, the type and severity of the target disease, and the circumstances of the subject (sex, age, body weight, etc.). When administered systemically to adults in the form of nucleic acids, the single dose of the expression inhibitor is usually 2 nmol/kg or more and 50 nmol/kg or less, and when locally administered, the dose is 1 pmol/kg or more and 10 nmol/kg or less. desirable. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times. The dose may be increased or decreased depending on the symptoms.
　本発明の医薬は、神経変性疾患に対する他の予防または治療薬（以下、「既存薬」と称することがある。）（例：アデュカヌマブ、ドネペジル、リバスチグミン、ガランタミン、ドネペジル、メマンチン等）と組み合わせて用いることもできる。従って、本発明の一態様において、KLHL32遺伝子の発現抑制薬と、１種以上の既存薬とを含有する、神経変性疾患の予防または治療薬が提供される。 The drug of the present invention is used in combination with other preventive or therapeutic drugs (hereinafter sometimes referred to as "existing drugs") for neurodegenerative diseases (e.g., aducanumab, donepezil, rivastigmine, galantamine, donepezil, memantine, etc.). You can also do that. Therefore, in one embodiment of the present invention, a preventive or therapeutic drug for neurodegenerative diseases is provided, which contains a drug that suppresses the expression of the KLHL32 gene and one or more existing drugs.
　併用薬剤として用いる場合には、かかる併用薬剤は、KLHL32遺伝子の発現抑制薬とともに製剤化して単一の製剤として投与することもできるし、あるいは、KLHL32遺伝子の発現抑制薬とは別個に製剤化して（例えば、キットとして）、本発明の医薬と同一若しくは別ルートで、同時若しくは時間差をおいて投与することもできる。また、これらの併用薬剤の投与量は、該薬剤を単独投与する場合に通常用いられる量であってよく、あるいは通常用いられる量より減量することもできる。 When used as a combination drug, such a combination drug can be formulated with a KLHL32 gene expression inhibitor and administered as a single formulation, or alternatively, it can be formulated separately from the KLHL32 gene expression inhibitor. It can also be administered (for example, as a kit) by the same or different route as the medicament of the invention, either simultaneously or at a staggered time. Further, the dosage of these combined drugs may be the amount normally used when the drugs are administered alone, or may be reduced from the amount normally used.
２．神経変性疾患の予防または治療薬のスクリーニング方法
　前述の通り、KLHL32遺伝子の発現抑制により、神経変性疾患を予防または治療することができるため、KLHL32遺伝子の発現を抑制する物質をスクリーニングできれば、該物質を神経変性疾患の予防または治療薬（換言すれば、神経変性疾患の予防または治療活性を有する物質）の候補として用いることができる。従って、別の態様において、本発明は、以下の工程を含む、神経変性疾患の予防または治療薬のスクリーニング方法（以下、「本発明のスクリーニング方法１」と称する場合がある。）を提供する。
　（１）KLHL32遺伝子を発現する細胞に、被験物質を接触させる工程、および
　（２）KLHL32遺伝子の発現量を減少させた被験物質を神経変性疾患の予防または治療薬の候補として選択する工程。 2. Method for screening drugs for preventing or treating neurodegenerative diseases As mentioned above, neurodegenerative diseases can be prevented or treated by suppressing the expression of the KLHL32 gene. It can be used as a candidate for a preventive or therapeutic agent for neurodegenerative diseases (in other words, a substance having preventive or therapeutic activity for neurodegenerative diseases). Accordingly, in another aspect, the present invention provides a method for screening for preventive or therapeutic agents for neurodegenerative diseases (hereinafter sometimes referred to as "Screening method 1 of the present invention"), which includes the following steps.
(1) A step of bringing a test substance into contact with cells expressing the KLHL32 gene, and (2) A step of selecting a test substance that reduces the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases.
　また、神経変性に先立って、KLHL32遺伝子が発現する、あるいは該遺伝子の発現量が増加するため、神経変性のモデル細胞において、KLHL32遺伝子の発現量を増加させない物質をスクリーニングできれば、該物質を神経変性疾患の予防または治療薬の候補として用いることができる。従って、さらに別の態様において、本発明は、以下の工程を含む、神経変性疾患の予防または治療薬のスクリーニング方法（以下、「本発明のスクリーニング方法２」と称する場合がある。）を提供する。
　（１’）細胞に、被験物質を接触させる工程、および
　（２’）KLHL32遺伝子の発現量の増加を抑制した被験物質を神経変性疾患の予防または治療薬の候補として選択する工程。
　以下では、本発明のスクリーニング方法１と本発明のスクリーニング方法２とを包含するものとして、「本発明のスクリーニング方法」との用語を用いることがある。 In addition, since the KLHL32 gene is expressed or the expression level of the gene increases prior to neurodegeneration, if we can screen for a substance that does not increase the expression level of the KLHL32 gene in model cells of neurodegeneration, it would be possible to screen for a substance that does not increase the expression level of the KLHL32 gene in neurodegeneration model cells. It can be used as a candidate for a preventive or therapeutic drug for diseases. Therefore, in yet another aspect, the present invention provides a method for screening for preventive or therapeutic agents for neurodegenerative diseases (hereinafter sometimes referred to as "Screening method 2 of the present invention"), which includes the following steps. .
(1') A step of contacting cells with a test substance; and (2') A step of selecting a test substance that suppresses an increase in the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases.
Below, the term "screening method of the present invention" may be used to include screening method 1 of the present invention and screening method 2 of the present invention.
　被験物質を接触させる細胞は、KLHL32遺伝子を発現する細胞、または培養等を行うことでKLHL32遺伝子を発現することとなる細胞であれば特に制限はなく、培養細胞でもよく、哺乳動物（例：ヒト、マウス、ラット、ハムスター、ウサギ、ネコ、イヌ、ウシ、ヒツジ、サル等）から単離した細胞であってもよく、哺乳動物内の細胞であってもよい。具体的には、本発明で用いる細胞として、例えば、（1）神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）、（2）組織前駆細胞、（3）神経細胞、リンパ球、上皮細胞、内皮細胞、筋肉細胞、線維芽細胞（皮膚細胞等）、毛細胞、肝細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞(膵外分泌細胞等)、脳細胞、肺細胞、腎細胞および脂肪細胞等の分化した細胞などが挙げられる。中でも、神経細胞が好ましく、ヒト神経細胞がより好ましい。一態様において、本発明に用いる神経細胞は、β-III tubulin、NeuN、N-CAM（neural cell adhesion molecule）、MAP2（microtubule-associated protein 2）からなる群から選択される神経細胞に特異的なマーカー遺伝子を少なくとも１以上発現し、かつβ-III tubulin陽性の突起（即ち、神経突起）を有する細胞である。 The cells to be contacted with the test substance are not particularly limited as long as they are cells that express the KLHL32 gene or cells that will express the KLHL32 gene by culturing, etc.; cultured cells may be used; , mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), or cells within a mammal. Specifically, the cells used in the present invention include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; (3) Nerve cells, lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain Examples include differentiated cells such as cells, lung cells, kidney cells, and adipocytes. Among these, nerve cells are preferred, and human nerve cells are more preferred. In one embodiment, the nerve cells used in the present invention are nerve cell-specific cells selected from the group consisting of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2). These cells express at least one marker gene and have β-III tubulin-positive processes (ie, neurites).
　また、別の態様において、細胞として、神経変性疾患のモデル細胞が挙げられる。神経変性疾患のモデル細胞としては、例えば、神経変性疾患の患者由来の細胞、神経変性疾患のモデル動物由来の細胞、神経変性疾患の原因である遺伝子に変異を有する多能性幹細胞から分化誘導した細胞などが挙げられる。前記神経変性疾患の原因である遺伝子に変異を有する多能性幹細胞は、神経変性疾患の患者から採取した体細胞を用いて、自体公知の方法により樹立した人工多能性幹細胞（iPS細胞）であることが好ましい。多能性幹細胞は、自体公知の方法により、任意の細胞（好ましくは、神経細胞）に分化することができる。神経変性疾患や神経変性疾患の原因である遺伝子、該遺伝子の変異体の具体例については、上記「１．神経変性疾患の予防または治療薬」で記載した内容が援用される。 In another embodiment, the cells include model cells for neurodegenerative diseases. Model cells for neurodegenerative diseases include, for example, cells derived from patients with neurodegenerative diseases, cells derived from model animals for neurodegenerative diseases, and cells induced to differentiate from pluripotent stem cells that have mutations in genes that cause neurodegenerative diseases. Examples include cells. The above-mentioned pluripotent stem cells that have mutations in genes that cause neurodegenerative diseases are induced pluripotent stem cells (iPS cells) that are established by a method known per se using somatic cells collected from patients with neurodegenerative diseases. It is preferable that there be. Pluripotent stem cells can be differentiated into any cells (preferably nerve cells) by a method known per se. Regarding specific examples of neurodegenerative diseases, genes that cause neurodegenerative diseases, and mutants of the genes, the contents described in the above "1. Preventive or therapeutic agents for neurodegenerative diseases" are cited.
　iPS細胞は、特定の初期化因子を、DNAまたはタンパク質の形態で体細胞に導入することによって作製することができる。初期化因子に含まれる遺伝子として、例えば、Oct3/4、Sox2、Sox1、Sox3、Sox15、Sox17、Klf4、Klf2、c-Myc、N-Myc、L-Myc、Nanog、Lin28、Fbx15、ERas、ECAT15-2、Tcl1、beta-catenin、Lin28b、Sall1、Sall4、Esrrb、Nr5a2、Tbx3またはGlis1等が例示され、これらの初期化因子は、単独で用いても良く、組み合わせて用いても良い。初期化因子の組み合わせとしては、WO2007/069666、WO2008/118820、WO2009/007852、WO2009/032194、WO2009/058413、WO2009/057831、WO2009/075119、WO2009/079007、WO2009/091659、WO2009/101084、WO2009/101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、WO2010/056831、WO2010/068955、WO2010/098419、WO2010/102267、WO2010/111409、WO2010/111422、WO2010/115050、WO2010/124290、WO2010/147395、WO2010/147612、Huangfu D, et al.（2008), Nat. Biotechnol., 26: 795-797、Shi Y, et al.（2008), Cell Stem Cell, 2: 525-528、Eminli S, et al.（2008), Stem Cells. 26:2467-2474、Huangfu D, et al.（2008), Nat Biotechnol. 26:1269-1275、Shi Y, et al.（2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al.（2008), Cell Stem Cell, 3:475-479、Marson A,（2008), Cell Stem Cell, 3, 132-135、Feng B, et al.（2009), Nat Cell Biol. 11:197-203、R.L. Judson et al.,（2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al.（2009), Proc Natl Acad Sci USA. 106:8912-8917、Kim JB, et al.（2009), Nature. 461:649-643、Ichida JK, et al.（2009), Cell Stem Cell. 5:491-503、Heng JC, et al.（2010), Cell Stem Cell. 6:167-74、Han J, et al.（2010), Nature. 463:1096-100、Mali P, et al.（2010), Stem Cells. 28:713-720、Maekawa M, et al.（2011), Nature. 474:225-9.に記載の組み合わせが例示される。 iPS cells can be created by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. Examples of genes included in the reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, and ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc., and these initialization factors may be used alone or in combination. Combinations of reprogramming factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091 659, WO2009/101084, WO2009/ 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO2010/0339 06, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO201 0/147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al ., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225- The combinations described in 9. are exemplified.
　iPS細胞などの多能性幹細胞を特定の細胞へ分化誘導する方法としては、種々の公知の分化誘導方法を適宜選択して用いることができ、例えば神経細胞への分化方法として、(1)Hester M.E. et al., Mol. Therapy, 19:1905-1912 (2011)に記載された、ヒト由来胚性幹細胞または人工多能性幹細胞からSFEB法で作製した神経前駆細胞に、３種類の（運動）神経細胞系譜特異的転写因子（Ngn2,Lhx3,およびIsl1）を発現させることで、運動神経細胞へ分化させる方法、(2)WO 2014/148646に記載された、多能性幹細胞に上記３種類の遺伝子を発現させることで、運動神経細胞へ分化させる方法、(3)WO 2014/148646に記載された、多能性幹細胞にNgn2を発現させることで、大脳皮質のグルタミン酸作動性神経細胞へ分化させる方法、(4)Pang Z.P. et al., Nature, 476:220-223 (2012)に記載された、ヒト胚性幹細胞に3種類の神経細胞系譜特異的転写因子（Ascl1,Brn2,およびMytl1）を発現させることで、神経細胞へ分化させる方法などが挙げられるが、これらに限定されない。 As a method for inducing differentiation of pluripotent stem cells such as iPS cells into specific cells, various known differentiation inducing methods can be selected and used as appropriate. For example, as a method for differentiating into nerve cells, (1) Hester As described in M.E. et al., Mol. Therapy, 19:1905-1912 (2011), three types of (motile) A method for differentiating into motor neurons by expressing neuronal cell lineage-specific transcription factors (Ngn2, Lhx3, and Isl1), (2) described in WO 2014/148646, in which the above three types are applied to pluripotent stem cells. A method for differentiating into motor neurons by expressing a gene, (3) described in WO 2014/148646, by expressing Ngn2 in pluripotent stem cells, they are differentiated into glutamatergic neurons in the cerebral cortex. Method, (4) Pang Z.P. et al., Nature, 476:220-223 (2012) injected three types of neuronal lineage-specific transcription factors (Ascl1, Brn2, and Mytl1) into human embryonic stem cells. Examples include, but are not limited to, a method of causing expression to differentiate into nerve cells.
　アルツハイマー病モデル動物の場合、APP、PSEN1およびPSEN2からなる群から選択される１種以上の遺伝子に変異が導入された、あるいはこれらの遺伝子の変異体が外部から導入された動物などが挙げられる。より具体的には、(1)PSEN1トランスジェニックマウス（マウスPrPプロモーター制御下にてエクソン9欠損体（PSEN1dE9）を発現しているマウス、Jankowsky J.L. et al., Hum Mol Genet. 13(2):159-70 (2004)）、(2)PSEN2トランスジェニックマウス（ユビキタスCMV初期エンハンサーおよびニワトリβアクチンプロモーター制御下にてヒトPSEN2変異体（N141I）を発現しているマウス、Oyama F. et al., J. Neurochem. 71: 313-322 (1998)、(3)ヒト二重変異体APP695（KM670/671NL、スウェーデンタイプ）トランスジェニックマウス（Hsiao K. et al., Science, 274(5284):99-102 (1996)）、(4)5×FADマウス（スウェーデンタイプ（KM670/671NL）、フロリダタイプ（I716V）およびロンドンタイプ（V717I）の三重変異を有するヒトAPP695と、二重変異（M146LおよびL285V）を有するヒトPSEN1とが、マウスThy1制御下にて発現しているトランスジェニックマウス）（Oakley H. et al., J. Neurosci. 26, 10129-10140 (2006)）、(5)マウスPrPプロモーター制御下にて、ヒトtau変異タンパク質が発現しているトランスジェニックマウス（Yoshiyama Y. et al., Neuron. 53(3):337-351 (2007）)などが挙げられる。 Examples of Alzheimer's disease model animals include animals in which mutations have been introduced in one or more genes selected from the group consisting of APP, PSEN1, and PSEN2, or animals in which mutants of these genes have been introduced externally. More specifically, (1) PSEN1 transgenic mice (mouse expressing exon 9 deletion (PSEN1dE9) under the control of mouse PrP promoter, Jankowsky J.L. et al., Hum Mol Genet. 13(2): 159-70 (2004)), (2) PSEN2 transgenic mouse (mice expressing human PSEN2 mutant (N141I) under the control of the ubiquitous CMV early enhancer and chicken β-actin promoter, Oyama F. et al., J. Neurochem. 71: 313-322 (1998), (3) Human double mutant APP695 (KM670/671NL, Swedish type) transgenic mouse (Hsiao K. et al., Science, 274(5284):99- 102 (1996)), (4) 5×FAD mice (human APP695 with triple mutations of Swedish type (KM670/671NL), Florida type (I716V) and London type (V717I), and double mutations (M146L and L285V) transgenic mouse in which human PSEN1 with the same expression is expressed under the control of mouse Thy1) (Oakley H. et al., J. Neurosci. 26, 10129-10140 (2006)), (5) Mouse PrP promoter control Transgenic mice expressing human tau mutant protein (Yoshiyama Y. et al., Neuron. 53(3):337-351 (2007)) are listed below.
