WO2021107005A1 - Prophylactic or therapeutic agent for cytomegalovirus-related diseases - Google Patents

Prophylactic or therapeutic agent for cytomegalovirus-related diseases Download PDF

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WO2021107005A1
WO2021107005A1 PCT/JP2020/044004 JP2020044004W WO2021107005A1 WO 2021107005 A1 WO2021107005 A1 WO 2021107005A1 JP 2020044004 W JP2020044004 W JP 2020044004W WO 2021107005 A1 WO2021107005 A1 WO 2021107005A1
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protein
homolog
cytomegalovirus
family
factor
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Japanese (ja)
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中村　浩幸
拓実 渡邉
成悦 藤原
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国立研究開発法人国立成育医療研究センター
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Definitions

  • the present invention relates to an agent for preventing or treating cytomegalovirus-related diseases.
  • Cytomegalovirus is a general term for viruses belonging to the genus Cytomegalovirus of the herpesvirus family, and human cytomegalovirus (HCMV) that infects humans is known.
  • HCMV human cytomegalovirus
  • HCMV infection occurs frequently, but in most healthy individuals, the symptoms do not manifest themselves. On the other hand, in the fetal and those with weakened immune function, infection with HCMV often causes serious symptoms.
  • HCMV infects the foetation through the placenta of a pregnant woman (so-called congenital infection). As a result, it can cause a delay in fetal intrauterine development.
  • congenital HCMV presenting with various organ disorders such as microcephaly; mental retardation; and neurological / sensory disorders such as sensory hearing loss; after the fetal period (ie, fetal and postnatal).
  • HCMV causes opportunistic infections such as pneumonia, enteritis, and retinitis in patients with iatrogenic immunodeficiency associated with transplantation medicine and patients with AIDS.
  • antiviral drug targets DNA polymerase encoded by HCMV
  • drug-resistant HCMV strains appear.
  • the above-mentioned antiviral drugs may cause serious side effects, so that the indications are limited.
  • some antiviral drugs are contraindicated for maternal administration because they can cause very serious side effects on the foetation.
  • fomivirsen is an antisense RNA against IE2 encoded by HCMV, and it has been pointed out that there is a risk of drug-resistant HCMV strains appearing.
  • the present invention has been made in view of the above problems, and an object of the present invention is to provide a novel agent that can be used for the prevention or treatment of cytomegalovirus-related diseases, which has a different mechanism of action from conventional agents for HCMV infection. To provide.
  • cytomegalovirus-related diseases by suppressing the expression or function of a specific gene in humans infected with HCMV.
  • the present invention has been completed. That is, one aspect of the present invention is as follows. ⁇ 80.
  • An agent for preventing or treating a cytomegalovirus-related disease which comprises a component that suppresses the expression or function of at least one gene selected from the group consisting of the genes of. 1.
  • AHNAK2 AHNAK nucleoprotein 2
  • AIDA axin interactor, dorsalization associated
  • AKAP5 A kinase (PRKA) anchor protein 5
  • AP1S2 adaptive protein complex 1, sigma 2 subunit
  • APC adenomatous polyposis coli
  • C3orf74 chromosome 3 open reading frame 74
  • C9orf9 chromosome 9 open reading frame 9)
  • C11orf67 chromosome 11 open reading frame 67
  • C19orf33 chromosome 19 open reading frame 33
  • CHTF18 CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae)
  • CREB1 cAMP responsive element binding protein 1
  • CYTH1 cytohesin 1
  • EML1 echinoderm microtubule associated protein like 1
  • EMP2 epidermal membrane protein 2 15.
  • EPHB6 EPH receptor B6
  • FAM160A1 family with sequence similarity 160, member A1
  • FAM176A family with sequence similarity 176, member A
  • FXR2 fragment X mental retardation, autosomal homolog 2
  • GPR126 G protein-coupled receptor 126)
  • HDHD1A haloacid dehalogenase-like hydrolase domain containing 1A
  • HNRNPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9)
  • TESPA1 Thymocyte Expressed, Positive Selection Associated 1
  • PALD1 Phosphatase Domain Containing Paladin 1
  • KRTAP12-1 Keratin associated protein 12-1) 25.
  • MBD1 methyl-CpG binding domain protein 1 26.
  • MESDC2 mesoderm development candidate 2 27.
  • MUC5AC mucin 5AC, oligomeric mucus / gel-forming
  • NOL12 nucleolar protein 12
  • NRP1 neutral protein 1 30.
  • NUP153 nucleoporin 153kDa
  • PATL2 protein associated with topoisomerase II homolog 2 (yeast)
  • PCBD1 pterin-4 alpha-carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha 33.
  • PPKRIR protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor) 34.
  • RBM26 RNA binding motif protein 26
  • RSBN1 round spermatid basic protein 1
  • SCXA seroton homolog A (mouse)
  • SPINT3 serpin peptidase inhibitor, Kunitz type, 3)
  • TATDN3 TatD DNase domain containing 3
  • TBC1D3G TBC1 domain family, member 3G
  • TSC22D1 TSC22 domain family, member 1
  • UNC119B unc-119 homolog B (C. elegans) 42.
  • WBP4 WW domain binding protein 4 (formin binding protein 21)) 43.
  • ZNF354A zinc finger protein 354A 44.
  • ATF1 Activating Transcription Factor 1
  • PGAM5 PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase
  • IPO8 Importin 8 47.
  • OGA O-GlcNA case
  • PROX1 Prospero Homeobox 1
  • CIDEC Cell Death Inducing DFFA Like Effector C
  • JPH2 Junctophilin 2
  • RNF208 Ring Finger Protein 208)
  • STEAP1B STEAP Family Member 1B 53.
