JP2014128249A - USE OF miRNA OR TARGET GENE THEREOF IN INSPECTION AND THERAPY OF NEURODEGENERATIVE DISEASE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an miRNA as being a biomarker which can be used in inspection diagnosis and therapy for an intractable neurodegenerative disease.SOLUTION: By measuring a gene product of an miRNA specified by the present invention or a target gene thereof, an intractable neurodegenerative disease, particularly amyotrophic lateral sclerosis and multiple system atrophy can be detected. In addition, the gene product of the miRNA of the present invention or a target gene thereof can be used in inspection diagnosis and therapy for the disease. Further, it can be used in screening a compound as being a therapeutic agent for the disease.

Description

  The present invention relates to microRNA (miRNA) or a target gene thereof useful as a diagnostic diagnosis for detecting the presence of a neurodegenerative disease and a therapeutic target for the disease, and use thereof.

  Since neurodegenerative diseases including dementia are generally progressive and involve irreversible degeneration of brain neurons, it is important to diagnose early and start treatment. However, an effective biomarker has not been established at present, a diagnostic method using a serological test method has not been established, and a reliable treatment method has not been established. As a result, it is refractory and the situation in which daily life is destroyed, not only the physical burden of the patient himself, but also a great burden on the family. Neurodegenerative diseases are not only for patients themselves but also for caregivers, and there is a real situation that leads to a great loss of vitality for the whole society. In Japan, where a super-aging society is advancing, countermeasures against neurodegenerative diseases have become an important national issue. That is, (1) It is a fundamental issue of medical welfare administration in an aging society, (2) To reduce huge economic loss due to dementia (equivalent to 5 trillion yen per year), (3) Japan through medical innovation Promote the development of innovative treatments from Japan, eliminate the import deficit in Japan (over 2 trillion yen), and (4) focus diseases (cancer, It is designated as intractable disease / rare disease, hepatitis, infection, diabetes, cerebrocardiovascular disease, neuropsychiatric disease, pediatric disease). Thus, the development of new biomarkers, the development of diagnostic reagents, and the development of therapeutic drugs and treatment methods are urgent issues.

  With regard to laboratory diagnostic methods, if disease-related biomarkers are identified and serological diagnostic methods enable simple early diagnosis and monitoring of pathological conditions, early treatment becomes possible, and many patients are irreversible. Damage can be minimized. If early diagnosis becomes possible, there is no doubt that it will greatly contribute to improving the QOL of not only patients but also caregivers, and thus maintaining the vitality of the entire Japanese society that has become super-aged. Regarding the treatment method, not only the pathologic-improving drug but also the progression inhibitor is sufficiently medically significant, and it is important to analyze the involvement of the biomarker in the nerve function.

  In vivo factors related to the onset and pathogenesis of neurodegenerative diseases have been extensively studied from genes to cells, but there are so many unexplained parts, and no useful biomarkers have yet been found to lead to diagnosis or treatment . Among refractory neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS) and Multiple System Atrophy (MSA) are serious neurological dysfunction and death, and are useful. Development of biomarkers is an urgent issue.

  In recent years, microRNA (abbreviated as miRNA) has attracted attention as an important factor of in vivo factors. miRNA is a protein non-coding RNA of about 22 bases, and it has been suggested that there are about 1000 or more types in humans. miRNA attracts attention as a molecule that suppresses the expression of various genes in vivo. A region of each miRNA gene exists on the genome and is transcribed by RNA polymerase II to form a miRNA initial transcription product of about several hundred bases. The miRNA initial transcript is processed by two RNase III enzymes called Drosha in the nucleus and Dicer in the cytoplasm to form mature miRNA. It is known that a mature miRNA interacts with mRNAs of a plurality of target genes having complementary sequences while cooperating with a regulatory protein complex (RNA-induced silencing complex: RISC) to suppress gene expression.