　筋萎縮性側索硬化症モデル動物の場合、SOD1、C9ORF72、TDP43、FUS、PRN1、EPH4N、ANG、UBQLNおよびHNPNPAからなる群から選択される１種以上の遺伝子に変異が導入された、あるいはこれらの遺伝子の変異体が外部から導入された動物などが挙げられる。より具体的には、変異型SOD1（例：A4V、G37R、G41D、H46R、G85R、D90A、G93A、G93S、I112T、I113T、L114FおよびS134Nからなる群から選択される１以上の変異）を導入して得られたトランスジェニックマウスが挙げられる。また、MAPT遺伝子に変異が導入された、あるいはこの遺伝子の変異体が外部から導入されたタウオパチーモデル動物などが挙げられる。より具体的には、G272V、N297K、P301L、P301S、V337MおよびR406Wからなる群から選択される1以上の変異型MAPT遺伝子を導入したタウオパチーモデルマウスが挙げられる。ここで挙げたモデル動物は例示であり、これら以外の神経変性モデルも本発明のスクリーニング方法に用いることができる。 In the case of amyotrophic lateral sclerosis model animals, mutations have been introduced into one or more genes selected from the group consisting of SOD1, C9ORF72, TDP43, FUS, PRN1, EPH4N, ANG, UBQLN, and HNPNPA, or Examples include animals into which mutant genes have been introduced from outside. More specifically, mutant SOD1 (e.g., one or more mutations selected from the group consisting of A4V, G37R, G41D, H46R, G85R, D90A, G93A, G93S, I112T, I113T, L114F and S134N) is introduced. Examples include transgenic mice obtained by Other examples include tauopathy model animals in which a mutation has been introduced into the MAPT gene, or a variant of this gene has been introduced externally. More specifically, examples include tauopathy model mice into which one or more mutant MAPT genes selected from the group consisting of G272V, N297K, P301L, P301S, V337M and R406W have been introduced. The model animals mentioned here are just examples, and neurodegeneration models other than these can also be used in the screening method of the present invention.
　本発明のスクリーニング方法では、KLHL32遺伝子の発現量（即ち、KLHL32転写産物の発現量またはKLHL32タンパク質の発現量）を指標とすることができる。これらの指標となる発現量が、被験物質を接触させる前の細胞における発現量、被験物質を接触させていない細胞における発現量、あるいは神経変性疾患の予防または治療効果が認められないことが公知である物質（対照物質）を接触させた細胞における現量と比較して低い場合に、該被験物質はKLHL32遺伝子の発現量を減少させた、あるいは該被験物質はKLHL32遺伝子の発現量の増加を抑制したと評価することができる。 In the screening method of the present invention, the expression level of the KLHL32 gene (ie, the expression level of the KLHL32 transcript or the expression level of the KLHL32 protein) can be used as an indicator. It is known that these expression levels, which serve as indicators, are the expression levels in cells before contact with the test substance, the expression levels in cells that have not been contacted with the test substance, or that no preventive or therapeutic effect on neurodegenerative diseases has been observed. If the amount is low compared to the current amount in cells that have been contacted with a certain substance (control substance), the test substance has decreased the expression level of the KLHL32 gene, or the test substance has suppressed the increase in the expression level of the KLHL32 gene. It can be evaluated that
　KLHL32転写産物の発現量は、例えば、細胞から抽出した転写産物を用いて、公知の方法で検出または定量でき、該方法としては、例えば、RT-qPCR法やデジタルPCR法などが挙げられる。具体的には、RT-qPCRは、周知の方法で行うことができ、例えば、全RNAを鋳型に逆転写酵素によりcDNAを合成し、標的の遺伝子に特異的な一組のプライマー、DNAポリメラーゼ、さらに、発現量を定量するためのDNAインターカレーターとして機能できる色素またはプローブ（例：TaqMan（登録商標）プローブ等）の存在下でPCRを行うことにより実施できる。 The expression level of the KLHL32 transcript can be detected or quantified by a known method using, for example, the transcript extracted from cells, and examples of such methods include RT-qPCR and digital PCR. Specifically, RT-qPCR can be performed using a well-known method. For example, cDNA is synthesized using reverse transcriptase using total RNA as a template, and a set of primers specific to the target gene, DNA polymerase, Furthermore, it can be carried out by performing PCR in the presence of a dye or probe (eg, TaqMan® probe, etc.) that can function as a DNA intercalator for quantifying the expression level.
　RT-qPCRに用いるプライマーは、合成したcDNA上に存在し、増幅の対象とする遺伝子に特異的一組のプライマーとすることができる。プライマーは、増幅のサイズ（例えば、80-150bpが好ましい）、プライマーのサイズ（例えば、17-25塩基とする）、GC含量（例えば、40-60%とする）、3’末端の配列（例えば、3’末端の塩基をなるべくGまたはCとする、3’末端の近傍のGC含量が多すぎるプライマーは避ける）、配列の隔たり（例えば、配列のリピートがないようにする）、配列の相補性（例えば、上流プライマー内部やプライマー間で3塩基以上相補しないようにする）、Tm値（例えば、上流プライマーと下流プライマーのTm値を揃える。Tm値とは2(A+T)+4(G+C)）等を考慮して設計でき、また当業者に周知のプライマー設計ソフトウエアを使用して設計することができる。PCRプライマーの設計は、また、Applied Biosysytems Inc.等に依頼することができる。 The primers used for RT-qPCR can be a set of primers that are present on the synthesized cDNA and are specific to the gene to be amplified. The primers have various characteristics such as amplification size (e.g., preferably 80-150 bp), primer size (e.g., 17-25 bases), GC content (e.g., 40-60%), 3'-end sequence (e.g., , use G or C as the base at the 3' end, avoid primers with too much GC content near the 3' end), sequence gaps (e.g., avoid sequence repeats), sequence complementarity. (For example, make sure that there are no more than 3 bases complementary within the upstream primer or between primers), Tm value (For example, make sure that the Tm values of the upstream and downstream primers are the same. The Tm value is 2(A+T)+4(G +C)) etc., and can be designed using primer design software well known to those skilled in the art. Design of PCR primers can also be requested from Applied Biosystems Inc., etc.
　KLHL32タンパク質の発現量は、公知の方法により検出または定量することができる。例えば、KLHL32タンパク質を特異的に認識する抗体を用いて、ウエスタンブロット、免疫染色、酵素免疫測定法（例：EIA、ELISA）などにより検出または定量することができるが、目的のKLHL32タンパク質の発現量を検出または定量できれば、これらの方法に限定されない。 The expression level of KLHL32 protein can be detected or quantified by known methods. For example, using an antibody that specifically recognizes the KLHL32 protein, it can be detected or quantified by Western blotting, immunostaining, enzyme immunoassay (e.g., EIA, ELISA), etc. It is not limited to these methods as long as it can be detected or quantified.
　本発明のスクリーニング方法で用いる被験物質としては、例えば、細胞抽出物、細胞培養上清、微生物発酵産物、海洋生物由来の抽出物、植物抽出物、精製タンパク質または粗タンパク質、ペプチド、非ペプチド化合物、合成低分子化合物、および天然化合物が例示される。前記被験物質はまた、（1）生物学的ライブラリー、（2）デコンヴォルーションを用いる合成ライブラリー法、（3）「1ビーズ1化合物（one-bead one-compound）」ライブラリー法、および（4）アフィニティクロマトグラフィー選別を使用する合成ライブラリー法を含む当技術分野で公知のコンビナトリアルライブラリー法における多くのアプローチのいずれかを使用して得ることができる。アフィニティクロマトグラフィー選別を使用する生物学的ライブラリー法はペプチドライブラリーに限定されるが、その他の4つのアプローチはペプチド、非ペプチドオリゴマー、または化合物の低分子化合物ライブラリーに適用できる（Lam（1997）Anticancer Drug Des. 12:145-67）。分子ライブラリーの合成方法の例は、当技術分野において見出され得る（DeWitt et al.（1993）Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al.（1994）Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al.（1994）J. Med. Chem. 37:2678-85; Cho et al.（1993）Science 261:1303-5; Carell et al.（1994）Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.（1994）Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al.（1994）J. Med. Chem. 37:1233-51）。化合物ライブラリーは、溶液（Houghten（1992）Bio/Techniques 13:412-21を参照のこと）またはビーズ（Lam（1991）Nature 354:82-4）、チップ（Fodor（1993）Nature 364:555-6）、細菌（米国特許第5,223,409号）、胞子（米国特許第5,571,698号、同第5,403,484号、および同第5,223,409号）、プラスミド（Cull et al.（1992）Proc. Natl. Acad. Sci. USA 89:1865-9）若しくはファージ（Scott and Smith（1990）Science 249:386-90; Devlin（1990）Science 249:404-6; Cwirla et al.（1990）Proc. Natl. Acad. Sci. USA 87:6378-82; Felici（1991）J. Mol. Biol. 222:301-10; 米国特許出願第2002103360号）として作製され得る。 Test substances used in the screening method of the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, Examples include synthetic low-molecular compounds and natural compounds. The test substance can also be used in (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography selection. Although biological library methods using affinity chromatography selection are limited to peptide libraries, the other four approaches can be applied to peptides, non-peptide oligomers, or small molecule libraries of compounds (Lam (1997) ) Anticancer Drug Des. 12:145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem 37:1233-51). Compound libraries can be prepared in solution (see Houghten (1992) Bio/Techniques 13:412-21), beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555- 6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,403,484, and U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA Natl. Acad. Sci. USA 87 :6378-82; Felici (1991) J. Mol. Biol. 222:301-10; US Patent Application No. 2002103360).
　被験物質と細胞との接触は、培養細胞にあっては、細胞を含む培地または緩衝液などに被験物質を添加することにより、哺乳動物内の細胞にあっては、被験物質をヒト以外の哺乳動物に経口もしくは非経口的に投与することにより行われ得る。培地等への添加量、哺乳動物への投与量は、被験物質に応じて適宜選択することができる。被験物質と細胞との接触は、特に制限されないが、例えば、1日以上、2日以上、3日以上、4日以上、5日以上、6日以上、7日以上が例示される。添加される被験物質の濃度は化合物の種類（溶解性、毒性等）によって適宜調節可能である。 Contact between the test substance and cells can be achieved by adding the test substance to the culture medium or buffer containing the cells in the case of cultured cells, or by adding the test substance to cells in mammals, such as by adding the test substance to non-human mammals. It can be carried out by administering to animals orally or parenterally. The amount added to a medium etc. and the amount administered to mammals can be appropriately selected depending on the test substance. The contact between the test substance and the cells is not particularly limited, but examples include, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more. The concentration of the added test substance can be adjusted as appropriate depending on the type of compound (solubility, toxicity, etc.).
　本発明のスクリーニング方法において、被験物質と細胞を接触させる際に用いる、細胞の培養液は、当該細胞を培養できる培地であれば特に限定されず、基本培地でもよいが、好ましくは、神経細胞への分化誘導に適した培地（以下、「神経分化誘導培地」と称することがある。）である。神経分化誘導培地は、神経栄養因子を添加した基本培地に神経栄養因子を添加して調製することができる。神経栄養因子とは、神経細胞の生存と機能維持に重要な役割を果たしている膜受容体のリガンドであり、例えば、Nerve Growth Factor（NGF）、Brain-derived Neurotrophic Factor（BDNF）、Neurotrophin 3（NT-3）、Neurotrophin 4/5（NT-4/5）、Neurotrophin 6（NT-6）、basic FGF、acidic FGF、FGF-5、Epidermal Growth Factor（EGF）、Hepatocyte Growth Factor（HGF）、Insulin、Insulin Like Growth Factor 1（IGF 1）、Insulin Like Growth Factor 2（IGF 2）、Glia cell line-derived Neurotrophic Factor（GDNF）、TGF-b2、TGF-b3、Interleukin 6（IL-6）、Ciliary Neurotrophic Factor（CNTF）およびLIF等が挙げられる。このうち、本発明において好ましい神経栄養因子は、GDNF、BDNF、および／またはNT-3である。 In the screening method of the present invention, the cell culture medium used when bringing the test substance into contact with cells is not particularly limited as long as it is a medium that can culture the cells, and may be a basic medium, but preferably (hereinafter sometimes referred to as "neural differentiation induction medium"). A neuronal differentiation induction medium can be prepared by adding neurotrophic factors to a basal medium supplemented with neurotrophic factors. Neurotrophic factors are membrane receptor ligands that play an important role in maintaining the survival and function of nerve cells, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT -3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) and LIF. Among these, preferred neurotrophic factors in the present invention are GDNF, BDNF, and/or NT-3.
　前記基本培地としては、例えば、Glasgow's Minimal Essential Medium(GMEM)培地、IMDM培地、Medium 199培地、Eagle's Minimum Essential Medium（EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium(DMEM)培地、Ham's F12(F12)培地、Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12(DMEM/F-12)培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium培地（Lifetechnologies社）、およびこれらの混合培地などが包含される。基本培地には血清が含有されていてもよいし、無血清でもよい。 Examples of the basic medium include Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 (F12). Media, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium medium (Lifetechnologies), and mixed media thereof are included. The basal medium may contain serum or may be serum-free.
　必要に応じて、培地は、例えば、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2 supplement（Invitrogen）、B27 supplement（Invitrogen）、アルブミン、トランスフェリン、アポトランスフェリン、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、3'-チオールグリセロールなどの1つ以上の血清代替物を含んでもよく、また、脂質、アミノ酸、L-グルタミン、Glutamax（Invitrogen）、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、セレン酸、プロゲステロンおよびプトレシンなどの1つ以上の物質も含有してもよい。 If necessary, the medium may contain, for example, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, May contain one or more serum replacements such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential It may also contain one or more substances such as amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine.
　被験物質と細胞を接触させる際の培養温度は、特に制限されないが、約30～40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2～5％である。 The culture temperature at which the test substance and cells are brought into contact is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed in an atmosphere containing CO2 , with a CO2 concentration of: Preferably it is about 2-5%.
３．神経変性疾患診断用のバイオマーカーおよびその用途
　下述の実施例で示される通り、AD患者の剖検脳を解析したところ、βアミロイドが蓄積している部位特異的にKLHL32の発現が認められること、アルツハイマー病マウスモデルでは、カスパーゼ-3の活性化に先行して神経細胞で発現することが確認された。従って、KLHL32タンパク質およびKLHL32転写産物は、神経変性疾患診断用のバイオマーカー（以下、「本発明のバイオマーカー」と称することがある。）として用いることができる。KLHL32タンパク質には、アイソフォームが複数（即ち、アイソフォームa～o、X1～X3）知られているが、特に断らない限り、以下では全てのアイソフォームを包含するものとして「KLHL32タンパク質」との用語を用いる。神経変性疾患診断用のバイオマーカーとしては、KLHL32タンパク質のアイソフォームの中でも、完全長KLHL32タンパク質（ヒトKLHL32タンパク質の場合にはアイソフォームa）が好ましい。従って、KLHL32転写産物についても、特に断らない限り、全てのアイソフォームを包含するものとして「KLHL32転写産物」との用語を用いる。神経変性疾患診断用のバイオマーカーとしては、KLHL32転写産物のアイソフォームの中でも、完全長KLHL32タンパク質をコードするKLHL32 mRNAが好ましい。 3. Biomarkers for diagnosing neurodegenerative diseases and their uses As shown in the examples below, when autopsy brains of AD patients were analyzed, expression of KLHL32 was found to be specific to areas where β-amyloid accumulates. In a mouse model of Alzheimer's disease, it was confirmed that it is expressed in neurons prior to caspase-3 activation. Therefore, KLHL32 protein and KLHL32 transcript can be used as a biomarker for diagnosing neurodegenerative diseases (hereinafter sometimes referred to as "biomarker of the present invention"). KLHL32 protein is known to have multiple isoforms (i.e., isoforms a to o, X1 to X3), but unless otherwise specified, the term "KLHL32 protein" will be used to include all isoforms. Use terminology. Among the isoforms of KLHL32 protein, full-length KLHL32 protein (isoform a in the case of human KLHL32 protein) is preferred as a biomarker for diagnosing neurodegenerative diseases. Therefore, regarding the KLHL32 transcript, unless otherwise specified, the term "KLHL32 transcript" is used to include all isoforms. Among the isoforms of KLHL32 transcripts, KLHL32 mRNA, which encodes the full-length KLHL32 protein, is preferred as a biomarker for diagnosing neurodegenerative diseases.
