WO2022085687A1 - Prophylactic or therapeutic agent for respiratory disease, examination method, examination reagent, method for screening active ingredient of prophylactic or therapeutic agent, and method for evaluating induction or exacerbation - Google Patents

Prophylactic or therapeutic agent for respiratory disease, examination method, examination reagent, method for screening active ingredient of prophylactic or therapeutic agent, and method for evaluating induction or exacerbation Download PDF

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WO2022085687A1
WO2022085687A1 PCT/JP2021/038632 JP2021038632W WO2022085687A1 WO 2022085687 A1 WO2022085687 A1 WO 2022085687A1 JP 2021038632 W JP2021038632 W JP 2021038632W WO 2022085687 A1 WO2022085687 A1 WO 2022085687A1
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fchsd1
sequence
protein
test
respiratory
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静男 審良
荘 佐藤
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国立大学法人大阪大学
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Definitions

  • the present invention relates to a preventive or therapeutic agent for respiratory diseases, a test method, a test agent, a screening method for an active ingredient of a preventive or therapeutic agent, a method for evaluating inducing or exacerbation, and the like.
  • Respiratory diseases can be caused by various factors such as viruses, bacteria, in-vivo immune system, smoking and the like.
  • respiratory diseases many inflammatory lung diseases such as acute bronchitis, bronchitis, interstitial pneumonia, and chronic obstructive pulmonary disease (COPD: Chronic Obstructive Pulmonary Disease) are known.
  • COPD chronic Obstructive Pulmonary Disease
  • COPD chronic obstructive pulmonary disease
  • Emphysema a major component of COPD, is caused by smoking and other environmental risk factors.
  • Oxidative stress induced by cigarette smoke is said to play an important role in the early stages of emphysema onset. Oxidative stress has been reported to enhance inflammation and extracellular matrix proteolysis, followed by alveolar cell and septal destruction apoptosis.
  • Oxidative stress is also involved in the development of elastase-induced emphysema.
  • impaired alveolar maintenance mechanisms trigger apoptosis and autophagy.
  • Lost control of autophagy increases cellular stress such as ER stress and induces apoptosis.
  • the chain of injury including apoptosis, oxidative stress, and inflammation, promotes disease progression while enhancing each other.
  • cell aging is accelerated in the lungs of COPD, resulting in decreased cell proliferation, increased inflammation and decreased cell regeneration.
  • Non-Patent Document 1 The current treatment method for COPD is symptomatic treatment aimed at suppressing the progression (Non-Patent Document 1), and the development of a more effective treatment technique is desired. In addition, if there is an effective diagnostic technique, earlier treatment can be performed.
  • the present inventor can prevent or treat / improve respiratory diseases by suppressing the expression or function of Fchsd1 (FCH and double SH3 domains protein 1).
  • Fchsd1 can be used as a biomarker for diagnosing respiratory diseases.
  • the present inventor has completed the present invention as a result of further research based on these findings. That is, the present invention includes the following aspects.
  • a preventive or therapeutic agent for respiratory diseases containing at least one component selected from the group consisting of an Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitor and an Fchsd1 function inhibitor.
  • Item 2 Prevention or treatment according to Item 1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody. Agent.
  • Item 3. The prophylactic or therapeutic agent according to Item 1 or 2, wherein the respiratory disease is a respiratory disease that can develop or worsen due to oxidative stress.
  • the respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure.
  • the prophylactic or therapeutic agent according to any one of Items 1 to 3, which is at least one of the above.
  • Item 4A The prophylactic or therapeutic agent according to any one of Items 1 to 4, wherein the respiratory disease is chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • Item 5 A method for examining a respiratory disease, which comprises a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject.
  • Item 5A A method for determining the risk of contracting a respiratory disease, which comprises a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject.
  • the item comprising (2) a step of determining that the subject has a respiratory disease when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value.
  • the inspection method according to 5. Item 6A.
  • the inspection method according to Item 5A The inspection method according to Item 5A.
  • Item 7. The method according to Item 5, 5A, 6 or 6A, wherein the Fchsd1 is a protein.
  • Item 8. The method according to any one of Items 5, 5A, 6, 6A, and 7, wherein the test sample is a test sample that may contain lung tissue of the subject.
  • Item 9 The examination method according to Item 8, wherein the examination sample that may contain the lung tissue includes at least one selected from the group consisting of a lung biopsy sample and a lung lavage fluid.
  • Item 10 A test drug for respiratory diseases, including a detection agent for Fchsd1 (FCH and double SH3 domains protein 1).
  • Item 11 A method for screening an active ingredient of a preventive or therapeutic agent for respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
  • Item 11A A method for screening an active ingredient of a preventive or therapeutic agent for respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from cells treated with a test substance as an index.
  • Item 12 A method for evaluating the induction or exacerbation of respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
  • Item 12A A method for evaluating the induction or exacerbation of respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in cells treated with a test substance as an index.
  • Item A1 Patients in need of prevention or treatment of respiratory diseases include at least one ingredient selected from the group consisting of Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitor and Fchsd1 function inhibitor. How to prevent or treat respiratory illness.
  • Item A2. Item 6. The method according to Item A1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody.
  • Item 6. The method according to Item A1 or A2, wherein the respiratory disease is a respiratory disease that can develop or be exacerbated by oxidative stress.
  • the respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure.
  • Item 6. The method according to any one of Items A1 to A4, wherein the respiratory disease is chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • Item B1 Use of at least one ingredient selected from the group consisting of Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitors and Fchsd1 function inhibitors for the manufacture of prophylactic or therapeutic agents for respiratory diseases.
  • Item B2. Item 6. The use according to Item B1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody.
  • Item B3. The use according to Item B1 or B2, wherein the respiratory disease is a respiratory disease that can develop or be exacerbated by oxidative stress.
  • the respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure.
  • infectious respiratory disease airway obstruction disease
  • allergic lung disease interstitial lung disease
  • neoplastic lung disease pulmonary vascular lesion
  • pleural disease pulmonary failure.
  • COPD chronic obstructive pulmonary disease
  • the results of immunoblotting determination of the levels of FCHSD1 and other FCH family proteins in mouse lung 7 days after intratracheal administration of elastase are shown.
  • the results of immunoblotting determination of the levels of FCHSD1 and other FCH family proteins in MLE-12 cells exposed to 200 ⁇ M H 2 O 2 for 12 hours are shown.
  • the results of determining the level of FCHSD1 1 day and 3 days after intratracheal administration of LPS by immunoblotting are shown.
  • the results of determining the level of FCHSD1 in various tissue homogenates of unstimulated mice by immunoblotting are shown. MRI images of the lungs after intratracheal administration of elastase are shown.
  • the average alveolar diameter (M.L.I.) after intratracheal administration of elastase is shown. Data are expressed as mean ⁇ SEM. ** indicates P ⁇ 0.01. The percentage of emphysema area after intratracheal administration of elastase is shown. Data are expressed as mean ⁇ SEM. ** indicates P ⁇ 0.01. The results of flow cytometric analysis of cell influx of Fchsd1 -/- and WT mice into the BAL fluid after intratracheal administration of elastase are shown. The data are expressed as mean ⁇ SEM. * Indicates P ⁇ 0.05.
  • the results of flow cytometric analysis of the number of neutrophils in the BAL solution of Fchsd1 -/- and WT mice 24 hours after intratracheal administration of elastase are shown.
  • the data are expressed as mean ⁇ SEM. ** indicates P ⁇ 0.01.
  • the results of measuring the TNF ⁇ concentration in the BAL solution of Fchsd1 -/- and WT mice 24 hours after the intratracheal administration of elastase by the ELISA method are shown.
  • the data is expressed as mean ⁇ SEM. * Indicates P ⁇ 0.05.
  • the results of TUNEL staining of lung sections of Fchsd1 -/- and WT mice to which PBS or elastase were intratracheally administered are shown.
  • Lung TUNEL-positive cells are shown in brown. Scale bar: 200 ⁇ m.
  • the results of immunoblot analysis after treating lung fibroblasts (MLF) isolated from Fchsd1 -/- and WT mouse lungs with 250 ⁇ M H 2 O 2 for 12 hours are shown.
  • the results of immunoblot analysis of Fchsd1 -/- and WT MLF after treatment with 50 ⁇ M H 2 O 2 for 24 hours are shown.
  • Il 6 and Tnfa mRNA levels in response to oxidative stress caused by 250 ⁇ M H 2 O 2 in MLE-12 cells transfected with the retrovirus Fchsd1 or an empty control vector are shown. The data are expressed as mean ⁇ SEM.
  • results of immunoblot analysis of the nuclei and cytoplasmic fractions of Fchsd1 -/- and WT MLF treated with untreated or 275 ⁇ M H 2 O 2 for 10 minutes are shown.
  • Results of transfection of MLE12 cells with Flag-tagged FCHSD1, Myc-tagged SNX9, HA-tagged NRF2, or empty vector and immunoprecipitation (IP) with anti-Flag resin prior to Western analysis of bound NRF2 or SNX9. Is shown.
  • the lower panel shows the results of immunoblots of whole cell lysate (WCL).
  • MLE-12 cells expressing Flag-tagged FCHSD1 or empty vector in response to oxidative stress from 250 ⁇ M H 2 O 2 treatment (5, 15, or 60 minutes) were immunoprecipitated using immunoblot analysis and anti-Flag resin. The result is shown.
  • the results of immunoprecipitation of MLE-12 cells expressing Flag-tagged SNX9 in response to oxidative stress by 250 ⁇ M H 2 O 2 treatment (10 or 30 minutes) using immunoblot analysis and anti-Flag resin are shown.
  • FCHSD1 forms a complex with NRF2 and SNX9 in the cytoplasm and blocks NRF2 from translocating to the nucleus.
  • NRF2 binds to its endogenous inhibitor, KEAP1, and is a target for proteasome degradation.
  • KEAP1 is modified and stabilizes NRF2.
  • FCHSD1 then dissociates from the complex and SNX9 bound to Importin8 bound to the nuclear envelope targets NRF2.
  • Identity of amino acid sequences refers to the degree of coincidence of amino acid sequences with each other among two or more comparable amino acid sequences. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, with default parameters.
  • FASTA a tool for sequence analysis, with default parameters.
  • the algorithm BLAST by Karlin and Altschul Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessment the statistical signature of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA.
  • K It can be determined using "Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90: 5873-7 (1993). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. The "identity" of the base sequence is also defined according to the above.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution.
  • amino acid residues having acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine
  • Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan
  • amino acid residues with ⁇ -branched side chains such as threonine, valine and isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitutions between amino acid residues are also conservative substitutions.
  • nucleic acid and polynucleotide are not particularly limited and include both natural and artificial ones. Specifically, in addition to DNA, RNA and the like, known chemical modifications may be applied as illustrated below. Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolases such as nucleases. Can be done.
  • phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolases such as nucleases.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate phosphorodithionate
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is designated as -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.).
  • R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.
  • the base moiety pyrimidine, purine
  • examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • BNA LNA
  • the conformation of the sugar portion is fixed to N-type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be used.
  • the present invention comprises at least one selected from the group consisting of an Fchsd1 expression inhibitor and an Fchsd1 function inhibitor, which comprises at least one of them (the present invention).
  • the present invention comprises at least one selected from the group consisting of an Fchsd1 expression inhibitor and an Fchsd1 function inhibitor, which comprises at least one of them (the present invention).
  • the agent of the present invention it may be referred to as "the agent of the present invention”). This will be described below.
  • the Fchsd1 gene is a gene belonging to the F-BAR family.
  • Fchsd1 Fchsd1 protein, Fchsd1 mRNA
  • Fchsd1 protein or Fchsd1 mRNA which is the target of expression or function suppression, is an expression product of the Fchsd1 gene and is expressed in the organism or its cells (particularly cells of lung tissue) to be prevented or treated for respiratory diseases.
  • Fchsd1 protein or Fchsd1 mRNA Fchsd1 protein or Fchsd1 mRNA. Therefore, the Fchsd1 protein and Fchsd1 mRNA to be suppressed also change depending on the target organism species.
  • the organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • the amino acid sequence of Fchsd1 protein derived from various species and the base sequence of Fchsd1 mRNA are known.
  • the human Fchsd1 protein includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_258260.1), and the mouse Fchsd1 protein includes the amino acid sequence shown in SEQ ID NO: 2.
  • the human Fchsd1 mRNA includes an mRNA consisting of the base sequence shown in SEQ ID NO: 3 (NCBI Reference Sequence: NM_033449.3)
  • the mouse Fchsd1 mRNA includes Examples thereof include mRNA (NCBI Reference Sequence: NM_175684.4) consisting of the base sequence shown in SEQ ID NO: 4.
  • the Fchsd1 protein and Fchsd1 mRNA may also include the above splicing variants.
  • the Fchsd1 protein to be regulated has amino acid mutations such as substitutions, deletions, additions, and insertions as long as it has the original property, that is, the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex. May be.
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the activity is less likely to be impaired.
  • Fchsd1 mRNA which is the subject of regulation, also has the protein translated from the mRNA, which interacts with NRF2 and SNX9 in the cytoplasm to form a complex, and dissociates from the complex in response to oxidative stress.
  • it may have basic mutations such as substitutions, deletions, additions, and insertions.
  • a mutation that does not cause an amino acid substitution or a mutation that causes a conservative substitution of an amino acid in the protein translated from the mRNA is preferable.
  • Fchsd1 protein to be regulated include the protein described in (a) below and the protein described in (b) below: It consists of (a) a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 and 2, and (b) an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 and 2. And at least one selected from the group consisting of proteins having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
  • the identity is more preferably 90% or more, further preferably 95% or more, still more preferably 98% or more.
  • one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any one of (b') SEQ ID NOs: 1 and 2.
  • examples thereof include proteins having the amino acid sequence obtained and having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
  • the plurality is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.
  • Fchsd1 mRNA to be regulated include the mRNA described in (c) below and the mRNA described in (d) below: (C) mRNA consisting of the base sequence shown in any of SEQ ID NOs: 3 to 4, and (d) consisting of a base sequence having 85% or more identity with the base sequence shown in any of SEQ ID NOs: 3 to 4. And mRNA encoding a protein that has the property of interacting with NRF2 and SNX9 to form a complex in the cytoplasm. At least one species selected from the group consisting of.
  • the identity is more preferably 90% or more, further preferably 95% or more, still more preferably 98% or more.
  • mRNA described in (d) above for example, one or more bases are substituted, deleted, added, or inserted into the base sequence shown in any one of (d') SEQ ID NOs: 3 to 4.
  • An mRNA encoding a protein consisting of the base sequence and having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex. Can be mentioned.
  • the plurality is, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, and even more preferably 2 to 10.
  • the active ingredient of the agent of the present invention is at least one ingredient selected from the group consisting of an Fchsd1 expression inhibitor and an Fchsd1 function inhibitor.
  • the component include a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, an antibody and the like.
  • the Fchsd1 expression inhibitor and the Fchsd1 function inhibitor will be specifically described below.
  • Fchsd1 expression inhibitor Fchsd1 expression inhibitor can suppress the expression level of Fchsd1 protein and / or Fchsd1 mRNA expressed in the organism to be prevented or treated for respiratory diseases or its cells (particularly cells of lung tissue). As long as it is a thing, there is no particular limitation.
  • the Fchsd1 expression inhibitor can be used alone or in combination of two or more.
  • Fchsd1 expression inhibitor examples include Fchsd1-specific small interfering RNA (siRNA), Fchsd1-specific microRNA (miRNA), Fchsd1-specific antisense nucleic acid, their expression cassettes; Fchsd1-specific ribozyme; Fchsd1 gene by CRISPR / Cas system. Examples include editorial agents.
  • expression suppression means that the expression levels of Fchsd1 protein, Fchsd1 mRNA, etc. are, for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and includes setting the expression level of these to 0.
  • siRNA, miRNA, antisense nucleic acid, and ribozyme Fchsd1-specific siRNA are not particularly limited as long as they are double-stranded RNA molecules that specifically suppress the expression of the gene encoding Fchsd1.
  • the siRNA is preferably, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more in length.
  • the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is assumed that the upper and lower limits of the siRNA lengths described here can be arbitrarily combined.
  • the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases.
  • a length; a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 A combination of lengths that are bases is assumed.
  • the siRNA may be shRNA (small hairpin RNA).
  • shRNA can be designed so that a part of it forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand in the order of sequence a, spacer, and sequence b. It can be designed to have a total length of 45 to 60 bases.
  • the sequence a is a sequence of a part of the base sequence encoding the target Fchsd1, and the target region is not particularly limited, and any region can be a candidate.
  • the length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
  • the Fchsd1-specific siRNA may have an additional base at the 5'or 3'end.
  • the length of the additional base is usually about 2 to 4 bases.
  • the additional base may be DNA or RNA, but DNA may be used to improve the stability of the nucleic acid.
  • the sequences of such additional bases include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Sequences such as', uuuuuu-3'can be mentioned, but are not limited to these.
  • the siRNA may have a protruding sequence (overhang) at the 3'end, and specific examples thereof include those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end without end addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand, for example, "asymmetrical interfering RNA" in which the antisense strand has a protruding sequence (overhang) at the 3'end and the 5'end. aiRNA) ”.
  • a typical aiRNA consists of 21 bases in the antisense strand and 15 bases in the sense strand, and has an overhang structure of 3 bases at both ends of the antisense strand.
  • the position of the target sequence of the Fchsd1-specific siRNA is not particularly limited, but in one embodiment, the target sequence is selected from 5'-UTR and from the start codon to about 50 bases, and from regions other than 3'-UTR. It is desirable to do.
  • BLAST http://www.ncbi.nlm.nih.gov/BLAST/
  • Etc. to confirm the specificity of the selected target sequence.
  • a sense strand having a 3'end overhang of TT or UU at 19-21 bases after AA (or NA), and a sequence complementary to the 19-21 base and TT or A double-stranded RNA consisting of an antisense strand with a 3'end overhang of UU may be designed as a siRNA.
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are passed through the linker sequence. It can be designed by connecting.
  • SiRNA and / or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include the following. SiRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) pSilencer® Expression Vector Insert Design Tool (http://www.ambion.com/) provided by Ambion jp / techlib / misc / psilencer_converter.html) GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.
  • the sense strand and antisense strand of the target sequence on mRNA are synthesized by a DNA / RNA automatic synthesizer, respectively, and denatured in an appropriate annealing buffer at about 90 to about 95 ° C. for about 1 minute. It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA as a precursor of siRNA and cleaving it with an RNA-cleaving protein dicer.
  • Fchsd1-specific siRNA for example, SASI_Hs01_00242324, SASI_Hs02_00307980, SASI_Mm01_00134930 and the like sold by Merck can be purchased and used.
  • Fchsd1-specific miRNAs are optional as long as they inhibit translation of the gene encoding Fchsd1.
  • miRNAs may pair with the target's 3'untranslated region (UTR) and inhibit its translation, rather than cleaving the target mRNA as with siRNA.
  • the miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA.
  • the length of the miRNA is not particularly limited, the length of the pri-miRNA is usually hundreds to thousands of bases, the length of the pre-miRNA is usually 50-80 bases, and the length of the mature miRNA is usually 18 ⁇ 30 bases.
  • the Fchsd1-specific miRNA is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA.
  • Such Fchsd1-specific miRNAs may be synthesized by known methods or may be purchased from companies that provide synthetic RNAs.
  • the Fchsd1-specific antisense nucleic acid is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the gene encoding Fchsd1, or a part thereof, and is specific and stable to the mRNA. It is a nucleic acid having a function of suppressing Fchsd1 protein synthesis by forming a double chain and binding to it.
  • the antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera.
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA is recognized by the endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the Fchsd1 gene.
  • the intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the Fchsd1 gene using a homology search program such as BLAST or FASTA.
  • the length of the target region of the Fchsd1-specific antisense nucleic acid is not limited as long as the hybridization of the antisense nucleic acid results in inhibition of translation into the Fchsd1 protein.
  • the Fchsd1-specific antisense nucleic acid may be a full sequence or a partial sequence of the mRNA encoding Fchsd1. Considering the ease of synthesis, antigenicity, intracellular transferability, and the like, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases are preferable, but the oligonucleotide is not limited thereto.
  • 'End hairpin loops and the like can be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.
  • Fchsd1-specific antisense nucleic acids not only hybridize with the mRNA and early transcripts of the Fchsd1 gene to inhibit translation into proteins, but also bind to these double-stranded DNA genes to triple-strand (triplex). ) And can inhibit transcription into RNA (antigene).
  • the target sequence of mRNA or early transcript is determined based on the cDNA sequence or genomic DNA sequence of the Fchsd1 gene, and commercially available DNA / RNA automation is performed. It can be prepared by synthesizing a sequence complementary to this using a synthesizer. In addition, all antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
  • the Fchsd1-specific siRNA, Fchsd1-specific miRNA, or Fchsd1-specific antisense nucleic acid is incorporated in a state in which it can be expressed. It is not particularly limited as long as it is a polynucleotide.
  • the expression cassette is a polynucleotide comprising a promoter sequence and a coding sequence of an Fchsd1-specific siRNA, an Fchsd1-specific miRNA, or an Fchsd1-specific antisense nucleic acid (and optionally a transcription termination signal sequence).
  • the promoter is not particularly limited, and is an RNA polymerase II (polII) promoter such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter; mouse and human U6-snRNA promoters, Examples include human H1-RNase P RNA promoters, RNA polymerase III (polIII) promoters such as human valine-tRNA promoters, and among these, polIII promoters are available from the viewpoint of accurate transcription of short RNAs. preferable. In addition, various promoters that can be induced by a drug can also be used.
  • the other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include origins of replication, drug resistance genes, and the like. Moreover, the above-mentioned types can be exemplified as the type of drug resistance gene and the type of vector.
  • Fchsd1-specific ribozyme Another example of an Fchsd1 expression inhibitor is an Fchsd1-specific ribozyme.
  • ribozyme in a narrow sense means RNA having an enzymatic activity of cleaving nucleic acid, but in the present application, DNA is also included as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • ribozyme when used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked to facilitate the transfer of transcripts to the cytoplasm. You can also [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • the Fchsd1 gene editing agent is not particularly limited as long as the expression of the Fchsd1 gene can be suppressed by a target sequence-specific nuclease system (eg, CRISPR / Cas system).
  • the expression of the Fchsd1 gene can be suppressed by, for example, disruption of the Fchsd1 gene or suppression of the activity of the promoter by modifying the promoter of the Fchsd1 gene.
  • a vector containing a guide RNA expression cassette targeting the Fchsd1 gene or its promoter and a Cas protein expression cassette (for Fchsd1 gene editing) is typically used.
  • Vector can be used, but is not limited to this.
  • a combination of a vector containing a guide RNA targeting the Fchsd1 gene or its promoter and / or an expression cassette thereof and a vector containing a Cas protein and / or its expression cassette can be used as an Fchsd1 gene editing agent. Can be used as.
  • the guide RNA expression cassette is not particularly limited as long as it is a polynucleotide used for the purpose of expressing the guide RNA in the target organism.
  • Typical examples of the expression cassette include a promoter and a polynucleotide containing a coding sequence of all or part of a guide RNA placed under the control of the promoter.
  • "arranged under the control of a promoter” means that the guide RNA coding sequence is arranged so that the transcription of the sequence is controlled by the promoter.
  • the guide RNA coding sequence is arranged directly under the 3'side of the promoter (for example, between the base at the 3'end of the promoter and the base at the 5'end of the guide RNA coding sequence.
  • the base pair number (bp) of the above is, for example, 100 bp or less, preferably 50 bp or less).
  • the promoter of the guide RNA expression cassette is not particularly limited, and a pol II promoter can be used, but a pol III promoter is preferable from the viewpoint of more accurately transcribing a relatively short RNA.
  • the pol III promoter is not particularly limited, and examples thereof include mouse and human U6-snRNA promoters, human H1-RNase PRNA promoters, and human valine-tRNA promoters.
  • various promoters that can be induced by a drug can also be used.
  • the guide RNA coding sequence is not particularly limited as long as it is a base sequence encoding the guide RNA.
  • the guide RNA is not particularly limited as long as it is used in the CRISPR / Cas system, for example, the Cas protein by binding to a target site of genomic DNA (for example, the Fchsd1 gene, its promoter, etc.) and by binding to the Cas protein.
  • a target site of genomic DNA for example, the Fchsd1 gene, its promoter, etc.
  • Various substances that can induce the gene into the target site of genomic DNA can be used.
  • the target site is a PAM (Proto-spacer Adjacent Motif) sequence and a 17 to 30 base length (preferably 18 to 25 base length, more preferably 19 to 22 base length) adjacent to the 5'side thereof.
  • PAM Proto-spacer Adjacent Motif
  • it is a site on genomic DNA consisting of a DNA strand (target strand) consisting of a sequence having a length of about 20 bases) and its complementary DNA strand (non-target strand).
  • the PAM sequence differs depending on the type of Cas protein used.
  • the PAM sequence corresponding to the Cas9 protein (type II) derived from S. pyogenes is 5'-NGG
  • the PAM sequence corresponding to the Cas9 protein (type I-A1) derived from S. solfataricus is 5'-CCN
  • the PAM sequence corresponding to the Cas9 protein (I-A2 type) derived from S. solfataricus is 5'-TCN
  • the PAM sequence corresponding to the Cas9 protein (I-B type) derived from H. walsbyl is 5'-TTC.
  • coli-derived Cas9 protein (I-E type) is 5'-AWG
  • PAM sequence corresponding to the E. coli-derived Cas9 protein (I-F type) is 5'-CC
  • the PAM sequence corresponding to the Cas9 protein (I-F type) derived from P. aeruginosa is 5'-CC
  • the PAM sequence corresponding to the Cas9 protein (II-A type) derived from S. Thermophilus is 5'-NNAGAA.
  • the PAM sequence corresponding to the Cas9 protein (II-A type) derived from S. agalactiae is 5'-NGG
  • aureus is 5'-NGRRT or 5'-NGRRN.
  • the PAM sequence corresponding to the Cas9 protein derived from N. meningitidis is 5'-NNNNGATT, and the PAM sequence corresponding to the Cas9 protein derived from T. denticola is 5'-NAAAAC.
  • the guide RNA has a sequence involved in the binding of genomic DNA to the target site (sometimes referred to as a crRNA (CRISPRRNA) sequence), and this crRNA sequence excludes the PAM sequence complementary sequence of the non-target strand.
  • CRISPRRNA crRNA
  • the guide RNA can bind to the target site of genomic DNA.
  • complementary binding is not limited to the case of binding based on a complete complementary relationship (A and T, and G and C), but also a complementary relationship to the extent that hybridization can be performed under stringent conditions.
  • Stringent conditions are the melting temperature of the nucleic acid that binds the complex or probe, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on (Tm). For example, as cleaning conditions after hybridization, conditions of about "1 x SSC, 0.1% SDS, 37 ° C.” can be mentioned.
  • the hybridized state is maintained even after washing under such conditions.