  • TENM4 Teeurin Transmembrane Protein 4
  • GCDH Glutaryl-CoA Dehydrogenase
  • INTS6L Integrator Complex Subunit 6 Like
  • MAN1A2 Mannosidase Alpha Class 1A Member 2
  • RBM5 RNA Binding Motif Protein 5
  • PIGW Phosphatidylinositol Glycan Anchor Biosynthesis Class W
  • SMARCA2 SWI / SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
  • MYCBP2 MYC Binding Protein 2
  • SEC24B SEC24 Homolog B, COPII Coat Complex Component
  • RNF13 Ring Finger Protein 13
  • DUSP22 Dual Specificity Phosphatase 22
  • SMNDC1 Sudvival Motor Neuron Domain Containing 1
  • SYF2 SYF2 Pre-MRNA Splicing Factor
  • SDHB Succinate Dehydrogenase Complex Iron Sulfur Subunit B
  • RASA1 RASA1 (RAS P21 Protein Activator 1)
  • ALKBH1 AlkB Homolog 1, Histone H2A Dioxygenase
  • CEP85 Cerrosomal Protein 85
  • PXN Paxillin
  • PEX12 Peroxisomal Biogenesis Factor 12
  • COMMD8 COMM Domain Containing 8
  • RHOQ Ras Homolog Family Member Q
  • SIL1 SIL1 Nucleotide Exchange Factor
  • F8 Coagulation Factor VIII
  • TCHP Trichoplein Keratin Filament Binding
  • NUP210 Nucleoporin 210)
  • MIR7-3HG MIR7-3 Host Gene
  • MFAP5 Microfibril Associated Protein 5 80.
  • TUG1 (Taurine Up-Regulated 1)
  • ⁇ 80 This is a method for screening candidate substances for the prevention or treatment of cytomegalovirus-related diseases, wherein one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the genes of.
  • Cytomegalovirus-related diseases [1. Drugs for the prevention or treatment of cytomegalovirus-related diseases] (Cytomegalovirus-related diseases)
  • cytomegalovirus (CMV) is a general term for viruses belonging to the genus Cytomegalovirus of the herpesvirus family in terms of taxonomy.
  • HCMV human cytomegalovirus
  • cytomegalovirus-related disease is a general term for diseases caused by infecting humans with cytomegalovirus.
  • Cytomegalovirus-related diseases are also referred to as “cytomegalovirus infections”.
  • the category of "cytomegalovirus-related diseases” includes, for example, 1) delayed fetal intrauterine development, miscarriage, stillbirth, etc., 2) microcephaly, 3) mental retardation, 4) caused by CMV infection.
  • Neuro-sensory disorders such as sensory deafness, 5) Hepatic splenoma, 6) Pneumonia, enteritis, esophagitis, retinitis, and 7) Degeneration of tumor cells (oncomodulation).
  • infected with CMV means a state in which the cells of an individual are infected with CMV, whether temporarily or persistently.
  • Periodically infected with CMV means a state of persistent CMV infection in an individual's cells.
  • the persistently infected individual exhibits or does not exhibit the symptoms associated with CMV infection (the latter individual is also referred to as a carrier).
  • prevention or treatment of cytomegalovirus-related diseases is intended to reduce the risk of CMV infection in humans as compared to the case where the agent according to one embodiment of the present invention is not administered. Particularly preferably, it means that the infection of CMV in humans is substantially completely prevented as compared with the case where the agent according to one embodiment of the present invention is not administered.
  • the agent according to one embodiment of the present invention can also be regarded as an agent that suppresses CMV infection in humans.
  • the target for prevention of cytomegalovirus-related diseases is not particularly limited, but in one example, it may be a foetation in the mother's body. That is, in one aspect of the present invention, infection of the foetation through the placenta of a pregnant woman (so-called congenital infection) is targeted for prevention.
  • the mother to whom the agent according to one embodiment of the present invention is administered may or may not be infected with CMV, but in a typical example, the mother is a carrier of CMV.
  • the human being targeted for prevention of cytomegalovirus-related disease may be a human with reduced immune function.
  • the human with reduced immune function is, for example, a patient with iatrogenic immunodeficiency associated with transplantation medicine, a patient with AIDS, and the like.
  • the term "treatment of a cytomegalovirus-related disease” means that the progression of symptoms of a cytomegalovirus-related disease is at least delayed as compared with the case where the agent according to one embodiment of the present invention is not administered. Preferably, at least one symptom is intended to be ameliorated.
  • the human beings to be treated for cytomegalovirus-related diseases are not particularly limited as long as they are infected with CMV.
  • the effect of the agent of the present invention can be evaluated by an existing method such as real-time PCR of the CMV genome.
  • Agents for the prevention or treatment of cytomegalovirus-related diseases according to one embodiment of the present invention are described in the column of (Means for Solving Problems). ⁇ 80. It contains a component (hereinafter, "active ingredient") that suppresses the expression or function of at least one gene selected from the group consisting of the genes of. All of these genes are targets present in human cells. Therefore, it is expected that the agent according to the present embodiment is less likely to develop drug-resistant CMV as compared with conventional antiviral agents and the like.
  • the agent according to the present embodiment exhibits an anti-CMV effect by a mechanism of action mediated by a cell factor, which is different from conventional antiviral drugs and the like. Therefore, the agent according to the present embodiment is also expected to be an alternative drug that exerts a complementary therapeutic effect on drug-resistant CMV.
  • the agent according to the present embodiment has the above-mentioned 1. ⁇ 80.
  • genes of it may be preferable to contain a component that suppresses the expression or function of at least one gene selected from the group consisting of the following 14 genes. 5.
  • APC adenomatous polyposis coli
  • C3orf74 chromosome 3 open reading frame 74
  • CREB1 cAMP responsive element binding protein 1
  • FAM160A1 family with sequence similarity 160, member A1
  • GPR126 G protein-coupled receptor 126) 27.
  • MUC5AC mofetin 5AC, oligomeric mucus / gel-forming 29.
  • NRP1 neuroropilin 1 36.
  • SCXA salivary homolog A (mouse) 37.
  • SPINT3 serotonin inhibitor, Kunitz type, 3 42.
  • WBP4 WW domain binding protein 4 (formin binding protein 21)).
  • ATF1 Activating Transcription Factor 1 45.
  • PGAM5 PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase 46.
  • IPO8 Importin 8) 47.
  • OGA O-GlcNA case
  • the agent according to this embodiment is 1. ⁇ 80.
  • An active ingredient (one type) that suppresses the expression or function of 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 gene selected from the group consisting of the genes of Also, there may be multiple species depending on the number of genes). Typically, one active ingredient suppresses the expression or function of one corresponding gene, but one active ingredient may suppress the expression or function of multiple genes.