  Although miRNA has been suggested to be widely related to human diseases, the expression of many miRNAs differs between normal and cancerous tissues in various organs, particularly regarding the relationship between cancer and miRNA. The involvement of abnormal expression is strongly suggested. That is, it has been strongly suggested that a decrease in tumor suppressor miRNA expression or an increase in carcinogenic miRNA expression may be involved in the human carcinogenesis process (Non-patent Document 1). Regarding the industrial use of miRNA, it is mainly used in the field of cancer, but finally its use for drug discovery for various diseases has begun to be reported (Non-patent Document 2). In relation to neurological diseases, a method using miRNA for regeneration of nerves after injury (Patent Document 1) and a method for stimulating neuronal differentiation of pluripotent stromal cells (Patent Document 2) have been reported.

  However, although it is highly likely that miRNA is widely involved in various diseases, there is little information on the application technology of miRNA to ALS and MSA, and miRNA-206 is involved in the suppression of ALS progression. (Non-document information 3), reports of changes in miRNAs in leukocytes of ALS patients (non-document information 4), and documents suggesting involvement in spinal muscular atrophy (SMA) (non-document information 3) There are only a few reports such as literature information5). In ALS and MSA, which are considered to be associated with progressive and intrinsic factors, development of disease biomarkers useful for laboratory diagnosis or drug discovery using miRNA is eagerly desired.

Special table 2011-515407 Special table 2012-530054 gazette

MicroRNA regulation and cancer, experimental medicine vol.27, No.8, 2009. MicroRNA, gene medicine MOOK 23 2012 Williams AH et al., Science. 2009; 326 (5959): 1549-54. De Felice B et al., Gene. 2012; 508 (1): 35-40. Haramati S et al., Proc Natl Acad Sci USA. 2010 Jul 20; 107 (29): 13111-6.

As described above, testing methods and treatment methods for refractory ALS and MSA have not been established, which is a major medical problem. If miRNA that can be used as a biomarker for neurodegenerative diseases of miRNA that is said to be involved in diseases is found and its usage is established, it will be possible to apply it to new diagnostic methods, pathological monitoring methods, and therapeutic drugs. .
The object of the present invention is the use of miRNA for the examination and treatment of neurodegenerative diseases, and in particular, miRNA that is a biomarker of ALS and MSA, or the gene product of its target gene, for clinical examination, pathological monitoring, and further treatment It is to apply.

The present invention relates to the following inventions:
[1] A step of measuring a miRNA in a sample collected from a subject or a gene product of a target gene thereof, wherein the miRNA is at least one of the miRNAs shown in Tables 1 to 4, and the target gene is represented by A method for detecting a neurodegenerative disease, which is at least one of the target genes shown in Tables 5 to 8.
[2] Detection of a neurodegenerative disease that is a miRNA selected from the group consisting of miRNAs described in Tables 1 to 4 or a gene product of a target gene selected from the group consisting of target genes described in Tables 5 to 8 Biomarker to do.
[3] A step of measuring a miRNA in a sample collected from a subject or a gene product of the target gene thereof, wherein the miRNA is at least one of the miRNAs described in Tables 1 to 2, and the target gene is represented by A method for detecting multiple system atrophy, which is at least one of the target genes described in Tables 5 to 6.
[4] including a step of measuring miRNA in a sample collected from a subject or a gene product of the target gene, wherein the miRNA is at least one of the miRNAs described in Tables 3 to 4, and the target gene is represented by A method for detecting amyotrophic lateral sclerosis, which is at least one of the target genes shown in Tables 7 to 8.
[5] The method includes collecting a sample from a subject treated for neurodegenerative disease, and measuring a miRNA in the sample or a gene product of a target gene thereof, wherein the miRNA is described in Tables 1 to 4 The method for determining the therapeutic effect, wherein the target gene is at least one of the target genes listed in Tables 5 to 8.
[6] A pharmaceutical composition comprising at least one full length or a part of the miRNA described in Tables 1 to 4 as an active ingredient.
[7] A pharmaceutical composition comprising an oligonucleotide that suppresses at least one of the miRNAs described in Tables 1 to 4 as an active ingredient.
[8] The pharmaceutical composition according to [6] or [7], which is used for treatment of multisystem atrophy or amyotrophic lateral sclerosis.
[9] A method for screening a therapeutic agent for a neurodegenerative disease, using as an index the expression of at least one of the miRNAs listed in Tables 1 to 4.
[10] A method for screening for a therapeutic agent for a neurodegenerative disease using the gene product of at least one target gene of miRNA described in Tables 1 to 4 as an index.