  • cleaning conditions of "0.5 x SSC, 0.1% SDS, 42 ° C” are listed as stricter hybridization conditions, and "0.1 x SSC, 0.1% SDS, 65 ° C” are listed as stricter hybridization conditions. Can be done.
  • the sequence that binds to the target sequence is, for example, 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, and particularly preferably 100% or more with the target strand. Has% identity. It is said that 12 bases on the 3'side of the sequence that binds to the target sequence are important for the binding of the guide RNA to the target site. Therefore, if the sequence that binds to the target sequence in the crRNA sequence is not exactly the same as the target strand, the base different from the target strand is 12 bases on the 3'side of the sequence that binds to the target sequence in the crRNA sequence. It is preferable that it exists in other than.
  • the guide RNA has a sequence involved in binding to the Cas protein (sometimes referred to as a tracrRNA (trans-activating crRNA) sequence), and this tracrRNA sequence binds to the Cas protein to form the Cas protein. It can be directed to the target site of genomic DNA.
  • tracrRNA trans-activating crRNA
  • the tracrRNA sequence is not particularly limited.
  • the tracrRNA sequence is typically an RNA consisting of a sequence having a length of about 50 to 100 bases capable of forming multiple (usually three) stem loops, and the sequence varies depending on the type of Cas protein used. ..
  • As the tracrRNA sequence various known sequences can be adopted depending on the type of Cas protein to be used.
  • the guide RNA usually contains the above-mentioned crRNA sequence and tracrRNA sequence.
  • the mode of the guide RNA may be a single-stranded RNA (sgRNA) containing a crRNA sequence and a tracrRNA sequence, or an RNA complex in which an RNA containing a crRNA sequence and an RNA containing a tracrRNA sequence are complementarily linked. It may be a body.
  • the Cas protein expression cassette is not particularly limited as long as it is a polynucleotide used for the purpose of expressing Cas protein in a target organism.
  • Typical examples of the expression cassette include a promoter and a polynucleotide containing a Cas protein coding sequence arranged under the control of the promoter. Note that "placed under the control of a promoter" is the same as the definition in the guide RNA expression cassette.
  • the promoter of the Cas protein expression cassette is not particularly limited, and for example, various polII promoters can be used.
  • the polII promoter is not particularly limited, but for example, CMV promoter, EF1 promoter, SV40 promoter, etc. Examples include the MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter and the like.
  • various promoters that can be induced by a drug can also be used.
  • the Cas protein coding sequence is not particularly limited as long as it is a base sequence encoding the amino acid sequence of the Cas protein.
  • the Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system, and various proteins that can bind to the target site of genomic DNA in a complex with the guide RNA and cleave the target site are used. can do.
  • Cas protein those derived from various organisms are known, for example, Cas9 protein derived from S. pyogenes (type II), Cas9 protein derived from S. solfataricus (type I-A1), and Cas9 protein derived from S. solfataricus. (I-A2 type), H. walsbyl-derived Cas9 protein (I-B type), E. coli-derived Cas9 protein (I-E type), E.
  • Cas9 protein is preferably mentioned, and more preferably Cas9 protein inherently possessed by a bacterium belonging to the genus Streptococcus. Information on the amino acid sequences of various Cas proteins and their coding sequences can be easily obtained on various databases such as NCBI.
  • the Cas protein may be a wild-type double-strand break type Cas protein or a nickase-type Cas protein.
  • the Cas protein may have an amino acid sequence variation (eg, substitution, deletion, insertion, addition, etc.) as long as its activity is not impaired, and known protein tags, signal sequences, enzyme proteins, etc. It may be the one to which a protein such as is added.
  • protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the signal sequence include a cytoplasmic transfer signal and the like.
  • the Fchsd1 gene editing vector may have other sequences.
  • the other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include origins of replication, drug resistance genes, and the like.
  • Examples of the drug resistance gene include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a canamycin resistance gene, a hyglomycin resistance gene, and a puromycin resistance gene.
  • the type of vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus; and agrobacterium vectors. Be done.
  • the Fchsd1 gene editor can be easily prepared according to a known genetic engineering method. For example, it can be produced by using PCR, restriction enzyme cleavage, DNA linkage technology, in vitro transcription / translation technology, recombinant protein production technology, and the like.
  • Fchsd1 function inhibitor is one that can regulate the function of Fchsd1 protein and / or Fchsd1 mRNA expressed in the organism to be prevented or treated for respiratory diseases or its cells (particularly cells of lung tissue). As long as it is, there is no particular limitation.
  • the Fchsd1 function inhibitor can be used alone or in combination of two or more.
  • the Fchsd1 function inhibitor is not particularly limited as long as it can reduce the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
  • Fchsd1 function inhibitor examples include Fchsd1 antibody and the like.
  • the Fchsd1 antibody is preferably an antibody having antigen-binding property to the amino acid sequence of the binding site of Fchsd1 with NRF2 and / or SNX9. By using such an antibody, the Fchsd1 function can be suppressed more reliably.
  • the binding site can be determined based on known information and / or inferred based on known information (eg, by building a docking model, etc.).
  • Antibodies include some of the above antibodies having antigen-binding properties, such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or fragments produced by Fab fragments and Fab expression libraries. Antibodies having antigen-binding activity to a polypeptide consisting of at least consecutive 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of Fchsd1 are also included in the antibody of the present invention. These antibodies are available, and for example, as Anti-FCHSD1 antibody, ab67017 manufactured by abcam, STJ117738 manufactured by St Johns Laboratory, LS-C766558-60 manufactured by LifeSpan Biosciences, and the like are known.
  • the methods for producing these antibodies are already well known and can be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 to 11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • Fchsd1 expressed and purified in E. coli or the like according to a conventional method is used, or an oligopeptide having a partial amino acid sequence of Fchsd1 is synthesized according to a conventional method. It is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method.
  • Fchsd1 expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of Fchsd1 is immunized against a non-human animal such as a mouse to obtain spleen cells and myeloma. It can be obtained from hybridoma cells prepared by cell fusion with cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).
  • Fchsd1 Used as an immune antigen for antibody production, Fchsd1 is based on known gene sequence information for DNA cloning, plasmid construction, host transfection, transformant culture and protein recovery from cultures. It can be obtained by operation. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
  • a recombinant DNA capable of expressing the gene encoding Fchsd1 in a desired host cell is prepared, introduced into the host cell for transformation, and the transformant is cultured.
  • a protein as an immune antigen for producing the antibody of the present invention can be obtained.
  • the partial peptide of Fchsd1 can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of Fchsd1.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have functional biological activity, but is preferably one having immunogenogenic properties similar to Fchsd1.
  • An oligo (poly) peptide having this immunogenetic property and consisting of at least consecutive 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of Fchsd1 can be exemplified.
  • an antibody against such an oligo (poly) peptide can also be carried out by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocianine and dinitrophenol.
  • active substances include active substances, human adjuvants such as BCG (Carmet-Guerin bacillus) and Corinebacterium-Palvum.
  • Fchsd1 function inhibitor in addition to the above-mentioned Fchsd1 antibody, a molecule having binding property to Fchsd1 (preferably having specific binding property) (for example, a low molecular weight compound, a peptide, a protein, an artificial antibody, an aptamer, etc.) can be used. If so, it can be used. Small molecule compounds also include compounds optimized to degrade the target protein (Proteolysis targeting chimera). When a protein or peptide such as an antibody is used as the Fchsd1 function inhibitor, an expression cassette thereof can be used instead. The expression cassette is the same as the definition in "2-2-1. Fchsd1 expression inhibitor" above.
  • Fchsd1 is highly expressed in the respiratory tract (especially lung) and interacts with NRF2 and SNX9 in the cytoplasm to form a complex. It suppresses NRF2 translocation to the nucleus and causing an oxidative stress response. Therefore, by suppressing the expression and / or function of Fchsd1, the oxidative stress response can be promoted.
  • various respiratory diseases such as COPD
  • oxidative stress is involved in the onset and / or exacerbation of the disease. Therefore, at least one selected from the group consisting of the Fchsd1 expression inhibitor and the Fchsd1 function inhibitor (active ingredient) is effective for the prevention or treatment (or improvement) of respiratory diseases.
  • This active ingredient can be used as an active ingredient of, for example, pharmaceuticals, reagents, food compositions, health promoters, nutritional supplements (supplements, etc.), etc.).
  • the active ingredient can be applied to animals, humans, and various cells (eg, administration, ingestion, inoculation, etc.) as it is or in combination with conventional ingredients in various compositions.
  • the respiratory disease is preferably a respiratory disease (preferably lung disease) that can develop or worsen due to oxidative stress, for example, acute bronchitis, bacterial pneumonia, pulmonary abscess, pulmonary tuberculosis, and non-tuberculous pulmonary acid bacterium.
  • oxidative stress for example, acute bronchitis, bacterial pneumonia, pulmonary abscess, pulmonary tuberculosis, and non-tuberculous pulmonary acid bacterium.
  • Infectious respiratory diseases such as illness, pulmonary fungal disease, pulmonary parasite disease, opportunistic infection (pneumocystis pneumonia, cytomegalovirus pneumonia), aspiration pneumonia, cold syndrome, influenza; chronic obstructive pulmonary disease (COPD), pulmonary emphysema Bronchial asthma, irritable pneumonia, eosinophil pneumonia, allergic bronchopulmonary aspergillosis, drug-induced pneumonia, eosinophilia polyangiitis granulomatosis, etc.
  • Neoplastic lung diseases such as mediastinal tumors; Pulmonary vascular lesions such as pulmonary thromboembolism, pulmonary arterial hypertension, and pulmonary edema; Examples include respiratory insufficiency such as chronic respiratory insufficiency.
  • the cell type of the target cell of the agent of the present invention is not particularly limited as long as it is a cell type capable of expressing Fchsd1, but is mainly cells of respiratory tissue (particularly lung tissue).
  • the target organism of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. ..
  • the form of the agent of the present invention is not particularly limited, and the form usually used in each application can be taken depending on the use of the agent of the present invention.
  • the form includes, for example, tablets (intraoral disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drop agents, etc.) when the uses are pharmaceuticals, health promoters, nutritional supplements (supplements, etc.), etc. ), Rounds, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly, etc.
  • Form oral formulation form
  • liquid, gel or solid foods such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, milk powder for childcare.
  • Cake mixes, powdered or liquid dairy products, breads, cookies and the like.
  • the agent of the present invention may further contain other components, if necessary.
  • the other ingredients are not particularly limited as long as they are ingredients that can be blended in, for example, pharmaceuticals, food compositions, health enhancers, nutritional supplements (supplements, etc.), but are not particularly limited, and are, for example, bases, carriers, solvents, and the like. Dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents and the like can be mentioned.
  • the content of the active ingredient of the agent of the present invention depends on the type, use, mode of use, application target, state of application target, etc. of the active ingredient, and is not limited, but is, for example, 0.0001 to 100% by weight. It can be preferably 0.001 to 50% by weight.
  • the application amount (for example, administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and the weight of the active ingredient is generally 0.1 to 1000 mg per day. / kg weight.
  • the above dose is preferably administered once a day or divided into 2 to 3 times, and may be appropriately increased or decreased depending on the age, pathological condition, and symptoms.
  • Fchsd1 FCH and double SH3 domains protein 1
  • the present invention relates to a method for inspecting a respiratory disease including a step (in the present specification, it may be referred to as "inspection method of the present invention”).
  • there is a method for examining the risk of contracting a respiratory disease which comprises (1) a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject. (In the present specification, it may be referred to as "the determination method of the present invention”).
  • Process (1) The types of "respiratory diseases" to be inspected or judged are as described in "2-3" above. All classes, grades, and stages of respiratory illness in the various classification criteria for the progression of respiratory illness can be tested or determined.
  • the subject is the target organism of the test method or determination method of the present invention, and the species thereof is not particularly limited.
  • the organism species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer, and humans are preferable. ..
  • the condition of the subject is not particularly limited.
  • the subjects include, for example, a sample whose presence or absence is unknown whether or not they have a respiratory disease, a sample which has already been determined to have a respiratory disease by another method, and a sample which has not already suffered from a respiratory disease. Examples thereof include a sample determined by the above method, a sample during treatment for a respiratory disease, a sample after treatment for a respiratory disease, and the like.
  • the test sample in the step (1) is not particularly limited as long as it is a sample in which Fchsd1 derived from the respiratory organ (particularly lung) of the subject can be detected.
  • the test sample preferably includes a test sample that may contain the lung tissue of the subject. Specific examples thereof include a lung biopsy sample and a lung lavage fluid.
  • the detection target in step (1) is Fchsd1, that is, Fchsd1 mRNA and / or Fchsd1 protein (in the present specification, these may be collectively referred to as “target biomarker”).
  • the detection target Fchsd1 is the same as in "2-1" above.
  • Fchsd1 to be detected is preferably a protein from the viewpoint of inspection accuracy.
  • Detection is usually performed by measuring the amount or concentration of the target biomarker.
  • concentration is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, raw data measured to know the absolute concentration, or the like.
  • the method for detecting the target biomarker is not particularly limited as long as it can specifically detect a part or all of the target biomarker.
  • Specific examples of the detection method include a mass analysis method for detecting peptides constituting the target biomarker, and an immunological measurement method using an antibody that specifically recognizes the target biomarker (for example, ELISA method, EIA). Method, RIA method, Western blotting method, etc.), Northern hybridization method, DNA microarray method, PCR method, etc. can be mentioned.
  • step (1) gene products other than the target biomarker can be detected.
  • the number of genes detected in the step (1) is smaller, for example, 1 to 50 types including the target biomarker (1 type of Fchsd1), preferably 1 to 20 types, and more. It is preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and particularly preferably 1 type.
  • the test method or determination target of the present invention including the step (1), it is possible to provide the amount and / or concentration of the target biomarker which is a detection index of a respiratory disease or a disease risk index, whereby the respiratory organs can be provided. It can assist in disease detection / diagnosis / disease risk determination.
  • the results of the test method or determination method of the present invention including step (1) include elucidation of the pathophysiology of respiratory diseases, prediction of prognosis of respiratory diseases, stratification of subjects, and selection of treatment methods (individualized medicine, treatment response). Gender), intractability in respiratory diseases, evaluation of remodeling, differentiation of respiratory phenotype, etc. can be used.
  • the test method of the present invention further determines that the subject suffers from a respiratory disease when (2) the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value. It is preferable to include a step of performing. According to the inspection method of the present invention including the step 2, it is possible to determine a respiratory disease. Further, in the determination method of the present invention, (2) when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value, the subject becomes a respiratory disease or a respiratory disease. It is preferable to include a step of determining that there is a risk (or high) of suffering.
  • the cutoff value can be appropriately set by those skilled in the art from the viewpoints of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, a subject collected from a subject not suffering from a respiratory disease. Based on the amount and / or concentration of the target biomarker in the test sample, it can be a predetermined value or a predetermined value each time.
  • the cutoff value is, for example, the amount and / or concentration of the target biomarker in the test sample collected from the test subject not suffering from respiratory disease (mean value, median value, etc. when there are multiple subjects). For example, the value can be 0.7 to 1.5 times.
  • the cutoff value can be determined, for example, the amount of target biomarker in past samples for the same specimen and / /.
  • the therapeutic effect can be determined by setting the value based on the concentration.
  • the test method of the present invention is further applied to the respiratory organs.
  • the respiratory disease can be diagnosed with higher accuracy.
  • the test method of the present invention can detect respiratory diseases more accurately, by combining the above steps with the test method of the present invention, it is said that "they are suffering from respiratory diseases" more efficiently and more accurately. Can be diagnosed.
  • the test method of the present invention is further applied, or the above-mentioned "4. Respiratory disease”. If a person is diagnosed with a respiratory disease as described in "Diagnosis with higher accuracy of organ disease", the combination of the test method of the present invention and the step of applying the diagnosis by a doctor is further applied. (3) By performing a step of treating a subject who has been determined or diagnosed to have a respiratory disease, the disease of the subject can be treated.
  • test method of the present invention can detect respiratory diseases more accurately, a step (for the test method of the present invention, or for a combination of the test method of the present invention and a step of applying a diagnosis by a doctor) ( By combining 3), a subject suffering from a respiratory disease can be treated more efficiently and more reliably.
  • the treatment method for respiratory diseases is not particularly limited, but typical examples include medication treatment.
  • Drugs used for medication include, for example, steroid drugs, ⁇ 2 stimulants, theophylline preparations, leukotriene receptor antagonists, chemical mediators (substances with bronchial contractile action released from obese cells) release inhibitors, Th2 cytokines. Examples include inhibitors, histamine H 1 antagonists, thromboxane A 2 synthesis inhibitors / receptor antagonists, anticholinergic agents, sputum regulators, etc.
  • the medicine can be used as one type, two types, or a combination of three or more types.
  • the present invention may be referred to as a respiratory disease test agent (in the present specification, the "test agent of the present invention") containing a detection agent for Fchsd1. ). This will be described below.
  • Fchsd1, respiratory diseases, etc. are the same as the definitions above.
  • the detection agent is not particularly limited as long as it can specifically detect the target biomarker.
  • Examples of the detection agent include antibodies, primers, probes and the like against the target biomarker.
  • the detection agent may be modified as long as its function is not significantly impaired. Modifications include, for example, addition and introduction of labeled substances such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemical luminescent substances, colloidal gold, biotin and the like.
  • labeled substances such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemical luminescent substances, colloidal gold, biotin and the like.
  • the detection agent can also be used by immobilizing it on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which a probe is immobilized, and another example, an ELISA plate on which an antibody is immobilized).
  • a substrate on which a detection agent is immobilized for example, a microarray chip on which a probe is immobilized, and another example, an ELISA plate on which an antibody is immobilized.
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize an antibody or the like, and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. can. Immobilization of the detection agent on the solid phase is not particularly limited.
  • the antibody is not particularly limited as long as it selectively (specifically) recognizes the target biomarker.
  • “selectively (specifically) recognizing” means that the target biomarker can be specifically detected, for example, in Western blotting or ELISA, but the person skilled in the art is not limited thereto. It suffices as long as it can be determined that the above-mentioned detected substance is derived from the target biomarker.
  • the antibody is as described in "2-2-2" above.
  • the primer, probe, etc. are not particularly limited as long as they selectively (specifically) recognize the target biomarker and the nucleic acid derived from the target biomarker.
  • “selectively (specifically) recognizing” means, for example, that the target biomarker can be specifically detected in Northern blotting, and that the target biomarker or a nucleic acid derived from the target biomarker is used in the RT-PCR method. It means that (cDNA, etc.) is specifically amplified, but is not limited to this, as long as it can be determined by those skilled in the art that the above-mentioned detected substance or amplified substance is derived from the target biomarker. good.
  • primers and probes include the polynucleotide described in (a) below and the polynucleotide described in (b) below: (A) A polynucleotide having at least 15 consecutive bases in the base sequence of the target biomarker and / or a polynucleotide complementary to the polynucleotide, and (b) a base sequence of the target biomarker or a base complementary thereto. Included is at least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize to the sequence under stringent conditions.
  • a complementary polynucleotide or a complementary base sequence is a full-length sequence of a polynucleotide consisting of the base sequence of a target biomarker, or a base sequence having a length of at least 15 consecutive bases in the base sequence.
  • a polynucleotide or base that is basically complementary to its partial sequence here, for convenience, these are also referred to as "regular chains" based on the base pair relationships such as A: T and G: C. It means an array.
  • such a complementary strand is not limited to the case where it completely forms a complementary sequence with the base sequence of the target positive chain, and has a complementary relationship to the extent that it can hybridize with the target positive chain under stringent conditions.
  • the stringent condition here is to bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as cleaning conditions after hybridization, conditions of about "1 x SSC, 0.1% SDS, 37 ° C.” can be mentioned. It is preferable that the complementary strand maintains a hybridized state with the positive strand of interest even when washed under such conditions.
  • cleaning conditions of "0.5 x SSC, 0.1% SDS, 42 ° C” are listed as stricter hybridization conditions, and "0.1 x SSC, 0.1% SDS, 65 ° C” are listed as stricter hybridization conditions.
  • a complementary chain a chain consisting of a base sequence having a completely complementary relationship with the base sequence of the positive chain of interest, and at least 90%, preferably 95%, more preferably of the chain.
  • a chain consisting of a base sequence having 98% or more, more preferably 99% or more identity can be exemplified.
  • Primers, probes, etc. can be designed using, for example, various design programs based on the base sequence of the target biomarker. Specifically, a candidate sequence of a primer or a probe obtained by subjecting the base sequence of the target biomarker to a design program, or a sequence containing at least the sequence as a part thereof can be used as a primer or a probe.
  • the base length of the primer, probe, etc. is not particularly limited as long as it has a continuous length of at least 15 bases as described above, and can be appropriately set according to the intended use.
  • As the base length for example, when used as a primer, for example, 15 bases to 35 bases can be exemplified, and when used as a probe, for example, 15 bases to 35 bases can be exemplified.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other components, if necessary.
  • Other ingredients include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances. , Chelating agent and the like.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain one that can be used for preparation of a test sample or detection of a target biomarker.
  • a reagent include various reagents (for example, secondary antibody, buffer solution, reagent for collecting, purifying, separating, and concentrating a test sample), and an instrument (for example, collecting, purifying, separating, and concentrating a test sample). Equipment (for example, column etc.)) and the like can be mentioned.
  • a method for screening an active ingredient of a prophylactic or therapeutic agent for respiratory diseases In one embodiment of the present invention, the amount or concentration of Fchsd1 in a test sample collected from an animal or cell treated with a test substance is used as an index.
  • a method for screening an active ingredient of a prophylactic or therapeutic agent for respiratory diseases hereinafter, may be referred to as "the active ingredient screening method of the present invention”. This will be described below.
  • the species of animals is not particularly limited. Species of animals include various mammalian species such as monkeys, mice, rats, dogs, cats and rabbits.
  • the type of cell is not particularly limited.
  • the cell can be a somatic cell or a stem cell.
  • stem cells include ES cells and iPS cells.
  • the origin of the cells is not particularly limited, and examples thereof include various mammals such as monkeys, mice, rats, dogs, cats, and rabbits.
  • the tissue from which the cells are derived is arbitrary, and for example, cells derived from lung tissue can be used.
  • test substance it can be widely used regardless of whether it is a naturally occurring compound or an artificially produced compound. Further, not only the purified compound but also a composition in which various compounds are mixed and an extract of animals and plants can be used.
  • the compound includes not only low molecular weight compounds but also high molecular weight compounds such as proteins, nucleic acids and polysaccharides.
  • the active ingredient screening method of the present invention is used when the value of the index for Fchsd1 is lower than the amount or concentration of Fchsd1 in a test sample collected from an animal not treated with the test substance. , The step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for respiratory diseases.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • the present invention relates to a method for evaluating the induction or exacerbation of respiratory diseases using the concentration as an index (in the present specification, it may be referred to as “the toxicity evaluation method of the present invention”). This will be described below.
  • the measurement of the amount or concentration of Fchsd1, test sample, test substance, animal, cell, respiratory disease, target biomarker, etc. is the same as the above definition.
  • the toxicity evaluation method of the present invention is used when the value of the index for Fchsd1 is higher than the amount or concentration of Fchsd1 in a test sample collected from an animal not treated with the test substance. It includes a step of determining that the test substance has an inducing or exacerbating malignancy of respiratory disease.
  • High means, for example, that the value of the index is twice, five times, ten times, 20 times, 50 times, and 100 times the control value.
  • Test example 1 Examination of FCHSD1 amount of lung tissue / alveolar epithelial cells in emphysema model
  • the materials used in this test example and the method adopted are as follows.
  • Mouse type II alveolar epithelial cells are 2% fetal bovine serum (no.10270-106; Gibco), 100 U / ml penicillin G, 100 ⁇ g / ml streptomycin (both from Nacalai Tesque), 0.005 mg.
  • FCHSD1 FCH family proteins
  • CIP4, TOCA1, FER, WRP, and FCHSD2 FCH family proteins
  • FCHSD1 protein was increased in mouse type II alveolar epithelial cells (MLE-12 cells) after H 2 O 2 stimulation (Fig. 1B).
  • injection of LPS into the lung did not change FCHSD1 levels (Fig. 1C). It was also confirmed that FCHSD1 was highly expressed in lung tissue compared to other tissues (Fig. 2A).
  • Test example 2 Preparation of Fchsd1 -/- mouse The materials used in this test example and the method adopted are as follows.
  • the conjugate was removed from the electrode chamber and washed 4 times with M2 medium.
  • the eggs were then cultured in MWM medium at 37 ° C. in a 5% CO2 incubator to the 2 cell stage. These eggs were then transferred to the fallopian tubes of the fake pregnant female on the day of vaginal plugging.
  • Fchsd1 knockout mice born in Mendelian ratios were generated. There were no significant differences in the immune cell fractions of the spleen, bone marrow, blood and lungs between Fchsd1 -/- mouse and wild-type (WT) mice, and the overall appearance and histological morphology of these organs. There was no significant difference in.
  • Test example 3 Examination of protective effect against emphysema by suppressing FCHSD1
  • the materials used and the method adopted in this test example are as follows. The materials and methods not described below are the same as those in the above test example.
  • Test example 4 Examination of the effect of FCHSD1 on inflammation and cell death of elastase-treated mouse lung
  • the materials used and the method adopted in this test example are as follows. The materials and methods not described below are the same as those in the above test example.
  • Flow cytometry Cell suspensions were prepared by sieving and pipetting. Cells were washed with FACS buffer [0.5% bovine serum albumin (BSA) and 2 mM EDTA in PBS, pH 7.2], then incubated with antibody for 20 minutes, followed by washing twice with FACS buffer. Data were obtained with flow cytometers (FACS Canto II, BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo (Tree Star Inc.). Antibodies for flow cytometry were purchased from the following distributors.
  • FACS buffer 0.5% bovine serum albumin (BSA) and 2 mM EDTA in PBS, pH 7.2
  • FITCanti-Ly-6C (HK1.4; BioLegend); PE anti-Ly-6G (1A8; BioLegend); PerCP anti-Mac1 (M1 / 70; BioLegend); APC Anti-CD45.2 (104; BioLegend); Anti-CD11c (N418; BioLegend); Anti-CD3e (145-2C11; BioLegend); Anti-CD4 (GK1.5; BioLegend); Anti-F4 / 80 (BM8; BioLegend); Anti-SiglecF (S17007L; BioLegend); CD8 (53-6.7) Biolegend); CCR3; (J073E5; Biolegend); B220; (RA3-6B2; Biolegend).
  • MEF Primary lung fibroblasts
  • ⁇ Construction of expression plasmid> The cDNAs for Fchsd1, Nrf2, and Snx9 were obtained by PCR from the mouse cDNA library. The Fchsd1 cDNA was cloned into pLZR-ires-GFP for retrovirus production. Flag-tagged Fchsd1, Flag-tagged Nrf2, and Flag-tagged Snx9 were cloned to pcDNA3.1 (+) for immunoprecipitation.