　本発明のバイオマーカーとして用いるKLHL32タンパク質は公知のタンパク質であり、ヒトの場合には、NCBI Accession No.: NP_443136.2またはNP_443136.2（アイソフォームa；配列番号３）、NP_001273179.1（アイソフォームb）、NP_001273180.1（アイソフォームc）、NP_001273181.1（アイソフォームd）、NP_001273183.1（アイソフォームe）、NP_001310182.1（アイソフォームf）、NP_001310183.1（アイソフォームg）、NP_001310184.1（アイソフォームh）、NP_001310185.1（アイソフォームi）、NP_001310186.1（アイソフォームj）、NP_001310187.1（アイソフォームk）、NP_001310189.1（アイソフォームl）、NP_001310191.1（アイソフォームm）、NP_001310192.1（アイソフォームn）、NP_001310193.1（アイソフォームo）、XP_005266870.1（アイソフォームX1）およびXP_016865717.1（アイソフォームX2）、XP_016865718.1（アイソフォームX3）としてアミノ酸配列が開示されている。本発明において、KLHL32タンパク質は、NCBI Accession Noのいずれかで表されるアミノ酸配列であってもよく、これらと実質的に同一のアミノ酸配列を含むタンパク質であってよく、ヒト以外の動物のヒトKLHL32遺伝子のオーソログにコードされるタンパク質であってもよい。 The KLHL32 protein used as a biomarker of the present invention is a known protein, and in the case of humans, NCBI Accession No.: NP_443136.2 or NP_443136.2 (isoform a; SEQ ID NO: 3), NP_001273179.1 (isoform b). 1 (isoform h), NP_001310185.1 (isoform i), NP_001310186.1 (isoform j), NP_001310187.1 (isoform k), NP_001310189.1 (isoform l), NP_001310191.1 (isoform m ), NP_001310192.1 (isoform n), NP_001310193.1 (isoform o), XP_005266870.1 (isoform X1) and XP_016865717.1 (isoform X2), XP_016865718.1 (isoform X3). Disclosed. In the present invention, the KLHL32 protein may be an amino acid sequence represented by any of the NCBI Accession Nos. It may be a protein containing an amino acid sequence substantially the same as these, and the KLHL32 protein of an animal other than human It may also be a protein encoded by an ortholog of a gene.
　上記NCBI Accession Noのいずれかで表されるアミノ酸配列（例えば、配列番号３で示される配列）と実質的に同一のアミノ酸配列としては、例えば、これらのアミノ酸配列と60％以上、好ましくは70％以上、より好ましくは80％以上、さらに好ましくは90％以上、さらにより好ましくは、95%以上、最も好ましくは98％以上の類似性または同一性を有するアミノ酸配列などが挙げられる。ここで「類似性」とは、当該技術分野において公知の数学的アルゴリズムを用いて2つのアミノ酸配列をアラインさせた場合の、最適なアラインメント（好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方若しくは両方へのギャップの導入を考慮し得るものである）における、オーバーラップする全アミノ酸残基に対する同一アミノ酸および類似アミノ酸残基の割合（％）を意味する。「類似アミノ酸」とは物理化学的性質において類似したアミノ酸を意味し、例えば、芳香族アミノ酸（Phe、Trp、Tyr）、脂肪族アミノ酸（Ala、Leu、Ile、Val）、極性アミノ酸（Gln、Asn）、塩基性アミノ酸（Lys、Arg、His)、酸性アミノ酸(Glu、Asp）、水酸基を有するアミノ酸（Ser、Thr）、側鎖の小さいアミノ酸（Gly、Ala、Ser、Thr、Met）などの同じグループに分類されるアミノ酸が挙げられる。このような類似アミノ酸による置換はタンパク質の表現型に変化をもたらさない（即ち、保存的アミノ酸置換である）ことが予測される。保存的アミノ酸置換の具体例は当該技術分野で周知であり、種々の文献に記載されている（例えば、Bowieら，Science, 247: 1306-1310 (1990)を参照）。本明細書におけるアミノ酸配列の類似性または同一性は、相同性計算アルゴリズムNCBI BLAST(National Center for Biotechnology Information Basic Local Alignment Search Tool)を用い、以下の条件（期待値＝10；ギャップを許す；マトリクス＝BLOSUM62；フィルタリング＝OFF）にて計算することができる。 Examples of amino acid sequences that are substantially identical to the amino acid sequences represented by any of the above NCBI Accession Nos. (for example, the sequence shown in SEQ ID NO: 3) include, for example, 60% or more, preferably 70%, of these amino acid sequences. Examples include amino acid sequences having a similarity or identity of more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more. "Similarity" herein refers to the optimal alignment of two amino acid sequences using a mathematical algorithm known in the art (preferably, the algorithm refers to the ratio (%) of identical and similar amino acid residues to all overlapping amino acid residues (in which the introduction of a gap in one or both may be considered). "Similar amino acids" means amino acids similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn ), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is predicted that such a substitution with a similar amino acid will not result in a change in the protein phenotype (ie, it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and described in various publications (see, eg, Bowie et al., Science, 247: 1306-1310 (1990)). The similarity or identity of amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; matrix = It can be calculated with BLOSUM62; filtering = OFF).
　KLHL32タンパク質は、公知のタンパク質合成法、例えば、固相合成法、液相合成法等に従って製造することができる。得られたタンパク質は、公知の精製法、例えば、溶媒抽出、蒸留、カラムクロマトグラフィー、液体クロマトグラフィー、再結晶、これらの組み合わせ等により精製単離することができる。また、自体公知の方法により、生体試料から単離、精製してもよい。あるいは、KLHL32タンパク質は、それをコードする核酸を含有する形質転換体を培養し、得られる培養物からタンパク質を分離精製することによって製造することもできる。かかる核酸はDNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。該核酸は二本鎖であっても、一本鎖であってもよい。 KLHL32 protein can be produced according to known protein synthesis methods, such as solid phase synthesis and liquid phase synthesis. The obtained protein can be purified and isolated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and a combination thereof. Alternatively, it may be isolated and purified from a biological sample by a method known per se. Alternatively, the KLHL32 protein can also be produced by culturing a transformant containing a nucleic acid encoding it, and separating and purifying the protein from the resulting culture. Such a nucleic acid may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded.
　また、上記NCBI Accession Noのいずれかで表されるアミノ酸配列と実質的に同一のアミノ酸配列としては、これらのアミノ酸配列のうち1または2個以上（好ましくは、1～100個程度、好ましくは1～50個程度、さらに好ましくは1～10個程度、特に好ましくは1～数（2、3、4若しくは5）個）のアミノ酸が置換、挿入および／または欠失したアミノ酸配列を含有するタンパク質なども含まれる。 In addition, the amino acid sequence that is substantially the same as the amino acid sequence represented by any of the above NCBI Accession No. is one or two or more (preferably about 1 to 100, preferably 1 or more) of these amino acid sequences. Proteins containing an amino acid sequence in which ~50 amino acids, more preferably 1 to 10, particularly preferably 1 to several (2, 3, 4, or 5) amino acids have been substituted, inserted, and/or deleted, etc. Also included.
　KLHL32転写産物は公知の転写産物であり、例えば、上記KLHL32タンパク質をコードするRNAが挙げられる。具体的には、ヒトの場合には、配列番号１または２に示されるアイソフォームaをコードするRNAだけでなく、NM_001286250.2（アイソフォームbをコードするRNA）、NM_001286251.2（アイソフォームcをコードするRNA）、NM_001286252.2（アイソフォームdをコードするRNA）、NM_001286254.3（アイソフォームeをコードするRNA）、NM_001323253.2（アイソフォームfをコードするRNA）、NM_001323254.2（アイソフォームgをコードするRNA）、NM_001323255.2（アイソフォームhをコードするRNA）、NM_001323256.2（アイソフォームiをコードするRNA）、NM_001323257.2（アイソフォームjをコードするRNA）、NM_001323258.2（アイソフォームkをコードするRNA）、NM_001323260.2（アイソフォームlをコードするRNA）、NM_001323262.2（アイソフォームmをコードするRNA）、NM_001323263.2（アイソフォームnをコードするRNA）、NM_001323264.2（アイソフォームoをコードするRNA）、XM_005266813.5（アイソフォームX1をコードするRNA）、XM_017010228.2（アイソフォームX2をコードするRNA）およびXM_017010229.2（アイソフォームX3をコードするRNA）として塩基配列が開示されている。本発明において、KLHL32転写産物としては、上記の塩基配列（但し、TをUと読み替えるものとする）を有する核酸であればいずれでもよく、これらの塩基配列と同一または実質的に同一な塩基配列を含む核酸であってもよい。また、ヒト以外の動物のヒトKLHL32遺伝子のオーソログから転写される転写産物であってもよい。 The KLHL32 transcription product is a known transcription product, and includes, for example, RNA encoding the above-mentioned KLHL32 protein. Specifically, in the case of humans, not only RNA encoding isoform a shown in SEQ ID NO: 1 or 2, but also NM_001286250.2 (RNA encoding isoform b), NM_001286251.2 (isoform c NM_001286252.2 (RNA encoding isoform d), NM_001286254.3 (RNA encoding isoform e), NM_001323253.2 (RNA encoding isoform RNA encoding form g), NM_001323255.2 (RNA encoding isoform h), NM_001323256.2 (RNA encoding isoform i), NM_001323257.2 (RNA encoding isoform j), NM_001323258.2 (RNA encoding isoform k), NM_001323260.2 (RNA encoding isoform l), NM_001323262.2 (RNA encoding isoform m), NM_001323263.2 (RNA encoding isoform n), NM_001323264 .2 (RNA encoding isoform o), XM_005266813.5 (RNA encoding isoform X1), XM_017010228.2 (RNA encoding isoform X2) and XM_017010229.2 (RNA encoding isoform X3) The base sequence has been disclosed as . In the present invention, the KLHL32 transcript may be any nucleic acid having the above nucleotide sequence (T should be read as U), and any nucleotide sequence that is the same or substantially the same as these nucleotide sequences. It may be a nucleic acid containing. Alternatively, it may be a transcription product transcribed from an ortholog of the human KLHL32 gene of an animal other than humans.
　上記塩基配列と実質的に同一な塩基配列を含む核酸としては、例えば、これらの塩基配列と60％以上、好ましくは70％以上、さらに好ましくは80％以上、より好ましくは90％以上、さらにより好ましくは95%以上、最も好ましくは98%以上の同一性を有する塩基配列を含有し、且つKLHL32タンパク質と実質的に同質の活性を有するタンパク質をコードする核酸などが挙げられる。 Nucleic acids containing base sequences that are substantially identical to the above base sequences include, for example, 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and even more Examples include nucleic acids that contain a base sequence that preferably has an identity of 95% or more, most preferably 98% or more, and encode a protein that has substantially the same activity as the KLHL32 protein.
　KLHL32転写産物は、例えば、該転写産物を含有する生体試料から、自体公知の方法により単離、精製することにより得ることができる。 The KLHL32 transcription product can be obtained, for example, by isolating and purifying a biological sample containing the transcription product by a method known per se.
　また、さらに別に態様において、本発明は、被験者または該被験者由来の試料において、本発明のバイオマーカーを検出する工程を含む、該被験者が神経変性疾患であるかの診断等（例：診断、判定、判断、鑑別、検査等）を行う方法、該診断等を補助する方法、あるいは神経変性疾患の可能性若しくは確率を分析等（例：分析、評価、算定等）する方法（以下では、これらをまとめて「本発明の方法」と称する場合がある）を提供する。検出対象のバイオマーカーは、１種でもよいし、２種以上であってもよい（例えば、KLHL32タンパク質とKLHL32 mRNAの両方を検出する等）。本明細書において、「診断等を補助する」とは、被験者が神経変性疾患であるかの判断のための指標となる情報を提供することをいい、医療行為である、神経変性疾患であるか否かを診断等する工程自体を含まないことを意味する。また、本明細書において、「バイオマーカーを検出する」には、被験者の脳の少なくとも一部（例えば、老人斑、変性神経突起等）または被験者由来の試料において、KLHL32遺伝子の発現が認められる（即ち、検出方法における検出限界以上の量のタンパク質または転写産物が存在する）か否かを調べることだけでなく、その発現量を測定する（定量する）ことも包含されるものとする。 In yet another aspect, the present invention provides a method for diagnosing whether a subject has a neurodegenerative disease (e.g., diagnosis, , judgment, differentiation, testing, etc.), methods to assist in the diagnosis, etc., or methods to analyze the possibility or probability of neurodegenerative diseases (e.g., analysis, evaluation, calculation, etc.) may be collectively referred to as "the methods of the present invention"). The number of biomarkers to be detected may be one or two or more (for example, both KLHL32 protein and KLHL32 mRNA may be detected). As used herein, "assisting diagnosis, etc." refers to providing information that serves as an indicator for determining whether a subject has a neurodegenerative disease, and is a medical procedure. This means that it does not include the process itself of diagnosing whether or not it is true. In addition, in this specification, "detecting a biomarker" refers to the expression of the KLHL32 gene observed in at least a part of the subject's brain (e.g., senile plaques, degenerated neurites, etc.) or in a sample derived from the subject ( That is, it includes not only determining whether a protein or transcription product is present in an amount exceeding the detection limit of the detection method, but also measuring (quantifying) its expression level.
　本発明の方法の被験対象となり得る被験者は、ヒト以外の動物であってもよい。好ましくは、神経変性疾患が疑われる動物が挙げられる。動物の種類としては、例えば、哺乳動物（例：ヒト、サル、ウシ、ブタ、ウマ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、ハムスター、モルモット、マウス、ラット等）、鳥類（例：ニワトリ等）などが挙げられる。好ましくは、哺乳動物、より好ましくはヒトである。 Subjects that can be tested in the method of the present invention may be animals other than humans. Preferably, animals suspected of having neurodegenerative diseases are mentioned. Examples of types of animals include mammals (e.g., humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, rats, etc.), birds (e.g., chickens, etc.) Examples include. Preferably it is a mammal, more preferably a human.
　被験者由来の試料は、例えば、血液、血清、血漿、唾液、尿、涙、汗、乳汁、鼻汁、精液、胸水、消化管分泌液、脳脊髄液、組織間液、およびリンパ液などが挙げられ、臨床の観点からは好ましくは血液、血清、血漿および脳脊髄液であり、より好ましくは血清および血漿である。これらの試料は、自体公知の方法により得ることができ、例えば、血清や血漿は、常法に従って被験者から採血し、液性成分を分離することにより調製することができ、脳脊髄液は、脊椎穿刺等の公知の手段により採取することができる。 Samples derived from the subject include, for example, blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal discharge, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, lymph fluid, etc. From a clinical point of view, blood, serum, plasma and cerebrospinal fluid are preferred, and serum and plasma are more preferred. These samples can be obtained by methods known per se. For example, serum and plasma can be prepared by collecting blood from a subject according to a conventional method and separating the humoral components, and cerebrospinal fluid can be prepared by It can be collected by known means such as puncture.
　被験者由来の試料におけるKLHL32転写産物の検出は、該試料からRNA（例：全RNA、mRNA）画分を調製し、該画分中に含まれるKLHL32転写産物を検出することにより調べることができる。従って、一実施態様において、本発明の方法は、KLHL32転写産物をそれぞれ特異的に認識し得る核酸プローブまたは核酸プライマーを用いて検出することを含む。 Detection of KLHL32 transcripts in a sample derived from a subject can be investigated by preparing an RNA (e.g., total RNA, mRNA) fraction from the sample and detecting the KLHL32 transcripts contained in the fraction. Accordingly, in one embodiment, the method of the invention comprises detecting a KLHL32 transcript using a nucleic acid probe or a nucleic acid primer, each of which is capable of specifically recognizing the KLHL32 transcript.
　RNA画分の調製は、グアニジン－CsCl超遠心法、AGPC法など公知の手法を用いて行うことができ、市販のRNA抽出用キット（例：RNeasy Mini Kit；QIAGEN製等）を用いて、微量検体から迅速且つ簡便に高純度の全RNAを調製することもできる。RNA画分中のKLHL32転写産物を検出する手段としては、例えば、ハイブリダイゼーション（ノーザンブロット、ドットブロット等）を用いる方法、あるいはPCR（RT－PCR、競合PCR、リアルタイムPCR等）などを用いる方法などが挙げられる。微量試料から迅速且つ簡便に発現を検出できる点で、競合PCRやリアルタイムPCRなどの定量的PCR法が好ましい。 Preparation of RNA fractions can be performed using known methods such as guanidine-CsCl ultracentrifugation and AGPC methods. Highly pure total RNA can also be rapidly and easily prepared from a specimen. Examples of methods for detecting KLHL32 transcripts in RNA fractions include methods using hybridization (Northern blot, dot blot, etc.), or methods using PCR (RT-PCR, competitive PCR, real-time PCR, etc.). can be mentioned. Quantitative PCR methods such as competitive PCR and real-time PCR are preferred because expression can be detected quickly and easily from a trace amount of sample.