  • suppressing gene expression or function means 1) synthesis of protein from gene (transcription and translation process), 2) action of transcribed gene (mRNA), and 3) from gene. It refers to directly or indirectly suppressing any of the actions of synthesized proteins.
  • the active ingredient described above suppresses its expression or function in a gene-specific manner as a target.
  • Examples of such an active ingredient include, but are not limited to, low molecular weight compounds, nucleic acid polymers such as oligonucleotides, high molecular weight compounds, and combinations thereof.
  • Examples of low molecular weight compounds include inhibitors of proteins encoded by the above genes.
  • Nucleic acid polymers such as oligonucleotides include 1) antisense oligonucleotides of the target gene, 2) nucleic acid polymers for RNAi (oligonucleotides), 3) hetero double-stranded nucleic acids (DNA / RNA), 4). Examples thereof include miRNA target nucleic acids and 5) nucleic acid aptamers for proteins encoded by the above genes.
  • a molecule having a size capable of crossing the blood-brain barrier and a substance delivery means may be required.
  • Nucleic acid polymers for RNAi are typically RNA molecules with complementary double-stranded moieties.
  • the complementary double-stranded portion may be, for example, 16 bp or more, preferably 17 bp or more, and more preferably 20 bp or more.
  • the complementary double-stranded portion may be, for example, 30 pb or less, preferably 27 or 26 bp or less, and more preferably 25 bp or less.
  • the structure of the nucleic acid polymer for RNAi is not particularly limited as long as it causes RNA interference with the target mRNA.
  • the nucleic acid polymer for RNAi can be, for example, shRNA, dumbbell type, siRNA, miRNA, or the like, and in one example, shRNA.
  • the base sequence of the loop portion in the shRNA is not particularly limited and can be appropriately designed.
  • the loop portion can be "CTCGAG" (from 5'end to 3'end, indicated by the sequence of the template DNA).
  • the active ingredient in this embodiment may be a vector expressing the above-mentioned nucleic acid polymer.
  • a vector can be prepared using a commercially available kit.
  • a vector expressing shRNA can be prepared by inserting a DNA that produces shRNA by transcription into a commercially available vector (for example, an adenovirus vector).
  • the base sequences of the promoter and terminator to be included in the DNA to be inserted are not particularly limited and can be appropriately designed.
  • the promoter portion can be "CCGG" and the terminator portion can be "TTTTTG" (both from the 5'end to the 3'end).
  • the agent according to this embodiment may contain other ingredients in addition to the above-mentioned active ingredients.
  • RNA Ribonucleic acid
  • the DNA sequences of # 1 to # 80 are all ordered from the 5'side (from the left side in the table), (1) the promoter portion "CCGG”, and (2) the first strand forming a complementary double-stranded portion. Consists of (sense side), (3) loop portion "CTCGAG”, (4) second strand (antisense side) forming a complementary double-stranded portion, and (5) terminator portion "TTTTTG”.
  • RNA RNA corresponding to the first strand of (2) above (using the first strand as a template) and the RNA corresponding to the second strand of (4) above are complementary. Form a chain portion.
  • RNA The expression products (RNA) of the DNA sequences # 1 to # 80 shown in Table 1 are described in 1. above. ⁇ 80. It is possible to suppress the expression or function of the gene having the corresponding number among the genes of.
  • RNA molecule having a complementary double-stranded portion equivalent to the expression product (RNA) of the DNA sequence shown in Table 1 functions as an oligonucleotide for RNAi.
  • SEQ ID NOs: 1 to 80 in the sequence listing indicate the first strand (sense side) forming the complementary double-stranded portion of (2) above in the DNA sequences # 1 to # 80 shown in Table 1.
  • RNA molecules that functions as a nucleic acid polymer for RNAi include a) RNA using DNA containing 16 or more consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 80 as a template, and b). It contains at least one of RNA, which uses a DNA complementary to the DNA as a template.
  • the RNA molecule is preferably a nucleic acid polymer in which the RNAs of a) and b) above form a double-stranded structure. This RNA molecule can be administered in the form of DNA (vector, etc.).
  • the number of consecutive bases described above is preferably 17 or more, more preferably 18 or more or 19 or more, and even more preferably 20 or more.
  • the nucleic acid polymer as an active ingredient may be a natural nucleic acid polymer or a non-natural nucleic acid polymer.
  • Nucleic acid polymers may be modified, eg, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate oligonucleotides, 2'O-Me modified oligonucleotides, 2'. Examples include, but are not limited to, -O, 4'-C-ethylene cross-linked nucleic acid species (ENA). Such modification can enhance the stability of the nucleic acid polymer in vivo.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • EDA phosphorothioate oligonucleotides
  • EDA 4'-C-ethylene cross-linked nucleic acid species
  • Ingredients other than the active ingredient constituting the agent according to the present embodiment are not particularly limited, and for example, pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmoregulation, etc. , Buffers, colorants, flavors, sweeteners, antioxidants, viscosity modifiers and the like.
  • the pharmaceutically acceptable carrier is not particularly limited, but is a carrier that does not inhibit the efficacy of the active ingredient when co-administered with the active ingredient, and is substantially the same for a human to be administered. It is preferable to have the property of not having an adverse effect.
  • a carrier those conventionally known in this field can be widely used, and specifically, for example, water, various salt solutions, alcohol, vegetable oil, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, and talc. , Silica, paraffin, fatty acid monoglyceride, fatty acid diglyceride, hydroxymethyl cellulose, polyvinylpyrrolidone and the like, but are not particularly limited thereto.
  • the type of carrier may be appropriately selected according to the dosage form, administration method, etc. of the agent according to the present embodiment.
  • the agent according to this embodiment can also be used in combination with an existing anti-cytomegalovirus agent.
  • One form of the combination is a so-called combination drug in which the agent according to the present embodiment is formulated containing an existing anti-cytomegalovirus agent.
  • the agent according to the present embodiment and the anti-cytomegalovirus agent are separately formulated, and these agents are used in combination (separate agents are administered simultaneously or sequentially). It is a form.
  • Existing anti-cytomegalovirus agents include, for example, ganciclovir and its salts, foscarnet and its salts (such as foscarnet sodium hydrate), sodium phosphenitoin and its salts (such as hydrate), valganciclovir and Examples include the group consisting of its salts (such as valganciclovir hydrochloride), letermovir, cidofovir and its salts, and fomivirsen.