  • MLE-12 cells were transduced with retrovirus supernatant.
  • the virus was generated using PlatE packaging cells transfected with pLZR-ires-GFP containing a full-length Fchsd1 cDNA or an empty vector. After transduction, GFP-positive cells were screened using FACS Aria III (BD Bioscience).
  • RNA was isolated using RNA purification kit (No. 1828665; Roche) or TRIzol (No. 15596018; Thermo Fisher Scientific) and reverse transcribed with ReverTraAce (Toyobo) according to the manufacturer's instructions.
  • Quantitative reverse transcription PCR was performed using a real-time PCR master mix (Toyobo), and fluorescence from the TaqMan probe of each gene was detected by the 7500 real-time PCR system (Applied Biosystems). To determine the relative induction of mRNA, the mRNA level of each gene was calculated using the standard curve method normalized to 18S. Commercially available gene-specific primers and probe sets were obtained from Integrated DNA Technologies (Coralville).
  • the primers and probes used in this study are: Acta2 (Mm.PT.58.16320644), Col1a1 (Mm.PT.58.7562513), Il6 (Mm.PT.58.10005566), Tgfb1 (Mm.PT.58.11254750). , And Tnfa (Mm .PT.58.12575861).
  • Test example 5 Examination of Negative Control of Nuclear Translocation of Nuclear factor-like 2 (NRF2) in response to H 2 O 2 -induced oxidative stress by FCHSD1
  • NRF2 Nuclear factor-like 2
  • ⁇ Intracellular fraction> For intracellular fractionation, MLF or MLE-12 cells were plated with 1.2 ⁇ 10 6 cells per dish. Cells were stimulated with 275 ⁇ M H 2 O 2 (for MLF) or 250 ⁇ M H 2 O 2 (for MLE 12 cells). After stimulation, a Cell Extract was prepared using a Nuclear Extraction Kit (no.40010; Active Motif) according to the method of use.
  • a Nuclear Extraction Kit no.40010; Active Motif
  • MLE-12 cells are pcDNA3.1 (+) or empty vectors encoding Fchsd1, Flag-tagged Nrf2, HA-tagged Nrf2, Flag-tagged Snx9, and Myc-tagged Snx9, and are lipofectamine 2000 (no. 11668027; Thermo Fisher Scientific) was used for transfection. Twenty-four hours after transfection, cytolysis was collected. For immunoprecipitation with anti-Flag antibody, lysis buffer containing cOmplete Mini Protease Inhibitor Cocktail (Roche) [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% (vol / vol) Nonidet P. -40] dissolved.
  • lysis buffer containing cOmplete Mini Protease Inhibitor Cocktail (Roche) [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% (vol / vol) Nonidet P. -40] dissolved.
  • Cytolysis was incubated with anti-Flag M2 affinity gel (No. A2220; Sigma Aldrich) for 2 hours and washed with wash buffer [20 mM Tris-HCl (pH 7.5) and 150 mM NaCl].
  • wash buffer 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl.
  • Sepharose bead-conjugated Myc-tag antibody No. 3400; Cell Signaling Technology
  • FCHSD1 has two SH3 domains and mediates multiple interactions with other proteins.
  • Test example 6 Effects of FCHSD1 deficiency on sirtuin 1 (SIRT1) in elastase-treated mouse lungs and H 2 O 2 treated cells
  • SIRT1 levels in lungs treated with elastase were examined. investigated. SIRT1 levels in the lungs of WT mice treated intratracheally for 24 hours with elastase resulted in a marked decrease, whereas the decrease was suppressed in Fchsd1 -/- mice.
  • the levels of FOXO3a and PPAR ⁇ which are the target molecules for deacetylation by SIRT1, were higher in Fchsd1 -/- mice than in WT mice.
  • FCHSD1 plays an important role in the reduction of SIRT1 in response to oxidative stress.

Abstract

Provided is a technique for treating, preventing and diagnosing respiratory diseases. Respiratory diseases are prevented or treated by inhibiting the expression or function of FCH and double SH3 domains protein 1 (Fchsd1), and furthermore, Fchsd1 is utilized as a respiratory disease diagnostic biomarker.

Description

呼吸器疾患の予防又は治療剤、検査方法、検査薬、予防又は治療剤の有効成分のスクリーニング方法、及び誘発性又は増悪性の評価方法Prophylactic or therapeutic agents for respiratory diseases, testing methods, testing agents, screening methods for active ingredients of prophylactic or therapeutic agents, and evaluation methods for inducing or exacerbation.
　本発明は、呼吸器疾患の予防又は治療剤、検査方法、検査薬、予防又は治療剤の有効成分のスクリーニング方法、誘発性又は増悪性の評価方法等に関する。 The present invention relates to a preventive or therapeutic agent for respiratory diseases, a test method, a test agent, a screening method for an active ingredient of a preventive or therapeutic agent, a method for evaluating inducing or exacerbation, and the like.
　呼吸器疾患は、ウイルス、細菌、生体内免疫系、喫煙等の各種要因によって発症し得るものである。呼吸器疾患としては、例えば、急性気管支炎、気管支喘息、間質性肺炎、慢性閉塞性肺疾患（COPD：Chronic Obstructive Pulmonary Disease）等、多くの炎症性肺疾患が知られている。 Respiratory diseases can be caused by various factors such as viruses, bacteria, in-vivo immune system, smoking and the like. As respiratory diseases, many inflammatory lung diseases such as acute bronchitis, bronchitis, interstitial pneumonia, and chronic obstructive pulmonary disease (COPD: Chronic Obstructive Pulmonary Disease) are known.
　その中で、慢性閉塞性肺疾患（COPD）は、罹患率と死亡率が高い疾患であり、慢性気管支炎や肺気腫などの疾患の総称とされてきた。COPDは、肺胞壁の破壊と肺機能の低下が特徴である。COPDの主な構成要素である肺気腫は、喫煙やその他の環境的危険因子によって引き起こされる。特に、タバコの煙によって誘発される酸化ストレスが肺気腫発症の初期段階で重要な役割を果たしているといわれている。酸化ストレスは、炎症と細胞外マトリックスのタンパク質分解を増強し、その後、肺胞細胞と中隔破壊のアポトーシスを増強することが報告されている。また、酸化ストレスは、エラスターゼ誘発性肺気腫の発症にも関与している。進行期では、肺胞維持機構が障害されることを引き金に、アポトーシスとオートファジーが引き起こされる。制御を失ったオートファジーはERストレスなどの細胞性ストレスを増加させ、アポトーシスを誘導する。アポトーシス、酸化ストレス、及び炎症を含む傷害の連鎖は互いに増強しながら疾患の進行を促進する。さらに、COPDの肺では細胞の老化が加速しており、細胞増殖が低下し、炎症の増加と細胞再生の低下が惹起される。 Among them, chronic obstructive pulmonary disease (COPD) is a disease with a high morbidity and mortality rate, and has been regarded as a general term for diseases such as chronic bronchitis and emphysema. COPD is characterized by destruction of the alveolar wall and decreased lung function. Emphysema, a major component of COPD, is caused by smoking and other environmental risk factors. In particular, oxidative stress induced by cigarette smoke is said to play an important role in the early stages of emphysema onset. Oxidative stress has been reported to enhance inflammation and extracellular matrix proteolysis, followed by alveolar cell and septal destruction apoptosis. Oxidative stress is also involved in the development of elastase-induced emphysema. In the advanced phase, impaired alveolar maintenance mechanisms trigger apoptosis and autophagy. Lost control of autophagy increases cellular stress such as ER stress and induces apoptosis. The chain of injury, including apoptosis, oxidative stress, and inflammation, promotes disease progression while enhancing each other. In addition, cell aging is accelerated in the lungs of COPD, resulting in decreased cell proliferation, increased inflammation and decreased cell regeneration.
　COPDに対する現在の治療法は進行抑制を目指した対症療法であり（非特許文献１）、より有効な治療技術の開発が望まれている。また、有効な診断技術があれば、より早期の治療を行うことができる。 The current treatment method for COPD is symptomatic treatment aimed at suppressing the progression (Non-Patent Document 1), and the development of a more effective treatment technique is desired. In addition, if there is an effective diagnostic technique, earlier treatment can be performed.
　本発明は、COPD等の呼吸器疾患に対する治療、予防、及び診断技術を提供することを課題とする。 It is an object of the present invention to provide treatment, prevention, and diagnostic techniques for respiratory diseases such as COPD.
　本発明者は上記課題に鑑みて鋭意研究を行った結果、Fchsd1（FCH and double SH3 domains protein 1）の発現又は機能を抑制することにより呼吸器疾患を予防又は治療／改善することができ、さらにFchsd1が呼吸器疾患診断バイオマーカーとして利用できることを見出した。本発明者はこれらの知見に基づいてさらに研究を進めた結果、本発明を完成させた。即ち、本発明は、下記の態様を包含する。 As a result of diligent research in view of the above problems, the present inventor can prevent or treat / improve respiratory diseases by suppressing the expression or function of Fchsd1 (FCH and double SH3 domains protein 1). We found that Fchsd1 can be used as a biomarker for diagnosing respiratory diseases. The present inventor has completed the present invention as a result of further research based on these findings. That is, the present invention includes the following aspects.
　項１．　Fchsd1（FCH and double SH3 domains protein 1）発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種の成分を含有する、呼吸器疾患の予防又は治療剤。 Item 1. A preventive or therapeutic agent for respiratory diseases containing at least one component selected from the group consisting of an Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitor and an Fchsd1 function inhibitor.
　項２．　前記成分が、Fchsd1を標的としたポリヌクレオチド、該ポリヌクレオチドの発現カセット、低分子化合物、ペプチド、タンパク質、及び抗体からなる群より選択される少なくとも1種である、項1に記載の予防又は治療剤。 Item 2. Item 2. Prevention or treatment according to Item 1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody. Agent.
　項３．　前記呼吸器疾患が、酸化ストレスにより発症又は増悪し得る呼吸器疾患である、項１又は２に記載の予防又は治療剤。 Item 3. Item 3. The prophylactic or therapeutic agent according to Item 1 or 2, wherein the respiratory disease is a respiratory disease that can develop or worsen due to oxidative stress.
　項４．　前記呼吸器疾患が、感染性呼吸器疾患、気道閉塞性疾患、アレルギー性肺疾患、間質性肺疾患、腫瘍性肺疾患、肺血管性病変、胸膜疾患、及び呼吸不全からなる群より選択される少なくとも1種である、項１～３のいずれかに記載の予防又は治療剤。 Item 4. The respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure. The prophylactic or therapeutic agent according to any one of Items 1 to 3, which is at least one of the above.
　項４Ａ．　前記呼吸器疾患が、慢性閉塞性肺疾患（COPD）である、項１～４のいずれかに記載の予防又は治療剤。 Item 4A. Item 6. The prophylactic or therapeutic agent according to any one of Items 1 to 4, wherein the respiratory disease is chronic obstructive pulmonary disease (COPD).
　項５．　（1）被検体から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）を検出する工程を含む、呼吸器疾患を検査する方法。
　項５Ａ．（1）被検体から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）を検出する工程を含む、呼吸器疾患に罹患するリスクを判定する方法。 Item 5. (1) A method for examining a respiratory disease, which comprises a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject.
Item 5A. (1) A method for determining the risk of contracting a respiratory disease, which comprises a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject.
　項６．　さらに、（2）前記工程（1）で検出されたFchsd1の量又は濃度がカットオフ値以上である場合に、前記被検体が呼吸器疾患に罹患していると判定する工程、を含む、項５に記載の検査方法。
　項６Ａ．　さらに、（2）前記工程（1）で検出されたFchsd1の量又は濃度がカットオフ値以上である場合に、前記被検体が呼吸器疾患に呼吸器疾患に罹患するリスクがあると判定する工程、を含む、項５Ａに記載の検査方法。 Item 6. Further, the item comprising (2) a step of determining that the subject has a respiratory disease when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value. The inspection method according to 5.
Item 6A. Further, (2) a step of determining that the subject is at risk of suffering from a respiratory disease due to a respiratory disease when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value. , The inspection method according to Item 5A.
　項７．　前記Fchsd1がタンパク質である、項５、５Ａ、６又は６Ａに記載の方法。 Item 7. Item 6. The method according to Item 5, 5A, 6 or 6A, wherein the Fchsd1 is a protein.
　項８．　前記被検試料は、被検体の肺組織を含み得る被検試料である、項５、５Ａ、６、６Ａ、及び７のいずれかに記載の方法。 Item 8. Item 6. The method according to any one of Items 5, 5A, 6, 6A, and 7, wherein the test sample is a test sample that may contain lung tissue of the subject.
　項９．　前記肺組織を含み得る被検試料が、肺の生検試料、及び肺の洗浄液からなる群より選択される少なくとも1種を含む、項８に記載の検査方法。 Item 9. Item 8. The examination method according to Item 8, wherein the examination sample that may contain the lung tissue includes at least one selected from the group consisting of a lung biopsy sample and a lung lavage fluid.
　項１０．　Fchsd1（FCH and double SH3 domains protein 1）の検出剤を含む、呼吸器疾患の検査薬。 Item 10. A test drug for respiratory diseases, including a detection agent for Fchsd1 (FCH and double SH3 domains protein 1).
　項１１．　被検物質で処理された動物から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の予防又は治療剤の有効成分のスクリーニング方法。
　項１１Ａ．　被検物質で処理された細胞から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の予防又は治療剤の有効成分のスクリーニング方法。 Item 11. A method for screening an active ingredient of a preventive or therapeutic agent for respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
Item 11A. A method for screening an active ingredient of a preventive or therapeutic agent for respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from cells treated with a test substance as an index.
　項１２．　被検物質で処理された動物から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の誘発性又は増悪性の評価方法。
　項１２Ａ．　被検物質で処理された細胞におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の誘発性又は増悪性の評価方法。 Item 12. A method for evaluating the induction or exacerbation of respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
Item 12A. A method for evaluating the induction or exacerbation of respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in cells treated with a test substance as an index.
　項Ａ１．
　呼吸器疾患の予防または治療が必要な患者に、Fchsd1（FCH and double SH3 domains protein 1）発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種の成分を投与することを含む、呼吸器疾患の予防または治療方法。
　項Ａ２．
　前記成分が、Fchsd1を標的としたポリヌクレオチド、該ポリヌクレオチドの発現カセット、低分子化合物、ペプチド、タンパク質、及び抗体からなる群より選択される少なくとも1種である、項Ａ１に記載の方法。
　項Ａ３．
　前記呼吸器疾患が、酸化ストレスにより発症又は増悪し得る呼吸器疾患である、項Ａ１又はＡ２に記載の方法。
　項Ａ４．
　前記呼吸器疾患が、感染性呼吸器疾患、気道閉塞性疾患、アレルギー性肺疾患、間質性肺疾患、腫瘍性肺疾患、肺血管性病変、胸膜疾患、及び呼吸不全からなる群より選択される少なくとも1種である、項Ａ１～Ａ３のいずれかに記載の方法。
　項Ａ５．
　前記呼吸器疾患が、慢性閉塞性肺疾患（COPD）である、項Ａ１～Ａ４のいずれかに記載の方法。 Item A1.
Patients in need of prevention or treatment of respiratory diseases include at least one ingredient selected from the group consisting of Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitor and Fchsd1 function inhibitor. How to prevent or treat respiratory illness.
Item A2.
Item 6. The method according to Item A1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody.
Item A3.
Item 6. The method according to Item A1 or A2, wherein the respiratory disease is a respiratory disease that can develop or be exacerbated by oxidative stress.
Item A4.
The respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure. The method according to any one of Items A1 to A3, which is at least one of the above.
Item A5.
Item 6. The method according to any one of Items A1 to A4, wherein the respiratory disease is chronic obstructive pulmonary disease (COPD).
　項Ｂ１．
　呼吸器疾患の予防剤または治療剤の製造のための、Fchsd1（FCH and double SH3 domains protein 1）発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種の成分の使用。
　項Ｂ２．
　前記成分が、Fchsd1を標的としたポリヌクレオチド、該ポリヌクレオチドの発現カセット、低分子化合物、ペプチド、タンパク質、及び抗体からなる群より選択される少なくとも1種である、項Ｂ１に記載の使用。
　項Ｂ３．
　前記呼吸器疾患が、酸化ストレスにより発症又は増悪し得る呼吸器疾患である、項Ｂ１又はＢ２に記載の使用。
　項Ｂ４．
　前記呼吸器疾患が、感染性呼吸器疾患、気道閉塞性疾患、アレルギー性肺疾患、間質性肺疾患、腫瘍性肺疾患、肺血管性病変、胸膜疾患、及び呼吸不全からなる群より選択される少なくとも1種である、請求項Ｂ１～Ｂ３のいずれかに記載の使用。
　項Ｂ５．
　前記呼吸器疾患が、慢性閉塞性肺疾患（COPD）である、項Ｂ１～Ｂ４のいずれかに記載の使用。 Item B1.
Use of at least one ingredient selected from the group consisting of Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitors and Fchsd1 function inhibitors for the manufacture of prophylactic or therapeutic agents for respiratory diseases.
Item B2.
Item 6. The use according to Item B1, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody.
Item B3.
Item 6. The use according to Item B1 or B2, wherein the respiratory disease is a respiratory disease that can develop or be exacerbated by oxidative stress.
Item B4.
The respiratory disease is selected from the group consisting of infectious respiratory disease, airway obstruction disease, allergic lung disease, interstitial lung disease, neoplastic lung disease, pulmonary vascular lesion, pleural disease, and respiratory failure. The use according to any one of claims B1 to B3, which is at least one of the above.
Item B5.
Item 6. The use according to any one of Items B1 to B4, wherein the respiratory disease is chronic obstructive pulmonary disease (COPD).
　本発明によれば、呼吸器疾患に対する治療、予防、及び診断技術を提供することができる。 According to the present invention, it is possible to provide treatment, prevention, and diagnostic techniques for respiratory diseases.
エラスターゼの気管内投与の7日後のマウス肺におけるFCHSD1及び他のFCHファミリータンパク質のレベルを、イムノブロッティングにより決定した結果を示す。The results of immunoblotting determination of the levels of FCHSD1 and other FCH family proteins in mouse lung 7 days after intratracheal administration of elastase are shown. 200μMのH2O2に12時間さらされたMLE-12細胞におけるFCHSD1及び他のFCHファミリータンパク質のレベルを、イムノブロッティングによって決定した結果を示す。The results of immunoblotting determination of the levels of FCHSD1 and other FCH family proteins in MLE-12 cells exposed to 200 μM H 2 O 2 for 12 hours are shown. LPSの気管内投与の1日後及び3日後のFCHSD1のレベルを、イムノブロッティングによって決定した結果を示す。The results of determining the level of FCHSD1 1 day and 3 days after intratracheal administration of LPS by immunoblotting are shown. 非刺激マウスの様々な組織ホモジネートおけるFCHSD1のレベルを、イムノブロッティングによって決定した結果を示す。The results of determining the level of FCHSD1 in various tissue homogenates of unstimulated mice by immunoblotting are shown. エラスターゼの気管内投与後の肺のMRI画像を示す。MRI images of the lungs after intratracheal administration of elastase are shown. エラスターゼの気管内投与後の平均肺胞径（M.L.I.）を示す。データは平均±SEMで表される。**はP < 0.01を示す。The average alveolar diameter (M.L.I.) after intratracheal administration of elastase is shown. Data are expressed as mean ± SEM. ** indicates P <0.01. エラスターゼの気管内投与後の肺気腫面積の割合を示す。データは平均±SEMで表される。**はP < 0.01を示す。The percentage of emphysema area after intratracheal administration of elastase is shown. Data are expressed as mean ± SEM. ** indicates P <0.01. エラスターゼの気管内投与後のFchsd1-/-及びWTマウスのBAL液への細胞流入を、フローサイトメトリー解析により決定した結果を示す。データは、平均±SEMとして表される。*はP < 0.05を示す。The results of flow cytometric analysis of cell influx of Fchsd1 -/- and WT mice into the BAL fluid after intratracheal administration of elastase are shown. The data are expressed as mean ± SEM. * Indicates P <0.05. エラスターゼの気管内投与後の24時間後のFchsd1-/-及びWTマウスのBAL液中の好中球数を、フローサイトメトリー解析により決定した結果を示す。データは、平均±SEMとして表される。**はP < 0.01を示す。The results of flow cytometric analysis of the number of neutrophils in the BAL solution of Fchsd1 -/- and WT mice 24 hours after intratracheal administration of elastase are shown. The data are expressed as mean ± SEM. ** indicates P <0.01. エラスターゼの気管内投与後の24時間後のFchsd1-/-及びWTマウスのBAL液中のTNFα濃度を、ELISA法で測定した結果を示す。データは平均値±SEMで表される。*はP < 0.05を示す。The results of measuring the TNFα concentration in the BAL solution of Fchsd1 -/- and WT mice 24 hours after the intratracheal administration of elastase by the ELISA method are shown. The data is expressed as mean ± SEM. * Indicates P <0.05. PBS又はエラスターゼを気管内投与したFchsd1-/-及びWTマウスの肺切片をTUNEL染色した結果を示す。肺のTUNEL陽性細胞は茶色で示される。スケールバー：200μm。The results of TUNEL staining of lung sections of Fchsd1 -/- and WT mice to which PBS or elastase were intratracheally administered are shown. Lung TUNEL-positive cells are shown in brown. Scale bar: 200 μm. Fchsd1-/-及びWTマウス肺から分離した肺線維芽細胞（MLF）を250μMのH2O2で12時間処理した後のイムノブロット分析結果を示す。The results of immunoblot analysis after treating lung fibroblasts (MLF) isolated from Fchsd1 -/- and WT mouse lungs with 250 μM H 2 O 2 for 12 hours are shown. 50μM H2O2で24時間処理した後のFchsd1-/-及びWT MLFのイムノブロット分析結果を示す。The results of immunoblot analysis of Fchsd1 -/- and WT MLF after treatment with 50 μM H 2 O 2 for 24 hours are shown. レトロウイルスFchsd1又は空の対照ベクターでトランスフェクトしたMLE-12細胞における、250μM H2O2によって引き起こされる酸化ストレスに応答したIl6及びTnfa mRNAレベルを示す。データは、平均±SEMとして表される。 Il 6 and Tnfa mRNA levels in response to oxidative stress caused by 250 μM H 2 O 2 in MLE-12 cells transfected with the retrovirus Fchsd1 or an empty control vector are shown. The data are expressed as mean ± SEM. 未処理の又は275μMのH2O2で10分間処理したFchsd1-/-及びWT MLFの核及び細胞質画分のイムノブロット分析結果を示す。The results of immunoblot analysis of the nuclei and cytoplasmic fractions of Fchsd1 -/- and WT MLF treated with untreated or 275 μM H 2 O 2 for 10 minutes are shown. MLE12細胞を、Flagタグ付きFCHSD1、Mycタグ付きSNX9、HAタグ付きNRF2、又は空ベクターでトランスフェクトし、結合したNRF2又はSNX9のウェスタン分析の前に抗Flag樹脂で免疫沈降（IP）させた結果を示す。下のパネルは、全細胞溶解液（WCL）をイムノブロットした結果を示す。Results of transfection of MLE12 cells with Flag-tagged FCHSD1, Myc-tagged SNX9, HA-tagged NRF2, or empty vector and immunoprecipitation (IP) with anti-Flag resin prior to Western analysis of bound NRF2 or SNX9. Is shown. The lower panel shows the results of immunoblots of whole cell lysate (WCL). 250μM H2O2処理（5、15、又は60分間）による酸化ストレスに応答して、Flagタグ付きFCHSD1又は空ベクターを発現するMLE-12細胞をイムノブロット分析及び抗Flag樹脂を用いて免疫沈降した結果を示す。MLE-12 cells expressing Flag-tagged FCHSD1 or empty vector in response to oxidative stress from 250 μM H 2 O 2 treatment (5, 15, or 60 minutes) were immunoprecipitated using immunoblot analysis and anti-Flag resin. The result is shown. 250μM H2O2処理（10又は30分間）による酸化ストレスに応答して、Flagタグ付きSNX9を発現するMLE-12細胞をイムノブロット分析及び抗Flag樹脂を用いて免疫沈降した結果を示す。The results of immunoprecipitation of MLE-12 cells expressing Flag-tagged SNX9 in response to oxidative stress by 250 μM H 2 O 2 treatment (10 or 30 minutes) using immunoblot analysis and anti-Flag resin are shown. 非刺激状態では、FCHSD1は、細胞質中でNRF2及びSNX9と複合体を形成し、NRF2が核にトランスロケーションするのを阻止する。また、NRF2は、その内因性阻害剤であるKEAP1と結合し、プロテアソーム分解の標的となる。酸化ストレスに応答して、KEAP1は修飾され、NRF2を安定化する。その後、FCHSD1は複合体から解離し、核膜に結合したImportin 8に結合したSNX9はNRF2を標的化する。In the unstimulated state, FCHSD1 forms a complex with NRF2 and SNX9 in the cytoplasm and blocks NRF2 from translocating to the nucleus. In addition, NRF2 binds to its endogenous inhibitor, KEAP1, and is a target for proteasome degradation. In response to oxidative stress, KEAP1 is modified and stabilizes NRF2. FCHSD1 then dissociates from the complex and SNX9 bound to Importin8 bound to the nuclear envelope targets NRF2.
　1．定義
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 1. 1. Definitions In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".
　アミノ酸配列の「同一性」とは、2以上の対比可能なアミノ酸配列の、お互いに対するアミノ酸配列の一致の程度をいう。従って、ある2つのアミノ酸配列の一致性が高いほど、それらの配列の同一性又は類似性は高い。アミノ酸配列の同一性のレベルは、例えば、配列分析用ツールであるFASTAを用い、デフォルトパラメータを用いて決定される。若しくは、Karlin及びAltschulによるアルゴリズムBLAST（KarlinS， Altschul SF．“Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes”Proc Natl Acad Sci USA．87:2264－2268（1990）、KarlinS，Altschul SF．“Applications and statistics for multiple high－scoring segments in molecular sequences．”Proc Natl Acad Sci USA．90:5873－7（1993））を用いて決定できる。このようなBLASTのアルゴリズムに基づいたBLASTXと呼ばれるプログラムが開発されている。これらの解析方法の具体的な手法は公知であり、National Center of Biotechnology Information（NCBI）のウェエブサイト（http://www.ncbi.nlm.nih.gov/）を参照すればよい。また、塩基配列の『同一性』も上記に準じて定義される。 "Identity" of amino acid sequences refers to the degree of coincidence of amino acid sequences with each other among two or more comparable amino acid sequences. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessment the statistical signature of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), K It can be determined using "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90: 5873-7 (1993). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. The "identity" of the base sequence is also defined according to the above.