　ノーザンブロットまたはドットブロットハイブリダイゼーションによる場合、KLHL32転写産物の検出は、例えば、KLHL32転写産物を特異的に認識し得る核酸プローブを用いて行うことができる。核酸プローブはDNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、プローブとして用いられる核酸は、二本鎖であっても一本鎖であってもよい。二本鎖の場合は、二本鎖DNA、二本鎖RNAまたはDNA:RNAのハイブリッドでもよい。一本鎖の場合は、アンチセンス鎖配列を含むものを用いることができる。 When using Northern blot or dot blot hybridization, detection of KLHL32 transcripts can be performed, for example, using a nucleic acid probe that can specifically recognize KLHL32 transcripts. The nucleic acid probe may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA. Further, the nucleic acid used as a probe may be double-stranded or single-stranded. If it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single strand, one containing an antisense strand sequence can be used.
　上記核酸プローブは、市販のDNA/RNA自動合成機等を用いて化学的に合成することなどによって得ることができる。また、上記核酸プローブは、標的核酸の検出を可能とするために、標識剤により標識されていることが好ましい。 The above nucleic acid probe can be obtained by chemically synthesizing it using a commercially available automatic DNA/RNA synthesizer or the like. Furthermore, the nucleic acid probe is preferably labeled with a labeling agent to enable detection of the target nucleic acid.
　被験者由来の試料におけるKLHL32タンパク質の検出は、該試料からタンパク質画分を調製し、該画分中に含まれる該遺伝子の翻訳産物（即ち、KLHL32タンパク質）を検出または定量することにより調べることができる。これらのタンパク質の検出または定量は、各タンパク質を特異的に認識する抗体を用いて、免疫学的測定法（例：ELISA、FIA、RIA、ウエスタンブロット等）によって行うことができるが、好ましくは免疫学的測定法、特にELISAを用いた方法がより好ましい。 Detection of KLHL32 protein in a sample derived from a subject can be investigated by preparing a protein fraction from the sample and detecting or quantifying the translation product of the gene (i.e., KLHL32 protein) contained in the fraction. . Detection or quantification of these proteins can be performed by immunoassays (e.g. ELISA, FIA, RIA, Western blotting, etc.) using antibodies that specifically recognize each protein, but preferably immunoassays A method using a chemical measurement method, particularly ELISA, is more preferred.
　KLHL32タンパク質を特異的に認識し得る抗体は、これらのタンパク質またはエピトープを有する部分ペプチドを免疫原として用い、既存の一般的な製造方法によって製造することができる。本明細書において、抗体には、ポリクローナル抗体、モノクローナル抗体（mAb）等の天然型抗体、遺伝子組換技術を用いて製造され得るキメラ抗体、ヒト化抗体や一本鎖抗体、およびこれらの結合性断片などが含まれるが、これらに限定されない。好ましくは、抗体はポリクローナル抗体、モノクローナル抗体またはこれらの結合性断片である。結合性断片とは、特異的結合活性を有する前述の抗体の一部分の領域を意味し、具体的には例えばF(ab’)2、Fab’、Fab、Fv、sFv、dsFv、sdAbなどが挙げられる（Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996）。抗体のクラスは、特に限定されず、IgG、IgM、IgA、IgDあるいはIgE等のいずれのアイソタイプを有する抗体をも包含する。好ましくは、IgGまたはIgMであり、精製の容易性等を考慮するとより好ましくはIgGである。また、本発明において、KLHL32タンパク質を特異的に認識し得る抗体として、市販の抗体または抗体を含むキットやアレイ等を使用することもまた好ましい。 Antibodies that can specifically recognize KLHL32 protein can be produced by existing general production methods using these proteins or partial peptides having epitopes as immunogens. In this specification, antibodies include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), chimeric antibodies that can be produced using genetic recombination technology, humanized antibodies, and single chain antibodies, and their binding properties. This includes, but is not limited to, fragments. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment refers to a partial region of the above-mentioned antibody that has specific binding activity, and specifically includes F(ab') 2 , Fab', Fab, Fv, sFv, dsFv, sdAb, etc. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The class of antibodies is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. Preferably, it is IgG or IgM, and considering ease of purification, etc., IgG is more preferable. In the present invention, it is also preferable to use commercially available antibodies or kits, arrays, etc. containing the antibodies as antibodies that can specifically recognize the KLHL32 protein.
　被験者におけるバイオマーカーの検出は、例えば、放射性核種で直接的または間接的に標識した抗体を用いて行うこともできる。かかる放射性核種としては、PET（Positron Emission Tomography）検査に用いることできる、ポジトロンを放出する核種（例：68Ga、64Cu、86Y、89Zr等）や、SPECT（Single Photon Emission Computed Tomography）検査に用いることができる、γ線を放出する核種（例：111In等）などが挙げられる。放射性核種で標識した抗体の作製や、該抗体を用いたバイオマーカーの検出は、自体公知の方法（例えば、WO2013/157102、WO2021/075544に記載の方法等）により行うことができる。 Detection of a biomarker in a subject can also be performed using, for example, an antibody labeled directly or indirectly with a radionuclide. Such radionuclides include positron-emitting nuclides (e.g. 68 Ga, 64 Cu, 86 Y, 89 Zr, etc.) that can be used in PET (Positron Emission Tomography) examinations, and SPECT (Single Photon Emission Computed Tomography) examinations. Examples include nuclides that emit gamma rays (e.g. 111 In, etc.) that can be used for Preparation of an antibody labeled with a radionuclide and detection of a biomarker using the antibody can be performed by a method known per se (for example, the method described in WO2013/157102, WO2021/075544, etc.).
　必要に応じて、上記抗体は、マイクロカプセル（ヒドロキシメチルセルロース、ゼラチン、ポリメタクリル酸メチル等のマイクロカプセル）に封入され、コロイドドラッグデリバリーシステム（リポソーム、アルブミンミクロスフェア、マイクロエマルジョン、ナノ粒子およびナノカプセル等）の形態としてもよい（"Remington’s Pharmaceutical Science 16th edition", Oslo Ed. (1980)等）。また、抗体に細胞膜透過ペプチド（例：TATペプチド、該ペプチドの改変体等）などの機能性ペプチドが付加されていてもよい。 Optionally, the antibodies are encapsulated in microcapsules (microcapsules of hydroxymethyl cellulose, gelatin, polymethyl methacrylate, etc.) and colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, etc.). ) ("Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.). Further, a functional peptide such as a cell membrane-penetrating peptide (eg, TAT peptide, a modified version of the peptide, etc.) may be added to the antibody.
　また、本発明のバイオマーカーの量の比較により、神経変性疾患であるとの診断等を行うこともできる。具体的には、例えば、健常者または健常者由来の試料（以下「対照試料」と称する場合がある）と、本発明の対象とする被験者または被験者由来の試料における本発明のバイオマーカーの量を定量し、両者の量を比較することにより行うことができる。あるいは、バイオマーカーの量を基準値と比較することにより行ってもよい。本発明に用いる「基準値」としては、対照試料における本発明のバイオマーカーの量を用いてもよい。あるいは、対照試料におけるバイオマーカーの定量値からあらかじめ設定した値を用いてもよい。この場合、基準値として、例えば、複数個体を対照群として、複数個体の測定値の平均値や最頻値などを採用することもできる。また、本明細書において、「健常者」とは、神経変性疾患であるとの確定診断がなされていない者を意味する。 Further, by comparing the amounts of the biomarkers of the present invention, it is also possible to diagnose a neurodegenerative disease, etc. Specifically, for example, the amount of the biomarker of the present invention in a healthy person or a sample derived from a healthy person (hereinafter sometimes referred to as a "control sample") and a subject targeted by the present invention or a sample derived from a subject is determined. This can be done by quantifying and comparing the amounts of both. Alternatively, it may be performed by comparing the amount of the biomarker with a reference value. As the "reference value" used in the present invention, the amount of the biomarker of the present invention in a control sample may be used. Alternatively, a value preset from the quantitative value of the biomarker in the control sample may be used. In this case, the reference value may be, for example, the average value or the mode of the measured values of a plurality of individuals, with the plurality of individuals as a control group. Moreover, in this specification, a "healthy person" means a person who has not been definitively diagnosed with a neurodegenerative disease.
　上記基準値は、カットオフ値であってもよい。「カットオフ値」は、その値を基準として疾患の判定をした場合に、高い診断感度（有病正診率）および高い診断特異度（無病正診率）の両方を満足できる値である。例えば、神経変性疾患患者群で高い陽性率を示し、かつ、健常者群で高い陰性率を示す値をカットオフ値として設定することができる。 The above reference value may be a cutoff value. The "cutoff value" is a value that satisfies both high diagnostic sensitivity (prevalence rate) and high diagnostic specificity (predictability rate) when a disease is determined based on the value. For example, a value that shows a high positive rate in a neurodegenerative disease patient group and a high negative rate in a healthy person group can be set as a cutoff value.
　カットオフ値の算出方法は、この分野において周知である。例えば、神経変性疾患患者群および健常者群から採取した血清中の本発明のバイオマーカーの量を定量し、定量された値における診断感度および診断特異度を求め、これらの値に基づき、市販の解析ソフトを使用してROC（Receiver Operating Characteristic）曲線を作成する。そして、診断感度と診断特異度が可能な限り100％に近いときの値を求めて、その値をカットオフ値とすることができる。 Methods for calculating cutoff values are well known in the field. For example, the amount of the biomarker of the present invention in serum collected from neurodegenerative disease patients and healthy subjects is quantified, the diagnostic sensitivity and diagnostic specificity of the quantified values are determined, and based on these values, commercially available Create a ROC (Receiver Operating Characteristic) curve using analysis software. Then, the value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can be determined, and this value can be used as the cutoff value.
　上記本発明のバイオマーカーの量の比較の結果、例えば、被験者または被験者由来の試料において、本発明のバイオマーカーが、健常者または対照試料に比べて高値であった場合、または上記基準値以上である場合には、該被験者が神経変性疾患であると診断等することができ、本発明のバイオマーカーが、健常者または対照試料に比べて同程度若しくは低値であった場合、または上記基準値未満である場合には、該被験者が神経変性疾患ではないと診断等することができる。 As a result of the above comparison of the amount of the biomarker of the present invention, for example, if the biomarker of the present invention is higher in the subject or sample derived from the subject than in a healthy person or a control sample, or if it is higher than the above reference value. In some cases, the subject can be diagnosed with a neurodegenerative disease, and the biomarker of the present invention is at the same level or lower than that of a healthy person or a control sample, or the above standard value If it is below, it can be diagnosed that the subject does not have a neurodegenerative disease.
　また、バイオマーカーの量の比較の結果、例えば、被験者または被験者由来の試料において、本発明のバイオマーカーが検出された場合、あるいは、健常者または対照試料に比べて高値であった場合、または上記基準値以上である場合には、該被験者が神経変性疾患である可能性または確率が高いと分析等することができ、健常者または対照試料に比べて同程度または低値であった場合、または上記基準値未満である場合には、該被験者が神経変性疾患である可能性または確率が低いと分析等することができる。 Furthermore, as a result of comparing the amounts of biomarkers, for example, if the biomarker of the present invention is detected in the subject or a sample derived from the subject, or if the level is higher than that of a healthy person or a control sample, or if the above-mentioned If the value is above the standard value, it can be analyzed that there is a high possibility or probability that the subject has a neurodegenerative disease, and if the value is the same or lower than that of healthy subjects or control samples, or If it is less than the above reference value, it can be analyzed that the possibility or probability that the subject has a neurodegenerative disease is low.
　本発明の方法の結果、被験者が神経変性疾患であるとの診断等がされた場合に、該診断等の結果に基づき、該被験者に投与すべき神経変性疾患の予防または治療薬を選択または決定し、該被験者に治療上有効量の予防または治療薬を投与することにより、神経変性疾患を予防または治療することができる。即ち、以下の(i)～(iii)の工程を含む、神経変性疾患の予防または治療方法も提供される。
　（i）被験者または被験者由来の試料における、本発明のバイオマーカーを検出する工程、
　（ii）前記工程(i)の結果に基づき、該被験者が神経変性疾患であるかの診断を行う工程、
　（iii）（ii）により神経変性疾患であると診断された被験者に、神経変性疾患の予防または治療薬を投与する工程。 When a test subject is diagnosed with a neurodegenerative disease as a result of the method of the present invention, a preventive or therapeutic drug for the neurodegenerative disease to be administered to the test subject is selected or determined based on the results of the diagnosis, etc. Neurodegenerative diseases can be prevented or treated by administering a therapeutically effective amount of a prophylactic or therapeutic agent to the subject. That is, a method for preventing or treating neurodegenerative diseases is also provided, which includes the following steps (i) to (iii).
(i) detecting the biomarker of the present invention in a subject or a sample derived from a subject;
(ii) diagnosing whether the subject has a neurodegenerative disease based on the results of step (i);
(iii) A step of administering a preventive or therapeutic agent for a neurodegenerative disease to a subject diagnosed as having a neurodegenerative disease according to (ii).
　上記神経変性疾患の予防または治療薬としては、本発明の医薬、１．に記載の既存薬、これらの組み合わせなどが挙げられる。 The preventive or therapeutic agents for the above neurodegenerative diseases include the pharmaceutical of the present invention, 1. Examples include existing drugs described in , and combinations thereof.
　上記予防または治療薬は、有効成分をそのまま単独で、または薬学的に許容される担体、賦形剤、希釈剤等と混合し、適当な剤型の医薬組成物として経口的または非経口的に投与してもよい。経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。一方、非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含してもよい。また、予防または治療薬の投与量は、化合物の種類、投与対象の症状、齢、体重、薬物受容性等の種々の条件により、適宜設定することができる。 The above prophylactic or therapeutic drugs can be administered orally or parenterally as a pharmaceutical composition containing the active ingredients alone or in a suitable dosage form by mixing them with pharmaceutically acceptable carriers, excipients, diluents, etc. May be administered. Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. formulations, emulsions, suspensions, etc. On the other hand, as compositions for parenteral administration, for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may also be included. Furthermore, the dosage of the prophylactic or therapeutic drug can be appropriately determined depending on various conditions such as the type of compound, the symptoms of the subject, age, body weight, and drug acceptability.
４．神経変性疾患の診断キット
　さらに本発明は、KLHL32タンパク質を特異的に認識する抗体またはKLHL32転写産物を特異的に認識する核酸プローブ若しくは核酸プライマーを含む、神経変性疾患診断用キット（以下、「本発明の診断用キット」と称することがある。）を提供する。KLHL32タンパク質、該タンパク質を特異的に認識する抗体、KLHL32転写産物、該転写産物を特異的に認識する核酸プローブ若しくは核酸プライマーの定義や具体例、検出方法等については、上記「３．神経変性疾患診断用のバイオマーカーおよびその用途」で記載した内容が援用される。 4. Diagnostic kit for neurodegenerative diseases The present invention further provides a kit for diagnosing neurodegenerative diseases (hereinafter referred to as "the present invention (sometimes referred to as "diagnostic kit"). For definitions, specific examples, detection methods, etc. of the KLHL32 protein, antibodies that specifically recognize the protein, KLHL32 transcripts, and nucleic acid probes or primers that specifically recognize the transcripts, please refer to "3. Neurodegenerative Diseases" above. The contents described in "Diagnostic Biomarkers and Their Uses" are incorporated.
　一態様において、本発明の診断用キットは、ELISA法を利用して、試料を基板に接触させるだけで、該試料中に本発明のバイオマーカーの有無を検出できるキット（以下、「本発明の簡易キット」ともいう。）としても提供される。本発明の簡易キットは、KLHL32タンパク質を特異的に認識する抗体（以下、「第一の抗体」ともいう。）が固定された基板を含む。また、本発明の簡易キットには、上記抗体とは別の、KLHL32タンパク質を特異的に認識する抗体（以下、「第二の抗体」ともいう。）（典型的には、該抗体は標識された抗体である。）が含まれることが好ましく、かかる態様により、サンドイッチELISA法により本発明のバイオマーカーを検出することができる。 In one embodiment, the diagnostic kit of the present invention is a kit that can detect the presence or absence of the biomarker of the present invention in a sample by simply bringing the sample into contact with a substrate using the ELISA method (hereinafter referred to as "the diagnostic kit of the present invention"). Also available as a simple kit. The simple kit of the present invention includes a substrate on which an antibody that specifically recognizes the KLHL32 protein (hereinafter also referred to as "first antibody") is immobilized. The simple kit of the present invention also includes an antibody (hereinafter also referred to as "second antibody") that specifically recognizes the KLHL32 protein, which is different from the above-mentioned antibody (typically, the antibody is labeled. It is preferable that the antibody is an antibody that has been used in the present invention. According to such an embodiment, the biomarker of the present invention can be detected by a sandwich ELISA method.