  • the agent according to the present embodiment can be administered to an individual human for the purpose of preventing or treating a cytomegalovirus-related disease.
  • an amount of the active ingredient effective for the prevention or treatment of cytomegalovirus-related diseases is administered to the human.
  • the active ingredient may be administered alone or as a component of an agent suitable for the purpose of administration.
  • the dose of the active ingredient contained in the agent according to the present embodiment is not particularly limited, and depends on the disease state, body weight, response to treatment, administration form of the agent, stage of disease course, or administration interval. Can be adjusted as appropriate.
  • the administration may be divided into one to several times, for example, one to several times per day.
  • the administration route of the agent according to this embodiment is not particularly limited, and may be systemically administered by a method such as oral administration, intravenous or intraarterial administration, and intestinal administration.
  • the agent according to the present embodiment may be locally administered by a method such as injection (using a syringe or an infusion pump), transdermal administration, sublingual administration, and organ surface administration.
  • the dosage and administration method may be appropriately selected by those skilled in the art according to the purpose of treatment, the weight, age, symptoms and the like of the patient.
  • the dosage form of the agent according to the present embodiment is not particularly limited, and for example, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, patches, ointments, injections and the like. Can be mentioned.
  • the agent according to this embodiment can be a gene therapy agent.
  • the gene therapy agent may be in the form of directly administering the active ingredient to humans by injection, or in the form of directly administering the vector incorporating the nucleic acid polymer to humans by injection. There may be.
  • the vector is not particularly limited, and examples thereof include vectors applicable to gene therapy, such as an adenovirus vector, an adeno-related virus vector, an adeno-associated virus vector, a herpesvirus vector, a vaccinia virus vector, and a retrovirus vector.
  • the gene therapy agent may be a liposome preparation.
  • the vector constituting the gene therapy agent incorporates an expression regulatory sequence that specifically expresses the active ingredient at the target site.
  • the expression regulatory sequence is, for example, a promoter or an enhancer.
  • the screening method of the present embodiment is a screening method for a candidate substance for prevention or treatment of cytomegalovirus-related diseases, and 1. ⁇ 80.
  • This is a screening method in which one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the above genes.
  • One aspect of the screening method of the present invention is a step of contacting a cell sample with a test substance (contact step), 1. ⁇ 80.
  • the cell sample used in the above contact step is, for example, a sample containing cells to be infected with CMV.
  • the cell sample include non-human organisms, cultured cells of various cell lines, and biological samples isolated from the organism.
  • the cultured cells are, for example, living cells of human and non-human organisms, ES cells, iPS cells and the like, and more specifically, a human nervous system cell line (U373MG cell line), a human fibroblast cell line and the like can be mentioned. ..
  • test substance examples include, but are not limited to, low molecular weight compounds, nucleic acid polymers such as oligonucleotides, peptides, proteins, high molecular weight compounds, and combinations thereof.
  • the test substance may be naturally occurring or chemically synthesized.
  • the candidate substance can preferably be a substance that is harmless in vivo.
  • An example of a test substance is shRNA.
  • the specific method of contacting the cell sample with the test substance is not particularly limited.
  • the candidate substance when the cell sample is a cultured cell, the candidate substance can be directly administered to the culture medium in which the cell is cultured.
  • the test substance when the test substance is an oligonucleotide, the test substance can be more reliably introduced into the cell by using a lipofection method, an electroporation method, or the like.
  • the test substance when the test substance is a polypeptide or a polymer compound, the test substance can be more reliably introduced into the cell by using an injection method, a liposome method, or the like.
  • the concentration of the test substance added can be appropriately selected according to the type of the substance as long as it does not adversely affect the growth of cells.
  • the contact period, the temperature during the contact period, and the like can also be appropriately selected.
  • the evaluation of gene expression or function in the evaluation step can be evaluated by, for example, real-time PCR, quantitative PCR such as digital PCR, or analysis such as microarray analysis.
  • the protein transcribed from the gene may be targeted and evaluated using ELISA or Western blotting method, Southern blotting method, immunofluorescent staining, etc., or evaluated based on whether cell death is observed. May be good. These operations can be performed according to a conventional method.
  • the genes used as indicators are 1. ⁇ 80. It is at least one gene selected from the group consisting of genes of, preferably two or more, more preferably three or more, even more preferably four or more, particularly preferably five or more, most preferably ten. The above is preferable.
  • the degree of gene expression or functional activity is compared with a control (for example, the degree of gene expression or functional activity in a cell sample to which the test substance was not administered).
  • the comparison is preferably made in consideration of the presence or absence of a significant difference.
  • the test substance is selected as a candidate substance for prevention or treatment of CMV-related diseases.
  • the screening method as described above is very useful as a method for efficiently obtaining a candidate substance for prevention or treatment of CMV-related diseases.
  • the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments can be used. Embodiments obtained by appropriately combining them are also included in the technical scope of the present invention.
  • the present invention includes, for example, the following aspects.
  • ⁇ 80 An agent for the prevention or treatment of cytomegalovirus-related diseases, which comprises a component that suppresses the expression or function of at least one gene selected from the group consisting of the genes of.
  • the nucleic acid polymer 1) uses RNA as a template for DNA containing 16 or more consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 80, and DNA complementary to the DNA as a template.
  • the anti-cytomegalovirus agent is ganciclovir and its salt, foscarnet and its salt (foscarnet sodium hydrate, etc.), phosphenitoin sodium and its salt (hydrate, etc.), valganciclovir and its salt.
  • the agent of (F) which is at least one selected from the group consisting of salts (such as valganciclovir hydrochloride), letermovir, cidofovir and salts thereof, and homivirsen.
  • ⁇ 80 A method for screening candidate substances for the prevention or treatment of cytomegalovirus-related diseases, wherein one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the genes of.
  • U373MG cell line Human nervous system cells (U373MG cell line) were used as target cells.
  • U373MG cells contain 10% fetal bovine serum (BioWest) and an antibiotic-antifungal mixed solution (penicillin 100 u / mL, streptomycin 100 ⁇ g / mL and amphotericin B 250 ng / mL) as a medium. ) was used, and the cells were cultured in an incubator at 37 ° C. and 5% CO 2.
  • a library of lentivirus-incorporated shRNAs included in a commercially available kit was introduced into U373MG cells.