　本明細書中において、「保存的置換」とは、アミノ酸残基が類似の側鎖を有するアミノ酸残基に置換されることを意味する。例えば、リジン、アルギニン、ヒスチジンといった塩基性側鎖を有するアミノ酸残基同士で置換されることが、保存的な置換にあたる。その他、アスパラギン酸、グルタミン酸といった酸性側鎖を有するアミノ酸残基；グリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システインといった非帯電性極性側鎖を有するアミノ酸残基；アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファンといった非極性側鎖を有するアミノ酸残基；スレオニン、バリン、イソロイシンといったβ－分枝側鎖を有するアミノ酸残基；チロシン、フェニルアラニン、トリプトファン、ヒスチジンといった芳香族側鎖を有するアミノ酸残基同士での置換も同様に、保存的な置換にあたる。 As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. Other amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine; aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitutions between amino acid residues are also conservative substitutions.
　本明細書において、「核酸」及び「ポリヌクレオチド」は、特に制限されず、天然、人工のいずれのものも包含する。具体的には、DNA、RNA等の他にも、次に例示するように、公知の化学修飾が施されたものであってもよい。ヌクレアーゼなどの加水分解酵素による分解を防ぐために、各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（PS）、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換することができる。また、各リボヌクレオチドの糖（リボース）の2位の水酸基を、-OR（Rは、例えばCH3（2´-O-Me）、CH2CH2OCH3（2´-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等を示す）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などが挙げられる。さらには、リン酸部分やヒドロキシル部分が、例えば、ビオチン、アミノ基、低級アルキルアミン基、アセチル基等で修飾されたものなどを挙げることができるが、これに限定されない。また、ヌクレオチドの糖部の2´酸素と4´炭素を架橋することにより、糖部のコンフォーメーションをN型に固定したものであるBNA（LNA）等もまた、用いられ得る。 In the present specification, "nucleic acid" and "polynucleotide" are not particularly limited and include both natural and artificial ones. Specifically, in addition to DNA, RNA and the like, known chemical modifications may be applied as illustrated below. Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolases such as nucleases. Can be done. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is designated as -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned. Further, examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto. Further, BNA (LNA) or the like in which the conformation of the sugar portion is fixed to N-type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be used.
　2．呼吸器疾患の予防又は治療剤
　本発明は、その一態様において、Fchsd1発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種を含有する、呼吸器疾患の予防又は治療剤（本明細書において、「本発明の剤」と示すこともある。）に関する。以下に、これについて説明する。 2. Prophylactic or Therapeutic Agent for Respiratory Disease In one aspect of the present invention, the present invention comprises at least one selected from the group consisting of an Fchsd1 expression inhibitor and an Fchsd1 function inhibitor, which comprises at least one of them (the present invention). In the specification, it may be referred to as "the agent of the present invention"). This will be described below.
　2-1．調節対象（Fchsd1）
　Fchsd1遺伝子はF-BARファミリーに属する遺伝子である。発現又は機能抑制対象であるFchsd1（Fchsd1タンパク質、Fchsd1 mRNA）は、Fchsd1遺伝子の発現産物であり、呼吸器疾患の予防又は治療対象の生物又はその細胞（特に肺組織の細胞）内で発現しているFchsd1タンパク質又はFchsd1 mRNAである。よって、該対象の生物種に応じて、抑制対象であるFchsd1タンパク質及びFchsd1 mRNAも変わる。該生物種としては、特に制限されず、動物、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類が挙げられる。 2-1. Adjustment target (Fchsd1)
The Fchsd1 gene is a gene belonging to the F-BAR family. Fchsd1 (Fchsd1 protein, Fchsd1 mRNA), which is the target of expression or function suppression, is an expression product of the Fchsd1 gene and is expressed in the organism or its cells (particularly cells of lung tissue) to be prevented or treated for respiratory diseases. Fchsd1 protein or Fchsd1 mRNA. Therefore, the Fchsd1 protein and Fchsd1 mRNA to be suppressed also change depending on the target organism species. The organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
　種々の生物種由来Fchsd1タンパク質のアミノ酸配列及びFchsd1 mRNAの塩基配列は公知である。具体的には、例えば、ヒトFchsd1タンパク質としては配列番号1に示されるアミノ酸配列からなるタンパク質（NCBI Reference Sequence：NP_258260.1）が挙げられ、マウスFchsd1タンパク質としては配列番号2に示されるアミノ酸配列からなるタンパク質（NCBI Reference Sequence：NP_783615.2）などが挙げられ、ヒトFchsd1 mRNAとしては配列番号3に示される塩基配列からなるmRNA（NCBI Reference Sequence：NM_033449.3）が挙げられ、マウスFchsd1 mRNAとしては配列番4に示される塩基配列からなるmRNA（NCBI Reference Sequence：NM_175684.4）などが挙げられる。また、Fchsd1タンパク質及びFchsd1 mRNAとしては、上記のスプライシングバリアントも包含され得る。 The amino acid sequence of Fchsd1 protein derived from various species and the base sequence of Fchsd1 mRNA are known. Specifically, for example, the human Fchsd1 protein includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_258260.1), and the mouse Fchsd1 protein includes the amino acid sequence shown in SEQ ID NO: 2. (NCBI Reference Sequence: NP_783615.2) and the like, and the human Fchsd1 mRNA includes an mRNA consisting of the base sequence shown in SEQ ID NO: 3 (NCBI Reference Sequence: NM_033449.3), and the mouse Fchsd1 mRNA includes Examples thereof include mRNA (NCBI Reference Sequence: NM_175684.4) consisting of the base sequence shown in SEQ ID NO: 4. The Fchsd1 protein and Fchsd1 mRNA may also include the above splicing variants.
　調節対象であるFchsd1タンパク質は、その本来の性質、すなわち細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を有する限りにおいて、置換、欠失、付加、挿入などのアミノ酸変異を有していてもよい。変異としては、活性がより損なわれ難いという観点から、好ましくは置換、より好ましくは保存的置換が挙げられる。 The Fchsd1 protein to be regulated has amino acid mutations such as substitutions, deletions, additions, and insertions as long as it has the original property, that is, the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex. May be. The mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the activity is less likely to be impaired.
　調節対象であるFchsd1 mRNAも、該mRNAから翻訳されるタンパク質が、その本来の性質、すなわち細胞質でNRF2及びSNX9と相互作用して複合体を形成し、酸化ストレスに応答して複合体から解離する性質を有する限りにおいて、置換、欠失、付加、挿入などの塩基変異を有していてもよい。変異としては、該mRNAから翻訳されるタンパク質においてアミノ酸置換が生じない変異やアミノ酸の保存的置換が生じる変異が好ましい。 Fchsd1 mRNA, which is the subject of regulation, also has the protein translated from the mRNA, which interacts with NRF2 and SNX9 in the cytoplasm to form a complex, and dissociates from the complex in response to oxidative stress. As long as it has properties, it may have basic mutations such as substitutions, deletions, additions, and insertions. As the mutation, a mutation that does not cause an amino acid substitution or a mutation that causes a conservative substitution of an amino acid in the protein translated from the mRNA is preferable.
　調節対象であるFchsd1タンパク質の好ましい具体例としては、下記（a）に記載するタンパク質及び下記（b）に記載するタンパク質：
　（a）配列番号1～2のいずれかに示されるアミノ酸配列からなるタンパク質、及び　（b）配列番号1～2のいずれかに示されるアミノ酸配列と85％以上の同一性を有するアミノ酸配列からなり、且つ細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を有するタンパク質
からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of the Fchsd1 protein to be regulated include the protein described in (a) below and the protein described in (b) below:
It consists of (a) a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 and 2, and (b) an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 and 2. And at least one selected from the group consisting of proteins having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
　上記（b）において、同一性は、より好ましくは90％以上、さらに好ましくは95％以上、よりさらに好ましくは98％以上である。 In (b) above, the identity is more preferably 90% or more, further preferably 95% or more, still more preferably 98% or more.
　上記（b）に記載するタンパク質の一例としては、例えば
（b’）配列番号1～2のいずれかに示されるアミノ酸配列に対して1若しくは複数個のアミノ酸が置換、欠失、付加、又は挿入されたアミノ酸配列からなり、且つ細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を有するタンパク質
が挙げられる。 As an example of the protein described in (b) above, for example, one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any one of (b') SEQ ID NOs: 1 and 2. Examples thereof include proteins having the amino acid sequence obtained and having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
　上記（b’）において、複数個とは、例えば2～20個であり、好ましくは2～10個であり、より好ましくは2～5個であり、よりさらに好ましくは2又は3個である。 In the above (b'), the plurality is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.
　調節対象であるFchsd1 mRNAの好ましい具体例としては、下記（c）に記載するmRNA及び下記（d）に記載するmRNA：
　（c）配列番号3～4のいずれかに示される塩基配列からなるmRNA、及び
　（d）配列番号3～4のいずれかに示される塩基配列と85％以上の同一性を有する塩基配列からなり、且つ細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を有するタンパク質をコードするmRNA
からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of Fchsd1 mRNA to be regulated include the mRNA described in (c) below and the mRNA described in (d) below:
(C) mRNA consisting of the base sequence shown in any of SEQ ID NOs: 3 to 4, and (d) consisting of a base sequence having 85% or more identity with the base sequence shown in any of SEQ ID NOs: 3 to 4. And mRNA encoding a protein that has the property of interacting with NRF2 and SNX9 to form a complex in the cytoplasm.
At least one species selected from the group consisting of.
　上記（d）において、同一性は、より好ましくは90％以上、さらに好ましくは95％以上、よりさらに好ましくは98％以上である。 In (d) above, the identity is more preferably 90% or more, further preferably 95% or more, still more preferably 98% or more.
　上記（d）に記載するmRNAの一例としては、例えば
（d’）配列番号3～4のいずれかに示される塩基配列に対して1若しくは複数個の塩基が置換、欠失、付加、又は挿入された塩基配列からなり、且つ細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を有するタンパク質をコードするmRNA
が挙げられる。 As an example of the mRNA described in (d) above, for example, one or more bases are substituted, deleted, added, or inserted into the base sequence shown in any one of (d') SEQ ID NOs: 3 to 4. An mRNA encoding a protein consisting of the base sequence and having the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
Can be mentioned.
　上記（d’）において、複数個とは、例えば2～200個であり、好ましくは2～100個であり、より好ましくは2～50個であり、よりさらに好ましくは2～10個である。 In the above (d'), the plurality is, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, and even more preferably 2 to 10.
　2-2．有効成分
　本発明の剤の有効成分は、Fchsd1発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種の成分である。当該成分としては、好ましくはFchsd1を標的としたポリヌクレオチド、該ポリヌクレオチドの発現カセット、低分子化合物、ペプチド、タンパク質、抗体等が挙げられる。以下に、Fchsd1発現抑制剤及びFchsd1機能抑制剤について具体的に説明する。 2-2. Active Ingredient The active ingredient of the agent of the present invention is at least one ingredient selected from the group consisting of an Fchsd1 expression inhibitor and an Fchsd1 function inhibitor. Examples of the component include a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, an antibody and the like. The Fchsd1 expression inhibitor and the Fchsd1 function inhibitor will be specifically described below.
　2-2-1．Fchsd1発現抑制剤
　Fchsd1発現抑制剤は、呼吸器疾患の予防又は治療対象の生物又はその細胞（特に肺組織の細胞）内で発現しているFchsd1タンパク質及び／又はFchsd1 mRNAの発現量を抑制可能なものである限り、特に制限されない。Fchsd1発現抑制剤は、1種単独で用いることもできるし、2種以上を組み合わせて用いることもできる。 2-2-1. Fchsd1 expression inhibitor Fchsd1 expression inhibitor can suppress the expression level of Fchsd1 protein and / or Fchsd1 mRNA expressed in the organism to be prevented or treated for respiratory diseases or its cells (particularly cells of lung tissue). As long as it is a thing, there is no particular limitation. The Fchsd1 expression inhibitor can be used alone or in combination of two or more.
　Fchsd1発現抑制剤としては、例えばFchsd1特異的small interfering RNA（siRNA）、Fchsd1特異的microRNA（miRNA）、Fchsd1特異的アンチセンス核酸、これらの発現カセット；　Fchsd1特異的リボザイム；　CRISPR/CasシステムによるFchsd1遺伝子編集剤などが挙げられる。 Examples of the Fchsd1 expression inhibitor include Fchsd1-specific small interfering RNA (siRNA), Fchsd1-specific microRNA (miRNA), Fchsd1-specific antisense nucleic acid, their expression cassettes; Fchsd1-specific ribozyme; Fchsd1 gene by CRISPR / Cas system. Examples include editorial agents.
　なお、発現抑制とは、Fchsd1タンパク質、Fchsd1 mRNAなどの発現量を、例えば1/2、1/3、1/5、1/10、1/20、1/30、1/50、1/100、1/200、1/300、1/500、1/1000、1/10000以下に抑制することを意味し、これらの発現量を0とすることをも包含する。 In addition, expression suppression means that the expression levels of Fchsd1 protein, Fchsd1 mRNA, etc. are, for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and includes setting the expression level of these to 0.
　2-2-1-1．siRNA、miRNA、アンチセンス核酸、リボザイム
　Fchsd1特異的siRNAは、Fchsd1をコードする遺伝子の発現を特異的に抑制する二本鎖RNA分子である限り特に制限されない。一実施形態において、siRNAは、例えば、18塩基以上、19塩基以上、20塩基以上、又は21塩基以上の長さであることが好ましい。また、siRNAは、例えば、25塩基以下、24塩基以下、23塩基以下、又は22塩基以下の長さであることが好ましい。ここに記載するsiRNAの長さの上限値及び下限値は任意に組み合わせることが想定される。例えば、下限が18塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が19塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が20塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が21塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さの組み合わせが想定される。 2-2-1-1. The siRNA, miRNA, antisense nucleic acid, and ribozyme Fchsd1-specific siRNA are not particularly limited as long as they are double-stranded RNA molecules that specifically suppress the expression of the gene encoding Fchsd1. In one embodiment, the siRNA is preferably, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more in length. Further, the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is assumed that the upper and lower limits of the siRNA lengths described here can be arbitrarily combined. For example, the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases. A length; a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 A combination of lengths that are bases is assumed.
　siRNAは、shRNA(small hairpin RNA)であっても良い。shRNAは、その一部がステムループ構造を形成するように設計することができる。例えば、shRNAは、ある領域の配列を配列aとし、配列aに対する相補鎖を配列bとすると、配列a、スペーサー、配列bの順になるようにこれらの配列が一本のRNA鎖に存在するようにし、全体で45～60塩基の長さとなるように設計することができる。配列aは、標的となるFchsd1をコードする塩基配列の一部の領域の配列であり、標的領域は特に限定されず、任意の領域を候補にすることが可能である。そして配列aの長さは19～25塩基、好ましくは19～21塩基である。 The siRNA may be shRNA (small hairpin RNA). shRNA can be designed so that a part of it forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand in the order of sequence a, spacer, and sequence b. It can be designed to have a total length of 45 to 60 bases. The sequence a is a sequence of a part of the base sequence encoding the target Fchsd1, and the target region is not particularly limited, and any region can be a candidate. The length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
　Fchsd1特異的siRNAは、5’又は3’末端に、付加的な塩基を有していてもよい。該付加的塩基の長さは、通常2～4塩基程度である。該付加的塩基は、DNAでもRNAでもよいが、DNAを用いると核酸の安定性を向上させることができる場合がある。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’などの配列が挙げられるが、これらに限定されるものではない。 The Fchsd1-specific siRNA may have an additional base at the 5'or 3'end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but DNA may be used to improve the stability of the nucleic acid. The sequences of such additional bases include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Sequences such as', uuuuu-3'can be mentioned, but are not limited to these.
　siRNAは、3'末端に突出部配列（オーバーハング）を有していてもよく、具体的には、dTdT（dTはデオキシチミジンを表わす）を付加したものが挙げられる。また、末端付加がない平滑末端（ブラントエンド）であってもよい。siRNAは、センス鎖とアンチセンス鎖が異なる塩基数であってもよく、例えば、アンチセンス鎖が3'末端及び5'末端に突出部配列（オーバーハング）を有している「asymmetrical interfering RNA（aiRNA）」を挙げることができる。典型的なaiRNAは、アンチセンス鎖が21塩基からなり、センス鎖が15塩基からなり、アンチセンス鎖の両端で各々3塩基のオーバーハング構造をとる。 The siRNA may have a protruding sequence (overhang) at the 3'end, and specific examples thereof include those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end without end addition. The siRNA may have a different number of bases in the sense strand and the antisense strand, for example, "asymmetrical interfering RNA" in which the antisense strand has a protruding sequence (overhang) at the 3'end and the 5'end. aiRNA) ”. A typical aiRNA consists of 21 bases in the antisense strand and 15 bases in the sense strand, and has an overhang structure of 3 bases at both ends of the antisense strand.
　Fchsd1特異的siRNAの標的配列の位置は特に制限されるわけではないが、一実施形態において、5’-UTR及び開始コドンから約50塩基まで、並びに3’-UTR以外の領域から標的配列を選択することが望ましい。選択された標的配列の候補群について、標的以外のmRNAにおいて16-17塩基の連続した配列に相同性がないかどうかを、BLAST（http://www.ncbi.nlm.nih.gov/BLAST/）などのホモロジー検索ソフトを用いて調べ、選択した標的配列の特異性を確認することが好ましい。特異性が確認された標的配列について、AA（もしくはNA）以降の19－21塩基にTTもしくはUUの3’末端オーバーハングを有するセンス鎖と、該19－21塩基に相補的な配列及びTTもしくはUUの3’末端オーバーハングを有するアンチセンス鎖とからなる2本鎖RNAをsiRNAとして設計してもよい。また、siRNAの前駆体であるshRNAは、ループ構造を形成しうる任意のリンカー配列（例えば、5-25塩基程度）を適宜選択し、上記センス鎖とアンチセンス鎖とを該リンカー配列を介して連結することにより設計することができる。 The position of the target sequence of the Fchsd1-specific siRNA is not particularly limited, but in one embodiment, the target sequence is selected from 5'-UTR and from the start codon to about 50 bases, and from regions other than 3'-UTR. It is desirable to do. For the selected target sequence candidate group, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) is checked to see if there is homology in the consecutive sequences of 16-17 bases in the non-target mRNA. ), Etc., to confirm the specificity of the selected target sequence. For the target sequence whose specificity was confirmed, a sense strand having a 3'end overhang of TT or UU at 19-21 bases after AA (or NA), and a sequence complementary to the 19-21 base and TT or A double-stranded RNA consisting of an antisense strand with a 3'end overhang of UU may be designed as a siRNA. Further, for the shRNA that is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are passed through the linker sequence. It can be designed by connecting.
　siRNA及び／又はshRNAの配列は、種々のwebサイト上に無料で提供される検索ソフトを用いて検索が可能である。このようなサイトとしては、例えば、以下を挙げることができる。Ambionが提供するsiRNA Target Finder（http://www.ambion.com/jp/techlib/misc/siRNA_finder.html）pSilencer（登録商標）Expression Vector用インサートデザインツール（http://www.ambion.com/jp/techlib/misc/psilencer_converter.html）RNAi Codexが提供するGeneSeer（http://codex.cshl.edu/scripts/newsearchhairpin.cgi）。 SiRNA and / or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include the following. SiRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) pSilencer® Expression Vector Insert Design Tool (http://www.ambion.com/) provided by Ambion jp / techlib / misc / psilencer_converter.html) GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.
　siRNAは、mRNA上の標的配列のセンス鎖及びアンチセンス鎖をDNA/RNA自動合成機でそれぞれ合成し、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、siRNAの前駆体となるshRNAを合成し、これを、RNA切断タンパク質ダイサー（dicer）を用いて切断することにより調製することもできる。このようなFchsd1特異的siRNAとしては、例えば、Merck社から販売されるSASI_Hs01_00242324、SASI_Hs02_00307980、SASI_Mm01_00134930などを購入して使用することができる。 For siRNA, the sense strand and antisense strand of the target sequence on mRNA are synthesized by a DNA / RNA automatic synthesizer, respectively, and denatured in an appropriate annealing buffer at about 90 to about 95 ° C. for about 1 minute. It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA as a precursor of siRNA and cleaving it with an RNA-cleaving protein dicer. As such Fchsd1-specific siRNA, for example, SASI_Hs01_00242324, SASI_Hs02_00307980, SASI_Mm01_00134930 and the like sold by Merck can be purchased and used.
　Fchsd1特異的miRNAは、Fchsd1をコードする遺伝子の翻訳を阻害する限り任意である。例えば、miRNAは、siRNAのように標的mRNAを切断するのではなく、標的の3’非翻訳領域（UTR）に対合してその翻訳を阻害してもよい。miRNAは、pri-miRNA（primary miRNA）、pre-miRNA（precursor miRNA）、及び成熟miRNAのいずれでもよい。miRNAの長さは特に制限されず、pri-miRNAの長さは通常数百～数千塩基であり、pre-miRNAの長さは通常50～80塩基であり、成熟miRNAの長さは通常18～30塩基である。一実施形態において、Fchsd1特異的miRNAは、好ましくはpre-miRNA又は成熟miRNAであり、より好ましくは成熟miRNAである。このようなFchsd1特異的miRNAは、公知の手法で合成してもよく、合成RNAを提供する会社から購入してもよい。 Fchsd1-specific miRNAs are optional as long as they inhibit translation of the gene encoding Fchsd1. For example, miRNAs may pair with the target's 3'untranslated region (UTR) and inhibit its translation, rather than cleaving the target mRNA as with siRNA. The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of the miRNA is not particularly limited, the length of the pri-miRNA is usually hundreds to thousands of bases, the length of the pre-miRNA is usually 50-80 bases, and the length of the mature miRNA is usually 18 ~ 30 bases. In one embodiment, the Fchsd1-specific miRNA is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA. Such Fchsd1-specific miRNAs may be synthesized by known methods or may be purchased from companies that provide synthetic RNAs.
　Fchsd1特異的アンチセンス核酸とは、Fchsd1をコードする遺伝子のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列又はその一部を含む核酸であって、該mRNAと特異的かつ安定した二重鎖を形成して結合することにより、Fchsd1タンパク質合成を抑制する機能を有する核酸である。アンチセンス核酸はDNA、RNA、DNA/RNAキメラのいずれでもよい。アンチセンス核酸がDNAの場合、標的RNAとアンチセンスDNAとによって形成されるRNA:DNAハイブリッドは、内在性リボヌクレアーゼH（RNase H）に認識されて標的RNAの選択的な分解を引き起こす。したがって、RNase Hによる分解を指向するアンチセンスDNAの場合、標的配列は、mRNA中の配列だけでなく、Fchsd1遺伝子の初期翻訳産物におけるイントロン領域の配列であってもよい。イントロン配列は、ゲノム配列と、Fchsd1遺伝子のcDNA塩基配列とをBLAST、FASTAなどのホモロジー検索プログラムを用いて比較することにより、決定することができる。 The Fchsd1-specific antisense nucleic acid is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the gene encoding Fchsd1, or a part thereof, and is specific and stable to the mRNA. It is a nucleic acid having a function of suppressing Fchsd1 protein synthesis by forming a double chain and binding to it. The antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA is recognized by the endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the Fchsd1 gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the Fchsd1 gene using a homology search program such as BLAST or FASTA.
　Fchsd1特異的アンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果としてFchsd1タンパク質への翻訳が阻害されるものであればその長さは制限されない。Fchsd1特異的アンチセンス核酸は、Fchsd1をコードするmRNAの全配列であっても部分配列であってもよい。合成の容易さや抗原性、細胞内移行性の問題などを考慮すれば、約10～約40塩基、特に約15～約30塩基からなるオリゴヌクレオチドが好ましいが、これらに限定されるものではない。より具体的には、Fchsd1遺伝子の5’端ヘアピンループ、5’端非翻訳領域、翻訳開始コドン、タンパク質コード領域、ORF翻訳終止コドン、3’端非翻訳領域、3’端パリンドローム領域又は3’端ヘアピンループなどをアンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されるものではない。 The length of the target region of the Fchsd1-specific antisense nucleic acid is not limited as long as the hybridization of the antisense nucleic acid results in inhibition of translation into the Fchsd1 protein. The Fchsd1-specific antisense nucleic acid may be a full sequence or a partial sequence of the mRNA encoding Fchsd1. Considering the ease of synthesis, antigenicity, intracellular transferability, and the like, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases are preferable, but the oligonucleotide is not limited thereto. More specifically, the 5'end hairpin loop, 5'end untranslated region, translation initiation codon, protein coding region, ORF translation stop codon, 3'end untranslated region, 3'end parindrome region or 3 of the Fchsd1 gene. 'End hairpin loops and the like can be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.
　Fchsd1特異的アンチセンス核酸は、Fchsd1遺伝子のmRNAや初期転写産物とハイブリダイズしてタンパク質への翻訳を阻害するだけでなく、二本鎖DNAであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、RNAへの転写を阻害し得るもの（アンチジーン（antigene））であってもよい。 Fchsd1-specific antisense nucleic acids not only hybridize with the mRNA and early transcripts of the Fchsd1 gene to inhibit translation into proteins, but also bind to these double-stranded DNA genes to triple-strand (triplex). ) And can inhibit transcription into RNA (antigene).
　Fchsd1特異的siRNA、Fchsd1特異的miRNA、及びFchsd1特異的アンチセンス核酸などは、Fchsd1遺伝子のcDNA配列もしくはゲノミックDNA配列に基づいてmRNAもしくは初期転写産物の標的配列を決定し、市販のDNA/RNA自動合成機を用いて、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。また、各種修飾を含むアンチセンス核酸も、いずれも公知の手法により、化学的に合成することができる。 For Fchsd1-specific siRNA, Fchsd1-specific miRNA, Fchsd1-specific antisense nucleic acid, etc., the target sequence of mRNA or early transcript is determined based on the cDNA sequence or genomic DNA sequence of the Fchsd1 gene, and commercially available DNA / RNA automation is performed. It can be prepared by synthesizing a sequence complementary to this using a synthesizer. In addition, all antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
　Fchsd1特異的siRNA、Fchsd1特異的miRNA、又はFchsd1特異的アンチセンス核酸の発現カセットについては、Fchsd1特異的siRNA、Fchsd1特異的miRNA、又はFchsd1特異的アンチセンス核酸が発現可能な状態で組み込まれているポリヌクレオチドである限りにおいて特に限定されない。典型的には、該発現カセットは、プロモーター配列、及びFchsd1特異的siRNA、Fchsd1特異的miRNA、又はFchsd1特異的アンチセンス核酸のコード配列（必要に応じて、さらに転写終結シグナル配列）を含むポリヌクレオチド、必要に応じて他の配列を含む。プロモーターは、特に制限されず、例えばCMVプロモーター、EF1プロモーター、SV40プロモーター、MSCVプロモーター、hTERTプロモーター、βアクチンプロモーター、CAGプロモーターなどのRNA polymerase II（polII）系プロモーター；　マウス及びヒトのU6-snRNAプロモーター、ヒトH1-RNase P RNAプロモーター、ヒトバリン-tRNAプロモーターなどのRNA polymerase III（polIII）系プロモーターなどが挙げられ、これらの中でも、短いRNAの転写を正確に行うことができるという観点から、polIII系プロモーターが好ましい。また、薬剤により誘導可能な各種プロモーターも使用することができる。他の配列としては、特に制限されず、発現ベクターが含み得る公知の配列を各種採用することができる。このような配列の一例としては、例えば複製起点、薬剤耐性遺伝子などが挙げられる。また、薬剤耐性遺伝子の種類及びベクターの種類は上述のものを例示できる。 For the expression cassette of Fchsd1-specific siRNA, Fchsd1-specific miRNA, or Fchsd1-specific antisense nucleic acid, the Fchsd1-specific siRNA, Fchsd1-specific miRNA, or Fchsd1-specific antisense nucleic acid is incorporated in a state in which it can be expressed. It is not particularly limited as long as it is a polynucleotide. Typically, the expression cassette is a polynucleotide comprising a promoter sequence and a coding sequence of an Fchsd1-specific siRNA, an Fchsd1-specific miRNA, or an Fchsd1-specific antisense nucleic acid (and optionally a transcription termination signal sequence). , Includes other sequences as needed. The promoter is not particularly limited, and is an RNA polymerase II (polII) promoter such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter; mouse and human U6-snRNA promoters, Examples include human H1-RNase P RNA promoters, RNA polymerase III (polIII) promoters such as human valine-tRNA promoters, and among these, polIII promoters are available from the viewpoint of accurate transcription of short RNAs. preferable. In addition, various promoters that can be induced by a drug can also be used. The other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include origins of replication, drug resistance genes, and the like. Moreover, the above-mentioned types can be exemplified as the type of drug resistance gene and the type of vector.