  According to the detection method of the present invention, a neurodegenerative disease, particularly multiple system atrophy (MSA) or amyotrophic lateral sclerosis (ALS) can be detected by a simple genetic test method or protein analysis method. . In addition, the miRNA in the present invention or the gene product of its target gene can be used for examination diagnosis and treatment of the same disease. Furthermore, it can utilize for the screening of the compound used as the therapeutic agent of the disease.

Except as specifically described below, the detection method, therapeutic effect determination method, therapeutic method, pharmaceutical composition, and therapeutic drug screening method of the present invention can be referred to each other as appropriate.
In the detection method of the present invention, as a biomarker for detecting a neurodegenerative disease, particularly multisystem atrophy (MSA) or amyotrophic lateral sclerosis (ALS), miRNAs described in Tables 1 to 4, or The gene products (polypeptides or messenger RNA (hereinafter referred to as mRNA), preferably polypeptides) described in Tables 5 to 8 can be used.
The gene products of the miRNA or its target gene can be used singly or in plural. In order to increase accuracy, it is preferable to use a plurality of them in combination. Specifically, it is desirable to use a gene product of miRNA and / or its target gene in combination of 1 to 20, preferably 2 to 10.
In addition, known miRNA and / or gene products of target genes thereof can be used in combination.

As shown in the specific experimental data in the examples described below, MSA patients have increased miRNA in Table 1 at the site of brain disease and decreased miRNA in Table 2 compared to healthy subjects, whereas patients with ALS Then, the miRNAs in Table 3 increased compared to healthy subjects at the brain disease site, and the miRNAs in Table 4 decreased. Therefore, these miRNAs (hereinafter referred to as MSA, respectively) in samples of organ tissues, cells, blood, cerebrospinal fluid, saliva, tears, etc. collected from subjects, particularly patients with suspected neurodegenerative diseases or patients with neurodegenerative diseases. It is possible to determine whether it is MSA or ALS by measuring at least one of them (referred to as ALS detection miRNA) according to a method known per se. In particular, it is preferable to use a brain disease site (such as a cerebellar lesion site for MSA patients or a brain motor field lesion site for ALS patients) or a peripheral site (cerebrospinal fluid or the like) as a sample.
In addition, all or part of the reduced miRNA is administered as a pharmaceutical composition, and miRNA is supplemented, or an oligonucleotide nucleic acid drug that acts to reduce the increased miRNA is administered, and the action of excess miRNA is reduced. Inhibiting both provides agents useful for the treatment of MSA or ALS.

  In the case of miRNA having an increased abundance, a more preferable target gene tends to have a high signal ratio. Therefore, miRNA having a change rate (Log 2 notation) of +0.1 or more, preferably +0.5 or more can be used as a neurodegenerative disease biomarker. On the other hand, in the case of miRNA having a reduced abundance, the signal ratio tends to be low in a more preferred target gene. Therefore, miRNA having a change rate (Log 2 notation) of less than −0.5, preferably less than −1.0 can be used as a biomarker for neurodegenerative diseases. However, since the rate of change may vary depending on the measurement conditions, miRNAs that serve as biomarkers can be selected in descending order of the range of variation regardless of the reference value of the rate of change. The sequences of miRNAs in Tables 1 to 4 can be obtained from a known database (miRBase: http://www.mirbase.org/).