　本発明の判定キットが前記の核酸プローブまたは核酸プライマー（単に「核酸」ともいう。）を構成として含む場合、これらの核酸は、乾燥した状態若しくはアルコール沈澱の状態で、固体として提供することもできるし、水若しくは適当な緩衝液（例：TE緩衝液等）中に溶解した状態で提供することもできる。標識プローブとして用いられる場合、核酸は予め上記のいずれかの標識物質で標識した状態で提供することもできるし、標識物質とそれぞれ別個に提供され、用時標識して用いることもできる。あるいは、該核酸は、適当な基板に固定された（担持された、または固相化されたともいう。）状態で提供することもできる。基板としては、例えば、ガラス、シリコン、プラスチック、ニトロセルロース、ナイロン、ポリビニリデンジフロリド等が挙げられるが、これらに限定されない。また、固定化手段としては、予め核酸にアミノ基、アルデヒド基、SH基、ビオチンなどの官能基を導入しておき、一方、基板上にも該核酸と反応し得る官能基（例：アルデヒド基、アミノ基、SH基、ストレプトアビジンなど）を導入し、両官能基間の共有結合で基板と核酸を架橋したり、ポリアニオン性の核酸に対して、基板をポリカチオンコーティングして静電結合を利用して核酸を固定化するなどの方法が挙げられるが、これらに限定されない。 When the determination kit of the present invention contains the aforementioned nucleic acid probe or nucleic acid primer (also simply referred to as "nucleic acid"), these nucleic acids can also be provided as a solid in a dry state or in an alcohol-precipitated state. However, it can also be provided in a state dissolved in water or an appropriate buffer (eg, TE buffer, etc.). When used as a labeled probe, the nucleic acid can be provided in a state that has been labeled in advance with any of the above-mentioned labeling substances, or it can be provided separately with each labeling substance and labeled before use. Alternatively, the nucleic acid can also be provided in a state immobilized (also referred to as supported or immobilized) on a suitable substrate. Examples of the substrate include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like. In addition, as an immobilization means, a functional group such as an amino group, an aldehyde group, an SH group, or a biotin is introduced into the nucleic acid in advance, and a functional group (e.g., an aldehyde group) that can react with the nucleic acid is also placed on the substrate. , amino groups, SH groups, streptavidin, etc.) and cross-link the substrate and nucleic acid with covalent bonds between both functional groups.For polyanionic nucleic acids, electrostatic bonding can be achieved by coating the substrate with polycations. Examples include, but are not limited to, methods such as immobilizing nucleic acids using a method of immobilizing nucleic acids.
　本発明の診断用キットには、上記核酸や抗体に加えて、本発明のバイオマーカーの発現を検出するための反応において必要な他の物質を含んでいてもよい。これらの他の物質は、反応に悪影響を及ぼさない限り、核酸や抗体等と共存状態で提供されてもよく、あるいは、別個の試薬とともに提供されてもよい。例えば、本発明のバイオマーカーの発現を検出するための反応がPCRの場合、当該他の物質としては、例えば、反応緩衝液、dNTPs、耐熱性DNAポリメラーゼ等が挙げられる。競合PCRやリアルタイムPCRを用いる場合は、competitor核酸や蛍光試薬（上記インターカレーターや蛍光プローブ等）などをさらに含むことができる。また、本発明のバイオマーカーの発現を検出するための反応が抗原抗体反応の場合、当該他の物質としては、例えば、反応緩衝液、competitor抗体、標識された二次抗体（例えば、一次抗体がウサギ抗体の場合、ペルオキシダーゼやアルカリホスファターゼ等で標識されたマウス抗ウサギIgGなど）、ブロッキング液、ELISA用プレートなどが挙げられる。また、本発明の判定キットには、キットや試薬の使用方法や、疾患の判定基準等の説明が記載された説明書を含んでいてもよい。また、上記判定キットには、例えばポジティブコントロールとして用いるため、本発明のバイオマーカーを１種以上含んでいてもよい。 In addition to the above nucleic acids and antibodies, the diagnostic kit of the present invention may contain other substances necessary for the reaction for detecting the expression of the biomarker of the present invention. These other substances may be provided in coexistence with the nucleic acid, antibody, etc., or may be provided together with separate reagents, as long as they do not adversely affect the reaction. For example, when the reaction for detecting the expression of the biomarker of the present invention is PCR, examples of the other substances include reaction buffer, dNTPs, thermostable DNA polymerase, and the like. When competitive PCR or real-time PCR is used, a competitor nucleic acid, a fluorescent reagent (the above-mentioned intercalator, fluorescent probe, etc.), etc. can be further included. Furthermore, when the reaction for detecting the expression of the biomarker of the present invention is an antigen-antibody reaction, the other substances include, for example, a reaction buffer, a competitor antibody, a labeled secondary antibody (for example, if the primary antibody is In the case of rabbit antibodies, examples include mouse anti-rabbit IgG labeled with peroxidase or alkaline phosphatase, etc.), blocking solutions, and ELISA plates. Furthermore, the determination kit of the present invention may include an instruction manual that describes how to use the kit and reagents, disease determination criteria, and the like. Moreover, the above-mentioned determination kit may contain one or more types of biomarkers of the present invention, for example, for use as a positive control.
　以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに何ら限定されるものではない。 The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these in any way.
＜材料および方法＞
（倫理）
　すべてのヒト組織の収集、ヒト幹細胞の研究、手順、および書面による同意は、京都大学医学部・医学研究科（Department of Medicine and Graduate School of Medicine, Kyoto University）の倫理委員会によって承認された。 <Materials and methods>
(ethics)
All human tissue collection, human stem cell research, procedures, and written consent were approved by the Ethics Committee of the Department of Medicine and Graduate School of Medicine, Kyoto University.
（ヒトiPSCの樹立）
　ヒト末梢血単核細胞（PBMC）由来のiPSC（iPS細胞）の場合、初期化因子用のヒトcDNAは、エピソーマルベクター（SOX2、KLF4、OCT4、L-MYC、LIN28、p53ドミナントネガティブ体）を使用してヒトPBMCに形質導入した。形質導入の数日後、PBMCを回収し、iMatrixでコーティングしたディッシュに再播種した。翌日、培地をStemFit AK03またはAK02Nに交換した。その後、1日おきに培地を交換した。形質導入の20日後、iPSCコロニーをピックアップした。樹立したPBMC由来のiPSCを拡大培養した（Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)）。 (Establishment of human iPSC)
In the case of iPSCs (iPS cells) derived from human peripheral blood mononuclear cells (PBMCs), human cDNA for reprogramming factors is an episomal vector (SOX2, KLF4, OCT4, L-MYC, LIN28, p53 dominant negative). was used to transduce human PBMC. Several days after transduction, PBMCs were collected and replated onto iMatrix-coated dishes. The next day, the medium was replaced with StemFit AK03 or AK02N. Thereafter, the medium was replaced every other day. Twenty days after transduction, iPSC colonies were picked. The established PBMC-derived iPSCs were expanded and cultured (Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)).
（iN-iPSCの作製）
　堅牢で迅速な分化方法を確立するために、直接変換技術を利用した。テトラサイクリン誘導性プロモーター（tetO）下のヒトニューロゲニン2（NGN2）cDNAを、piggyBacトランスポゾンシステムとLipofectamine LTX（Thermo Fisher Scientific Inc.、ウォルサム、マサチューセッツ州）を用いてiPSCにトランスフェクトした。tetO::NGN2を含むpiggyBacベクターを使用した（Kim et al., 2016）。G418二硫酸塩(Nacalai-Tesque、京都、日本)の抗生物質選択後、コロニーを選択し、MAP2/DAPI（純度96％未満）でNGN2の一過的発現を誘導することにより神経細胞に効率的に分化できるサブクローンを選択した（Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)）。 (Preparation of iN-iPSCs)
A direct conversion technique was utilized to establish a robust and rapid differentiation method. Human neurogenin 2 (NGN2) cDNA under the tetracycline-inducible promoter (tetO) was transfected into iPSCs using the piggyBac transposon system and Lipofectamine LTX (Thermo Fisher Scientific Inc., Waltham, MA). The piggyBac vector containing tetO::NGN2 was used (Kim et al., 2016). After antibiotic selection with G418 disulfate (Nacalai-Tesque, Kyoto, Japan), select colonies and efficiently target neuronal cells by inducing transient expression of NGN2 with MAP2/DAPI (<96% purity). We selected subclones that can differentiate into (Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)).
（活性化カスパーゼ-3のライブイメージング検出）
　アポトーシス神経細胞（apoptotic neuron）における活性化カスパーゼ-3を、核酸結合色素がコンジュゲートした4アミノ酸ペプチドDEVDからなる蛍光発生基質（CellEvent Caspase-3/7 Green Detection Reagent、Thermo Fisher）を使用して検出した。DEVDペプチドが、色素のDNAに結合する能力を阻害するため、試薬は本来非蛍光性である。しかし、死細胞における活性化カスパーゼ-3がDEVDペプチドを切断すると、蛍光色素がDNAに結合できるようになり、蛍光発生応答が発生する。この応答は、従来のタイムラプスイメージングシステムの標準的なFITC-グリーンフィルターセットを使用して検出できる（露光時間= 400 ms、Incucyte ZOOM、Essen BioScience、Sartorius group）。画像を、薬の処理後6時間ごとに慢性的にキャプチャした。 (Live imaging detection of activated caspase-3)
Activated caspase-3 in apoptotic neurons was detected using a fluorogenic substrate consisting of the 4-amino acid peptide DEVD conjugated with a nucleic acid-binding dye (CellEvent Caspase-3/7 Green Detection Reagent, Thermo Fisher) did. The reagent is non-fluorescent in nature because the DEVD peptide inhibits the dye's ability to bind to DNA. However, when activated caspase-3 in dead cells cleaves the DEVD peptide, the fluorescent dye can bind to the DNA, resulting in a fluorogenic response. This response can be detected using a standard FITC-green filter set in a conventional time-lapse imaging system (exposure time = 400 ms, Incucyte ZOOM, Essen BioScience, Sartorius group). Images were captured chronically every 6 hours after drug treatment.
（データの準備）
　Incucyte ZOOMにより、384ウェルプレートのウェルから2つの画像をキャプチャした。キャプチャした画像はグレースケールで、元のサイズは1,392 x 1,040ピクセルであった。元の画像を4つの領域に均等に分割し、各領域の中央から正方形の画像をクロッピングした。クロッピングされた画像の幅x高さのサイズを、128 x 128、256 x 256、512 x 512、768 x 768、1,024 x 1,024から選択した。元の画像のサイズは1,392 x 1,038であったため、クロッピングサイズが519 x 519より大きい場合、クロッピング領域は元の画像からはみ出す。その場合、元の画像の端に合うようにクロッピング領域をシフトした。次に、クロッピングされた画像のサイズを256 x 256に変更した。トレーニング用の256 x 256のサイズの画像を、50％の確率で水平方向にランダムに反転し、224 x 224ピクセルの領域を画像からランダムにクロッピングした。224 x 224のクロッピングされた領域を、最終的にネットワークへの入力として使用した。検証データとテストデータでは、224 x 224サイズの画像を反転せずにクロッピングした。 (Data preparation)
Two images were captured from the wells of a 384-well plate by Incucyte ZOOM. The captured image was grayscale and had an original size of 1,392 x 1,040 pixels. The original image was divided equally into four regions, and a square image was cropped from the center of each region. The width x height size of the cropped image was selected from 128 x 128, 256 x 256, 512 x 512, 768 x 768, and 1,024 x 1,024. The original image size was 1,392 x 1,038, so if the cropping size is larger than 519 x 519, the cropping area will extend beyond the original image. In that case, I shifted the cropping area to match the edges of the original image. Next, I resized the cropped image to 256 x 256. A training image of size 256 x 256 was randomly flipped horizontally with a probability of 50%, and a region of 224 x 224 pixels was randomly cropped from the image. The 224 x 224 cropped region was finally used as input to the network. For validation and test data, 224 x 224 images were cropped without flipping.
　マイクロモラースケールでのZ-VAD-FMKの対数スケール濃度を標的として定義した。独立した実験の3つのバッチを実行し、各バッチで2つの384ウェルプレートを使用した。2つの実験バッチからのDMSO/Z-VAD-FMK処理後0～12時間での画像（各クラスにつき768画像）をトレーニングと検証（トレーニングデータセット）に使用し、残りの実験バッチからの処理後特定のタイミングの画像をテストに使用した（各クラスにつき128枚の画像；テストデータセット）。細胞運命分類分析で決定したものと同じクロッピングサイズも採用した。他の化合物を投与した細胞の画像と、CRISPRで修正した細胞の画像の評価では、プレートのすべてのウェルをテストデータセットとして使用した。 The logarithmic scale concentration of Z-VAD-FMK on the micromolar scale was defined as the target. Three independent batches of experiments were performed, and two 384-well plates were used in each batch. Images from 0 to 12 hours after DMSO/Z-VAD-FMK processing from two experimental batches (768 images for each class) were used for training and validation (training dataset), and after processing from the remaining experimental batches. Images at specific times were used for testing (128 images for each class; test dataset). The same cropping size as determined in the cell fate classification analysis was also employed. For evaluation of images of cells treated with other compounds and CRISPR-modified cells, all wells of the plate were used as the test data set.
（畳み込みニューラルネットワークの構成）
　ディープラーニングフレームワークであるPyTorch（https://pytorch.org/）(Paszke A. et al., Automatic differentiation in PyTorch. In NIPS (2017))を、ネットワークの構築、トレーニング、検証、およびテストに使用した。ニューラルネットワークアーキテクチャとして、densenet-121モデルを利用した(Huang G. et al., Proc. - 30th IEEE Conf. Comput. Vis. Pattern Recognition, CVPR 2017 2017-January, 2261-2269 (2016))。医学的または生物学的データでよくあるような、トレーニングサンプルの数が限られている場合に効果的な微調整アプローチを採用した(Esteva A. et al., Nature 542, 115-118 (2017))。ネットワークはImageNetデータセットで事前にトレーニングされており、該データセットには、1,000のカテゴリのオブジェクトの120万のラベル付き画像がある。事前トレーニング済みのネットワークの最終層のノード数は、細胞運命のクラスの数（= 2）、または予測されたZVAD-FMKの同等濃度（predicted ZVAD-FMK-equivalent concentration）もしくはDL予測スコアを出力するノードの数に調整した。入力ノードの数は、画像サイズに合わせて調整した。トレーニングフェーズ中に、すべての層の重みが再最適化された。平均二乗誤差（回帰分析）を損失関数として使用した。誤差はネットワークを介して逆伝播され、重みはミニバッチを使用した確率的勾配降下法（SGD）によって最適化された。 (Configuration of convolutional neural network)
The deep learning framework PyTorch (https://pytorch.org/) (Paszke A. et al., Automatic differentiation in PyTorch. In NIPS (2017)) was used to build, train, validate, and test the network. did. We used the densenet-121 model as the neural network architecture (Huang G. et al., Proc. - 30th IEEE Conf. Comput. Vis. Pattern Recognition, CVPR 2017 2017-January, 2261-2269 (2016)). We adopted a fine-tuning approach that is effective when the number of training samples is limited, as is often the case with medical or biological data (Esteva A. et al., Nature 542, 115-118 (2017) ). The network was pre-trained on the ImageNet dataset, which has 1.2 million labeled images of objects in 1,000 categories. The number of nodes in the final layer of the pre-trained network outputs the number of cell fate classes (= 2) or the predicted ZVAD-FMK-equivalent concentration or DL prediction score. Adjusted to the number of nodes. The number of input nodes was adjusted according to the image size. During the training phase, the weights of all layers were reoptimized. Mean squared error (regression analysis) was used as the loss function. Errors were backpropagated through the network and weights were optimized by stochastic gradient descent (SGD) using mini-batches.