  • U373MG cells into which shRNA was introduced were cultured in a 10 ⁇ g / mL Puromycin (Invivogen) medium at 37 ° C. in a 5% CO 2 incubator for 14 days, and a cell population showing Puromycin resistance was selected.
  • a U373MG cell population into which shRNAs that do not interfere with the survival and proliferation of U373MG cells were introduced (hereinafter referred to as shRNA-introduced U373MG cell population) was acquired.
  • the shRNA-introduced U373MG cell population was infected with the human cytomegalovirus Towne strain (ATCC, product number VR-977), and the culture was continued to observe resistance to cell death.
  • U373MG cells into which the shRNA library had not been introduced were similarly infected with the human cytomegalovirus Towne strain.
  • FIG. 1 cell population A shows a strength-tolerant clone and cell population B shows a moderately resistant clone.
  • the cell populations that survive after about 3 weeks are the cell populations that are resistant to the human cytomegalovirus Towne strain (hereinafter referred to as human CMV resistance). Cell population). Most of the U373MG cells used in the control experiment died about 3 weeks after infection.
  • Single-cell cloning was performed on a human CMV-resistant cell population using the limiting dilution method using a 96-well plate. Subsequently, genomic DNA was extracted from each single cell clone according to a conventional method. The shRNA region of the extracted genomic DNA was amplified by the Polymerase Chain Reaction (PCR) method. Subsequently, each PCR product was introduced into a plasmid, and the nucleotide sequence of shRNA was determined by the dideoxy method. Furthermore, based on the information in the shRNA library provided by the seller of the kit, the cell gene targeted by each shRNA was identified from the determined nucleotide sequence of shRNA. The nucleotide sequences of shRNA and the cell genes targeted by each shRNA are shown in Tables 1 and 2 below.
  • Samples A to F Six clones (referred to as Samples A to F) of the single cell clones obtained by single cell cloning from the above-mentioned human CMV resistant cell population and U373MG cells (control) into which negative control shRNA was introduced were used. The expression of the HCMV gene in each cell was compared. More specifically, the expression level of the HCMV gene was compared using the expression level of the IE2 protein transcribed from the HCMV gene as an index. Western blotting and immunofluorescence staining were used to detect the IE2 protein. In the western blotting method, 3 out of 6 clones were used, and ⁇ -actin was used as a loading control.
  • the remaining 3 clones were used for immunofluorescence staining.
  • the cell nuclei were stained with DAPI.
  • suppression of IE2 protein expression was observed in U373MG cells resistant to human cytomegalovirus into which a specific shRNA was introduced, suggesting suppression of HCMV gene expression (results not shown). ).
  • RT-PCR Reverse Transcription-PCR
  • U373MG cells were newly prepared and cultured under the above conditions (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and an antibiotic-antifungal mixed solution, 37 ° C., 5% CO 2 incubator).
  • U373MG cells newly introduced with # 5 and # 11 shRNA and U373MG cells (control) introduced with negative control shRNA were used in a 10 ⁇ g / mL Puromycin medium at 37 ° C. in a 5% CO 2 incubator for 14 days. After culturing, a cell population showing Puromycin resistance was selected. Selected cell populations were infected with the human cytomegalovirus Towne strain and their resistance to cell death was compared. As shown in FIG. 2, it was confirmed that U373MG cells into which a specific shRNA was introduced showed resistance to cell death associated with CMV infection.

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Abstract

The present invention provides a novel agent that can be used for the prevention or treatment of cytomegalovirus-related diseases, and has an action mechanism different from that of existing drugs for HCMV infectious diseases. A prophylactic or therapeutic agent for cytomegalovirus-related diseases, said agent including a component that suppresses the expression or function of at least one gene selected from a group consisting of 80 genes.

Description

サイトメガロウイルス関連疾患の予防又は治療用の剤Drugs for the prevention or treatment of cytomegalovirus-related diseases
　本発明は、サイトメガロウイルス関連疾患の予防又は治療用の剤に関する。 The present invention relates to an agent for preventing or treating cytomegalovirus-related diseases.
　サイトメガロウイルスは、ヘルペスウイルス科サイトメガロウイルス属に属するウイルスの総称であり、ヒトに感染するヒトサイトメガロウイルス（ＨＣＭＶ）等が知られている。 Cytomegalovirus is a general term for viruses belonging to the genus Cytomegalovirus of the herpesvirus family, and human cytomegalovirus (HCMV) that infects humans is known.
　ＨＣＭＶの感染は、頻繁に起こっているが、健常者では、症状が顕在化しないケースがほとんどである。一方、胎児及び免疫機能が低下した者等では、ＨＣＭＶの感染は、重篤な症状をしばしば引き起こす。 HCMV infection occurs frequently, but in most healthy individuals, the symptoms do not manifest themselves. On the other hand, in the fetal and those with weakened immune function, infection with HCMV often causes serious symptoms.
　例えば、ＨＣＭＶは、妊婦の胎盤を介して胎児に感染する（いわゆる、先天性感染）。その結果として、胎児の子宮内発達の遅延を引き起こし得る。さらに、胎児期以降（すなわち、胎児、及び出生後）における、小頭症；精神発達遅滞；及び、感音性難聴などの神経・感覚器障害；など、さまざまな臓器障害を呈する、先天性ＨＣＭＶ感染症を引き起こす。またＨＣＭＶは、移植医療にともなう医原性免疫不全患者、及びエイズ患者などにおいて、肺炎、腸炎、及び、網膜炎などの日和見感染症を引き起こす。 For example, HCMV infects the foetation through the placenta of a pregnant woman (so-called congenital infection). As a result, it can cause a delay in fetal intrauterine development. In addition, congenital HCMV presenting with various organ disorders such as microcephaly; mental retardation; and neurological / sensory disorders such as sensory hearing loss; after the fetal period (ie, fetal and postnatal). Causes infectious diseases. In addition, HCMV causes opportunistic infections such as pneumonia, enteritis, and retinitis in patients with iatrogenic immunodeficiency associated with transplantation medicine and patients with AIDS.