　Fchsd1発現抑制剤の別の例としては、Fchsd1特異的リボザイムなどが挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するRNAを意味するが、本願では配列特異的な核酸切断活性を有する限りDNAをも包含する。リボザイム核酸として最も汎用性の高いものは、ウイロイドやウイルソイドなどの感染性RNAに見られるセルフスプライシングRNAがあり、ハンマーヘッド型やヘアピン型などが知られている。ハンマーヘッド型は約40塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約10塩基程度）をmRNAの所望の切断部位と相補的な配列にすることにより、標的mRNAのみを特異的に切断することが可能である。このタイプのリボザイム核酸は、RNAのみを基質とするので、ゲノムDNAを攻撃することがないという利点を有する。Fchsd1遺伝子のmRNAが自身で二本鎖構造をとる場合には、RNAヘリカーゼと特異的に結合し得るウイルス核酸由来のRNAモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)］。さらに、リボザイムを、それをコードするDNAを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、tRNAを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる［Nucleic Acids Res., 29(13): 2780-2788 (2001)］。 Another example of an Fchsd1 expression inhibitor is an Fchsd1-specific ribozyme. The term "ribozyme" in a narrow sense means RNA having an enzymatic activity of cleaving nucleic acid, but in the present application, DNA is also included as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the advantage that it does not attack genomic DNA because it uses only RNA as a substrate. When the mRNA of the Fchsd1 gene has a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase. It can be [Proc. Natl. Acad. Sci. USA, 98 (10): 5722-5577 (2001)]. In addition, when the ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked to facilitate the transfer of transcripts to the cytoplasm. You can also [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
　2-2-1-2．遺伝子編集剤
　Fchsd1遺伝子編集剤は、標的配列特異的ヌクレアーゼシステム（例えばCRISPR/Casシステム）により、Fchsd1遺伝子の発現を抑制可能なものである限り特に制限されない。Fchsd1遺伝子の発現抑制は、例えばFchsd1遺伝子の破壊、Fchsd1遺伝子のプロモーターの改変による該プロモーターの活性抑制により可能である。 2-2-1-2. Gene editing agent The Fchsd1 gene editing agent is not particularly limited as long as the expression of the Fchsd1 gene can be suppressed by a target sequence-specific nuclease system (eg, CRISPR / Cas system). The expression of the Fchsd1 gene can be suppressed by, for example, disruption of the Fchsd1 gene or suppression of the activity of the promoter by modifying the promoter of the Fchsd1 gene.
　Fchsd1遺伝子編集剤としては、例えばCRISPR/Casシステムを採用する場合は、典型的には、Fchsd1遺伝子又はそのプロモーターを標的とするガイドRNA発現カセット、及びCasタンパク質発現カセットを含むベクター（Fchsd1遺伝子編集用ベクター）を用いることができるが、これに限定されない。この典型例以外にも、例えばFchsd1遺伝子又はそのプロモーターを標的とするガイドRNA及び／又はその発現カセットを含むベクターと、Casタンパク質及び／又はその発現カセットを含むベクターとの組み合わせを、Fchsd1遺伝子編集剤として用いることが可能である。 When the CRISPR / Cas system is adopted as the Fchsd1 gene editing agent, for example, a vector containing a guide RNA expression cassette targeting the Fchsd1 gene or its promoter and a Cas protein expression cassette (for Fchsd1 gene editing) is typically used. Vector) can be used, but is not limited to this. In addition to this typical example, a combination of a vector containing a guide RNA targeting the Fchsd1 gene or its promoter and / or an expression cassette thereof and a vector containing a Cas protein and / or its expression cassette can be used as an Fchsd1 gene editing agent. Can be used as.
　ガイドRNA発現カセットは、対象の生物内でガイドRNAを発現させる目的で用いられるポリヌクレオチドである限り特に制限されない。該発現カセットの典型例としては、プロモーター、及び該プロモーターの制御下に配置されたガイドRNA全体又は一部のコード配列を含むポリヌクレオチドが挙げられる。なお「プロモーターの制御下に配置」とは、換言すれば、ガイドRNAコード配列が、該配列の転写がプロモーターによって制御されるように配置されていることを意味する。具体的な配置の態様としては、例えばプロモーターの3´側直下にガイドRNAコード配列が配置されている態様（例えば、プロモーター3´末端の塩基からガイドRNAコード配列の5´末端の塩基までの間の塩基対数（bp）が、例えば100 bp以下、好ましくは50 bp以下である態様）が挙げられる。 The guide RNA expression cassette is not particularly limited as long as it is a polynucleotide used for the purpose of expressing the guide RNA in the target organism. Typical examples of the expression cassette include a promoter and a polynucleotide containing a coding sequence of all or part of a guide RNA placed under the control of the promoter. In other words, "arranged under the control of a promoter" means that the guide RNA coding sequence is arranged so that the transcription of the sequence is controlled by the promoter. As a specific arrangement mode, for example, the guide RNA coding sequence is arranged directly under the 3'side of the promoter (for example, between the base at the 3'end of the promoter and the base at the 5'end of the guide RNA coding sequence. The base pair number (bp) of the above is, for example, 100 bp or less, preferably 50 bp or less).
　ガイドRNA発現カセットのプロモーターとしては、特に制限されず、pol II系プロモーターを使用することもできるが、比較的短いRNAの転写をより正確に行わせるという観点から、pol III系プロモーターが好ましい。pol III系プロモーターとしては、特に制限されないが、例えばマウス及びヒトのU6-snRNAプロモーター、ヒトH1-RNase P RNAプロモーター、ヒトバリン-tRNAプロモーターなどが挙げられる。また、薬剤により誘導可能な各種プロモーターも使用することができる。 The promoter of the guide RNA expression cassette is not particularly limited, and a pol II promoter can be used, but a pol III promoter is preferable from the viewpoint of more accurately transcribing a relatively short RNA. The pol III promoter is not particularly limited, and examples thereof include mouse and human U6-snRNA promoters, human H1-RNase PRNA promoters, and human valine-tRNA promoters. In addition, various promoters that can be induced by a drug can also be used.
　ガイドRNAコード配列は、ガイドRNAをコードする塩基配列である限り特に制限されない。 The guide RNA coding sequence is not particularly limited as long as it is a base sequence encoding the guide RNA.
　ガイドRNAは、CRISPR/Casシステムにおいて用いられるものであれば特に制限されず、例えばゲノムDNAの標的部位（例えばFchsd1遺伝子、そのプロモーターなど）に結合し、且つCasタンパク質と結合することにより、Casタンパク質をゲノムDNAの標的部位に誘導可能なものを各種使用することができる。 The guide RNA is not particularly limited as long as it is used in the CRISPR / Cas system, for example, the Cas protein by binding to a target site of genomic DNA (for example, the Fchsd1 gene, its promoter, etc.) and by binding to the Cas protein. Various substances that can induce the gene into the target site of genomic DNA can be used.
　本明細書において、標的部位とは、PAM（Proto-spacer Adjacent Motif）配列及びその5´側に隣接する17～30塩基長（好ましくは18～25塩基長、より好ましくは19～22塩基長、特に好ましくは20塩基長）程度の配列からなるDNA鎖（標的鎖）とその相補DNA鎖（非標的鎖）からなる、ゲノムDNA上の部位である。 In the present specification, the target site is a PAM (Proto-spacer Adjacent Motif) sequence and a 17 to 30 base length (preferably 18 to 25 base length, more preferably 19 to 22 base length) adjacent to the 5'side thereof. Particularly preferably, it is a site on genomic DNA consisting of a DNA strand (target strand) consisting of a sequence having a length of about 20 bases) and its complementary DNA strand (non-target strand).
　PAM配列は、利用するCasタンパク質の種類によって異なる。例えば、S. pyogenes由来のCas9タンパク質（II型）に対応するPAM配列は5´-NGGであり、S. solfataricus由来のCas9タンパク質（I-A1型）に対応するPAM配列は5´-CCNであり、S. solfataricus由来のCas9タンパク質（I-A2型）に対応するPAM配列は5´-TCNであり、H. walsbyl由来のCas9タンパク質（I-B型）に対応するPAM配列は5´-TTCであり、E. coli由来のCas9タンパク質（I-E型）に対応するPAM配列は5´-AWGであり、E. coli由来のCas9タンパク質（I-F型）に対応するPAM配列は5´-CCであり、P. aeruginosa由来のCas9タンパク質（I-F型）に対応するPAM配列は5´-CCであり、S. Thermophilus由来のCas9タンパク質（II-A型）に対応するPAM配列は5´-NNAGAAであり、S. agalactiae由来のCas9タンパク質（II-A型）に対応するPAM配列は5´-NGGであり、S. aureus由来のCas9タンパク質に対応するPAM配列は、5´-NGRRT又は5´-NGRRNであり、N. meningitidis由来のCas9タンパク質に対応するPAM配列は、5´-NNNNGATTであり、T. denticola由来のCas9タンパク質に対応するPAM配列は、5´-NAAAACである。 The PAM sequence differs depending on the type of Cas protein used. For example, the PAM sequence corresponding to the Cas9 protein (type II) derived from S. pyogenes is 5'-NGG, and the PAM sequence corresponding to the Cas9 protein (type I-A1) derived from S. solfataricus is 5'-CCN. Yes, the PAM sequence corresponding to the Cas9 protein (I-A2 type) derived from S. solfataricus is 5'-TCN, and the PAM sequence corresponding to the Cas9 protein (I-B type) derived from H. walsbyl is 5'-TTC. Yes, the PAM sequence corresponding to the E. coli-derived Cas9 protein (I-E type) is 5'-AWG, and the PAM sequence corresponding to the E. coli-derived Cas9 protein (I-F type) is 5'-CC. The PAM sequence corresponding to the Cas9 protein (I-F type) derived from P. aeruginosa is 5'-CC, and the PAM sequence corresponding to the Cas9 protein (II-A type) derived from S. Thermophilus is 5'-NNAGAA. The PAM sequence corresponding to the Cas9 protein (II-A type) derived from S. agalactiae is 5'-NGG, and the PAM sequence corresponding to the Cas9 protein derived from S. aureus is 5'-NGRRT or 5'-NGRRN. The PAM sequence corresponding to the Cas9 protein derived from N. meningitidis is 5'-NNNNGATT, and the PAM sequence corresponding to the Cas9 protein derived from T. denticola is 5'-NAAAAC.
　ガイドRNAはゲノムDNAの標的部位への結合に関与する配列（crRNA（CRISPR RNA）配列といわれることもある）を有しており、このcrRNA配列が、非標的鎖のPAM配列相補配列を除いてなる配列に相補的（好ましくは、相補的且つ特異的）に結合することにより、ガイドRNAはゲノムDNAの標的部位に結合することができる。 The guide RNA has a sequence involved in the binding of genomic DNA to the target site (sometimes referred to as a crRNA (CRISPRRNA) sequence), and this crRNA sequence excludes the PAM sequence complementary sequence of the non-target strand. By binding complementaryly (preferably complementaryly and specifically) to the sequence, the guide RNA can bind to the target site of genomic DNA.
　なお、「相補的」に結合とは、完全な相補関係（AとT、及びGとC）に基づいて結合する場合のみならず、ストリンジェントな条件でハイブリダイズすることができる程度の相補関係に基づいて結合する場合も包含される。ストリンジェントな条件は、Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA) に教示されるように、複合体或いはプローブを結合する核酸の融解温度(Tm)に基づいて決定することができる。例えばハイブリダイズ後の洗浄条件として、通常「1×SSC、0.1% SDS、37℃」程度の条件を挙げることができる。かかる条件で洗浄してもハイブリダイズ状態を維持するものであることが好ましい。特に制限されないが、より厳しいハイブリダイズ条件として「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件として「0.1×SSC、0.1%SDS、65℃」程度の洗浄条件を挙げることができる。 It should be noted that "complementary" binding is not limited to the case of binding based on a complete complementary relationship (A and T, and G and C), but also a complementary relationship to the extent that hybridization can be performed under stringent conditions. The case of combining based on is also included. Stringent conditions are the melting temperature of the nucleic acid that binds the complex or probe, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on (Tm). For example, as cleaning conditions after hybridization, conditions of about "1 x SSC, 0.1% SDS, 37 ° C." can be mentioned. It is preferable that the hybridized state is maintained even after washing under such conditions. Although not particularly limited, cleaning conditions of "0.5 x SSC, 0.1% SDS, 42 ° C" are listed as stricter hybridization conditions, and "0.1 x SSC, 0.1% SDS, 65 ° C" are listed as stricter hybridization conditions. Can be done.
　具体的には、crRNA配列の内、標的配列に結合する配列は、標的鎖と例えば90％以上、好ましくは95％以上、より好ましくは98％以上、さらに好ましくは99％以上、特に好ましくは100％の同一性を有する。なお、ガイドRNAの標的部位への結合には、crRNA配列の内、標的配列に結合する配列の3´側の12塩基が重要であるといわれている。このため、crRNA配列の内、標的配列に結合する配列が、標的鎖と完全同一ではない場合、標的鎖と異なる塩基は、crRNA配列の内、標的配列に結合する配列の3´側の12塩基以外に存在することが好ましい。 Specifically, among the crRNA sequences, the sequence that binds to the target sequence is, for example, 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, and particularly preferably 100% or more with the target strand. Has% identity. It is said that 12 bases on the 3'side of the sequence that binds to the target sequence are important for the binding of the guide RNA to the target site. Therefore, if the sequence that binds to the target sequence in the crRNA sequence is not exactly the same as the target strand, the base different from the target strand is 12 bases on the 3'side of the sequence that binds to the target sequence in the crRNA sequence. It is preferable that it exists in other than.
　ガイドRNAは、Casタンパク質との結合に関与する配列（tracrRNA（trans-activating crRNA）配列といわれることもある）を有しており、このtracrRNA配列が、Casタンパク質に結合することにより、Casタンパク質をゲノムDNAの標的部位に誘導することができる。 The guide RNA has a sequence involved in binding to the Cas protein (sometimes referred to as a tracrRNA (trans-activating crRNA) sequence), and this tracrRNA sequence binds to the Cas protein to form the Cas protein. It can be directed to the target site of genomic DNA.
　tracrRNA配列は、特に制限されない。tracrRNA配列は、典型的には、複数（通常、3つ）のステムループを形成可能な50～100塩基長程度の配列からなるRNAであり、利用するCasタンパク質の種類に応じてその配列は異なる。tracrRNA配列としては、利用するCasタンパク質の種類に応じて、公知の配列を各種採用することができる。 The tracrRNA sequence is not particularly limited. The tracrRNA sequence is typically an RNA consisting of a sequence having a length of about 50 to 100 bases capable of forming multiple (usually three) stem loops, and the sequence varies depending on the type of Cas protein used. .. As the tracrRNA sequence, various known sequences can be adopted depending on the type of Cas protein to be used.
　ガイドRNAは、通常、上記したcrRNA配列とtracr RNA配列を含む。ガイドRNAの態様は、crRNA配列とtracr RNA配列を含む一本鎖RNA（sgRNA）であってもよいし、crRNA配列を含むRNAとtracrRNA配列を含むRNAとが相補的に結合してなるRNA複合体であってもよい。 The guide RNA usually contains the above-mentioned crRNA sequence and tracrRNA sequence. The mode of the guide RNA may be a single-stranded RNA (sgRNA) containing a crRNA sequence and a tracrRNA sequence, or an RNA complex in which an RNA containing a crRNA sequence and an RNA containing a tracrRNA sequence are complementarily linked. It may be a body.
　Casタンパク質発現カセットは、対象の生物内でCasタンパク質を発現させる目的で用いられるポリヌクレオチドである限り特に制限されない。該発現カセットの典型例としては、プロモーター、及び該プロモーターの制御下に配置されたCasタンパク質コード配列を含むポリヌクレオチドが挙げられる。なお「プロモーターの制御下に配置」とは、ガイドRNA発現カセットにおける定義と同様である。 The Cas protein expression cassette is not particularly limited as long as it is a polynucleotide used for the purpose of expressing Cas protein in a target organism. Typical examples of the expression cassette include a promoter and a polynucleotide containing a Cas protein coding sequence arranged under the control of the promoter. Note that "placed under the control of a promoter" is the same as the definition in the guide RNA expression cassette.
　Casタンパク質発現カセットのプロモーターとしては、特に制限されず、例えばpol II系プロモーターを各種使用することができる、pol II系プロモーターとしては、特に制限されないが、例えば例えばCMVプロモーター、EF1プロモーター、SV40プロモーター、MSCVプロモーター、hTERTプロモーター、βアクチンプロモーター、CAGプロモーターなどが挙げられる。また、薬剤により誘導可能な各種プロモーターも使用することができる。 The promoter of the Cas protein expression cassette is not particularly limited, and for example, various polII promoters can be used. The polII promoter is not particularly limited, but for example, CMV promoter, EF1 promoter, SV40 promoter, etc. Examples include the MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter and the like. In addition, various promoters that can be induced by a drug can also be used.
　Casタンパク質コード配列は、Casタンパク質のアミノ酸配列をコードする塩基配列である限り特に制限されない。 The Cas protein coding sequence is not particularly limited as long as it is a base sequence encoding the amino acid sequence of the Cas protein.
　Casタンパク質は、CRISPR/Casシステムにおいて用いられるものであれば特に制限されず、例えばガイドRNAと複合体を形成した状態でゲノムDNAの標的部位に結合し、該標的部位を切断できるものを各種使用することができる。Casタンパク質としては、各種生物由来のものが知られており、例えばS. pyogenes由来のCas9タンパク質（II型）、S. solfataricus由来のCas9タンパク質（I-A1型）、S. solfataricus由来のCas9タンパク質（I-A2型）、H. walsbyl由来のCas9タンパク質（I-B型）、E. coli由来のCas9タンパク質（I-E型）、E. coli由来のCas9タンパク質（I-F型）、P. aeruginosa由来のCas9タンパク質（I-F型）、S. Thermophilus由来のCas9タンパク質（II-A型）、S. agalactiae由来のCas9タンパク質（II-A型）、S. aureus由来のCas9タンパク質、N. meningitidis由来のCas9タンパク質、T. denticola由来のCas9タンパク質、F. novicida由来のCpf1タンパク質（V型）などが挙げられる。これらの中でも、好ましくはCas9タンパク質が挙げられ、より好ましくはストレプトコッカス属に属する細菌が内在的に有するCas9タンパク質が挙げられる。各種Casタンパク質のアミノ酸配列、及びそのコード配列の情報は、NCBIなどの各種データベース上で容易に得ることができる。 The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system, and various proteins that can bind to the target site of genomic DNA in a complex with the guide RNA and cleave the target site are used. can do. As Cas protein, those derived from various organisms are known, for example, Cas9 protein derived from S. pyogenes (type II), Cas9 protein derived from S. solfataricus (type I-A1), and Cas9 protein derived from S. solfataricus. (I-A2 type), H. walsbyl-derived Cas9 protein (I-B type), E. coli-derived Cas9 protein (I-E type), E. coli-derived Cas9 protein (I-F type), P. aeruginosa-derived Cas9 protein (I-F type), Cas9 protein derived from S. Thermophilus (II-A type), Cas9 protein derived from S. agalactiae (II-A type), Cas9 protein derived from S. aureus, Cas9 protein derived from N. meningitidis, T Examples include Cas9 protein derived from .denticola and Cpf1 protein (V type) derived from F. novicida. Among these, Cas9 protein is preferably mentioned, and more preferably Cas9 protein inherently possessed by a bacterium belonging to the genus Streptococcus. Information on the amino acid sequences of various Cas proteins and their coding sequences can be easily obtained on various databases such as NCBI.
　Casタンパク質は、野生型の2本鎖切断型Casタンパク質であってもよいし、ニッカーゼ型Casタンパク質であってもよい。また、Casタンパク質は、その活性を損なわない限りにおいて、アミノ酸配列の変異（例えば、置換、欠失、挿入、付加など）を有していてもよいし、公知のタンパク質タグ、シグナル配列、酵素タンパク質などのタンパク質が付加されたものであってもよい。タンパク質タグとしては、例えばビオチン、Hisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグなどが挙げられる。シグナル配列としては、例えば細胞質移行シグナルなどが挙げられる。 The Cas protein may be a wild-type double-strand break type Cas protein or a nickase-type Cas protein. In addition, the Cas protein may have an amino acid sequence variation (eg, substitution, deletion, insertion, addition, etc.) as long as its activity is not impaired, and known protein tags, signal sequences, enzyme proteins, etc. It may be the one to which a protein such as is added. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like. Examples of the signal sequence include a cytoplasmic transfer signal and the like.
　Fchsd1遺伝子編集用ベクターは、他の配列を有していてもよい。他の配列としては、特に制限されず、発現ベクターが含み得る公知の配列を各種採用することができる。このような配列の一例としては、例えば複製起点、薬剤耐性遺伝子などが挙げられる。 The Fchsd1 gene editing vector may have other sequences. The other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include origins of replication, drug resistance genes, and the like.
　薬剤耐性遺伝子としては、例えばクロラムフェニコール耐性遺伝子、テトラサイクリン耐性遺伝子、ネオマイシン耐性遺伝子、エリスロマイシン耐性遺伝子、スペクチノマイシン耐性遺伝子、カナマイシン耐性遺伝子、ハイグロマイシン耐性遺伝子、ピューロマイシン耐性遺伝子などが挙げられる。 Examples of the drug resistance gene include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a canamycin resistance gene, a hyglomycin resistance gene, and a puromycin resistance gene.
　ベクターの種類は、特に制限されず、例えば動物細胞発現プラスミドなどのプラスミドベクター；　レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、センダイウイルスなどのウイルスベクター；　アグロバクテリウムベクターなどが挙げられる。 The type of vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus; and agrobacterium vectors. Be done.
　Fchsd1遺伝子編集剤は、公知の遺伝子工学的手法に従って容易に作製することができる。例えば、PCR、制限酵素切断、DNA連結技術、in vitro転写・翻訳技術、リコンビナントタンパク質作製技術などを利用して作製することができる。 The Fchsd1 gene editor can be easily prepared according to a known genetic engineering method. For example, it can be produced by using PCR, restriction enzyme cleavage, DNA linkage technology, in vitro transcription / translation technology, recombinant protein production technology, and the like.
　2-2-2．Fchsd1機能抑制剤
　Fchsd1機能抑制剤は、呼吸器疾患の予防又は治療対象の生物又はその細胞（特に肺組織の細胞）内で発現しているFchsd1タンパク質及び／又はFchsd1 mRNAの機能を調節可能なものである限り、特に制限されない。Fchsd1機能抑制剤は、1種単独で用いることもできるし、2種以上を組み合わせて用いることもできる。 2-2-2. Fchsd1 function inhibitor Fchsd1 function inhibitor is one that can regulate the function of Fchsd1 protein and / or Fchsd1 mRNA expressed in the organism to be prevented or treated for respiratory diseases or its cells (particularly cells of lung tissue). As long as it is, there is no particular limitation. The Fchsd1 function inhibitor can be used alone or in combination of two or more.
　Fchsd1機能抑制剤としては、細胞質でNRF2及びSNX9と相互作用して複合体を形成する性質を低下させることができる限りにおいて特に制限されない。 The Fchsd1 function inhibitor is not particularly limited as long as it can reduce the property of interacting with NRF2 and SNX9 in the cytoplasm to form a complex.
　Fchsd1機能抑制剤としては、例えばFchsd1抗体等が挙げられる。Fchsd1抗体は、好ましくは、Fchsd1のNRF2及び／又はSNX9との結合部位のアミノ酸配列に対して、抗原結合性を有する抗体である。このような抗体を用いることにより、より確実に、Fchsd1機能を抑制することができる。結合部位は公知の情報に基づいて決定すること、及び／又は公知の情報に基づいて推測すること（例えば、ドッキングモデル構築等により）が可能である。 Examples of the Fchsd1 function inhibitor include Fchsd1 antibody and the like. The Fchsd1 antibody is preferably an antibody having antigen-binding property to the amino acid sequence of the binding site of Fchsd1 with NRF2 and / or SNX9. By using such an antibody, the Fchsd1 function can be suppressed more reliably. The binding site can be determined based on known information and / or inferred based on known information (eg, by building a docking model, etc.).
　抗体には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、またはFabフラグメントやFab発現ライブラリーによって生成されるフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。Fchsd1のアミノ酸配列のうち少なくとも連続する、通常8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体も、本発明の抗体に含まれる。これらの抗体は入手可能であり、例えば、Anti-FCHSD1抗体として、abcam社製のab67017、St Johns Laboratory社製のSTJ117738、LifeSpan Biosciences社製のLS-C766558-60等が知られている。 Antibodies include some of the above antibodies having antigen-binding properties, such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or fragments produced by Fab fragments and Fab expression libraries. Antibodies having antigen-binding activity to a polypeptide consisting of at least consecutive 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of Fchsd1 are also included in the antibody of the present invention. These antibodies are available, and for example, as Anti-FCHSD1 antibody, ab67017 manufactured by abcam, STJ117738 manufactured by St Johns Laboratory, LS-C766558-60 manufactured by LifeSpan Biosciences, and the like are known.