（トレーニングおよび評価）
　まず、ネットワークはSGDの固定ハイパーパラメータ、学習率lr = 0.001、モーメンタムm = 0.9でトレーニングし、平均二乗誤差によって評価し、入力画像のさまざまなクロッピングサイズの条件間（w x h = {256 × 256、512 × 512、768 × 768、1,024 × 1,024}）で比較した。クロッピングサイズを固定した後、学習率lr = {0.001、0.002、0.004}およびモーメンタムm = {0、0.5、0.9}に対してグリッドサーチを実行し、平均二乗誤差を4分割交差検定でトレーニングデータセットを評価した。最適化されたハイパーパラメータを使用して、ネットワークのトレーニングおよび評価を行った。ネットワークがトレーニングデータセットに対して最高の分類精度を示したときのエポックでのネットワークパラメータをテストに使用した。トレーニングおよびテストの実験バッチをローテーションし、3回の試行の結果の平均を計算した。トレーニングのミニバッチサイズはb = 8で、トレーニング中のエポックは10であった。データをデュアルGPUで並列処理した。DL予測スコアと実際のZ-VAD-FMK濃度の間に直線を当てはめ、ネットワークのパフォーマンスを決定係数（R二乗値）で評価した。 (Training and Evaluation)
First, the network was trained with fixed hyperparameters of SGD, learning rate lr = 0.001, momentum m = 0.9, and evaluated by mean squared error, and the difference between conditions of different cropping sizes of input images (w x h = {256 × 256, 512 × 512, 768 × 768, 1,024 × 1,024}). After fixing the cropping size, we perform a grid search for learning rate lr = {0.001, 0.002, 0.004} and momentum m = {0, 0.5, 0.9}, and calculate the mean squared error using 4-fold cross-validation on the training dataset. was evaluated. The optimized hyperparameters were used to train and evaluate the network. The network parameters at the epoch when the network showed the highest classification accuracy on the training dataset were used for testing. We rotated the training and testing experimental batches and calculated the average of the results of the three trials. The training mini-batch size was b = 8, and there were 10 epochs during training. Data was processed in parallel using dual GPUs. A straight line was fitted between the DL prediction score and the actual Z-VAD-FMK concentration, and the performance of the network was evaluated by the coefficient of determination (R-squared value).
（CNNでの画像処理の視覚化）
　入力セル画像を処理するときにCNNの焦点がどこにあったかを解釈するために、GradCAMをCNNに実装した。GradCAMは、CNNが決定を下すために利用されたローカライズされた領域の同定を可能にする勾配ベースのクラスアクティベーションマップを提供する(Selvaraju, R.R. et al., arXiv:1610.02391v1 (2016))。この手法をトレーニング済みの（trained）CNNに適用し、ネットワークが画像の予測スコアが高いと判断したときの、画像上のCNNの焦点を視覚化したマップを取得した。より詳細な解釈のために、ガイド付き誤差逆伝播法とGradCAMを組み合わせたものである、ガイド付き（Guided）GradCAMもCNNに適用した(Springenberg et al., 2014)。 (Visualization of image processing with CNN)
We implemented GradCAM into the CNN in order to interpret where the focus of the CNN was when processing the input cell images. GradCAM provides gradient-based class activation maps that allow identification of localized regions where CNNs are utilized to make decisions (Selvaraju, RR et al., arXiv:1610.02391v1 (2016)). They applied this method to a trained CNN to obtain a map that visualizes the CNN's focus on an image when the network determines that the image has a high prediction score. For a more detailed interpretation, we also applied Guided GradCAM to the CNN, which is a combination of guided error backpropagation and GradCAM (Springenberg et al., 2014).
（ヒト脳組織）
　予め登録し、死前に同意した健康な対照およびAD患者のすべてのヒトの死後の脳組織を、バナーサンヘルス研究所（Banner Sun Health Research Institute）の脳および体の寄付プログラム（Brain and Body Donation Program）（Beach T.G. et al., Cell Tissue Bank. 9, 229-245 (2008)）から入手し、標準的な研究プロトコルは施設内倫理審査委員会（Institutional Review Board）によって承認された。本研究は、国立病院機構鳥取医療センターの倫理委員会によって承認された。対照およびAD患者すべてを、生前の医療記録、ならびにADの登録を確立するためのコンソーシアム（CERAD）の病理学的基準（Mirra, S.S. et al. Neurology 41, 479-486 (1991)）およびブラーク病期分類（Braak H. et al., Acta Neuropathol. 112, 389-404 (2006)）を用いた死後の神経病理学的検査に基づいて診断した。 (human brain tissue)
Postmortem brain tissue from all pre-registered and consented antemortem healthy controls and AD patients will be donated to the Banner Sun Health Research Institute's Brain and Body Donation Program. The standard study protocol was approved by the Institutional Review Board. This study was approved by the Ethics Committee of the National Hospital Organization Tottori Medical Center. All controls and AD patients were compared with antemortem medical records and the pathological criteria of the Consortium for the Establishment of a Registry of AD (CERAD) (Mirra, SS et al. Neurology 41, 479-486 (1991)) and Braak's disease. Diagnosis was based on postmortem neuropathological examination using stage classification (Braak H. et al., Acta Neuropathol. 112, 389-404 (2006)).
（ヒトの脳対象のための免疫組織化学）
　組織染色のため、死後4時間以内に脳を摘出し、1 cm間隔で冠状切片を作製し、氷冷した4％緩衝パラホルムアルデヒド（pH 7.4）で固定し、40 μmの切片に切断し、凍結保護と共に2％DMSO/20％グリセロール中に-30℃で保存した。側頭皮質由来の固定した浮遊組織切片を、0.1 M クエン酸緩衝液（pH 6.0）中でマイクロ波照射下処理し、次いで0.5％ Triton X-100、3％ 過酸化水素、4％ BSAを含むPBSで室温、30分間処理し、1：200で希釈した抗KLHL32ウサギポリクローナル抗体（ab243805、Abcam、ケンブリッジ、マサチューセッツ州）で4℃、24時間処理し、次いでビオチンをコンジュゲートしたヤギ抗ウサギIgG（Jackson ImmunoResearch Laboratories、バーハーバー、メイン州）で処理した。続いて、標識した切片をアビジン-ビオチン-西洋ワサビペルオキシダーゼ複合体（Vectastain Elite ABCキット、Vector Laboratories、バーリンゲーム、カリフォルニア州）と反応させ、既報（Kume H. et al., Neuropathol. Appl. Neurobiol. 35, 178-188 (2009)；Tooyama I., et al., Dement. Geriatr. Cogn. Disord. 12, 237-242 (2001)）の通り、ペルオキシダーゼ活性を0.05％ 3,3'-ジアミノベンジジン四塩酸塩（Dojindo、熊本、日本）で可視化した。KLHL32で染色した切片を、BIOREVO BZ-X800顕微鏡（キーエンス、大阪、日本）でデジタル化した。得られた画像を、イメージングソフトウェアのcellSens（オリンパス、東京、日本）を使用して定量化した。 (Immunohistochemistry for human brain subjects)
For histological staining, brains were removed within 4 hours after death, coronal sections were prepared at 1 cm intervals, fixed in ice-cold 4% buffered paraformaldehyde (pH 7.4), cut into 40 μm sections, and frozen. Stored at -30°C in 2% DMSO/20% glycerol with protection. Fixed floating tissue sections from temporal cortex were treated under microwave irradiation in 0.1 M citrate buffer (pH 6.0) containing 0.5% Triton X-100, 3% hydrogen peroxide, 4% BSA. Treated with PBS for 30 min at room temperature, treated with anti-KLHL32 rabbit polyclonal antibody (ab243805, Abcam, Cambridge, MA) diluted 1:200 for 24 h at 4°C, and then treated with biotin-conjugated goat anti-rabbit IgG ( Jackson ImmunoResearch Laboratories, Bar Harbor, ME). The labeled sections were then reacted with an avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) as previously described (Kume H. et al., Neuropathol. Appl. Neurobiol. 35, 178-188 (2009); Tooyama I., et al., Dement. Geriatr. Cogn. Disord. 12, 237-242 (2001)), peroxidase activity was reduced by 0.05% Visualized with hydrochloride (Dojindo, Kumamoto, Japan). Sections stained with KLHL32 were digitized with a BIOREVO BZ-X800 microscope (Keyence, Osaka, Japan). The resulting images were quantified using the imaging software cellSens (Olympus, Tokyo, Japan).
実施例１：ディープラーニングベースのiPSCモデルによる、変性する神経細胞のin vitroでの予測
　本実施例の目的は、図に示されているように、位相差画像のみを使用してiPSC由来神経細胞の細胞運命を予測するディープラーニングベースのiPSCモデル（Deep-iモデル）を開発することであった（図1A）。神経細胞死は、ADを含む神経変性疾患の最も特徴的な組織病理学的特徴であり、活性化カスパーゼ-3はAD患者の脳における神経細胞死の分子マーカーである(Christie L.A. et al., Neurobiol. Dis. 26, 165-173 (2007); Louneva N. et al., Am. J. Pathol. 173, 1488-1495 (2008); Su J.H. et al., Brain Res. 898, 350-357 (2001))。神経変性疾患患者のiPS細胞から分化させた神経細胞は、時間の経過とともに細胞死を再現することが広く知られている(Imamura K. et al., Sci. Rep. 6, 34904 (2016); Imamura K. et al., Sci. Transl. Med. 9, eaaf3962 (2017); Nguyen H.N. et al., Cell Stem Cell 8, 267-280 (2011); Sanchez-Danes A. et al., EMBO Mol. Med. 4, 380-395 (2012))。家族性ADのiPSC由来の皮質神経細胞もin vitroで徐々に細胞死を示す(Kondo T. et al, Cell Stem Cell 12, 487-496 (2013))。 Example 1: In vitro prediction of degenerating neurons using a deep learning-based iPSC model The purpose of this example was to predict iPSC-derived neurons using only phase contrast images, as shown in the figure. The aim was to develop a deep learning-based iPSC model (Deep-i model) that predicts the cell fate of cells (Figure 1A). Neuronal cell death is the most characteristic histopathological feature of neurodegenerative diseases, including AD, and activated caspase-3 is a molecular marker of neuronal cell death in the brains of AD patients (Christie LA et al., Neurobiol. Dis. 26, 165-173 (2007); Louneva N. et al., Am. J. Pathol. 173, 1488-1495 (2008); Su JH et al., Brain Res. 898, 350-357 ( 2001)). It is widely known that neurons differentiated from iPS cells from patients with neurodegenerative diseases reproduce cell death over time (Imamura K. et al., Sci. Rep. 6, 34904 (2016); Imamura K. et al., Sci. Transl. Med. 9, eaaf3962 (2017); Nguyen HN et al., Cell Stem Cell 8, 267-280 (2011); Sanchez-Danes A. et al., EMBO Mol. Med. 4, 380-395 (2012)). Cortical neurons derived from iPSCs in familial AD also show gradual cell death in vitro (Kondo T. et al, Cell Stem Cell 12, 487-496 (2013)).
　まず、家族性AD患者から樹立されたiPSCを使用して神経細胞死を評価するライブイメージングシステムを構築した（図1B）。家族性AD患者由来iPSCにおいて、皮質神経細胞分化の特異的転写因子の1つであるヒトニューロゲニン2（NGN2）の外来性発現を誘導して皮質神経細胞へと分化誘導し、分化開始後わずか8日でiPSC由来皮質神経細胞を調製することに成功した（図1B）。NGN2の発現誘導期間は5日間（120時間）とした。当該分化法は、ヒトiPS細胞をほぼ100％の純度と高い再現性で皮質神経細胞に変換することができ(Kondo T. et al., Cell Rep. 21, 2304-2312 (2017))、この高い純度と再現性は異なる細胞運命間の微細な違いを分析および検出するために必要である。カスパーゼ-3活性化の時間依存的な変化を連続的に評価するために、核酸結合色素がコンジュゲートした4アミノ酸ペプチドDEVDを使用して、活性化カスパーゼ-3を緑色蛍光として検出した（図1C）。分化誘導から8日後（NGN2の発現誘導開始から192時間後）を解析の起点（すなわち、DEVDの添加を開始した時点）（Time 0）とした。6時間ごとの時系列画像から、カスパーゼ-3の活性化が7日間にわたって発生したことが示された（図1Dおよび1E）。活性化カスパーゼ-3を介した細胞死のこの結果は、神経変性疾患の患者からのiPSC由来神経細胞を利用した以前の研究と一致していた(Imamura K. et al., Sci. Transl. Med. 9, eaaf3962 (2017); Kondo T. et al, Cell Stem Cell 12, 487-496 (2013))。さらに、実験における緑色蛍光がカスパーゼ-3の活性化を反映していることを確認するために、カスパーゼに対する細胞膜透過性阻害剤である25 μMのZ-Val-Ala-Asp（OMe）-CH2F（Z-VAD-FMK）を添加した。緑色蛍光のほぼ完全な消失が観察できたが、これは、Z-VAD-FMKがカスパーゼ-3の活性化を阻害したことを意味する（図1Dおよび1E）。将来、細胞が死ぬコンディションと生存するコンディション間のカスパーゼ-3活性状態の違いは、アッセイ開始後30時間から統計的に有意であった（図1E）。 First, we constructed a live imaging system to assess neuronal cell death using iPSCs established from familial AD patients (Figure 1B). In iPSCs derived from familial AD patients, exogenous expression of human neurogenin 2 (NGN2), which is a specific transcription factor for cortical neuron differentiation, is induced to differentiate into cortical neurons, and only a short time after the initiation of differentiation. We successfully prepared iPSC-derived cortical neurons in 8 days (Figure 1B). The expression induction period of NGN2 was 5 days (120 hours). This differentiation method can convert human iPS cells into cortical neurons with almost 100% purity and high reproducibility (Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)), and this High purity and reproducibility are necessary to analyze and detect subtle differences between different cell fates. To continuously assess time-dependent changes in caspase-3 activation, activated caspase-3 was detected as green fluorescence using a 4-amino acid peptide DEVD conjugated with a nucleic acid-binding dye (Figure 1C ). Eight days after induction of differentiation (192 hours after the start of induction of NGN2 expression) was taken as the starting point of analysis (ie, the time when addition of DEVD was started) (Time 0). Time series images every 6 hours showed that caspase-3 activation occurred over 7 days (Figures 1D and 1E). This finding of activated caspase-3-mediated cell death was consistent with previous studies utilizing iPSC-derived neurons from patients with neurodegenerative diseases (Imamura K. et al., Sci. Transl. Med 9, eaaf3962 (2017); Kondo T. et al, Cell Stem Cell 12, 487-496 (2013)). Furthermore, to confirm that the green fluorescence in the experiment reflected the activation of caspase-3, 25 μM Z-Val-Ala-Asp(OMe) -CH2 , a cell membrane-permeable inhibitor of caspases, was added. F(Z-VAD-FMK) was added. Almost complete disappearance of green fluorescence could be observed, implying that Z-VAD-FMK inhibited the activation of caspase-3 (Figures 1D and 1E). In the future, the difference in caspase-3 activity status between conditions where cells die and survive was statistically significant from 30 hours after the start of the assay (Figure 1E).
　これらの結果から、構築されたライブイメージングシステムが、活性化カスパーゼ-3により引き起こされる神経細胞死の連続的な増加を時間依存的に評価できることが示された。このライブイメージングシステムを適用して、神経細胞の死の運命を予測するためのDeep-iモデルを確立した。 These results showed that the constructed live imaging system could evaluate the continuous increase in neuronal cell death caused by activated caspase-3 in a time-dependent manner. By applying this live imaging system, we established a Deep-i model for predicting the death fate of neurons.