  Factors presumed that gene expression is suppressed in miRNA target gene candidates increased by MSA are estimated in Table 5, and gene expression is estimated to be enhanced in miRNA target gene candidates decreased by MSA. These factors are shown in Table 6 (hereinafter, these may be referred to as MSA detection gene products). The gene products (polypeptides or mRNAs, preferably polypeptides) encoded by these target genes can not only be used as biomarkers for MSA, but can also be used as targets for MSA therapeutics or onset prevention drugs. Can also be used.

  Factors presumed that gene expression is suppressed in miRNA target gene candidates increased by ALS are shown in Table 7, and miRNA target gene candidates reduced by ALS are presumed to have enhanced gene expression. The factors are shown in Table 8 (hereinafter, these may be referred to as ALS detection gene products). The gene products (polypeptides or mRNAs, preferably polypeptides) encoded by these target genes can not only be used as biomarkers for detecting ALS, but can also be used as targets for ALS therapeutics or onset prevention drugs. Can also be used.

When miRNA or mRNA of its target gene is used as a biomarker in the detection method of the present invention, measurement is performed by a known genetic test method such as a hybridization method using a nucleic acid probe or a PCR method using a PCR primer. be able to.
When the polypeptide encoded by the target gene of miRNA is used as a biomarker in the detection method of the present invention, a protein analysis method known per se, for example, an immunological analysis method using an antibody, biochemistry such as electrophoresis, etc. An automatic analyzer for clinical examination can also be used.

  Samples used in the method of the present invention include, for example, brain disease sites, cerebrospinal fluid, blood samples (eg, peripheral blood, plasma, serum), saliva, tears, urine, lymph, and other body fluids, preferably brain diseases A part or its peripheral part (cerebrospinal fluid etc.) can be used. Furthermore, organ tissues, pathological tissues, cells collected by biopsy, or components extracted therefrom can also be used.

The miRNAs described in Tables 1 to 4 used as biomarkers in the detection method of the present invention, or the gene products (polypeptides or mRNAs, preferably polypeptides) of the target genes described in Tables 5 to 8 are neurodegenerative. It can also be used to determine the effects of various treatments performed on patients with a disease, particularly MSA or ALS.
The therapeutic effect determination method of the present invention includes a step of collecting a sample from a subject who has been treated for a neurodegenerative disease, and a step of measuring a gene product of miRNA or a target gene thereof in the sample.

In the therapeutic effect determination method of the present invention, when determining the therapeutic effect on MSA patients, the MSA detection miRNA described in Table 1 or Table 2, or the gene product of the target gene described in Table 5 or Table 6 ( Polypeptide or mRNA) can be used.
When miRNA for detection of MSA shown in Table 1 (miRNA whose abundance is increased in MSA patients) is used as a biomarker, the miRNA value is higher in healthy MSA patients than in healthy subjects before the treatment. There is a tendency to show. When the miRNA value decreases by treatment, it can be determined that the treatment was effective. On the other hand, if the treatment did not decrease, it can be determined that the treatment was ineffective.
In addition, when the miRNA for detection of MSA described in Table 2 (miRNA whose abundance is reduced in MSA patients) is used as a biomarker, the miRNA value in the MSA patient before the treatment is lower than that in healthy subjects There is a tendency to show numerical values. When the miRNA value increases as a result of treatment, it can be determined that the treatment was effective. On the other hand, when the treatment does not increase, it can be determined that the treatment has no effect.
However, since it is estimated that miRNAs individually control the expression of target genes in multiple types, the individual miRNA values do not have to fluctuate in a coordinated manner, and have a therapeutic effect in consideration of the state of clinical symptoms. Determined.

When using as a biomarker the gene product for detection of MSA described in Table 5 (the gene product of the target gene of miRNA presumed that gene expression is suppressed in MSA patients), in the MSA patient before the treatment, It is estimated that the expression level is lower than that of healthy subjects. When the expression level increases by performing treatment, it can be determined that the treatment was effective. On the other hand, when the treatment does not increase, it can be determined that the treatment has no effect.
When using the gene product for detection of MSA described in Table 6 (the gene product of the target gene of miRNA estimated to have enhanced gene expression in the MSA patient) as a biomarker, in the MSA patient before the treatment, The expression level is estimated to be higher than that of healthy subjects. When the expression level is decreased by treatment, it can be determined that the treatment was effective. On the other hand, if the treatment did not decrease, it can be determined that the treatment was ineffective.