　重篤な症状を引き起こすＨＣＭＶの感染に対する治療法としては、ＨＣＭＶの増殖を抑制するために、ＨＣＭＶのＤＮＡ複製に必要な遺伝子のＤＮＡあるいはそのｍＲＮＡに対するアンチセンスＲＮＡを用いる方法があり（例えば、特許文献１）、臨床では、ガンシクロビル、バルガンシクロビル、及び、ホスカルネット等の抗ウイルス薬の投与が行われている。また、初の核酸医薬として上市されたホミビルセンの投与も、エイズ患者のＨＣＭＶ性網膜炎に対する治療法の一つの選択肢となっている。一方で、ＨＣＭＶに有効なワクチンは、未だ報告がなされていない。 As a treatment method for HCMV infection that causes serious symptoms, there is a method of using DNA of a gene required for DNA replication of HCMV or antisense RNA against its mRNA in order to suppress the proliferation of HCMV (for example, patent). Document 1), clinically, antiviral drugs such as ganciclovir, valganciclovir, and foscarnet are administered. The administration of fomivirsen, which was launched as the first nucleic acid drug, is also an option for the treatment of HCMV retinitis in AIDS patients. On the other hand, no effective vaccine for HCMV has been reported yet.
日本国公開公報「特表平１０－５００８５１号公報」Japan Public Gazette "Special Table No. 10-58051 Gazette"
　しかしながら、上記の抗ウイルス薬は、ＨＣＭＶがコードするＤＮＡポリメラーゼなどを標的としているため、薬剤耐性ＨＣＭＶ株が出現する。また、上記の抗ウイルス薬は、重篤な副作用を引き起こす場合があるため、適応疾患が限定的である。とりわけ、胎児に対して、非常に重篤な副作用を引き起こし得るので、母体への投与自体が禁忌となっている抗ウイルス薬も存在する。 However, since the above antiviral drug targets DNA polymerase encoded by HCMV, drug-resistant HCMV strains appear. In addition, the above-mentioned antiviral drugs may cause serious side effects, so that the indications are limited. In particular, some antiviral drugs are contraindicated for maternal administration because they can cause very serious side effects on the foetation.
　また、ホミビルセンは、ＨＣＭＶがコードするＩＥ２に対するアンチセンスＲＮＡであり、薬剤耐性ＨＣＭＶ株が出現する危険性が指摘されている。 In addition, fomivirsen is an antisense RNA against IE2 encoded by HCMV, and it has been pointed out that there is a risk of drug-resistant HCMV strains appearing.
　本発明は上記の問題点に鑑みてなされたものであり、その目的は、ＨＣＭＶ感染症の従来薬とは作用機序が異なる、サイトメガロウイルス関連疾患の予防又は治療に用い得る新規な剤を提供することにある。 The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel agent that can be used for the prevention or treatment of cytomegalovirus-related diseases, which has a different mechanism of action from conventional agents for HCMV infection. To provide.
　発明者らは上記課題を解決するために鋭意検討した結果、ＨＣＭＶの感染対象となるヒトにおける特定の遺伝子の発現又は機能を抑制することで、サイトメガロウイルス関連疾患を予防又は治療することができることを見出し、本発明を完成させるに至った。すなわち、本発明の一態様は、以下の１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制する成分を含む、サイトメガロウイルス関連疾患の予防又は治療用の剤である。
１．　AHNAK2 (AHNAK nucleoprotein 2)
２．　AIDA (axin interactor, dorsalization associated)
３．　AKAP5 (A kinase (PRKA) anchor protein 5)
４．　AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
５．　APC (adenomatous polyposis coli)
６．　C3orf74 (chromosome 3 open reading frame 74)
７．　C9orf9 (chromosome 9 open reading frame 9)
８．　C11orf67 (chromosome 11 open reading frame 67)
９．　C19orf33 (chromosome 19 open reading frame 33)
１０．　CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
１１．　CREB1 (cAMP responsive element binding protein 1)
１２．　CYTH1 (cytohesin 1)
１３．　EML1 (echinoderm microtubule associated protein like 1)
１４．　EMP2 (epithelial membrane protein 2)
１５．　EPHB6 (EPH receptor B6)
１６．　FAM160A1 (family with sequence similarity 160, member A1)
１７．　FAM176A (family with sequence similarity 176, member A)
１８．　FXR2 (fragile X mental retardation, autosomal homolog 2)
１９．　GPR126 (G protein-coupled receptor 126)
２０．　HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
２１．　HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
２２．　TESPA1 (Thymocyte Expressed, Positive Selection Associated 1) 
２３．　PALD1 (Phosphatase Domain Containing Paladin 1) 
２４．　KRTAP12-1 (keratin associated protein 12-1)
２５．　MBD1 (methyl-CpG binding domain protein 1)
２６．　MESDC2 (mesoderm development candidate 2)
２７．　MUC5AC (mucin 5AC, oligomeric mucus/gel-forming)
２８．　NOL12 (nucleolar protein 12)
２９．　NRP1 (neuropilin 1)
３０．　NUP153 (nucleoporin 153kDa)
３１．　PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
３２.　PCBD1 (pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha)
３３．　PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
３４．　RBM26 (RNA binding motif protein 26)
３５．　RSBN1 (round spermatid basic protein 1)
３６．　SCXA (scleraxis homolog A (mouse))
３７．　SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
３８．　TATDN3 (TatD DNase domain containing 3)
３９．　TBC1D3G (TBC1 domain family, member 3G)
４０．　TSC22D1 (TSC22 domain family, member 1)
４１．　UNC119B (unc-119 homolog B (C. elegans))
４２．　WBP4 (WW domain binding protein 4 (formin binding protein 21))
４３．　ZNF354A (zinc finger protein 354A)
４４．　ATF1 (Activating Transcription Factor 1)
４５．　PGAM5 (PGAM Family Member 5, Mitochondrial Serine/Threonine Protein Phosphatase)
４６．　IPO8 (Importin 8)
４７．　OGA (O-GlcNAcase)
４８．　PROX1 (Prospero Homeobox 1)
４９．　CIDEC (Cell Death Inducing DFFA Like Effector C)
５０．　JPH2 (Junctophilin 2)
５１．　RNF208 (Ring Finger Protein 208)
５２．　STEAP1B (STEAP Family Member 1B)
５３．　TENM4 (Teneurin Transmembrane Protein 4)
５４．　GCDH (Glutaryl-CoA Dehydrogenase)
５５．　INTS6L (Integrator Complex Subunit 6 Like)
５６．　MAN1A2 (Mannosidase Alpha Class 1A Member 2)
５７．　RBM5 (RNA Binding Motif Protein 5)
５８．　PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
５９．　SMARCA2 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
６０．　MYCBP2 (MYC Binding Protein 2)
６１．　SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
６２．　RNF13 (Ring Finger Protein 13)
６３．　DUSP22 (Dual Specificity Phosphatase 22)