　また、これらの抗体の製造方法は、すでに周知であり、これらの常法に従って製造することができる（Current protocols in Molecular Biology , Chapter 11.12～11.13(2000)）。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したFchsd1を用いて、あるいは常法に従って当該Fchsd1の部分アミノ酸配列を有するオリゴペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したFchsd1、あるいはFchsd1の部分アミノ酸配列を有するオリゴペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる（Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4～11.11）。 In addition, the methods for producing these antibodies are already well known and can be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, Fchsd1 expressed and purified in E. coli or the like according to a conventional method is used, or an oligopeptide having a partial amino acid sequence of Fchsd1 is synthesized according to a conventional method. It is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, Fchsd1 expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of Fchsd1 is immunized against a non-human animal such as a mouse to obtain spleen cells and myeloma. It can be obtained from hybridoma cells prepared by cell fusion with cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).
　抗体の作製に免疫抗原として使用されるFchsd1は、公知の遺伝子配列情報に基づいて、DNAクローニング、各プラスミドの構築、宿主へのトランスフェクション、形質転換体の培養および培養物からのタンパク質の回収の操作により得ることができる。これらの操作は、当業者に既知の方法、あるいは文献記載の方法（Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)）などに準じて行うことができる。 Used as an immune antigen for antibody production, Fchsd1 is based on known gene sequence information for DNA cloning, plasmid construction, host transfection, transformant culture and protein recovery from cultures. It can be obtained by operation. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
　具体的には、Fchsd1をコードする遺伝子が所望の宿主細胞中で発現できる組み換えDNA（発現ベクター）を作製し、これを宿主細胞に導入して形質転換し、該形質転換体を培養して、得られる培養物から、目的タンパク質を回収することによって、本発明抗体の製造のための免疫抗原としてのタンパク質を得ることができる。またFchsd1の部分ペプチドは、公知の遺伝子配列情報に従って、一般的な化学合成法（ペプチド合成）によって製造することもできる。 Specifically, a recombinant DNA (expression vector) capable of expressing the gene encoding Fchsd1 in a desired host cell is prepared, introduced into the host cell for transformation, and the transformant is cultured. By recovering the target protein from the obtained culture, a protein as an immune antigen for producing the antibody of the present invention can be obtained. Further, the partial peptide of Fchsd1 can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
　また本発明の抗体は、Fchsd1の部分アミノ酸配列を有するオリゴペプチドを用いて調製されるものであってよい。かかる抗体の製造のために用いられるオリゴ（ポリ）ペプチドは、機能的な生物活性を有することは要しないが、Fchsd1と同様な免疫原特性を有するものであることが望ましい。好ましくはこの免疫原特性を有し、且つFchsd1のアミノ酸配列において少なくとも連続する8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるオリゴ（ポリ）ペプチドを例示することができる。 Further, the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of Fchsd1. The oligo (poly) peptide used for the production of such an antibody does not need to have functional biological activity, but is preferably one having immunogenogenic properties similar to Fchsd1. An oligo (poly) peptide having this immunogenetic property and consisting of at least consecutive 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of Fchsd1 can be exemplified.
　かかるオリゴ（ポリ）ペプチドに対する抗体の製造は、宿主に応じて種々のアジュバントを用いて免疫学的反応を高めることによって行うこともできる。限定はされないが、そのようなアジュバントには、フロイントアジュバント、水酸化アルミニウムのようなミネラルゲル、並びにリゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油乳剤、キーホールリンペットヘモシアニン及びジニトロフェノールのような表面活性物質、BCG（カルメット－ゲラン桿菌）やコリネバクテリウム-パルヴムなどのヒトアジュバントが含まれる。 The production of an antibody against such an oligo (poly) peptide can also be carried out by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocianine and dinitrophenol. Includes active substances, human adjuvants such as BCG (Carmet-Guerin bacillus) and Corinebacterium-Palvum.
　Fchsd1機能抑制剤としては、上記Fchsd1抗体以外にも、Fchsd1に結合性を有する（好ましくは、特異的結合性を有する）分子（例えば、低分子化合物、ペプチド、タンパク質、人工抗体、アプタマー等）であれば、使用することが可能である。低分子化合物としては、標的タンパク質を分解するように最適化された化合物（Proteolysis targeting chimera）も含まれる。また、Fchsd1機能抑制剤として抗体等のタンパク質又はペプチドを採用する場合は、それに代えて、その発現カセットを採用することもできる。発現カセットについては、上記「2-2-1．Fchsd1発現抑制剤」における定義と同様である。 As the Fchsd1 function inhibitor, in addition to the above-mentioned Fchsd1 antibody, a molecule having binding property to Fchsd1 (preferably having specific binding property) (for example, a low molecular weight compound, a peptide, a protein, an artificial antibody, an aptamer, etc.) can be used. If so, it can be used. Small molecule compounds also include compounds optimized to degrade the target protein (Proteolysis targeting chimera). When a protein or peptide such as an antibody is used as the Fchsd1 function inhibitor, an expression cassette thereof can be used instead. The expression cassette is the same as the definition in "2-2-1. Fchsd1 expression inhibitor" above.
　2-3．用途、その他の成分
　後述の実施例で明らかにされているように、Fchsd1は、呼吸器（特に肺）において多く発現し、細胞質でNRF2及びSNX9と相互作用して複合体を形成しており、NRF2が核移行して酸化ストレス応答を引き起こすことを抑制している。よって、Fchsd1の発現及び／又は機能を抑制することにより、酸化ストレス応答を促進することができる。一方、COPD等の各種呼吸器疾患は、その発症及び／又は増悪に、酸化ストレスが関与している。このため、Fchsd1発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種（有効成分）は、呼吸器疾患の予防又は治療（若しくは改善）に有効である。この有効成分は、例えば医薬、試薬の他、食品組成物、健康増進剤、栄養補助剤（サプリメントなど）など）の有効成分としての利用が可能である。有効成分は、これをそのまま、あるいは慣用の成分とともに各種組成物となし、動物、ヒト、及び各種細胞に適用（例えば、投与、摂取、接種など）することができる。 2-3. Applications, Other Ingredients As will be revealed in the examples below, Fchsd1 is highly expressed in the respiratory tract (especially lung) and interacts with NRF2 and SNX9 in the cytoplasm to form a complex. It suppresses NRF2 translocation to the nucleus and causing an oxidative stress response. Therefore, by suppressing the expression and / or function of Fchsd1, the oxidative stress response can be promoted. On the other hand, in various respiratory diseases such as COPD, oxidative stress is involved in the onset and / or exacerbation of the disease. Therefore, at least one selected from the group consisting of the Fchsd1 expression inhibitor and the Fchsd1 function inhibitor (active ingredient) is effective for the prevention or treatment (or improvement) of respiratory diseases. This active ingredient can be used as an active ingredient of, for example, pharmaceuticals, reagents, food compositions, health promoters, nutritional supplements (supplements, etc.), etc.). The active ingredient can be applied to animals, humans, and various cells (eg, administration, ingestion, inoculation, etc.) as it is or in combination with conventional ingredients in various compositions.
　呼吸器疾患としては、酸化ストレスにより発症又は増悪し得る呼吸器疾患（好ましくは肺疾患）であることが好ましく、例えば急性気管支炎、細菌性肺炎、肺膿瘍、肺結核、非結核性肺抗酸菌症、肺真菌症、肺寄生虫症、日和見感染（ニューモシスチス肺炎、サイトメガロウイルス肺炎）、誤嚥性肺炎、かぜ症候群、インフルエンザ等の感染性呼吸器疾患；慢性閉塞性肺疾患（COPD）、肺気腫、びまん性汎細気管支炎等の気道閉塞性疾患；気管支喘息、過敏性肺炎、好酸球肺炎、アレルギー性気管支肺アスペルギルス症、薬剤性肺炎、好酸球性多発血管炎性肉芽腫症等のアレルギー性肺疾患；肺線維症、特発性間質性肺炎、放射線肺炎、サルコイドーシス、特発性器質化肺炎、膠原病肺等の間質性肺疾患；肺癌、転移性肺腫瘍、肺の良性腫瘍、縦隔腫瘍等の腫瘍性肺疾患；肺血栓塞栓症、肺動脈性肺高血圧症、肺水腫等の肺血管性病変；胸膜炎、膿胸、胸膜腫瘍、気胸等の胸膜疾患；急性呼吸不全（ARDS）、慢性呼吸不全等の呼吸不全等が挙げられる。 The respiratory disease is preferably a respiratory disease (preferably lung disease) that can develop or worsen due to oxidative stress, for example, acute bronchitis, bacterial pneumonia, pulmonary abscess, pulmonary tuberculosis, and non-tuberculous pulmonary acid bacterium. Infectious respiratory diseases such as illness, pulmonary fungal disease, pulmonary parasite disease, opportunistic infection (pneumocystis pneumonia, cytomegalovirus pneumonia), aspiration pneumonia, cold syndrome, influenza; chronic obstructive pulmonary disease (COPD), pulmonary emphysema Bronchial asthma, irritable pneumonia, eosinophil pneumonia, allergic bronchopulmonary aspergillosis, drug-induced pneumonia, eosinophilia polyangiitis granulomatosis, etc. Allergic lung disease; pulmonary fibrosis, idiopathic interstitial pneumonia, radiation pneumonia, sarcoidosis, idiopathic organizing pneumonia, collagen disease lung, etc. Neoplastic lung diseases such as mediastinal tumors; Pulmonary vascular lesions such as pulmonary thromboembolism, pulmonary arterial hypertension, and pulmonary edema; Examples include respiratory insufficiency such as chronic respiratory insufficiency.
　本発明の剤の対象細胞の細胞種は、Fchsd1を発現し得る細胞種である限り特に限定されないが、主に呼吸器組織（特に肺組織）の細胞である。 The cell type of the target cell of the agent of the present invention is not particularly limited as long as it is a cell type capable of expressing Fchsd1, but is mainly cells of respiratory tissue (particularly lung tissue).
　本発明の剤の対象生物は特に限定されず、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物などが挙げられる。 The target organism of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. ..
　本発明の剤の形態は、特に限定されず、本発明の剤の用途に応じて、各用途において通常使用される形態をとることができる。 The form of the agent of the present invention is not particularly limited, and the form usually used in each application can be taken depending on the use of the agent of the present invention.
　形態としては、用途が医薬、健康増進剤、栄養補助剤（サプリメントなど）などである場合は、例えば錠剤（口腔内側崩壊錠、咀嚼可能錠、発泡錠、トローチ剤、ゼリー状ドロップ剤などを含む）、丸剤、顆粒剤、細粒剤、散剤、硬カプセル剤、軟カプセル剤、ドライシロップ剤、液剤（ドリンク剤、懸濁剤、シロップ剤を含む）、ゼリー剤などの経口摂取に適した製剤形態（経口製剤形態）、点鼻剤、吸入剤、肛門坐剤、挿入剤、浣腸剤、ゼリー剤、注射剤、貼付剤、ローション剤、クリーム剤などの非経口摂取に適した製剤形態（非経口製剤形態）が挙げられる。 The form includes, for example, tablets (intraoral disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drop agents, etc.) when the uses are pharmaceuticals, health promoters, nutritional supplements (supplements, etc.), etc. ), Rounds, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly, etc. Form (oral formulation form), nasal drop, inhalant, anal suppository, insert, enema, jelly, injection, patch, lotion, cream, etc. Suitable for parenteral ingestion (non-formal) Oral formulation form).
　形態としては、用途が食品組成物の場合は、液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、豆乳、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキーなどが挙げられる。 As a form, when the use is a food composition, liquid, gel or solid foods such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, milk powder for childcare. , Cake mixes, powdered or liquid dairy products, breads, cookies and the like.
　本発明の剤は、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、例えば医薬、食品組成物、健康増進剤、栄養補助剤（サプリメントなど）などに配合され得る成分である限り特に限定されるものではないが、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤などが挙げられる。 The agent of the present invention may further contain other components, if necessary. The other ingredients are not particularly limited as long as they are ingredients that can be blended in, for example, pharmaceuticals, food compositions, health enhancers, nutritional supplements (supplements, etc.), but are not particularly limited, and are, for example, bases, carriers, solvents, and the like. Dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents and the like can be mentioned.
　本発明の剤の有効成分の含有量は、有効成分の種類、用途、使用態様、適用対象、適用対象の状態などに左右されるものであり、限定はされないが、例えば0.0001～100重量％、好ましくは0.001～50重量％とすることができる。 The content of the active ingredient of the agent of the present invention depends on the type, use, mode of use, application target, state of application target, etc. of the active ingredient, and is not limited, but is, for example, 0.0001 to 100% by weight. It can be preferably 0.001 to 50% by weight.
　本発明の剤の適用（例えば、投与、摂取、接種など）量は、薬効を発現する有効量であれば特に限定されず、通常は、有効成分の重量として、一般に一日あたり0.1～1000 mg/kg体重である。上記投与量は1日1回又は2～3回に分けて投与するのが好ましく、年齢、病態、症状により適宜増減することもできる。 The application amount (for example, administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and the weight of the active ingredient is generally 0.1 to 1000 mg per day. / kg weight. The above dose is preferably administered once a day or divided into 2 to 3 times, and may be appropriately increased or decreased depending on the age, pathological condition, and symptoms.
　3．呼吸器疾患の検査方法及び呼吸器疾患の罹患リスク判定方法
　本発明は、その一態様において、（1）被検体から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）を検出する工程を含む、呼吸器疾患を検査する方法（本明細書において、「本発明の検査方法」と示すこともある。）に関する。本発明は、その一態様において、（1）被検体から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）を検出する工程を含む、呼吸器疾患に罹患するリスクを検査する方法（本明細書において、「本発明の判定方法」と示すこともある。）に関する。以下、これらについて説明する。 3. 3. Respiratory disease testing method and respiratory disease morbidity risk determination method In one aspect of the present invention, (1) Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a test subject is detected. The present invention relates to a method for inspecting a respiratory disease including a step (in the present specification, it may be referred to as "inspection method of the present invention"). In one aspect of the present invention, there is a method for examining the risk of contracting a respiratory disease, which comprises (1) a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject. (In the present specification, it may be referred to as "the determination method of the present invention"). These will be described below.
　3-1．工程（1）
　検査対象又は判定対象である「呼吸器疾患」の種類は、上記「2-3」の通りである。呼吸器疾患の進行度に関する各種分類基準における全てのクラス、グレード、ステージの呼吸器疾患が検査対象又は判定対象となり得る。 3-1. Process (1)
The types of "respiratory diseases" to be inspected or judged are as described in "2-3" above. All classes, grades, and stages of respiratory illness in the various classification criteria for the progression of respiratory illness can be tested or determined.
　被検体は、本発明の検査方法又は判定方法の対象生物であり、その生物種は特に制限されない。被検体の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物が挙げられ、好ましくはヒトが挙げられる。 The subject is the target organism of the test method or determination method of the present invention, and the species thereof is not particularly limited. Examples of the organism species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer, and humans are preferable. ..
　被検体の状態は、特に制限されない。被検体としては、例えば呼吸器疾患に罹患しているかどうか不明な検体、呼吸器疾患に罹患していると既に別の方法により判定されている検体、呼吸器疾患に罹患していないと既に別の方法により判定されている検体、呼吸器疾患の治療中の検体、呼吸器疾患の治療後の検体等が挙げられる。 The condition of the subject is not particularly limited. The subjects include, for example, a sample whose presence or absence is unknown whether or not they have a respiratory disease, a sample which has already been determined to have a respiratory disease by another method, and a sample which has not already suffered from a respiratory disease. Examples thereof include a sample determined by the above method, a sample during treatment for a respiratory disease, a sample after treatment for a respiratory disease, and the like.
　工程（1）における被検試料は、被検体の呼吸器（特に肺）由来のFchsd1を検出可能な試料である限り、特に制限されない。被検試料として、好ましくは被検体の肺組織を含み得る被検試料が挙げられる。具体的には、例えば肺の生検試料、肺の洗浄液等が挙げられる。 The test sample in the step (1) is not particularly limited as long as it is a sample in which Fchsd1 derived from the respiratory organ (particularly lung) of the subject can be detected. The test sample preferably includes a test sample that may contain the lung tissue of the subject. Specific examples thereof include a lung biopsy sample and a lung lavage fluid.
　工程（1）の検出対象は、Fchsd1、すなわちFchsd1 mRNA及び／又はFchsd1タンパク質（本明細書において、これらをまとめて「対象バイオマーカー」と示すこともある。）である。検出対象であるFchsd1については、上記「2-1」と同様である。検出対象であるFchsd1は、検査精度の観点から、タンパク質であることが好ましい。 The detection target in step (1) is Fchsd1, that is, Fchsd1 mRNA and / or Fchsd1 protein (in the present specification, these may be collectively referred to as “target biomarker”). The detection target Fchsd1 is the same as in "2-1" above. Fchsd1 to be detected is preferably a protein from the viewpoint of inspection accuracy.
　検出は、通常は、対象バイオマーカーの量又は濃度を測定することによって行われる。「濃度」とは、絶対濃度に限らず、相対濃度や、単位体積辺りの重量や、絶対濃度を知るために測定した生データなどでもよい。 Detection is usually performed by measuring the amount or concentration of the target biomarker. The "concentration" is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, raw data measured to know the absolute concentration, or the like.
　対象バイオマーカーを検出する方法としては、対象バイオマーカーの一部又は全部を特異的に検出できる方法であれば特に制限されない。検出方法としては、具体的には、例えば、対象バイオマーカーを構成するペプチドを検出する質量分析法、対象バイオマーカーを特異的に認識する抗体を用いた免疫学的測定法（例えばELISA法、EIA法、RIA法、ウェスタンブロッティング法等）、ノーザンハイブリダイゼーション法、DNAマイクロアレイ法、PCR法等を挙げることができる。 The method for detecting the target biomarker is not particularly limited as long as it can specifically detect a part or all of the target biomarker. Specific examples of the detection method include a mass analysis method for detecting peptides constituting the target biomarker, and an immunological measurement method using an antibody that specifically recognizes the target biomarker (for example, ELISA method, EIA). Method, RIA method, Western blotting method, etc.), Northern hybridization method, DNA microarray method, PCR method, etc. can be mentioned.
　工程（1）においては、対象バイオマーカー以外の遺伝子産物を検出することができる。ただ、検査効率の観点から、工程（1）で検出する遺伝子は、より少ないことが好ましく、例えば、対象バイオマーカー（Fchsd1の1種）を含む1～50種、好ましくは1～20種、より好ましくは1～10種、さらに好ましくは1～5種、よりさらに好ましくは1～3種、特に好ましくは1種である。 In step (1), gene products other than the target biomarker can be detected. However, from the viewpoint of test efficiency, it is preferable that the number of genes detected in the step (1) is smaller, for example, 1 to 50 types including the target biomarker (1 type of Fchsd1), preferably 1 to 20 types, and more. It is preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and particularly preferably 1 type.
　工程（1）を含む本発明の検査方法又は判定対象によれば、呼吸器疾患の検出指標又は罹患リスク指標である対象バイオマーカーの量及び／又は濃度を提供することができ、これにより呼吸器疾患の検出／診断／罹患リスク判定などを補助することができる。 According to the test method or determination target of the present invention including the step (1), it is possible to provide the amount and / or concentration of the target biomarker which is a detection index of a respiratory disease or a disease risk index, whereby the respiratory organs can be provided. It can assist in disease detection / diagnosis / disease risk determination.
　工程（1）を含む本発明の検査方法又は判定方法による結果は、呼吸器疾患の病態解明、呼吸器疾患の予後予測、被検体の層別化、治療方法の選択（個別化医療、治療反応性）、呼吸器疾患における難治化や、リモデリングの評価、呼吸器疾患の表現型等の鑑別等に利用し得る。 The results of the test method or determination method of the present invention including step (1) include elucidation of the pathophysiology of respiratory diseases, prediction of prognosis of respiratory diseases, stratification of subjects, and selection of treatment methods (individualized medicine, treatment response). Gender), intractability in respiratory diseases, evaluation of remodeling, differentiation of respiratory phenotype, etc. can be used.
　3-2．工程（2）
　本発明の検査方法は、さらに、（2）前記工程（1）で検出されたFchsd1の量又は濃度がカットオフ値以上である場合に、前記被検体が呼吸器疾患に罹患していると判定する工程、を含むことが好ましい。該工程2を含む本発明の検査方法によれば、呼吸器疾患を判定することが可能となる。また、本発明の判定方法は、さらに、（2）前記工程（1）で検出されたFchsd1の量又は濃度がカットオフ値以上である場合に、前記被検体が呼吸器疾患に呼吸器疾患に罹患するリスクがある（又は高い）と判定する工程、を含むことが好ましい。 3-2. Process (2)
The test method of the present invention further determines that the subject suffers from a respiratory disease when (2) the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value. It is preferable to include a step of performing. According to the inspection method of the present invention including the step 2, it is possible to determine a respiratory disease. Further, in the determination method of the present invention, (2) when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value, the subject becomes a respiratory disease or a respiratory disease. It is preferable to include a step of determining that there is a risk (or high) of suffering.
　カットオフ値は、感度、特異度、陽性的中率、陰性的中率などの観点から当業者が適宜設定することができ、例えば、呼吸器疾患に罹患していない被検体から採取された被検試料における対象バイオマーカーの量及び／又は濃度に基づいて、その都度定められた値、或いは予め定められた値とすることができる。カットオフ値は、例えば、呼吸器疾患に罹患していない被検体から採取された被検試料における対象バイオマーカーの量及び／又は濃度（被検体が複数の場合は、平均値、中央値など）の、例えば0.7～1.5倍の値とすることができる。 The cutoff value can be appropriately set by those skilled in the art from the viewpoints of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, a subject collected from a subject not suffering from a respiratory disease. Based on the amount and / or concentration of the target biomarker in the test sample, it can be a predetermined value or a predetermined value each time. The cutoff value is, for example, the amount and / or concentration of the target biomarker in the test sample collected from the test subject not suffering from respiratory disease (mean value, median value, etc. when there are multiple subjects). For example, the value can be 0.7 to 1.5 times.
　工程（2）の好ましい一態様においては、被検体が呼吸器疾患の治療中又は治療後の検体である場合、カットオフ値を、例えば同一検体についての過去の試料における対象バイオマーカーの量及び／又は濃度に基づいた値とすることにより、治療効果を判定することができる。 In a preferred embodiment of step (2), if the subject is a specimen during or after treatment for respiratory disease, the cutoff value can be determined, for example, the amount of target biomarker in past samples for the same specimen and / /. Alternatively, the therapeutic effect can be determined by setting the value based on the concentration.
　4．呼吸器疾患のより高い精度での診断
　工程（2）を含む本発明の検査方法により、被検体が呼吸器疾患に罹患していると判定された場合、本発明の検査方法に、さらに呼吸器疾患の医師による診断を適用する工程を組み合わせることによって、より高い精度で呼吸器疾患を診断することができる。また、本発明の検査方法はより正確に呼吸器疾患を検出できるので、本発明の検査方法に上記工程を組み合わせることによって、より効率的且つより正確に「呼吸器疾患に罹患している」と診断できる。 Four. When it is determined by the test method of the present invention including the diagnosis step (2) with higher accuracy of the respiratory disease that the subject has the respiratory disease, the test method of the present invention is further applied to the respiratory organs. By combining the steps of applying the diagnosis by the disease doctor, the respiratory disease can be diagnosed with higher accuracy. In addition, since the test method of the present invention can detect respiratory diseases more accurately, by combining the above steps with the test method of the present invention, it is said that "they are suffering from respiratory diseases" more efficiently and more accurately. Can be diagnosed.
　5．呼吸器疾患の治療
　工程（2）を含む本発明の検査方法により被検体が呼吸器疾患に罹患していると判定された場合は本発明の検査方法に対してさらに、或いは上記「4．呼吸器疾患のより高い精度での診断」に記載の様に呼吸器疾患に罹患していると診断された場合は本発明の検査方法と医師による診断を適用する工程との組合せに対してさらに、（3）呼吸器疾患に罹患していると判定又は診断された被検体に対して、該疾患の治療を行う工程を行うことによって、被検体の該疾患を治療することが可能となる。また、本発明の検査方法はより正確に呼吸器疾患を検出できるので、本発明の検査方法に対して、或いは本発明の検査方法と医師による診断を適用する工程との組合せに対して工程（3）を組み合わせることによって、呼吸器疾患に罹患している被検体をより効率的に、より確実に治療できる。 Five. If the subject is determined to have a respiratory disease by the test method of the present invention including the treatment step (2) of the respiratory disease, the test method of the present invention is further applied, or the above-mentioned "4. Respiratory disease". If a person is diagnosed with a respiratory disease as described in "Diagnosis with higher accuracy of organ disease", the combination of the test method of the present invention and the step of applying the diagnosis by a doctor is further applied. (3) By performing a step of treating a subject who has been determined or diagnosed to have a respiratory disease, the disease of the subject can be treated. In addition, since the test method of the present invention can detect respiratory diseases more accurately, a step (for the test method of the present invention, or for a combination of the test method of the present invention and a step of applying a diagnosis by a doctor) ( By combining 3), a subject suffering from a respiratory disease can be treated more efficiently and more reliably.
　呼吸器疾患の治療方法は、特に制限されないが、代表的には投薬治療が挙げられる。投薬治療に用いる医薬としては、例えば、ステロイド薬、β2刺激薬、テオフィリン製剤、ロイコトリエン受容体拮抗薬、化学伝達物質（肥満細胞から放出される気管支収縮作用を有する物質）遊離抑制薬、Th2サイトカイン阻害薬、ヒスタミンH1拮抗薬、トロンボキサンA2合成阻害薬・受容体拮抗薬、抗コリン薬、喀痰調整薬等が挙げられる。医薬は、1種、2種、又は3種以上を組み合わせて用いることができる。 The treatment method for respiratory diseases is not particularly limited, but typical examples include medication treatment. Drugs used for medication include, for example, steroid drugs, β 2 stimulants, theophylline preparations, leukotriene receptor antagonists, chemical mediators (substances with bronchial contractile action released from obese cells) release inhibitors, Th2 cytokines. Examples include inhibitors, histamine H 1 antagonists, thromboxane A 2 synthesis inhibitors / receptor antagonists, anticholinergic agents, sputum regulators, etc. The medicine can be used as one type, two types, or a combination of three or more types.
　6．呼吸器疾患の検査薬、検査キット
　本発明は、その一態様において、Fchsd1の検出剤を含む、呼吸器疾患の検査薬（本明細書において、「本発明の検査薬」と示すこともある。）に関する。以下、これについて説明する。 6. Respiratory Disease Test Agent, Test Kit In one aspect of the present invention, the present invention may be referred to as a respiratory disease test agent (in the present specification, the "test agent of the present invention") containing a detection agent for Fchsd1. ). This will be described below.
　Fchsd1、呼吸器疾患等については、上記における定義と同様である。 Fchsd1, respiratory diseases, etc. are the same as the definitions above.
　検出剤は、対象バイオマーカーを特異的に検出できるものである限り特に制限されない。該検出剤としては、例えば対象バイオマーカーに対する抗体、プライマー、プローブ等が挙げられる。 The detection agent is not particularly limited as long as it can specifically detect the target biomarker. Examples of the detection agent include antibodies, primers, probes and the like against the target biomarker.