In the therapeutic effect determination method of the present invention, when determining the therapeutic effect on ALS patients, the ALS detection miRNA described in Table 3 or Table 4, or the gene product of the target gene described in Table 7 or Table 8 ( Polypeptide or mRNA) can be used.
When miRNA for ALS detection shown in Table 3 (miRNA whose abundance is increased in ALS patients) is used as a biomarker, the ALS patient before performing the treatment has a higher miRNA value than that of healthy subjects. There is a tendency to show. When the miRNA value decreases by treatment, it can be determined that the treatment was effective. On the other hand, if the treatment did not decrease, it can be determined that the treatment was ineffective.
In addition, when the miRNA for detection of ALS shown in Table 4 (miRNA whose abundance is reduced in ALS patients) is used as a biomarker, the miRNA value in ALS patients before the treatment is lower than that in healthy subjects There is a tendency to show numerical values. When the miRNA value increases as a result of treatment, it can be determined that the treatment was effective. On the other hand, when the treatment does not increase, it can be determined that the treatment has no effect.
However, since it is estimated that miRNAs individually control the expression of target genes in multiple types, the individual miRNA values do not have to fluctuate in a coordinated manner, and have a therapeutic effect in consideration of the state of clinical symptoms. Determined.

When using the gene product for detection of ALS shown in Table 7 (the gene product of the target gene of miRNA that gene expression is presumed to be suppressed in ALS patients) as a biomarker, in ALS patients before the treatment, It is estimated that the expression level is lower than that of healthy subjects. When the expression level increases by performing treatment, it can be determined that the treatment was effective. On the other hand, when the treatment does not increase, it can be determined that the treatment has no effect.
When the gene product for detection of ALS shown in Table 8 (the gene product of the target gene of miRNA estimated to have enhanced gene expression in an ALS patient) is used as a biomarker, in the ALS patient before the treatment, The expression level is estimated to be higher than that of healthy subjects. When the expression level is decreased by treatment, it can be determined that the treatment was effective. On the other hand, if the treatment did not decrease, it can be determined that the treatment was ineffective.

  In the treatment method of the present invention, the presence or absence of a neurodegenerative disease, in particular, the full-length or part of the miRNA described in Table 2 or Table 4 in which the abundance is decreased in MSA or ALS, or the abundance in the disease. Neurodegenerative diseases can be treated using the increasing inhibitory oligonucleotides to miRNAs listed in Table 1 or Table 3. In addition, in a neurodegenerative disease patient in which gene expression of a miRNA target gene is suppressed, by replenishing the gene product, conversely, in a neurodegenerative disease patient in which gene expression is enhanced, the expression of the gene product is Alternatively, neurodegenerative diseases can be treated by suppressing the activity. In this specification, the term “treatment” includes “treatment” in a narrow sense for treating a patient after the onset of a disease and “prevention” for treating a patient before the onset of the disease.

  Patients with neurodegenerative diseases in which at least one of the miRNAs listed in Table 2 or Table 4 is reduced (especially MSA patients in which at least one of the miRNAs listed in Table 2 is reduced, or miRNAs listed in Table 4) In ALS patients, at least one of which is reduced, neurodegenerative diseases can be treated by supplementing the patient with that reduced miRNA. As a method of administering miRNA to a patient, it can be performed according to a method known per se, a method of encapsulating miRNA or a derivative thereof in a liposome imitating exosome, a method of slow release as a complex with collagen, Utilization of chemical modification by 4′-thioRNA conversion in which O (oxygen) in the sugar part of RNA is replaced with S (sulfur), 2′-F, 2′-O-methyl, 2′-F It can be stably replenished in vivo by using a chemically modified product modified with '-O-methoxyethyl. At the same time, since miRNA has a high possibility of controlling a target gene with a plurality of molecules, replenishment with a plurality of nucleic acid cocktails can be preferably used for the above-described nucleic acid supplementation method.