　次に、Z-VAD-FMKを8点の濃度（0、0.0016、0.008、0.04、0.2、1、5、25 μM）で皮質神経細胞培養物に添加し、それに続くカスパーゼ-3の活性化をモニターした。6時間ごとに異なる条件下で位相差画像と緑色蛍光画像をキャプチャした（図2A）。高感度緑色蛍光（enhanced green fluorescence）により検出された神経細胞の活性化カスパーゼ-3は、Z-VAD-FMKの用量依存的に減少した（図2A）。さらに、384ウェルプレートの異なる列の間で、活性化カスパーゼ-3に由来する緑色蛍光シグナルの位置効果がないことも確認した（図3A）。実験スキームの再現性を確認するために、元のバッチ1と同様の結果を示す2つの追加バッチを準備した（図4Aおよび4B）。細胞死シミュレーションシステムとして、合計3つのバッチからの位相差画像のデータセットを利用した。細胞死が有意に現れる前に神経細胞の運命を予測するために、Deep-iモデルをトレーニングした（図2B）。トレーニングステップでは、細胞死または細胞生存状態の単純な分類器はなく、継続的な尺度として死の可能性を計算する方法として、ディープラーニングベースの予測法を構築しようとした。ニューラルネットワークアーキテクチャとして、DenseNet121ネットワークを活用し(Huang G. et al., Proc. - 30th IEEE Conf. Comput. Vis. Pattern Recognition, CVPR 2017 2017-January, 2261-2269 (2016))、ネットワークはImageNetデータセットで事前トレーニングした(Deng J. et al., In 2009 IEEE Conference on Computer Vision and Pattern Recognition, (IEEE), pp. 248-255 (2009))。ネットワークを、Z-VAD-FMKの濃度を予測するために再トレーニングし、予測濃度をDL予測スコアとして定義した。追跡開始後0～72時間の各時間枠（図5A）の位相差画像のデータセットを準備した。実験の3つのバッチを実施し（図5B）、各バッチで2つの384ウェルプレートを使用した（図5B）。 Z-VAD-FMK was then added to cortical neuronal cultures at eight concentrations (0, 0.0016, 0.008, 0.04, 0.2, 1, 5, 25 μM) and subsequent activation of caspase-3 I monitored it. Phase contrast images and green fluorescence images were captured under different conditions every 6 hours (Figure 2A). Activated caspase-3 in neurons, detected by enhanced green fluorescence, decreased in a Z-VAD-FMK dose-dependent manner (Figure 2A). Furthermore, we confirmed that there was no position effect of the green fluorescent signal derived from activated caspase-3 between different rows of the 384-well plate (Figure 3A). To confirm the reproducibility of the experimental scheme, two additional batches were prepared showing similar results to the original batch 1 (Figures 4A and 4B). A dataset of phase contrast images from a total of three batches was utilized as a cell death simulation system. We trained a Deep-i model to predict neuronal fate before significant cell death appeared (Figure 2B). In the training step, we sought to build a deep learning-based prediction method, rather than a simple classifier of cell death or cell survival status, as a way to calculate the probability of death as a continuous measure. As a neural network architecture, we utilize DenseNet121 network (Huang G. et al., Proc. - 30th IEEE Conf. Comput. Vis. Pattern Recognition, CVPR 2017 2017-January, 2261-2269 (2016)), and the network uses ImageNet data. (Deng J. et al., In 2009 IEEE Conference on Computer Vision and Pattern Recognition, (IEEE), pp. 248-255 (2009)). The network was retrained to predict the concentration of Z-VAD-FMK, and the predicted concentration was defined as the DL prediction score. We prepared a dataset of phase contrast images for each time frame (Figure 5A) from 0 to 72 hours after the start of tracking. Three batches of experiments were performed (Fig. 5B), and two 384-well plates were used in each batch (Fig. 5B).
　トレーニングデータセットのため、開始後0～12時間での2つの実験バッチから画像を採用した（図4B）。合計で、時間窓（time-window）内の1,536枚の画像を準備した（図4A）。豊富なサンプルを学習するために、それぞれ2つの培養プレートで構成される、3つの異なる実験バッチすべてを使用し、これらの独立したバッチ間でトレーニング、検証、およびテストのステップをローテーションして、再現性を確認し、バッチに依存する結果を回避した（図5B）。 For the training dataset, images were taken from two experimental batches from 0 to 12 hours after initiation (Figure 4B). In total, 1,536 images within a time-window were prepared (Figure 4A). To learn a rich sample, we used all three different experimental batches, each consisting of two culture plates, and rotated the training, validation, and testing steps between these independent batches for replication. consistency and avoided batch-dependent results (Figure 5B).
　画像の異なるピクセルサイズの影響を評価するために、各フルサイズ画像から4つの正方形の画像のさまざまなピクセルサイズをクロッピングした（図5C）。画像サイズは、トレーニングデータセットの4分割交差検定から得られた平均二乗誤差に従って最適化した。実験バッチの3つの組み合わせすべてにおいて（図5B）、最大のピクセル画像である1,024 × 1,024の結果は、対数スケールの実際のZ-VAD-FMK濃度とDL予測スコアとして定義される予測濃度との間の平均二乗誤差が最小であった（図2C）。従って、さらに分析するために、1,024 × 1,024ピクセルのクロッピングを選択した（図4D）。 To evaluate the influence of different pixel sizes of images, we cropped different pixel sizes of four square images from each full-size image (Figure 5C). Image size was optimized according to the mean squared error obtained from 4-fold cross-validation of the training dataset. For all three combinations of experimental batches (Figure 5B), the largest pixel image, 1,024 × 1,024 results, are between the actual Z-VAD-FMK concentration on a logarithmic scale and the predicted concentration, defined as the DL prediction score. had the smallest mean squared error (Figure 2C). Therefore, we chose a cropping of 1,024 × 1,024 pixels for further analysis (Figure 4D).
　入力画像サイズおよびハイパーパラメータを最適化した後、トレーニング済みのネットワークのパフォーマンスをテストデータセットに対して評価した。ネットワークを、トレーニングデータセットの主要部分を使用してトレーニングし、各エポックで残りのトレーニングデータセットの損失を評価した。トレーニングエポック全体でのネットワークの最良の学習状態を保存した。テストでは、トレーニングステップで使用した2つのバッチの独立したバッチからの画像を使用した。テストデータセットは、追跡開始から24時間後に収集した画像であり、カスパーゼ-3シグナルは、DMSOとZ-VAD-FMK条件との間で有意差は認められなかった（図1E）。トレーニング済みのネットワークは、テスト画像からのDL予測スコアを生成した。DL予測スコアは、アッセイ開始から24時間後の実際のZ-VAD-FMK濃度と線形相関を示した（図2D）。 After optimizing the input image size and hyperparameters, the performance of the trained network was evaluated on the test dataset. The network was trained using the main part of the training dataset and the loss was evaluated on the remaining training dataset at each epoch. The best learning state of the network across training epochs was saved. The tests used images from independent batches of the two batches used in the training step. The test data set was images collected 24 hours after the start of tracking, and caspase-3 signals were not significantly different between DMSO and Z-VAD-FMK conditions (Figure 1E). The trained network produced DL prediction scores from test images. The DL prediction score showed a linear correlation with the actual Z-VAD-FMK concentration 24 hours after the start of the assay (Fig. 2D).
　トレーニングデータが取得されたときの異なる時点での予測の精度を比較した。スコアに対する線形回帰モデルのR二乗は、3つのバッチすべてで約0.7であった（図5D）。テスト画像が後の時点から描画された場合、平均二乗誤差は増加し（図2E）、近似線形モデルのR二乗は減少した（図2F）。また、DL予測スコアは、主に細胞の特徴に由来するが、ウェルの位置に起因するアーティファクトには由来しないことも確認した。DL予測スコアを、任意の位置のすべてのウェルがDMSO状態にあるプレートからの画像から推測した。すべてのDMSOで調整されたプレートのスコアは、ウェルの位置に応じた明らかな変化を示さなかった（図5E）。 We compared the accuracy of predictions at different times when the training data was acquired. The R-squared of the linear regression model for scores was approximately 0.7 for all three batches (Figure 5D). When test images were drawn from later time points, the mean squared error increased (Fig. 2E) and the R-squared of the fitted linear model decreased (Fig. 2F). We also confirmed that the DL prediction score is primarily derived from cell characteristics, but not from artifacts due to well position. DL prediction scores were estimated from images from plates where all wells at any location were in DMSO. Scores for all DMSO-adjusted plates showed no obvious changes depending on well position (Fig. 5E).
　次に、確立したシステムを疾患モデリングに適用して検証した。FAD患者iPSCのPSEN1 G384A変異を遺伝的に修正し、CRISPR-Cas9システムを使用して遺伝的に編集されたPSEN1の配列を除いて、元のFAD iPSCと同一のゲノムを保持するアイソジェニックなiPSCクローンを作製した(Kondo T. et al., Cell Rep. 21, 2304-2312 (2017))。トレーニング済みのCNNは、FADとアイソジェニックなiPSC由来神経細胞の両方からの画像を処理し、DL予測スコアを異なるクローン間で比較した（図2Gおよび5F）。PSEN1変異のない神経細胞は、DL予測スコアがより高く、細胞が生存する未来に向かう傾向がより高くなっていた。さらに、ADの最も重要な原因の1つであり、FAD神経細胞で変化するAβ産生を改変できる化合物をテストした。用量依存的にAβ産生を抑制する、0～25 μMのβ-セクレターゼ阻害剤であるBSI IVを使用した。DL予測スコアは、BSI IVの用量依存的に増加したが、これは、Z-VAD-FMKの結果と比較して、細胞死の軽度な抑制と同様の傾向を示している（図2H）。これらの結果から、Deep-iモデルが疾患モデリングに適用可能であり、DL予測スコアがDeep-iモデルのトレーニングに使用したZ-VAD-FMKの濃度だけに固有ではないことが示された。 Next, we applied the established system to disease modeling and verified it. Genetically correct the PSEN1 G384A mutation in FAD patient iPSCs and create isogenic iPSCs that retain the genome identical to the original FAD iPSCs, except for the genetically edited PSEN1 sequence using the CRISPR-Cas9 system. A clone was created (Kondo T. et al., Cell Rep. 21, 2304-2312 (2017)). The trained CNN processed images from both FAD and isogenic iPSC-derived neurons, and DL prediction scores were compared between different clones (Figures 2G and 5F). Neurons without PSEN1 mutations had higher DL prediction scores and were more likely to move toward a future in which they would survive. Additionally, they tested compounds that can modify Aβ production, which is one of the most important causes of AD and is altered in FAD neurons. BSI IV, a β-secretase inhibitor, was used at 0-25 μM, which suppresses Aβ production in a dose-dependent manner. The DL prediction score increased in a dose-dependent manner of BSI IV, which shows a similar trend with mild inhibition of cell death compared to the results of Z-VAD-FMK (Fig. 2H). These results showed that the Deep-i model is applicable to disease modeling and that the DL prediction score is not specific only to the concentration of Z-VAD-FMK used to train the Deep-i model.
　さらに、Z-VAD-FMKに基づくDL予測スコアのその他の化合物への適応可能性を評価した。最初に、RIP1キナーゼの強力な阻害剤であり、壊死性細胞死を抑制するネクロスタチン-1を評価した（Degterev A. et al., Nat. Chem. Biol. 1, 112-119 (2005)）。予想通り、Z-VAD-FMKと比較して、ネクロスタチン-1は神経変性を部分的にレスキューし、中濃度から高濃度の状態でより高いDL予測スコアを示した（図2Iおよび2J）。また、高濃度条件でのみ神経変性を抑制できるネクロスタチン-1アナログを評価し（Takahashi N. et al., Cell Death Dis. 2012 311 3, e437-e437 (2012)）、ネクロスタチン-1アナログは5～25μMの高濃度でのみDL予測スコアを改善することを見出した（図2Kおよび2L）。さらに、非カスパーゼ阻害または非RIPK経路を介して神経変性を改善するDHAを評価し、異なる濃度でのDL予測スコアの変化が、蛍光シグナルとして示される実際の神経変性の変化と同様のパターンを示すことを見出した（図2Mおよび2N）。DL予測スコアの用量依存性は、スコアがカスパーゼ3に依存しない細胞変性の起源を反映していることを示唆している。これらの結果から、DL予測スコアは、異なる化合物と可変的な濃度に起因する神経変性状態の微細な変化を反映できることが確認できた。 Furthermore, we evaluated the applicability of the DL prediction score based on Z-VAD-FMK to other compounds. First, we evaluated necrostatin-1, which is a potent inhibitor of RIP1 kinase and suppresses necrotic cell death (Degterev A. et al., Nat. Chem. Biol. 1, 112-119 (2005)). . As expected, compared to Z-VAD-FMK, necrostatin-1 partially rescued neurodegeneration and showed higher DL prediction scores at intermediate to high concentration conditions (Figures 2I and 2J). In addition, we evaluated necrostatin-1 analogs that can suppress neurodegeneration only under high concentration conditions (Takahashi N. et al., Cell Death Dis. 2012 311 3, e437-e437 (2012)). We found that only high concentrations of 5-25 μM improved the DL prediction score (Figures 2K and 2L). Furthermore, we evaluated DHA to ameliorate neurodegeneration through non-caspase inhibition or non-RIPK pathways, and the changes in DL prediction scores at different concentrations show a similar pattern to the actual neurodegenerative changes shown as fluorescent signals. We found that (Figures 2M and 2N). The dose dependence of the DL prediction score suggests that the score reflects a caspase-3-independent cytopathic origin. These results confirmed that the DL prediction score can reflect subtle changes in neurodegenerative conditions caused by different compounds and variable concentrations.
実施例２：単一細胞RNAシーケンス解析による、神経変性がプログラムされた細胞の分子マーカーの同定
　前記のように、Deep-iモデルは、神経細胞死までの過程で隠れた神経変性を検出することができた。しかしながら、Deep-iモデルは、どの細胞が変性し始めるかを検出できるが、細胞の分子シグネチャーは検出できない。そこで、in vitroで神経変性がプログラムされた神経細胞の分子シグネチャーを見つけるため、同時に単一細胞RNAシーケンス解析を行った。アッセイ開始（Time 0）から12時間後は、Z-VAD-FMKを添加しない陰性コントロール群でもカスパーゼ-3の活性化の増加がまだ検出されないステージだが、そのステージで神経細胞をサンプリングし、DMSO対照条件、Z-VAD-FMK 1 μM条件、およびZ-VAD-FMK 25μM条件の3つの異なる条件からのデータを比較した（図6A）。厳格な品質管理フィルタリングの後、条件ごとに3,937～6,214個の細胞を取得し（図7Aを参照）、各条件で細胞ごとに中央値2,753、2,484、および2,202個の遺伝子を検出した（図6B）。3つの異なる条件間で全体の遺伝子またはミトコンドリアの遺伝子の発現パターンに明らかな違いはなく、人工の細胞損傷に通常は起因する実験的なノイズはないと判断した（図7A）。詳細な不均一性を可視化するために、各条件に対して教師なしクラスタリング分析を行い、単一核のトランスクリプトームを15のクラスターに分類した（UMAPプロット、図6B）。3つの異なる条件間で各クラスターの存在割合（％）を比較した結果、「クラスター12」のクラスターサイズ（総細胞数のうち、クラスター12に含まれる細胞の割合）が、Z-VAD-FMKの濃度の増加とともに用量依存的に減少（アポトーシスした神経細胞の数の減少に等しい）することが明らかになった（図6C）。クラスター12には、DMSO対照群の1.42％の細胞、Z-VAD-FMK 1 μM処理群の1.22％の細胞、Z-VAD-FMK 25 μM処理群の0.75％の細胞がそれぞれ含まれている（図6Cおよび2B）。この結果は、クラスター12の神経細胞の集団が、数時間後に神経変性を起こす運命にある細胞であることを示す。 Example 2: Identification of molecular markers of cells programmed for neurodegeneration by single-cell RNA-sequencing analysis As mentioned above , the Deep-i model can detect hidden neurodegeneration during the process leading up to neuronal cell death. was completed. However, while the Deep-i model can detect which cells begin to degenerate, it cannot detect the molecular signature of the cells. To find the molecular signature of neurons programmed for neurodegeneration in vitro, we simultaneously performed single-cell RNA-sequencing analysis. Twelve hours after the start of the assay (Time 0), an increase in caspase-3 activation was not yet detected even in the negative control group without the addition of Z-VAD-FMK. We compared data from three different conditions: Z-VAD-FMK 1 μM condition, and Z-VAD-FMK 25 μM condition (Figure 6A). After strict quality control filtering, we obtained between 3,937 and 6,214 cells per condition (see Figure 7A) and detected medians of 2,753, 2,484, and 2,202 genes per cell in each condition (Figure 6B ). There were no obvious differences in global or mitochondrial gene expression patterns between the three different conditions, and we determined that there was no experimental noise normally attributed to artificial cell damage (Figure 7A). To visualize detailed heterogeneity, we performed unsupervised clustering analysis for each condition and classified the single-nuclei transcriptome into 15 clusters (UMAP plot, Figure 6B). As a result of comparing the abundance ratio (%) of each cluster between three different conditions, the cluster size of "cluster 12" (the percentage of cells included in cluster 12 out of the total number of cells) was smaller than that of Z-VAD-FMK. A dose-dependent decrease (equivalent to a decrease in the number of apoptotic neurons) with increasing concentration was revealed (Figure 6C). Cluster 12 contains 1.42% cells in the DMSO control group, 1.22% cells in the Z-VAD-FMK 1 μM treated group, and 0.75% cells in the Z-VAD-FMK 25 μM treated group, respectively ( Figures 6C and 2B). This result indicates that the population of neurons in cluster 12 is one that is destined to undergo neurodegeneration after a few hours.
