WO2022085687A1 - Agent de prévention ou de traitement de maladies des voies respiratoires, procédé de détection, agent de détection, procédé de criblage de principe actif d'agent de prévention ou de traitement, et procédé d'évaluation d'inductibilité ou de risques d'aggravation - Google Patents

Agent de prévention ou de traitement de maladies des voies respiratoires, procédé de détection, agent de détection, procédé de criblage de principe actif d'agent de prévention ou de traitement, et procédé d'évaluation d'inductibilité ou de risques d'aggravation 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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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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.

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Abstract

L'invention fournit une technique de traitement, de prévention et de diagnostique de maladies des voies respiratoires. Selon l'invention, des maladies des voies respiratoires sont prévenues ou traitées par inhibition de l'expression ou des fonctions d'une Fchsd1 (protéine 1 à domaines FCH et double SH3), et cette Fchsd1 est mise en œuvre en tant que biomarqueur de diagnostique de maladies des voies respiratoires.
PCT/JP2021/038632 2020-10-19 2021-10-19 Agent de prévention ou de traitement de maladies des voies respiratoires, procédé de détection, agent de détection, procédé de criblage de principe actif d'agent de prévention ou de traitement, et procédé d'évaluation d'inductibilité ou de risques d'aggravation WO2022085687A1 (fr)

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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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