  On the other hand, patients with neurodegenerative diseases in which at least one of the miRNAs described in Table 1 or Table 3 is increased (in particular, MSA patients in which at least one of the miRNAs described in Table 1 is increased, or those described in Table 3 In ALS patients in which at least one of the miRNAs is increased, neurodegenerative diseases can be treated by suppressing the increased miRNA in the patient. As a method for suppressing miRNA in a living body, it can be carried out according to a method known per se, for example, an oligonucleotide (antisense oligonucleotide) comprising a complementary sequence capable of hybridizing with the full length or a part of the target miRNA. Can be administered. In this case, the antisense oligonucleotide is a method of encapsulating in a liposome imitating an exosome, a method of slow release as a complex with collagen, and 4 ′ in which O (oxygen) in the sugar portion of RNA is replaced with S (sulfur). -Use of chemically modified products by thioRNA conversion, use of chemically modified products in which the sugar portion of the oligonucleotide is modified with 2'-F, 2'-O-methyl, 2'-O-methoxyethyl, etc. Can be replenished stably. At the same time, since miRNA has a high possibility of controlling a target gene with a plurality of molecules, replenishment with a plurality of nucleic acid cocktails can be preferably used for the above-described nucleic acid supplementation method.

The pharmaceutical composition of the present invention can be used for the treatment of neurodegenerative diseases, particularly MSA or ALS. As an active ingredient, at least one miRNA described in Table 2 or Table 4 (full length or a part thereof), or Inhibitory oligonucleotides to at least one of the miRNAs listed in Table 1 or Table 3 can be included, and optionally a pharmaceutically acceptable carrier.
A pharmaceutical composition of the present invention comprising at least one miRNA listed in Table 2 (full length or a part thereof), or a derivative thereof, or an inhibitory oligonucleotide against at least one of the miRNA listed in Table 1 is MSA. Can be used to treat patients.
A pharmaceutical composition of the present invention comprising at least one of the miRNAs described in Table 4 (full length or a part thereof), or a derivative thereof, or an inhibitory oligonucleotide against at least one of the miRNAs described in Table 3. Can be used to treat patients.

The miRNA described in Tables 1 to 4 used as a biomarker in the detection method of the present invention, or the gene product (polypeptide or mRNA, preferably polypeptide) encoded by the target gene is used for neurodegenerative diseases, particularly It can be used to screen for MSA or ALS therapeutics.
In the present invention, a method for screening a candidate substance (for example, a compound) that targets a gene product of miRNA or a target gene thereof is a method or culture in which a candidate substance that directly binds to a target is examined by spectroscopic changes in vitro. A method of examining a target substance (miRNA or gene product of the target gene) in a cultured cell by exposing a candidate substance to cells is applied, and in vivo, a neurodegenerative symptom or a neurodegenerative pathological image in a model animal The method using the improvement of the index as an index is applied.

  For example, in the screening method of the present invention, in the embodiment using the polypeptides encoded by the target genes listed in Tables 5 to 8, it can be carried out using the various in vitro assays or the in vivo assays described above. . In this aspect, for example, the step of bringing the polypeptide or a cell, tissue, or animal individual (particularly a non-human animal) expressing the polypeptide into contact with the candidate substance, and the binding of the candidate substance to the polypeptide, It can be carried out by a method comprising a step of analyzing a change in the expression level or activity of a polypeptide, or a symptom change in an animal individual.

  In the screening method of the present invention, in the embodiment using miRNAs described in Table 1 to Table 4 or mRNA expression of the target gene described in Tables 5 to 8 as an index, for example, the candidate substance is a cultured cell, It can be carried out by a method comprising the steps of administering to a tissue or an animal individual (particularly a non-human animal), collecting a sample from the individual, and measuring miRNA or mRNA in the sample.