６４．　SMNDC1 (Survival Motor Neuron Domain Containing 1)
６５．　SYF2 (SYF2 Pre-MRNA Splicing Factor)
６６．　SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
６７．　RASA1 (RAS P21 Protein Activator 1)
６８．　ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
６９．　CEP85 (Centrosomal Protein 85)
７０．　PXN (Paxillin)
７１．　PEX12 (Peroxisomal Biogenesis Factor 12)
７２．　COMMD8 (COMM Domain Containing 8)
７３．　RHOQ (Ras Homolog Family Member Q)
７４．　SIL1 (SIL1 Nucleotide Exchange Factor)
７５．　F8 (Coagulation Factor VIII)
７６．　TCHP (Trichoplein Keratin Filament Binding)
７７．　NUP210 (Nucleoporin 210)
７８．　MIR7-3HG (MIR7-3 Host Gene)
７９．　MFAP5 (Microfibril Associated Protein 5)
８０．　TUG1 (Taurine Up-Regulated 1)
　また、本発明の他の態様は、上述の１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制するかを一つの指標とする、サイトメガロウイルス関連疾患の予防又は治療用の候補物質のスクリーニング方法である。 As a result of diligent studies to solve the above problems, the inventors can prevent or treat cytomegalovirus-related diseases by suppressing the expression or function of a specific gene in humans infected with HCMV. The present invention has been completed. That is, one aspect of the present invention is as follows. ~ 80. An agent for preventing or treating a cytomegalovirus-related disease, which comprises a component that suppresses the expression or function of at least one gene selected from the group consisting of the genes of.
1. 1. AHNAK2 (AHNAK nucleoprotein 2)
2. AIDA (axin interactor, dorsalization associated)
3. 3. AKAP5 (A kinase (PRKA) anchor protein 5)
4. AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
5. APC (adenomatous polyposis coli)
6. C3orf74 (chromosome 3 open reading frame 74)
7. C9orf9 (chromosome 9 open reading frame 9)
8. C11orf67 (chromosome 11 open reading frame 67)
9. C19orf33 (chromosome 19 open reading frame 33)
10. CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
11. CREB1 (cAMP responsive element binding protein 1)
12. CYTH1 (cytohesin 1)
13. EML1 (echinoderm microtubule associated protein like 1)
14. EMP2 (epithelial membrane protein 2)
15. EPHB6 (EPH receptor B6)
16. FAM160A1 (family with sequence similarity 160, member A1)
17. FAM176A (family with sequence similarity 176, member A)
18. FXR2 (fragile X mental retardation, autosomal homolog 2)
19. GPR126 (G protein-coupled receptor 126)
20. HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
21. HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
22. TESPA1 (Thymocyte Expressed, Positive Selection Associated 1)
23. PALD1 (Phosphatase Domain Containing Paladin 1)
24. KRTAP12-1 (keratin associated protein 12-1)
25. MBD1 (methyl-CpG binding domain protein 1)
26. MESDC2 (mesoderm development candidate 2)
27. MUC5AC (mucin 5AC, oligomeric mucus / gel-forming)
28. NOL12 (nucleolar protein 12)
29. NRP1 (neuropilin 1)
30. NUP153 (nucleoporin 153kDa)
31. PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
32. PCBD1 (pterin-4 alpha-carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha)
33. PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
34. RBM26 (RNA binding motif protein 26)
35. RSBN1 (round spermatid basic protein 1)
36. SCXA (scleraxis homolog A (mouse))
37. SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
38. TATDN3 (TatD DNase domain containing 3)
39. TBC1D3G (TBC1 domain family, member 3G)
40. TSC22D1 (TSC22 domain family, member 1)
41. UNC119B (unc-119 homolog B (C. elegans))
42. WBP4 (WW domain binding protein 4 (formin binding protein 21))
43. ZNF354A (zinc finger protein 354A)
44. ATF1 (Activating Transcription Factor 1)
45. PGAM5 (PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase)
46. IPO8 (Importin 8)
47. OGA (O-GlcNA case)
48. PROX1 (Prospero Homeobox 1)
49. CIDEC (Cell Death Inducing DFFA Like Effector C)
50. JPH2 (Junctophilin 2)
51. RNF208 (Ring Finger Protein 208)
52. STEAP1B (STEAP Family Member 1B)
53. TENM4 (Teneurin Transmembrane Protein 4)
54. GCDH (Glutaryl-CoA Dehydrogenase)
55. INTS6L (Integrator Complex Subunit 6 Like)
56. MAN1A2 (Mannosidase Alpha Class 1A Member 2)
57. RBM5 (RNA Binding Motif Protein 5)
58. PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
59. SMARCA2 (SWI / SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
60. MYCBP2 (MYC Binding Protein 2)
61. SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
62. RNF13 (Ring Finger Protein 13)
63. DUSP22 (Dual Specificity Phosphatase 22)
64. SMNDC1 (Survival Motor Neuron Domain Containing 1)
65. SYF2 (SYF2 Pre-MRNA Splicing Factor)
66. SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
67. RASA1 (RAS P21 Protein Activator 1)
68. ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
69. CEP85 (Centrosomal Protein 85)
70. PXN (Paxillin)
71. PEX12 (Peroxisomal Biogenesis Factor 12)
72. COMMD8 (COMM Domain Containing 8)
73. RHOQ (Ras Homolog Family Member Q)
74. SIL1 (SIL1 Nucleotide Exchange Factor)
75. F8 (Coagulation Factor VIII)
76. TCHP (Trichoplein Keratin Filament Binding)
77. NUP210 (Nucleoporin 210)
78. MIR7-3HG (MIR7-3 Host Gene)
79. MFAP5 (Microfibril Associated Protein 5)
80. TUG1 (Taurine Up-Regulated 1)
In addition, another aspect of the present invention is described in 1. ~ 80. This is a method for screening candidate substances for the prevention or treatment of cytomegalovirus-related diseases, wherein one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the genes of.