　検出剤は、その機能が著しく損なわれない限りにおいて、修飾が施されていてもよい。修飾としては、例えば、標識物、例えば蛍光色素、発光物質、色素、酵素、タンパク質、放射性同位体、化学発光物質、金コロイド、ビオチン等の付加、導入等が挙げられる。 The detection agent may be modified as long as its function is not significantly impaired. Modifications include, for example, addition and introduction of labeled substances such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemical luminescent substances, colloidal gold, biotin and the like.
　検出剤は、任意の固相に固定化して用いることもできる。このため本発明の検査薬は、検出剤を固定化した基板（例えばプローブを固定化したマイクロアレイチップ等。別の例として、抗体を固定化したELISAプレート等）の形態として提供することができる。 The detection agent can also be used by immobilizing it on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which a probe is immobilized, and another example, an ELISA plate on which an antibody is immobilized).
　固定化に使用される固相は、抗体等を固定化できるものであれば特に制限されることなく、例えばガラス板、ナイロンメンブレン、マイクロビーズ、シリコンチップ、キャピラリーまたはその他の基板等を挙げることができる。固相への検出剤の固定は、特に制限されない。 The solid phase used for immobilization is not particularly limited as long as it can immobilize an antibody or the like, and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. can. Immobilization of the detection agent on the solid phase is not particularly limited.
　抗体は、対象バイオマーカーを選択的に（特異的に）認識するものであれば、特に限定されない。ここで、「選択的に（特異的に）認識する」とは、例えばウェスタンブロット法やELISA法において、対象バイオマーカーが特異的に検出できることを意味するが、それに限定されることなく、当業者が上記検出物が対象バイオマーカーに由来するものであると判断できるものであればよい。その他、抗体については、上記「2-2-2」の通りである。 The antibody is not particularly limited as long as it selectively (specifically) recognizes the target biomarker. Here, "selectively (specifically) recognizing" means that the target biomarker can be specifically detected, for example, in Western blotting or ELISA, but the person skilled in the art is not limited thereto. It suffices as long as it can be determined that the above-mentioned detected substance is derived from the target biomarker. In addition, the antibody is as described in "2-2-2" above.
　プライマーやプローブ等は、対象バイオマーカーやそれに由来する核酸等を選択的に（特異的に）認識するものであれば、特に限定されない。ここで、「選択的に（特異的に）認識する」とは、例えばノーザンブロット法においては、対象バイオマーカーが特異的に検出できること、またRT-PCR法においては対象バイオマーカー若しくはそれに由来する核酸（cDNA等）が特異的に増幅されることを意味するが、それに限定されることなく、当業者が上記検出物または増幅物が対象バイオマーカーに由来するものであると判断できるものであればよい。 The primer, probe, etc. are not particularly limited as long as they selectively (specifically) recognize the target biomarker and the nucleic acid derived from the target biomarker. Here, "selectively (specifically) recognizing" means, for example, that the target biomarker can be specifically detected in Northern blotting, and that the target biomarker or a nucleic acid derived from the target biomarker is used in the RT-PCR method. It means that (cDNA, etc.) is specifically amplified, but is not limited to this, as long as it can be determined by those skilled in the art that the above-mentioned detected substance or amplified substance is derived from the target biomarker. good.
　プライマーやプローブの具体例としては、下記（a）に記載するポリヌクレオチド並びに下記（b）に記載するポリヌクレオチド：
　（a）対象バイオマーカーが有する塩基配列において連続する少なくとも15塩基を有するポリヌクレオチド及び／若しくは当該ポリヌクレオチドに相補的なポリヌクレオチド、並びに
　（b）対象バイオマーカーが有する塩基配列若しくはそれに相補的な塩基配列にストリンジェントな条件下でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチドからなる群より選択される少なくとも1種が挙げられる。 Specific examples of primers and probes include the polynucleotide described in (a) below and the polynucleotide described in (b) below:
(A) A polynucleotide having at least 15 consecutive bases in the base sequence of the target biomarker and / or a polynucleotide complementary to the polynucleotide, and (b) a base sequence of the target biomarker or a base complementary thereto. Included is at least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize to the sequence under stringent conditions.
　相補的なポリヌクレオチド又は相補的な塩基配列（相補鎖、逆鎖）とは、対象バイオマーカーの塩基配列からなるポリヌクレオチドの全長配列、または該塩基配列において少なくとも連続した15塩基長の塩基配列を有するその部分配列（ここでは便宜上、これらを「正鎖」ともいう）に対して、A:TおよびG:Cといった塩基対関係に基づいて、塩基的に相補的な関係にあるポリヌクレオチド又は塩基配列を意味するものである。ただし、かかる相補鎖は、対象とする正鎖の塩基配列と完全に相補配列を形成する場合に限らず、対象とする正鎖とストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有するものであってもよい。なお、ここでストリンジェントな条件は、Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA) に教示されるように、複合体或いはプローブを結合する核酸の融解温度(Tm)に基づいて決定することができる。例えばハイブリダイズ後の洗浄条件として、通常「1×SSC、0.1%SDS、37℃」程度の条件を挙げることができる。相補鎖はかかる条件で洗浄しても対象とする正鎖とハイブリダイズ状態を維持するものであることが好ましい。特に制限されないが、より厳しいハイブリダイズ条件として「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件として「0.1×SSC、0.1%SDS、65℃」程度の洗浄条件を挙げることができる。具体的には、このような相補鎖として、対象の正鎖の塩基配列と完全に相補的な関係にある塩基配列からなる鎖、並びに該鎖と少なくとも90％、好ましくは95％、より好ましくは98％以上、さらに好ましくは99％以上の同一性を有する塩基配列からなる鎖を例示することができる。 A complementary polynucleotide or a complementary base sequence (complementary chain, reverse chain) is a full-length sequence of a polynucleotide consisting of the base sequence of a target biomarker, or a base sequence having a length of at least 15 consecutive bases in the base sequence. A polynucleotide or base that is basically complementary to its partial sequence (here, for convenience, these are also referred to as "regular chains") based on the base pair relationships such as A: T and G: C. It means an array. However, such a complementary strand is not limited to the case where it completely forms a complementary sequence with the base sequence of the target positive chain, and has a complementary relationship to the extent that it can hybridize with the target positive chain under stringent conditions. It may have. The stringent condition here is to bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as cleaning conditions after hybridization, conditions of about "1 x SSC, 0.1% SDS, 37 ° C." can be mentioned. It is preferable that the complementary strand maintains a hybridized state with the positive strand of interest even when washed under such conditions. Although not particularly limited, cleaning conditions of "0.5 x SSC, 0.1% SDS, 42 ° C" are listed as stricter hybridization conditions, and "0.1 x SSC, 0.1% SDS, 65 ° C" are listed as stricter hybridization conditions. Can be done. Specifically, as such a complementary chain, a chain consisting of a base sequence having a completely complementary relationship with the base sequence of the positive chain of interest, and at least 90%, preferably 95%, more preferably of the chain. A chain consisting of a base sequence having 98% or more, more preferably 99% or more identity can be exemplified.
　プライマーやプローブ等は、例えば対象バイオマーカーが有する塩基配列をもとに、例えば各種設計プログラムを利用して設計することができる。具体的には前記対象バイオマーカーの塩基配列を設計プログラムにかけて得られる、プライマーまたはプローブの候補配列、若しくは少なくとも該配列を一部に含む配列を、プライマーまたはプローブとして使用することができる。 Primers, probes, etc. can be designed using, for example, various design programs based on the base sequence of the target biomarker. Specifically, a candidate sequence of a primer or a probe obtained by subjecting the base sequence of the target biomarker to a design program, or a sequence containing at least the sequence as a part thereof can be used as a primer or a probe.
　プライマーやプローブ等の塩基長は、上述のように連続する少なくとも15塩基の長さを有するものであれば特に制限されず、用途に応じて適宜設定することができる。塩基長としては、例えばプライマーとして用いる場合であれば、例えば15塩基～35塩基が例示でき、例えばプローブとして用いる場合であれば、例えば15塩基～35塩基が例示できる。 The base length of the primer, probe, etc. is not particularly limited as long as it has a continuous length of at least 15 bases as described above, and can be appropriately set according to the intended use. As the base length, for example, when used as a primer, for example, 15 bases to 35 bases can be exemplified, and when used as a probe, for example, 15 bases to 35 bases can be exemplified.
　本発明の検査薬は、組成物の形態であってもよい。該組成物には、必要に応じて他の成分が含まれていてもよい。他の成分としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 The test agent of the present invention may be in the form of a composition. The composition may contain other components, if necessary. Other ingredients include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances. , Chelating agent and the like.
　本発明の検査薬は、キットの形態であってもよい。該キットには、上記検出剤或いはこれを含む上記組成物のほかに、被検試料の調製や対象バイオマーカーの検出に使用し得るものを含んでいてもよい。このようなものの具体例としては、各種試薬（例えば二次抗体、緩衝液、被検試料の採取、精製、分離、濃縮用試薬等）、器具（例えば被検試料の採取、精製、分離、濃縮用器具（例えばカラム等））等が挙げられる。 The test agent of the present invention may be in the form of a kit. In addition to the above-mentioned detection agent or the above-mentioned composition containing the same, the kit may contain one that can be used for preparation of a test sample or detection of a target biomarker. Specific examples of such a reagent include various reagents (for example, secondary antibody, buffer solution, reagent for collecting, purifying, separating, and concentrating a test sample), and an instrument (for example, collecting, purifying, separating, and concentrating a test sample). Equipment (for example, column etc.)) and the like can be mentioned.
　7．呼吸器疾患の予防又は治療剤の有効成分のスクリーニング方法
　本発明は、その一態様において、被検物質で処理された動物又は細胞から採取された被検試料におけるFchsd1の量又は濃度を指標とする、呼吸器疾患の予防又は治療剤の有効成分のスクリーニング方法（本明細書において、「本発明の有効成分スクリーニング方法」と示すこともある。）に関する。以下、これについて説明する。 7. 7. A method for screening an active ingredient of a prophylactic or therapeutic agent for respiratory diseases In one embodiment of the present invention, the amount or concentration of Fchsd1 in a test sample collected from an animal or cell treated with a test substance is used as an index. , A method for screening an active ingredient of a prophylactic or therapeutic agent for respiratory diseases (hereinafter, may be referred to as "the active ingredient screening method of the present invention"). This will be described below.
　Fchsd1、被検試料、呼吸器疾患、対象バイオマーカーの量又は濃度の測定等については、上記における定義と同様である。 The measurement of Fchsd1, test sample, respiratory disease, amount or concentration of target biomarker, etc. is the same as the above definition.
　動物の生物種は特に制限されない。動物の生物種としては、例えばサル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられる。 The species of animals is not particularly limited. Species of animals include various mammalian species such as monkeys, mice, rats, dogs, cats and rabbits.
　細胞の種類は特に制限されない。細胞は、体細胞又は幹細胞であり得る。幹細胞としては、例えば、ＥＳ細胞及びｉＰＳ細胞を挙げることができる。細胞の由来は特に制限されず、例えば、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物を挙げることができる。細胞が由来する組織は任意であり、例えば、肺組織に由来する細胞を使用できる。 The type of cell is not particularly limited. The cell can be a somatic cell or a stem cell. Examples of stem cells include ES cells and iPS cells. The origin of the cells is not particularly limited, and examples thereof include various mammals such as monkeys, mice, rats, dogs, cats, and rabbits. The tissue from which the cells are derived is arbitrary, and for example, cells derived from lung tissue can be used.
　被検物質としては、天然に存在する化合物又は人工に作られた化合物を問わず広く使用することができる。また、精製された化合物に限らず、多種の化合物を混合した組成物や、動植物の抽出液も使用することができる。化合物には、低分子化合物に限らず、タンパク質、核酸、多糖類等の高分子化合物も包含される。 As the test substance, it can be widely used regardless of whether it is a naturally occurring compound or an artificially produced compound. Further, not only the purified compound but also a composition in which various compounds are mixed and an extract of animals and plants can be used. The compound includes not only low molecular weight compounds but also high molecular weight compounds such as proteins, nucleic acids and polysaccharides.
　本発明の有効成分スクリーニング方法は、より具体的には、Fchsd1に関する前記指標の値が、被検物質で処理されていない動物から採取された被検試料におけるFchsd1の量又は濃度よりも低い場合に、前記被検物質を呼吸器疾患の予防又は治療剤の有効成分として選択する工程、を含む。 More specifically, the active ingredient screening method of the present invention is used when the value of the index for Fchsd1 is lower than the amount or concentration of Fchsd1 in a test sample collected from an animal not treated with the test substance. , The step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for respiratory diseases.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 "Low" means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　8．呼吸器疾患の誘発性又は増悪性の評価方法
　本発明は、その一態様において、被検物質で処理された動物から採取された被検試料又は被検物質で処理された細胞におけるFchsd1の量又は濃度を指標とする、呼吸器疾患の誘発性又は増悪性の評価方法（本明細書において、「本発明の毒性評価方法」と示すこともある。）に関する。以下、これについて説明する。 8. Method for Evaluating Inducible or Exacerbation of Respiratory Disease In one embodiment of the present invention, the amount of Fchsd1 in a test sample or cells treated with a test substance collected from an animal treated with the test substance or The present invention relates to a method for evaluating the induction or exacerbation of respiratory diseases using the concentration as an index (in the present specification, it may be referred to as “the toxicity evaluation method of the present invention”). This will be described below.
　Fchsd1、被検試料、被検物質、動物、細胞、呼吸器疾患、対象バイオマーカーの量又は濃度の測定等については、上記における定義と同様である。 The measurement of the amount or concentration of Fchsd1, test sample, test substance, animal, cell, respiratory disease, target biomarker, etc. is the same as the above definition.
　本発明の毒性評価方法は、より具体的には、Fchsd1に関する前記指標の値が、被検物質で処理されていない動物から採取された被検試料におけるFchsd1の量又は濃度よりも高い場合に、前記被検物質を呼吸器疾患の誘発性又は増悪性があると判定する工程、を含む。 More specifically, the toxicity evaluation method of the present invention is used when the value of the index for Fchsd1 is higher than the amount or concentration of Fchsd1 in a test sample collected from an animal not treated with the test substance. It includes a step of determining that the test substance has an inducing or exacerbating malignancy of respiratory disease.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 "High" means, for example, that the value of the index is twice, five times, ten times, 20 times, 50 times, and 100 times the control value.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
　2つの独立したグループのデータは、スチューデントのt検定によって分析した。すべてのテストで、0.05未満のP値は統計的に有意であると見なした。 Data from two independent groups were analyzed by Student's t-test. In all tests, P-values less than 0.05 were considered statistically significant.
　試験例1．肺気腫モデルにおける肺組織・肺胞上皮細胞のFCHSD1量の検討
　本試験例で使用した材料と採用した方法は以下の通りである。 Test example 1. Examination of FCHSD1 amount of lung tissue / alveolar epithelial cells in emphysema model The materials used in this test example and the method adopted are as follows.
　＜エラスターゼ処理（肺気腫モデルの作製）＞
　8～12週齢の雄マウスに、4.2 U / kg体重のブタ膵臓エラスターゼ（PPE、富士フィルム和光純薬株式会社）又は生理食塩水のみを50μlの量で気管内に投与した。 <Elastase treatment (preparation of emphysema model)>
Male mice aged 8 to 12 weeks were intratracheally administered with only 4.2 U / kg body weight of porcine pancreatic elastase (PPE, Wako Pure Chemical Industries, Ltd.) or saline in an amount of 50 μl.
　＜細胞培養＞
　マウスII型肺胞上皮細胞（MLE-12）は、2％ウシ胎児血清（no.10270-106; Gibco）、100 U / mlペニシリンG、100μg/ mlストレプトマイシン（どちらもNacalai Tesqueから）、0.005 mg / mlインスリン（no.2585; Gibco）、0.01 mg / mlトランスフェリン（no.T5391; Sigma Aldrich）、30 nM亜セレン酸ナトリウム（no.S9133; Sigma Aldrich）、10 nMヒドロコルチゾン（no.H0135; Sigma Aldrich）、10 nM βエストラジオール（no.E2257; Sigma Aldrich）、10 mM HEPES（no.H0887; Sigma Aldrich）、及び2 mM L-グルタミン（no. 250030; Gibco）を添加したDMEM Ham's F12培地（no.08460-95; Nacalai Tesque）で、5％CO2、95％O2雰囲気、37℃で培養した。 <Cell culture>
Mouse type II alveolar epithelial cells (MLE-12) are 2% fetal bovine serum (no.10270-106; Gibco), 100 U / ml penicillin G, 100 μg / ml streptomycin (both from Nacalai Tesque), 0.005 mg. / ml insulin (no.2585; Gibco), 0.01 mg / ml transferase (no.T5391; Sigma Aldrich), 30 nM sodium selenate (no.S9133; Sigma Aldrich), 10 nM hydrocortisone (no.H0135; Sigma Aldrich) ), 10 nM β estradiol (no.E2257; Sigma Aldrich), 10 mM HEPES (no.H0887; Sigma Aldrich), and 2 mM L-glutamine (no. 250030; Gibco) in DMEM Ham's F12 medium (no. 08460-95; Nacalai Tesque), cultured at 5% CO2, 95% O2 atmosphere, 37 ° C.
　＜イムノブロッティング＞
　培養細胞を、cOmplete Mini Protease Inhibitor Cocktail（Roche）を含む溶解バッファー[20 mM Tris-HCl（pH 7.5）、150 mM NaCl、1 mM EDTA及び1％（vol / vol）Nonidet P-40] で溶解し、組織サンプルをRIPAバッファー(no.08714; Nacalai Tesque)中で1:10 (w/v)でホモジナイズした後、遠心分離して残った細胞/組織をペレット化した。溶解物を標準のSDS-PAGEで分離した後に、次の抗体を使用してイムノブロッティングで分析した。：SIRT1（no. 9475; Cell Signaling Technology）、FCHSD1（no. 23362-1-AP; Proteintech）、PPARγ（no. 2443; Cell Signaling Technology）、NRF2（no. 12721; Cell Signaling Technology）、Lamin A/C（no. 2032; Cell Signaling Technology）、β-チューブリン（No.2128；Cell Signaling Technology）、WRP（No.E-AB-11600；Elabscience）、TOCA1（No.E-AB-19876；Elabscience）、FER（No.E-AB-19864；Elabscience）、CIP4（No.E-AB-61483；Elabscience）、FCHSD2（No.E-AB-183665；bior．orb183665; biorbyt）、Importin8（no. ab208162; Abcam）、Importinα5（no. 18137-1-AP; Proteintech）、SNX9（no. 15721-1-AP; Proteintech）、Importinβ1（no. 51186; Cell Signaling Technology）、KPNA2（no. 14372; Cell Signaling Technology）、Importinβ1（no. 51186; Cell Signaling Technology）、KPNA2（14372；Cell Signaling Technology）、β-アクチン（no.sc-47778；Santa Cruz Biotechnology）、Cleaved Caspase-3（no.9664；Cell Signaling Technology）、Caspase-3（no.9662；Cell Signaling Technology）、及びアセチル-p53（no.2570；Cell Signaling Technology）。 <Immunoblotting>
Cultured cells were lysed with lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and 1% (vol / vol) Nonidet P-40] containing cOmplete Mini Protease Inhibitor Cocktail (Roche). , Tissue samples were homogenized in RIPA buffer (no.08714; Nacalai Tesque) at 1:10 (w / v) and then centrifuged to pellet the remaining cells / tissues. The lysates were separated by standard SDS-PAGE and then analyzed by immunoblotting using the following antibodies. : SIRT1 (no. 9475; Cell Signaling Technology), FCHSD1 (no. 23362-1-AP; Proteintech), PPARγ (no. 2443; Cell Signaling Technology), NRF2 (no. 12721; Cell Signaling Technology), Lamin A / C (no. 2032; Cell Signaling Technology), β-tubulin (No.2128; Cell Signaling Technology), WRP (No.E-AB-11600; Elabscience), TOCA1 (No.E-AB-19876; Elabscience) , FER (No.E-AB-19864; Elabscience), CIP4 (No.E-AB-61483; Elabscience), FCHSD2 (No.E-AB-183665; bior.orb183665; biorbyt), Importin8 (no. Ab208162; Abcam), Importinα5 (no. 18137-1-AP; Proteintech), SNX9 (no. 15721-1-AP; Proteintech), Importinβ1 (no. 51186; Cell Signaling Technology), KPNA2 (no. 14372; Cell Signaling Technology) , Importin β1 (no. 51186; Cell Signaling Technology), KPNA2 (14372; Cell Signaling Technology), β-actin (no.sc-47778; Santa Cruz Biotechnology), Cleaved Caspase-3 (no.9664; Cell Signaling Technology), Caspase-3 (no.9662; Cell Signaling Technology), and Acetyl-p53 (no.2570; Cell Signaling Technology).
　＜LPS誘発肺炎症モデルの作製＞
　雄マウス（8～12週齢）に、サルモネラ菌ミネソタR595由来のLPS（10 mg / kg体重; Invivogen）を気管内投与した。 <Preparation of LPS-induced pneumonia model>
Male mice (8-12 weeks old) were intratracheally administered with LPS (10 mg / kg body weight; Invivogen) derived from Salmonella Minnesota R595.
　＜結果＞
　エラスターゼ処理後のマウス肺における FCH ファミリー蛋白質の発現を調べた。その結果、エラスターゼ処理後の肺では、FCHSD1 の発現量が増加していたのに対し、CIP4、TOCA1、FER、WRP、FCHSD2 などの他の FCH ファミリー蛋白質の発現量は変化していなかった（図 1A）。また、H2O2刺激後のマウスII型肺胞上皮細胞（MLE-12細胞）において、FCHSD1タンパク質が増加していることを見出した（図1B）。対照的に、肺にLPSを注入してもFCHSD1レベルは変化しなかった（図1C）。また、FCHSD1は他組織に比べ肺組織で高く発現していることが確認された（図2A）。 <Result>
We investigated the expression of FCH family proteins in mouse lungs after elastase treatment. As a result, the expression level of FCHSD1 was increased in the lung after elastase treatment, while the expression level of other FCH family proteins such as CIP4, TOCA1, FER, WRP, and FCHSD2 did not change (Fig.). 1A). We also found that FCHSD1 protein was increased in mouse type II alveolar epithelial cells (MLE-12 cells) after H 2 O 2 stimulation (Fig. 1B). In contrast, injection of LPS into the lung did not change FCHSD1 levels (Fig. 1C). It was also confirmed that FCHSD1 was highly expressed in lung tissue compared to other tissues (Fig. 2A).
　試験例2．Fchsd1 -/- マウスの作製
　本試験例で使用した材料と採用した方法は以下の通りである。 Test example 2. Preparation of Fchsd1 -/- mouse The materials used in this test example and the method adopted are as follows.
　＜マウスの作製＞
　C57BL6/Jマウスから卵子と精子を採取し、改変ヒト尿管液培地で3時間一緒に培養した。受精卵をM2培地を用いて洗浄し、次いで30個の接合体を採取し、Opti-MEM Iで3回洗浄し、培地中の血清を除去した。次いで、この接合体を、F501PT1-10白金板電極（長さ：10mm、幅：3mm、高さ：0.5mm、ギャップ：1mm）を備えたCUY21EDIT IIエレクトロポレーターを用いてエレクトロポレーションした（BEX社）。次いで、Cas9タンパク質とsgRNAの混合物を含む5μlのOpti-MEM Iを充填した電極上に配置し、エレクトロポレーションを行った。エレクトロポレーション後、直ちに接合体を電極チャンバーから回収し、M2培地で4回洗浄した。次いで、この卵をMWM培地で37℃、5％CO2インキュベーター中で2細胞期まで培養した。次いで、これらの卵を、仮妊娠雌の卵管に膣栓の日に移した。 <Making a mouse>
Eggs and sperms were collected from C57BL6 / J mice and cultured together in modified human ureteral medium for 3 hours. Fertilized eggs were washed with M2 medium, then 30 zygotes were collected and washed 3 times with Opti-MEM I to remove serum in the medium. The junction was then electroporated using a CUY21EDIT II electroporator equipped with an F501PT1-10 platinum plate electrode (length: 10 mm, width: 3 mm, height: 0.5 mm, gap: 1 mm) (BEX). Company). It was then placed on an electrode filled with 5 μl Opti-MEM I containing a mixture of Cas9 protein and sgRNA and electroporated. Immediately after electroporation, the conjugate was removed from the electrode chamber and washed 4 times with M2 medium. The eggs were then cultured in MWM medium at 37 ° C. in a 5% CO2 incubator to the 2 cell stage. These eggs were then transferred to the fallopian tubes of the fake pregnant female on the day of vaginal plugging.
　肺気腫の発生におけるFCHSD1の役割を決定するために、メンデル比で生まれたFchsd1ノックアウト（Fchsd1-/-）マウスを作製した。Fchsd1-/-マウスと野生型（WT）マウスとの間には、脾臓、骨髄、血液、肺の免疫細胞画分に有意な差はなく、これらの臓器の総体的な外観及び組織学的形態にも有意な差はなかった。 To determine the role of FCHSD1 in the development of emphysema, Fchsd1 knockout (Fchsd1 -/- ) mice born in Mendelian ratios were generated. There were no significant differences in the immune cell fractions of the spleen, bone marrow, blood and lungs between Fchsd1 -/- mouse and wild-type (WT) mice, and the overall appearance and histological morphology of these organs. There was no significant difference in.
　試験例3．FCHSD1抑制による肺気腫に対する保護作用の検討
　本試験例で使用した材料と採用した方法は以下の通りである。以下の記載の無い材料及び方法については、上記試験例と同様である。 Test example 3. Examination of protective effect against emphysema by suppressing FCHSD1 The materials used and the method adopted in this test example are as follows. The materials and methods not described below are the same as those in the above test example.
　＜磁気共鳴画像＞
　1H QDコイルを備えたAvanceIII BioSpec 117/11システム（Bruker）をMRIイメージングに使用した。画像取得中にマウスをセボフルランで麻酔し、温水循環チューブでマウスの体温を維持した。超短エコー時間イメージング法は、次のパラメータを使用して肺イメージングに使用した：TR / TE = 5 / 0.1ミリ秒、視野3 cm、スライス厚0.15 mm。画像マトリックスは200×200×200に設定した。 <Magnetic resonance image>
An Avance III BioSpec 117/11 system (Bruker) with 1H QD coils was used for MRI imaging. Mice were anesthetized with sevoflurane during image acquisition and the temperature of the mice was maintained with a warm water circulation tube. The ultrashort echo time imaging method was used for lung imaging using the following parameters: TR / TE = 5 / 0.1 ms, field of view 3 cm, slice thickness 0.15 mm. The image matrix was set to 200 x 200 x 200.