  Candidate substances to be evaluated by the screening method of the present invention are not particularly limited, but in addition to various compounds, various extracts such as culture supernatants of microorganisms, natural organisms or extracts derived from various organisms or tissues thereof Can be mentioned. Furthermore, the cells used are cultured cells isolated from tissues, established cell lines, ES cells, iPS cells, Muse cells, etc. In non-human animals, specific genes were knocked out or knocked in. Model mice and model rats can be mentioned.

  In the screening method of the present invention, when measuring at least one of the miRNAs described in Table 2 or Table 4, a substance capable of increasing the miRNA amount can be selected as a candidate for a therapeutic agent for neurodegenerative diseases. . In particular, substances that can increase the amount of miRNA listed in Table 2 are selected as candidates for ALS therapeutic agents, and substances that can increase the amount of miRNA listed in Table 4 are selected as candidates for MSA therapeutic agents. be able to.

  On the other hand, when measuring at least one of the miRNAs listed in Table 1 or Table 3, a substance capable of reducing the amount of the miRNA can be selected as a candidate for a therapeutic agent for neurodegenerative diseases. In particular, substances that can reduce the amount of miRNA listed in Table 1 are selected as candidates for ALS therapeutic agents, and substances that can reduce the amount of miRNA listed in Table 3 are selected as candidates for MSA therapeutic agents. be able to.

  In addition, when measuring at least one of the gene products of the target genes listed in Table 6 or Table 8, a substance capable of reducing the expression level or activity of the gene product is selected as a candidate for a therapeutic drug for neurodegenerative diseases. can do. In particular, substances capable of reducing the expression level or activity of the target gene gene product described in Table 6 are candidates for ALS therapeutic agents, and the target gene gene product expression level or activity described in Table 8 Substances that can be reduced can be selected as candidates for MSA therapeutics.

  On the other hand, when measuring at least one of the gene products of the target genes listed in Table 5 or Table 7, a substance capable of increasing the expression level or activity of the gene product is selected as a candidate for a therapeutic agent for neurodegenerative diseases. can do. In particular, substances capable of increasing the expression level or activity of the target gene gene product described in Table 6 are candidates for ALS therapeutic agents, and the target gene gene product expression level or activity described in Table 8 is increased. Substances that can be increased can be selected as candidates for MSA therapeutics.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Analysis of miRNA in brain lesion tissue
Cerebellar lesion site in a patient with multisystem atrophy (MSA) diagnosed definitely, Brain motor field lesion site in a patient with definitive amyotrophic lateral sclerosis (ALS), and normal brain as a healthy control group The site was collected after autopsy, fixed with 10% formalin, embedded in paraffin, and used as a FFPE (formalin-fixed paraffin embedded) specimen. Using FFPE specimens (5x5mm, 5μm, 2-4 pieces) as materials, miRNA analysis by microarray after RNA extraction from 5 MSA patients, 3 MSA healthy controls, 3 ALS patients, and 3 ALS healthy controls. Went.

The microarray uses a 3D-gene chip (manufactured by Toray Industries, Inc.), a miRNA extract designated by the manufacturer, a hybridization reagent set, and a human miRNA Oligo chip (about 1,800 human miRNA probes selected from the database miRBase Release 17.0). The change in the signal intensity of the patient group relative to the target group was determined.
The rate of change in miRNA increase / decrease was determined, and the top 20 miRNAs increased relative to healthy controls were defined as “increased miRNA (upregulated miRNA)” and the decreased top 30 miRNAs were defined as “downregulated miRNA”. did.

As a result, the results shown in Tables 1 to 4 were obtained. Table 1 shows miRNA groups increased in cerebellar FFPE specimens of MSA patients, and Table 2 shows miRNA groups reduced in cerebellar FFPE specimens of MSA patients. Table 3 shows miRNA groups increased in the brain motor cortex FFPE of ALS patients, and Table 4 shows miRNA groups decreased in the brain motor cortex FFPE of ALS patients.
These miRNA groups can be used as test diagnostic biomarkers for each disease or miRNA information for treatment.