　次に、クラスター12に含まれる細胞を遺伝的に特徴づけるために、クラスター12が発現する遺伝子セットを分析した。数種類の遺伝子と関連する分子経路がクラスター12に特異的であり（図6D）、クラスター12を標識するためのマーカーとなり得ることが明らかになった。特に、KLHL32はクラスター12の最も特異的な遺伝子であり、クラスター12の個々の神経細胞の約97％はKLHL32の高発現を示した（図6E）。さらに、クラスター12の最も特異的な遺伝子（上位50）についてパスウェイ分析を行い、クラスター12に関連する上位2つのパスウェイがシナプス形成と細胞死であることを見出した（図6D）。活性化カスパーゼ-3を介した細胞死イベントにおけるKLHL32の重要性を確認するために、siRNAノックダウン実験を行い、KLHL32の抑制がカスパーゼ-3の活性化と細胞死の運命を抑制することを見出した（図7Cおよび7D）。用いたsiRNAの標的配列を表１に示す。標的遺伝子（Non-targeting Controlも含む）あたり、4種類のsiRNAを混ぜて使用した。一方、Z-Val-Ala-Asp(OMe)-CH2F（以下、Z-VAD-FMK）は、KLHL32の発現も用量依存的に抑制した（図6Fおよび6G）。KLHL32陽性細胞の細胞体は拡大し、丸みを帯びた形状を示したが、この形状は、死にかけている神経細胞の形状に似ている。KLHL32の陽性率は約数パーセントであり（図6G）、この陽性率は単一細胞RNAシーケンス解析におけるクラスター12の存在量と一致していた。これらの結果から、単一細胞レベルでの解析より、KLHL32が細胞死の運命の前の初期マーカーとして特定された。 Next, to genetically characterize the cells included in cluster 12, we analyzed the gene set expressed by cluster 12. It was revealed that several types of genes and associated molecular pathways were specific to cluster 12 (Figure 6D) and could serve as markers for labeling cluster 12. Notably, KLHL32 was the most specific gene in cluster 12, with approximately 97% of individual neurons in cluster 12 showing high expression of KLHL32 (Fig. 6E). Furthermore, we performed pathway analysis on the most specific genes (top 50) of cluster 12 and found that the top two pathways associated with cluster 12 were synapse formation and cell death (Figure 6D). To confirm the importance of KLHL32 in activated caspase-3-mediated cell death events, we performed siRNA knockdown experiments and found that inhibition of KLHL32 suppressed caspase-3 activation and cell death fate. (Figures 7C and 7D). Table 1 shows the target sequences of the siRNAs used. A mixture of four types of siRNA was used for each target gene (including non-targeting control). On the other hand, Z-Val-Ala-Asp(OMe)-CH 2 F (hereinafter referred to as Z-VAD-FMK) also suppressed the expression of KLHL32 in a dose-dependent manner (FIGS. 6F and 6G). The cell bodies of KLHL32-positive cells were enlarged and had a rounded shape, similar to that of dying neurons. The positive rate for KLHL32 was approximately a few percent (Figure 6G), which was consistent with the abundance of cluster 12 in single-cell RNA-seq analysis. Based on these results and analysis at the single cell level, KLHL32 was identified as an early marker before cell death fate.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　さらに、深層学習ベースの方法と単一細胞RNAシーケンス解析の結果を組み合わせて、Deep-iモデルがKLHL32陽性集団を観察できるか否かを評価し、DLスコアを予測した。深層学習ベースの方法が観察し、DLスコアを決定する場所を視覚的に説明できる、GradCAM（Gradient-weighted Class Activation Mapping）法を適用した（Selvaraju, R.R. et al., arXiv:1610.02391v1 (2016)）。DenseNet-121アーキテクチャにおけるDense Block 3から神経変性の運命を予測するためのGradCAMマップを生成する（図8A）。原記載（Selvaraju, R.R. et al., arXiv:1610.02391v1 (2016)）に従い、最大強度の15％のしきい値でマップを二値化し、GradCAM陽性領域（「Grad」と命名した）のエリアを定義した（図8Bおよび3C）。また、神経細胞の細胞体（「Cell」と命名した）とKLHL32陽性領域（「Mark」と命名した）にもラベルを付けた。Grad、CellまたはMarkの間の重複領域を定量化することにより（図8Bおよび8C）、Deep-iモデルが、バックグラウンドノイズではなく神経細胞の細胞体を調べたことを明確にすることができた（図7Cおよび7D）。GradCAM陽性領域との重複を調べた結果、細胞体全体との重複（Cell-Grad/Cell）よりもKLHL32陽性細胞（Mark-Grad/Mark）との重複が多く観察された（図6H）。これらの結果から、Deep-iモデルが神経細胞の運命を予測するためにKLHL32陽性細胞に焦点を合わせていることが示された。以上より、Deep-iモデルが分子ネットワークとして観察したものを説明でき、また神経変性がプログラムされた細胞の分子シグネチャーとしてKLHL32を特定することができた。 Furthermore, by combining deep learning-based methods and the results of single-cell RNA sequencing analysis, we evaluated whether the Deep-i model could observe the KLHL32-positive population and predicted the DL score. We applied the GradCAM (Gradient-weighted Class Activation Mapping) method, which can visually explain where deep learning-based methods observe and determine DL scores (Selvaraju, R.R. et al., arXiv:1610.02391v1 (2016) ). Generate GradCAM maps for predicting neurodegenerative fate from Dense Block 3 in the DenseNet-121 architecture (Figure 8A). Following the original description (Selvaraju, R.R. et al., arXiv:1610.02391v1 (2016)), the map was binarized with a threshold of 15% of the maximum intensity and the area of GradCAM positive region (named “Grad”) was defined (Figures 8B and 3C). We also labeled the neuron's cell body (named "Cell") and the KLHL32-positive region (named "Mark"). By quantifying the overlap region between Grad, Cell or Mark (Figures 8B and 8C), we can make it clear that the Deep-i model is examining the neuronal cell body rather than background noise. (Figures 7C and 7D). As a result of examining the overlap with the GradCAM-positive region, more overlap with KLHL32-positive cells (Mark-Grad/Mark) was observed than overlap with the entire cell body (Cell-Grad/Cell) (Figure 6H). These results showed that the Deep-i model focuses on KLHL32-positive cells to predict neuronal cell fate. Thus, we were able to explain what the Deep-i model observed as a molecular network and identify KLHL32 as a molecular signature of cells programmed for neurodegeneration.
実施例３：アルツハイマー病（AD）のマウスモデルまたは死後のAD患者の脳組織における、神経変性がプログラムされた細胞の優先的な蓄積部位の特定
　Deep-iモデルは、神経変性の運命の間、いつ、どこで、何が起こっているかを知るためのマーカー情報を提供する。次に、同定されたマーカーであるKLHL32が、ADマウスモデルの脳または死後のAD患者の脳組織の神経変性の過程で発見されるか否かを調査した。 Example 3: Identification of preferential accumulation sites of neurodegeneratively programmed cells in a mouse model of Alzheimer's disease (AD) or postmortem AD patient brain tissue. Provide marker information to know what is happening, when and where. Next, we investigated whether the identified marker, KLHL32, is found in the process of neurodegeneration in the brain of an AD mouse model or in the brain tissue of postmortem AD patients.
　最初に、5×FADマウスを使用して、神経変性死が発生する前の前段階の脳サンプルを調査した（Eimer W.A. and Vassar R., Mol. Neurodegener. 8, 2 (2013); Oakley H. et al., J. Neurosci. 26, 10129-10140 (2006)）。5×FADマウスは最も広く使用されているADマウスの1種であり、神経変性は生後約4～6か月で観察される（Eimer W.A. and Vassar R., Mol. Neurodegener. 8, 2 (2013))。神経変性の3ヶ月前に、KLHL32陽性細胞の分布が観察された。以前報告されたように、Aβの病理は脳梁膨大後部皮質（RSP）と海馬台（SUB）に局在化していた（図9A）。KLHL-32陽性細胞の定量化された領域は、対照同腹仔（非トランスジェニックマウス）と比較して、5×FADマウスでのみ優勢に観察された（図9Bおよび10）。KLHL32陽性細胞の分布はAβ病理と同様であった（図9Aおよび9B）。一方、3ヶ月齢でAβ病理が認められなかった海馬CA1（CA1）と嗅内野（EN）では、上衣下の星状細胞を除いて、KLHL32陽性細胞は認められなかった（図9Aおよび9B）。これらの結果から、KLHL32は神経変性の発症前にADの病理に関連していることが観察され、細胞死とそれに続く脳萎縮を引き起こす、神経変性がプログラムされた細胞を早期に同定するためのマーカーとなり得ることが明らかになった。 First, we used 5×FAD mice to examine pre-stage brain samples before neurodegenerative death occurs (Eimer W.A. and Vassar R., Mol. Neurodegener. 8, 2 (2013); Oakley H. et al., J. Neurosci. 26, 10129-10140 (2006)). The 5×FAD mouse is one of the most widely used AD mice, and neurodegeneration is observed at approximately 4 to 6 months of age (Eimer W.A. and Vassar R., Mol. Neurodegener. 8, 2 (2013 )). A distribution of KLHL32-positive cells was observed 3 months before neurodegeneration. As previously reported, Aβ pathology was localized to the retrosplenial cortex (RSP) and subiculum (SUB) (Figure 9A). A quantified area of KLHL-32-positive cells was observed predominantly only in 5×FAD mice compared to control littermates (non-transgenic mice) (Figures 9B and 10). The distribution of KLHL32-positive cells was similar to Aβ pathology (Figures 9A and 9B). On the other hand, no KLHL32-positive cells were observed in hippocampal CA1 (CA1) and entorhinal cortex (EN), where Aβ pathology was not observed at 3 months of age, except for subependymal astrocytes (Figures 9A and 9B). . From these results, KLHL32 is observed to be associated with AD pathology before the onset of neurodegeneration, and may be useful for early identification of cells programmed for neurodegeneration, causing cell death and subsequent brain atrophy. It became clear that it could be used as a marker.
　次に、KLHL32がAD患者の脳にも分布しているか否かを調べた。神経病理学的に診断されたADおよび健康な高齢者対照からの死後の脳サンプルを使用した。KLHL32の免疫陽性を調べたところ、KLHL32が側頭皮質の神経細胞と星状細胞で陽性に染色されていることを見出した（図9C）。特にAD患者の脳の場合、健康な高齢者対照（図9E）と比較して、KLHL32は、ADの神経病理学的特徴である老人斑、および神経変性の前段階を示す変性神経突起に分布していた（図9D）。これらの結果から、Deep-iモデルが、AD病理の結果としての神経変性が運命づけられた細胞集団を同定するためのマーカーを明らかにできることが示される。 Next, we investigated whether KLHL32 is also distributed in the brains of AD patients. Postmortem brain samples from neuropathologically diagnosed AD and healthy elderly controls were used. When we examined immunopositivity for KLHL32, we found that KLHL32 was positively stained in neurons and astrocytes in the temporal cortex (Figure 9C). Particularly in the brains of AD patients, compared with healthy elderly controls (Figure 9E), KLHL32 is distributed in senile plaques, a neuropathological hallmark of AD, and in degenerating neurites, indicating a pre-stage of neurodegeneration. (Figure 9D). These results demonstrate that the Deep-i model can reveal markers to identify cell populations destined for neurodegeneration as a result of AD pathology.
　以上の結果より、KLHL32遺伝子の発現抑制薬は、神経変性の予防または治療効果を発揮することが強く示唆される。 The above results strongly suggest that drugs that suppress KLHL32 gene expression exert preventive or therapeutic effects on neurodegeneration.
　KLHL32遺伝子の発現抑制薬は、神経変性疾患の予防または治療に有用である。また、KLHL32遺伝子の発現抑制薬は、神経変性疾患の治療のための併用薬剤としても有用である。 Drugs that suppress KLHL32 gene expression are useful for preventing or treating neurodegenerative diseases. Furthermore, a drug that suppresses the expression of the KLHL32 gene is useful as a combination drug for the treatment of neurodegenerative diseases.
　本出願は、日本で出願された特願２０２２－０５２１１４（出願日：２０２２年３月２８日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2022-052114 (filing date: March 28, 2022) filed in Japan, the contents of which are fully included in this specification.

Claims (12)

  1. 　KLHL32遺伝子の発現抑制薬を含有してなる、神経変性疾患の予防または治療薬。 A drug for preventing or treating neurodegenerative diseases, which contains a drug that suppresses the expression of the KLHL32 gene.
  2. 　前記発現抑制薬が、siRNA、ヘテロ二本鎖核酸、アンチセンス核酸、shRNA、miRNA、アンチジーン核酸およびCRISPR-Casシステムからなる群から選択される、請求項１に記載の予防または治療薬。 The preventive or therapeutic drug according to claim 1, wherein the expression suppressing drug is selected from the group consisting of siRNA, heteroduplex nucleic acid, antisense nucleic acid, shRNA, miRNA, antigene nucleic acid, and CRISPR-Cas system.
  3. 　前記発現抑制薬がsiRNAである、請求項２に記載の予防または治療薬。 The prophylactic or therapeutic agent according to claim 2, wherein the expression inhibitor is siRNA.
  4. 　神経変性疾患が、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症（ＡＬＳ）、脊髄小脳変性症、前頭側頭葉変性症、レビー小体型認知症、多系統萎縮症、ハンチントン病、進行性核上性麻痺および大脳皮質基底核変性症からなる群から選択される、請求項１～３のいずれか１項に記載の予防または治療薬。 Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, and progressive The prophylactic or therapeutic agent according to any one of claims 1 to 3, which is selected from the group consisting of supranuclear palsy and corticobasal degeneration.
  5. 　神経変性疾患がアルツハイマー病である、請求項１～４のいずれか１項に記載の予防または治療薬。 The prophylactic or therapeutic agent according to any one of claims 1 to 4, wherein the neurodegenerative disease is Alzheimer's disease.
  6. 　（１）KLHL32遺伝子を発現する細胞に、被験物質を接触させる工程、および
    　（２）KLHL32遺伝子の発現量を減少させた被験物質を神経変性疾患の予防または治療薬の候補として選択する工程
    を含む、神経変性疾患の予防または治療薬のスクリーニング方法。     (1) A step of bringing a test substance into contact with cells expressing the KLHL32 gene; and (2) A step of selecting a test substance that reduces the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. , a screening method for preventive or therapeutic drugs for neurodegenerative diseases.
  7. 　（１）細胞に、被験物質を接触させる工程、および
    　（２）KLHL32遺伝子の発現量の増加を抑制した被験物質を神経変性疾患の予防または治療薬の候補として選択する工程
    を含む、神経変性疾患の予防または治療薬のスクリーニング方法。     Neurodegenerative diseases, including (1) a step of contacting cells with a test substance, and (2) a step of selecting a test substance that suppresses an increase in the expression level of the KLHL32 gene as a candidate for a preventive or therapeutic drug for neurodegenerative diseases. screening methods for preventive or therapeutic drugs.
  8. 　前記細胞が神経変性疾患のモデル細胞である、請求項６または７に記載の方法。 The method according to claim 6 or 7, wherein the cell is a model cell for a neurodegenerative disease.
  9. 　前記神経変性疾患のモデル細胞が、神経変性疾患の患者由来の細胞または神経変性疾患の原因である遺伝子に変異を有する多能性幹細胞から分化誘導した細胞である、請求項８に記載の方法。 The method according to claim 8, wherein the model cell for a neurodegenerative disease is a cell derived from a patient with a neurodegenerative disease or a cell induced to differentiate from a pluripotent stem cell having a mutation in a gene that causes the neurodegenerative disease.
  10. 　KLHL32タンパク質またはKLHL32転写産物からなる、神経変性疾患診断用バイオマーカー。 Biomarker for diagnosing neurodegenerative diseases consisting of KLHL32 protein or KLHL32 transcript.
  11. 　被験者または該被験者由来の試料において、請求項１０に記載のバイオマーカーを検出する工程を含む、該被験者が神経変性疾患であるかの診断を補助する方法。 A method for assisting in diagnosing whether a subject has a neurodegenerative disease, the method comprising the step of detecting the biomarker according to claim 10 in a subject or a sample derived from the subject.
  12. 　KLHL32タンパク質を特異的に認識する抗体またはKLHL32転写産物を特異的に認識する核酸プローブ若しくは核酸プライマーを含む、神経変性疾患診断用キット。
     

          A kit for diagnosing a neurodegenerative disease, comprising an antibody that specifically recognizes KLHL32 protein or a nucleic acid probe or primer that specifically recognizes KLHL32 transcript.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006138363A2 (en) * 2005-06-17 2006-12-28 Merck & Co., Inc. Method for identifying modulators of keah6 useful for treating alzheimer's disease

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