　＜組織学的分析＞
　各臓器を速やかに切除し、4％パラホルムアルデヒドで24時間固定し、パラフィンに包埋した。組織切片（厚さ5μm）は、パラフィンミクロトームを使用して調製し、キシレンで脱パラフィンし、段階的エタノールシリーズで脱水し、ヘマトキシリン及びエオシン（HE）染色用に処理した。HEで染色したスライドを使用して、肺気腫を評価した。全肺の気腫に冒された肺組織の割合は、BZ-X710蛍光顕微鏡（Keyence Corp.）を使用して測定し、画像はメーカーの指示に従ってBZ-Xアナライザーソフトウェアを使用して処理及び再構築した。すべての定量的測定は、同じ光学条件と光条件下で同等の領域で実行した。肺気腫の評価のために、HE染色後にImage Jソフトウェアを使用して平均線形切片（M.L.I.）及び肺気腫面積を測定した。 <Histological analysis>
Each organ was rapidly resected, fixed with 4% paraformaldehyde for 24 hours and embedded in paraffin. Tissue sections (5 μm thick) were prepared using a paraffin microtome, deparaffinized with xylene, dehydrated with a stepwise ethanol series and treated for hematoxylin and eosin (HE) staining. Emphysema was assessed using HE-stained slides. The proportion of lung tissue affected by emphysema of the entire lung is measured using a BZ-X710 fluorescence microscope (Keyence Corp.), and images are processed and reprocessed using BZ-X analyzer software according to the manufacturer's instructions. It was constructed. All quantitative measurements were performed in comparable regions under the same optical and optical conditions. For evaluation of emphysema, mean linear intercept (MLI) and emphysema area were measured using Image J software after HE staining.
　＜結果＞
　エラスターゼを気管内投与したFchsd1-/-マウスとWTマウスをMRIで解析した結果、Fchsd1-/-マウスはWTマウスに比べて肺の不透明度が減っていることが明らかになった（図2B）。これと一致するように、組織学的解析を行ったところ、Fchsd1-/-マウスはWTマウスに比べて平均肺胞径（Mean linear intercept（M.L.I.））と肺気腫面積が有意に減少していることが明らかになった（図2C-D）。 <Result>
MRI analysis of Fchsd1 -/- mice and WT mice that received elastase intratracheally revealed that Fchsd1 -/- mice had less lung opacity than WT mice (Fig. 2B). .. Histological analysis consistent with this showed that Fchsd1 -/- mice had significantly reduced mean alveolar diameter (Mean linear intercept (MLI)) and emphysema area compared to WT mice. Was clarified (Fig. 2C-D).
　試験例4．FCHSD1がエラスターゼ処理マウス肺の炎症と細胞死に及ぼす影響の検討
　本試験例で使用した材料と採用した方法は以下の通りである。以下の記載の無い材料及び方法については、上記試験例と同様である。 Test example 4. Examination of the effect of FCHSD1 on inflammation and cell death of elastase-treated mouse lung The materials used and the method adopted in this test example are as follows. The materials and methods not described below are the same as those in the above test example.
　＜気管支肺胞洗浄（BAL）及びフローサイトメトリーとELISAによる分析＞
　気管を切開した後、プラスチック製のカニューレを挿入し、空腔を1mlの生理食塩水で洗浄した。この手順を3回行った。遠心分離後、上清をサイトカイン測定に使用した。細胞は細胞測定に使用した。細胞を赤血球溶解緩衝液（Sigma-Aldrich）で処理し、FACS緩衝液で洗浄した。細胞測定データは、FACS Canto IIフローサイトメーターを使用して取得し、上記のようにFlowJoソフトウェアで分析した。市販のELISAアッセイキット（R＆D Biosystems）を使用して、製造元の指示に従って、示されたサンプル中の総TNFα及びIL6を測定した。 <Bronchoalveolar lavage (BAL) and flow cytometry and ELISA analysis>
After the trachea was incised, a plastic cannula was inserted and the void was washed with 1 ml saline. This procedure was performed 3 times. After centrifugation, the supernatant was used for cytokine measurement. The cells were used for cell measurement. Cells were treated with erythrocyte lysis buffer (Sigma-Aldrich) and washed with FACS buffer. Cell measurement data were acquired using a FACS Canto II flow cytometer and analyzed with FlowJo software as described above. Total TNFα and IL6 in the indicated samples were measured using a commercially available ELISA assay kit (R & D Biosystems) according to the manufacturer's instructions.
　＜フローサイトメトリー＞
　細胞懸濁液は、ふるいにかけ、ピペッティングすることにより調製した。細胞をFACS緩衝液［0.5％ウシ血清アルブミン（BSA）及びPBS中2mM EDTA、pH7.2］で洗浄し、次いで、抗体と20分間インキュベートし、続いてFACS緩衝液で2回洗浄した。データはフローサイトメーター（FACS Canto II、BD Bioscience、サンノゼ、カリフォルニア州、米国）で取得し、FlowJo（Tree Star Inc.）を使用して分析した。フローサイトメトリー用の抗体は、以下の販売元から購入した。FITCanti-Ly-6C（HK1.4; BioLegend）; PE anti-Ly-6G（1A8; BioLegend）; PerCP anti-Mac1（M1 / 70; BioLegend）; APCアンチCD45.2（104; BioLegend）;抗CD11c（N418; BioLegend）;抗CD3e（145-2C11; BioLegend）;抗CD4（GK1.5; BioLegend）;抗F4 / 80（BM8; BioLegend）; Anti-SiglecF（S17007L; BioLegend）; CD8（53-6.7; Biolegend）; CCR3; （J073E5; Biolegend）; B220; （RA3-6B2; Biolegend）。 <Flow cytometry>
Cell suspensions were prepared by sieving and pipetting. Cells were washed with FACS buffer [0.5% bovine serum albumin (BSA) and 2 mM EDTA in PBS, pH 7.2], then incubated with antibody for 20 minutes, followed by washing twice with FACS buffer. Data were obtained with flow cytometers (FACS Canto II, BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo (Tree Star Inc.). Antibodies for flow cytometry were purchased from the following distributors. FITCanti-Ly-6C (HK1.4; BioLegend); PE anti-Ly-6G (1A8; BioLegend); PerCP anti-Mac1 (M1 / 70; BioLegend); APC Anti-CD45.2 (104; BioLegend); Anti-CD11c (N418; BioLegend); Anti-CD3e (145-2C11; BioLegend); Anti-CD4 (GK1.5; BioLegend); Anti-F4 / 80 (BM8; BioLegend); Anti-SiglecF (S17007L; BioLegend); CD8 (53-6.7) Biolegend); CCR3; (J073E5; Biolegend); B220; (RA3-6B2; Biolegend).
　＜細胞の分離と培養＞
　WTマウスとFchsd1-/-マウスからトリプシン処理により初代肺線維芽細胞（MLF）を分離し、10％ウシ胎児血清（no.10270-106; Gibco）、100 U / mlペニシリンG、100μg/ mlストレプトマイシン（どちらもNacalai Tesque製）、及び2-メルカプトエタノール（no.21417-52; Nacalai Tesque）を補充したDMEMで、5％CO2、95％O2雰囲気、37℃で培養した。 <Cell separation and culture>
Primary lung fibroblasts (MLF) were isolated from WT and Fchsd1-/-mice by trypsinization, 10% fetal bovine serum (no.10270-106; Gibco), 100 U / ml penicillin G, 100 μg / ml streptomycin. (Both manufactured by Nacalai Tesque) and DMEM supplemented with 2-mercaptoethanol (no.21417-52; Nacalai Tesque) were cultured in 5% CO2, 95% O2 atmosphere, and 37 ° C.
　＜発現プラスミドの構築＞
　Fchsd1、Nrf2、及びSnx9のcDNAは、マウスのcDNAライブラリからPCRによって取得した。Fchsd1 cDNAは、レトロウイルス生産のためにpLZR-ires-GFPにクローニングした。Flagタグ付きFchsd1、Flagタグ付きNrf2、及びFlagタグ付きSnx9は、免疫沈降のためにpcDNA3.1（+）にクローン化した。 <Construction of expression plasmid>
The cDNAs for Fchsd1, Nrf2, and Snx9 were obtained by PCR from the mouse cDNA library. The Fchsd1 cDNA was cloned into pLZR-ires-GFP for retrovirus production. Flag-tagged Fchsd1, Flag-tagged Nrf2, and Flag-tagged Snx9 were cloned to pcDNA3.1 (+) for immunoprecipitation.
　＜レトロウイルスによる形質導入＞
　MLE-12細胞をレトロウイルス上清で形質導入した。ウイルスは、全長のFchsd1 cDNA又は空のベクターを含むpLZR-ires-GFPでトランスフェクトされたPlatEパッケージング細胞を使用して生成した。形質導入後、FACS Aria III（BD Bioscience）を使用してGFP陽性細胞を選別した。 <Transduction by retrovirus>
MLE-12 cells were transduced with retrovirus supernatant. The virus was generated using PlatE packaging cells transfected with pLZR-ires-GFP containing a full-length Fchsd1 cDNA or an empty vector. After transduction, GFP-positive cells were screened using FACS Aria III (BD Bioscience).
　＜RT-qPCR＞
　RNA精製キット（No. 1828665; Roche）又はTRIzol（No. 15596018; Thermo Fisher Scientific）を使用して全RNAを単離し、製造元の指示に従ってReverTraAce（Toyobo）で逆転写を行った。リアルタイムPCRマスターミックス（Toyobo）を使用して定量的逆転写PCR（RT-qPCR）を行い、各遺伝子のTaqManプローブからの蛍光を7500リアルタイムPCRシステム（Applied Biosystems）で検出した。mRNAの相対誘導を決定するために、各遺伝子のmRNAレベルは、18Sに正規化された標準曲線法を使用して計算した。市販の遺伝子特異的プライマー及びプローブセットは、Integrated DNA Technologies（Coralville）から入手した。この研究で使用したプライマーとプローブは次の通りである：Acta2（Mm.PT.58.16320644）、Col1a1（Mm.PT.58.7562513）、Il6（Mm.PT.58.10005566）、Tgfb1（Mm.PT.58.11254750）、及びTnfa（Mm .PT.58.12575861）。 <RT-qPCR>
Total RNA was isolated using RNA purification kit (No. 1828665; Roche) or TRIzol (No. 15596018; Thermo Fisher Scientific) and reverse transcribed with ReverTraAce (Toyobo) according to the manufacturer's instructions. Quantitative reverse transcription PCR (RT-qPCR) was performed using a real-time PCR master mix (Toyobo), and fluorescence from the TaqMan probe of each gene was detected by the 7500 real-time PCR system (Applied Biosystems). To determine the relative induction of mRNA, the mRNA level of each gene was calculated using the standard curve method normalized to 18S. Commercially available gene-specific primers and probe sets were obtained from Integrated DNA Technologies (Coralville). The primers and probes used in this study are: Acta2 (Mm.PT.58.16320644), Col1a1 (Mm.PT.58.7562513), Il6 (Mm.PT.58.10005566), Tgfb1 (Mm.PT.58.11254750). , And Tnfa (Mm .PT.58.12575861).
　＜結果＞
　FCHSD1欠損による肺気腫に対する保護が、早期の炎症低下と関連しているかどうかを調べた。その結果、Fchsd1-/-マウスは、エラスターゼによって誘発される急性炎症に対して防御効果を示すことが確認された（図3 A-C）。対照的に、レトロウイルス感染によるMLE-12細胞におけるFchsd1の異所性発現は、H2O2誘発酸化ストレスに応答してIL-6及びTNFαのmRNAの増加をもたらした（図3G）。しかしながら、TLRリガンドに応答するIL-6及びTNFのような炎症性サイトカインの産生においては、Fchsd1-/-とWTの肺胞マクロファージとの間で違いは示されなかった。さらに、TUNEL染色により、WTマウスと比較して、Fchsd1-/-マウスではエラスターゼ気管内投与による肺胞構成細胞の細胞死が抑えられることを明らかにした（図3D）。この結果と一致して、H2O2処理後のFchsd1-/-マウス肺線維芽細胞（MLF）において、WTのMLFと比較して、切断されたカスパーゼ3の発現が減少した（図3E）。さらに、アポトーシスのマーカーであるLys382におけるp53のアセチル化は、Fchsd1-/-マウス肺線維芽細胞（MLFs）においてWT MLFsと比較して減少した（図3F）。以上のことから、FCHSD1はエラスターゼやH2O2刺激後の炎症や細胞死を促進することが示唆された。 <Result>
We investigated whether protection against emphysema due to FCHSD1 deficiency was associated with early reduction in inflammation. As a result, it was confirmed that Fchsd1 -/- mice showed a protective effect against acute inflammation induced by elastase (Fig. 3 AC). In contrast, ectopic expression of Fchsd1 in MLE-12 cells by retroviral infection resulted in an increase in IL-6 and TNFα mRNA in response to H 2 O 2 -induced oxidative stress (Fig. 3G). However, no difference was shown between Fchsd1 -/- and WT alveolar macrophages in the production of inflammatory cytokines such as IL-6 and TNF in response to TLR ligands. Furthermore, TUNEL staining revealed that Fchsd1 -/- mice suppressed cell death of alveolar constituent cells by intratracheal elastase administration compared with WT mice (Fig. 3D). Consistent with this result, the expression of cleaved caspase 3 was reduced in Fchsd1 -/- mouse lung fibroblasts (MLF) after H 2 O 2 treatment compared to WT MLF (Fig. 3E). .. In addition, p53 acetylation in Lys382, a marker of apoptosis, was reduced in Fchsd1 -/- mouse lung fibroblasts (MLFs) compared to WT MLFs (Fig. 3F). From the above, it was suggested that FCHSD1 promotes inflammation and cell death after stimulation with elastase and H 2 O 2 .
　試験例5．FCHSD1による、H 2 O 2 誘発酸化ストレスに応答したNuclear factor-like 2 (NRF2)の核移行のネガティブ制御の検討
　本試験例で使用した材料と採用した方法は以下の通りである。以下の記載の無い材料及び方法については、上記試験例と同様である。 Test example 5. Examination of Negative Control of Nuclear Translocation of Nuclear factor-like 2 (NRF2) in response to H 2 O 2 -induced oxidative stress by FCHSD1 The materials used in this test example and the method adopted are as follows. The materials and methods not described below are the same as those in the above test example.
　＜細胞内分画＞
　細胞内分画のために、MLF又はMLE-12細胞をディッシュあたり1.2×106細胞でプレーティングした。細胞を275μM H2O2（MLFの場合）又は250μM H2O2（MLE12細胞の場合）で刺激した。刺激後、Nuclear抽出キット（no.40010; Active Motif）を用い、その使用方法に従い、細胞抽出物を調製した。 <Intracellular fraction>
For intracellular fractionation, MLF or MLE-12 cells were plated with 1.2 × 10 6 cells per dish. Cells were stimulated with 275 μM H 2 O 2 (for MLF) or 250 μM H 2 O 2 (for MLE 12 cells). After stimulation, a Cell Extract was prepared using a Nuclear Extraction Kit (no.40010; Active Motif) according to the method of use.
　＜トランスフェクションと免疫沈降＞
　MLE-12細胞は、Flagタグ付きFchsd1、Flagタグ付きNrf2、HAタグ付きNrf2、Flagタグ付きSnx9、及びMycタグ付きSnx9をコードするpcDNA3.1（+）又は空ベクターで、リポフェクタミン2000（no.11668027; Thermo Fisher Scientific）を用いてトランスフェクションした。トランスフェクションの24時間後、細胞溶解物を回収した。抗Flag抗体を用いた免疫沈降のために、cOmplete Mini Protease Inhibitor Cocktail（Roche）を含む溶解緩衝液[20 mM Tris-HCl（pH 7.5）、150 mM NaCl、及び1％（vol/vol）Nonidet P-40]で溶解した。細胞溶解物を抗Flag M2アフィニティーゲル（No. A2220; Sigma Aldrich）と2時間インキュベートし、洗浄バッファー[20 mM Tris-HCl（pH 7.5）及び150 mM NaCl]で洗浄した。抗Myc抗体を用いた免疫沈降には、製造元の指示に従い、セファロースビーズコンジュゲートMycタグ抗体（No. 3400; Cell Signaling Technology）を使用した。タンパク質を標準のSDS-PAGEで分離し、イムノブロッティングで分析した。 <Transfection and immunoprecipitation>
MLE-12 cells are pcDNA3.1 (+) or empty vectors encoding Fchsd1, Flag-tagged Nrf2, HA-tagged Nrf2, Flag-tagged Snx9, and Myc-tagged Snx9, and are lipofectamine 2000 (no. 11668027; Thermo Fisher Scientific) was used for transfection. Twenty-four hours after transfection, cytolysis was collected. For immunoprecipitation with anti-Flag antibody, lysis buffer containing cOmplete Mini Protease Inhibitor Cocktail (Roche) [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% (vol / vol) Nonidet P. -40] dissolved. Cytolysis was incubated with anti-Flag M2 affinity gel (No. A2220; Sigma Aldrich) for 2 hours and washed with wash buffer [20 mM Tris-HCl (pH 7.5) and 150 mM NaCl]. For immunoprecipitation using the anti-Myc antibody, Sepharose bead-conjugated Myc-tag antibody (No. 3400; Cell Signaling Technology) was used according to the manufacturer's instructions. Proteins were separated by standard SDS-PAGE and analyzed by immunoblotting.
　酸化ストレスに対する酸化ストレス感受性転写因子であるNRF2の細胞内局在を調べた。細胞質及び核画分のイムノブロット解析を行ったところ、H2O2処理に応答して、Fchsd1-/-MLFではWT MLFに比べてNRF2の核内取り込みが亢進していることが示された（図4）。FCHSD1は2つのSH3ドメインを有しており、他のタンパク質との複数の相互作用を媒介している。NRF2の核内移行の制御におけるFCHSD1の機能をさらに明らかにするために、我々は、エンドサイトーシス及び細胞内輸送におけるその役割のための鍵となるインタラクターとして、ソーティングネクシン9(SNX9)に注目した。SNX9がFCHSD1と相互作用するタンパク質であることを確認し、FCHSD1がNRF2に直接結合するかどうかを確認するために、我々はMLE-12細胞においてFCHSD1、SNX9、及びNRF2との共免疫沈降実験を行った。その結果、FCHSD1はSNX9と相互作用し、NRF2とも相互作用することが分かった（図5A）。次に、H2O2刺激がこれらの相互作用を変化させるかどうかについて調べた。NRF2及びSNX9へのFCHSD1の結合は、H2O2処理に応答して阻害された（図5B）。並行して、SNX9とNRF2及びSNX9と相互作用するImportinβの1つであるImportin8へのSNX9の結合は、H2O2刺激後に促進された（図5C）。FCHSD1は、SIRT1、KEAP1、又はImportinとは相互作用せず、SNX9、NRF2はImportinに結合しなかった。これらの結果は、FCHSD1が細胞質でNRF2及びSNX9と相互作用し、NRF2が核に移行するのを阻止することを示している。さらに、H2O2刺激に応答して、FCHSD1は複合体から解離し、NRF2の核内移行は、NRF2-SNX9及びSNX9-Importin8相互作用の増加と関連して起こることを示す。 The intracellular localization of NRF2, a transcription factor sensitive to oxidative stress, was investigated. Immunoblot analysis of cytoplasmic and nuclear fractions showed that Fchsd1 − / − MLF had enhanced nuclear uptake of NRF2 compared to WT MLF in response to H 2 O 2 treatment. (Fig. 4). FCHSD1 has two SH3 domains and mediates multiple interactions with other proteins. To further elucidate the function of FCHSD1 in controlling the nuclear translocation of NRF2, we have assigned Sorting Nexin 9 (SNX9) as a key interactor for its role in endocytosis and intracellular transport. noticed. To confirm that SNX9 is a protein that interacts with FCHSD1 and to confirm whether FCHSD1 binds directly to NRF2, we conducted co-immunoprecipitation experiments with FCHSD1, SNX9, and NRF2 in MLE-12 cells. gone. As a result, it was found that FCHSD1 interacts with SNX9 and also with NRF2 (Fig. 5A). Next, we investigated whether H 2 O 2 stimuli alter these interactions. Binding of FCHSD1 to NRF2 and SNX9 was inhibited in response to H 2 O 2 treatment (Fig. 5B). In parallel, the binding of SNX9 to Importin8, one of the Importinβ that interacts with SNX9 and NRF2 and SNX9, was promoted after H 2 O 2 stimulation (Fig. 5C). FCHSD1 did not interact with SIRT1, KEAP1, or Importin, and SNX9, NRF2 did not bind to Importin. These results indicate that FCHSD1 interacts with NRF2 and SNX9 in the cytoplasm and blocks NRF2 from translocating to the nucleus. Furthermore, in response to H 2 O 2 stimulation, FCHSD1 dissociates from the complex, indicating that nuclear translocation of NRF2 occurs in association with increased NRF2-SNX9 and SNX9-Importin8 interactions.
　試験例6．エラスターゼ処理マウス肺とH 2 O 2 処理細胞におけるFCHSD1欠損によるサーチュイン1（SIRT1）への影響
　急性肺炎やアポトーシス時にFCHSD1が及ぼすその他の影響を確認する目的で、エラスターゼ処理後の肺のSIRT1のレベルを検討した。エラスターゼを24時間気管内投与したWTマウス肺のSIRT1レベルは顕著な減少をもたらしたのに対し、Fchsd1-/-マウスにおいては、その減少が抑制された。同様に、SIRT1による脱アセチル化の標的分子であるFOXO3a及びPPARγレベルは、WTマウスよりもFchsd1-/-マウスの方が高かった。また、H2O2処理したWTのMLFにおけるSIRT1の減少も、Fchsd1-/-MLFにおいて抑制されることが確認された。対照的に、MLE-12細胞において、レトロウイルスを用いてFCHSD1を発現させた場合、非常に低いH2O2濃度においてもSIRT1の減少をもたらすことが確認された。これらの結果は、FCHSD1が酸化ストレスに応答したSIRT1の減少において、重要な役割を果たしていることを示す。 Test example 6. Effects of FCHSD1 deficiency on sirtuin 1 (SIRT1) in elastase-treated mouse lungs and H 2 O 2 treated cells To confirm other effects of FCHSD1 during acute pneumonia and apoptosis, SIRT1 levels in lungs treated with elastase were examined. investigated. SIRT1 levels in the lungs of WT mice treated intratracheally for 24 hours with elastase resulted in a marked decrease, whereas the decrease was suppressed in Fchsd1 -/- mice. Similarly, the levels of FOXO3a and PPARγ, which are the target molecules for deacetylation by SIRT1, were higher in Fchsd1 -/- mice than in WT mice. It was also confirmed that the decrease in SIRT1 in MLF of WT treated with H 2 O 2 was also suppressed in Fchsd1 − / − MLF. In contrast, expression of FCHSD1 in MLE-12 cells with a retrovirus was found to result in a reduction in SIRT1 even at very low H 2 O 2 concentrations. These results indicate that FCHSD1 plays an important role in the reduction of SIRT1 in response to oxidative stress.

Claims (10)

  1. （1）被検体から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）を検出する工程を含む、呼吸器疾患を検査する方法。 (1) A method for examining a respiratory disease, which comprises a step of detecting Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from a subject.
  2. さらに、（2）前記工程（1）で検出されたFchsd1の量又は濃度がカットオフ値以上である場合に、前記被検体が呼吸器疾患に罹患していると判定する工程、を含む、請求項１に記載の検査方法。 Further, the claim includes (2) a step of determining that the subject has a respiratory disease when the amount or concentration of Fchsd1 detected in the step (1) is equal to or higher than the cutoff value. Item 1. The inspection method according to Item 1.
  3. 前記Fchsd1がタンパク質である、請求項１又は２に記載の検査方法。 The test method according to claim 1 or 2, wherein Fchsd1 is a protein.
  4. 前記被検試料は、被検体の肺組織を含み得る被検試料である、請求項１～３のいずれかに記載の検査方法。 The test method according to any one of claims 1 to 3, wherein the test sample is a test sample that may include lung tissue of the test subject.
  5. 前記肺組織を含み得る被検試料が、肺の生検試料、及び肺の洗浄液からなる群より選択される少なくとも1種を含む、請求項４に記載の検査方法。 The test method according to claim 4, wherein the test sample that may contain the lung tissue includes at least one selected from the group consisting of a lung biopsy sample and a lung lavage fluid.
  6. Fchsd1（FCH and double SH3 domains protein 1）の検出剤を含む、呼吸器疾患の検査薬。 A test drug for respiratory diseases, including a detection agent for Fchsd1 (FCH and double SH3 domains protein 1).
  7. 被検物質で処理された動物から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の予防又は治療剤の有効成分のスクリーニング方法。 A method for screening an active ingredient of a preventive or therapeutic agent for respiratory diseases, using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
  8. 被検物質で処理された動物から採取された被検試料におけるFchsd1（FCH and double SH3 domains protein 1）の量又は濃度を指標とする、呼吸器疾患の誘発性又は増悪性の評価方法。 A method for evaluating the induction or exacerbation of respiratory diseases using the amount or concentration of Fchsd1 (FCH and double SH3 domains protein 1) in a test sample collected from an animal treated with a test substance as an index.
  9. Fchsd1（FCH and double SH3 domains protein 1）発現抑制剤及びFchsd1機能抑制剤からなる群より選択される少なくとも1種の成分を含有する、呼吸器疾患の予防又は治療剤  。 A preventive or therapeutic agent for respiratory diseases, which contains at least one component selected from the group consisting of an Fchsd1 (FCH and double SH3 domains protein 1) expression inhibitor and an Fchsd1 function inhibitor.
  10. 前記成分が、Fchsd1を標的としたポリヌクレオチド、該ポリヌクレオチドの発現カセット、低分子化合物、ペプチド、タンパク質、及び抗体からなる群より選択される少なくとも1種である、請求項９に記載の予防又は治療剤。 The prophylaxis or prophylaxis according to claim 9, wherein the component is at least one selected from the group consisting of a polynucleotide targeting Fchsd1, an expression cassette of the polynucleotide, a small molecule compound, a peptide, a protein, and an antibody. Therapeutic agent.
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* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
Title
KAWASAKI TAKAHIRO, SUGIHARA FUMINORI, FUKUSHIMA KIYOHARU, MATSUKI TAKANORI, NABESHIMA HIROSHI, MACHIDA TOMOHISA, MITSUI YUICHI, FU: "Loss of FCHSD1 leads to amelioration of chronic obstructive pulmonary disease", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 118, no. 26, 29 June 2021 (2021-06-29), XP055924581, ISSN: 0027-8424, DOI: 10.1073/pnas.2019167118 *
XIAO GUAN-YU, MOHANAKRISHNAN APARNA, SCHMID SANDRA L.: "Role for ERK1/2-dependent activation of FCHSD2 in cancer cell-selective regulation of clathrin-mediated endocytosis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 115, no. 41, 9 October 2018 (2018-10-09), XP055924576, ISSN: 0027-8424, DOI: 10.1073/pnas.1810209115 *

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