<< Example 2: Method for specifying target gene of miRNA >>
In order to identify the target gene group that the miRNA group detected in Example 1 controls in vivo, a two-stage analysis method was used.
First, in order to estimate target genes from the detected miRNA list (Tables 1 to 4 above), an analysis tool based on miRanda-mirSVR microRNA.org-Targets and Expression (http://www.microrna.org) /microrna/home.do), list target gene candidates by matching with miRNA sequence information (about 50 types). Among these, 10 to 30 types of factors that seem to be related to the nervous system are listed. Extracted.
Factors presumed to suppress gene expression in miRNA target gene candidates increased by MSA are shown in Table 5, and gene expression is estimated to be enhanced in miRNA target gene candidates decreased by MSA. Factors are listed in Table 6, miRNA target gene candidates increased by ALS and gene expression is estimated to be suppressed in Table 7, and gene expression is enhanced in miRNA target gene candidates decreased by ALS. Table 8 shows the factors estimated to be present.

  The miRNA described in the present specification uses a commercially available 3D-gene chip (manufactured by Toray Industries, Inc.) equipped with about 1,800 human miRNA probes selected from the database miRBase Release 17.0 as described above. Therefore, the name (ID) based on the database miRBase Release 17.0 is used. Tables 9 to 12 show the names of miRNAs described in the present specification and the Accession No. in the database miRBase (http://www.mirbase.org/).

  The present invention can be used for examination diagnosis and treatment of neurodegenerative diseases. Furthermore, it can utilize for the screening of the compound used as the therapeutic agent of the disease.

Claims (10)

  A step of measuring a miRNA in a sample collected from a subject or a gene product of the target gene thereof, wherein the miRNA is at least one of the miRNAs shown in Tables 1 to 4, and the target gene is shown in Tables 5 to 5 A method for detecting a neurodegenerative disease, which is at least one of the target genes according to claim 8.   For detecting a neurodegenerative disease, which is a miRNA selected from the group consisting of miRNAs described in Tables 1 to 4 or a gene product of a target gene selected from the group consisting of target genes described in Tables 5 to 8 Biomarker.   A step of measuring a miRNA in a sample collected from a subject or a gene product of the target gene thereof, wherein the miRNA is at least one of the miRNAs shown in Tables 1 and 2, and the target gene is shown in Tables 5 to 5 A method for detecting multiple system atrophy, which is at least one of the target genes according to 6.   A step of measuring a miRNA in a sample collected from a subject or a gene product of the target gene thereof, wherein the miRNA is at least one of the miRNAs described in Tables 3 to 4, and the target gene is Tables 7 to A method for detecting amyotrophic lateral sclerosis, which is at least one of the target genes according to claim 8.   A step of collecting a sample from a subject treated for neurodegenerative disease, and a step of measuring a miRNA in the sample or a gene product of a target gene thereof, wherein the miRNA is one of the miRNAs described in Tables 1 to 4 The method for determining a therapeutic effect, wherein the method is at least one and the target gene is at least one of the target genes shown in Tables 5 to 8.   A pharmaceutical composition comprising, as an active ingredient, at least one full length or a part of the miRNA described in Tables 1 to 4.   A pharmaceutical composition comprising as an active ingredient an oligonucleotide that suppresses at least one of the miRNAs listed in Tables 1 to 4.   The pharmaceutical composition according to claim 6 or 7, which is used for treatment of multiple system atrophy or amyotrophic lateral sclerosis.   A method for screening a therapeutic agent for a neurodegenerative disease, using as an index the expression of at least one of the miRNAs listed in Tables 1 to 4.   A method for screening a therapeutic agent for a neurodegenerative disease, using as an index the gene product of at least one target gene of miRNA described in Tables 1 to 4.