　本発明によれば、新規な作用機序に基づく、サイトメガロウイルス関連疾患の予防又は治療用の剤を提供することができる。 According to the present invention, it is possible to provide an agent for the prevention or treatment of cytomegalovirus-related diseases based on a novel mechanism of action.
本発明の実施例の結果を示す図である。It is a figure which shows the result of the Example of this invention. 本発明の実施例の結果を示す図である。It is a figure which shows the result of the Example of this invention.
　以下に、本発明の一実施形態について詳述するが、本発明はこれに限定されるものではない。 Hereinafter, one embodiment of the present invention will be described in detail, but the present invention is not limited thereto.
　〔１．サイトメガロウイルス関連疾患の予防又は治療用の剤〕
　（サイトメガロウイルス関連疾患）
　本明細書において、「サイトメガロウイルス（ＣＭＶ）」とは、分類学上、ヘルペスウイルス科サイトメガロウイルス属に属するウイルスの総称である。サイトメガロウイルスの中では、特に好ましくはヒトサイトメガロウイルス（ＨＣＭＶ）を指す。 [1. Drugs for the prevention or treatment of cytomegalovirus-related diseases]
(Cytomegalovirus-related diseases)
In the present specification, "cytomegalovirus (CMV)" is a general term for viruses belonging to the genus Cytomegalovirus of the herpesvirus family in terms of taxonomy. Among the cytomegaloviruses, human cytomegalovirus (HCMV) is particularly preferable.
　本明細書において、「サイトメガロウイルス関連疾患」とは、サイトメガロウイルスがヒトに感染することによって引き起こされる疾患の総称である。「サイトメガロウイルス関連疾患」は、「サイトメガロウイルス感染症」とも称される。「サイトメガロウイルス関連疾患」の範疇には、例えば、ＣＭＶの感染を原因とする、１）胎児の子宮内発達の遅延、流産、死産等、２）小頭症、３）精神発達遅滞、４）感音性難聴などの神経・感覚器障害、５）肝脾腫、６）肺炎、腸炎、食道炎、網膜炎、並びに、７）腫瘍細胞の変性（オンコモジュレーション）、なども含まれる。 In the present specification, "cytomegalovirus-related disease" is a general term for diseases caused by infecting humans with cytomegalovirus. "Cytomegalovirus-related diseases" are also referred to as "cytomegalovirus infections". The category of "cytomegalovirus-related diseases" includes, for example, 1) delayed fetal intrauterine development, miscarriage, stillbirth, etc., 2) microcephaly, 3) mental retardation, 4) caused by CMV infection. ) Neuro-sensory disorders such as sensory deafness, 5) Hepatic splenoma, 6) Pneumonia, enteritis, esophagitis, retinitis, and 7) Degeneration of tumor cells (oncomodulation).
　本明細書において、「ＣＭＶに感染している」は、一時的にか持続的にかを問わず、個体の細胞へＣＭＶが感染している状態を意味する。「ＣＭＶに持続感染している」は、個体の細胞におけるＣＭＶの感染が持続している状態を意味する。ここで、持続感染している個体は、ＣＭＶ感染にともなう症状を呈しているか、又は呈していない（後者の個体をキャリアとも称する）。 In the present specification, "infected with CMV" means a state in which the cells of an individual are infected with CMV, whether temporarily or persistently. "Persistently infected with CMV" means a state of persistent CMV infection in an individual's cells. Here, the persistently infected individual exhibits or does not exhibit the symptoms associated with CMV infection (the latter individual is also referred to as a carrier).
　（サイトメガロウイルス関連疾患の予防又は治療）
　本明細書において、「サイトメガロウイルス関連疾患の予防」とは、本発明の一実施形態にかかる剤を投与しない場合と比較して、ヒトにおけるＣＭＶの感染リスクが低減することを意図する。特に好ましくは、本発明の一実施形態にかかる剤を投与しない場合と比較して、ヒトにおけるＣＭＶの感染を、実質的に完全に防止することを意味する。本発明の一実施形態にかかる剤は、ヒトにおける、ＣＭＶの感染を抑制する剤と捉えることもできる。 (Prevention or treatment of cytomegalovirus-related diseases)
As used herein, "prevention of cytomegalovirus-related diseases" is intended to reduce the risk of CMV infection in humans as compared to the case where the agent according to one embodiment of the present invention is not administered. Particularly preferably, it means that the infection of CMV in humans is substantially completely prevented as compared with the case where the agent according to one embodiment of the present invention is not administered. The agent according to one embodiment of the present invention can also be regarded as an agent that suppresses CMV infection in humans.
　サイトメガロウイルス関連疾患の予防の対象となるヒトは、特に限定されないが、一例では、母体内にいる胎児であってもよい。すなわち、本発明の一態様では、妊婦の胎盤を介した胎児への感染（いわゆる、先天性感染）を、予防の対象とする。この場合、本発明の一実施形態にかかる剤が投与される母体は、ＣＭＶに感染していてもいなくてもよいが、典型的な一例では、母体は、ＣＭＶのキャリアである。 The target for prevention of cytomegalovirus-related diseases is not particularly limited, but in one example, it may be a foetation in the mother's body. That is, in one aspect of the present invention, infection of the foetation through the placenta of a pregnant woman (so-called congenital infection) is targeted for prevention. In this case, the mother to whom the agent according to one embodiment of the present invention is administered may or may not be infected with CMV, but in a typical example, the mother is a carrier of CMV.
　他の一例では、サイトメガロウイルス関連疾患の予防の対象となるヒトは、免疫機能が低下したヒトであってもよい。免疫機能が低下したヒトとは、例えば、移植医療にともなう医原性免疫不全患者、及びエイズ患者などである。 In another example, the human being targeted for prevention of cytomegalovirus-related disease may be a human with reduced immune function. The human with reduced immune function is, for example, a patient with iatrogenic immunodeficiency associated with transplantation medicine, a patient with AIDS, and the like.
　本明細書において、「サイトメガロウイルス関連疾患の治療」とは、本発明の一実施形態にかかる剤を投与しない場合と比較して、サイトメガロウイルス関連疾患の症状の進行が少なくとも遅延すること、好ましくは、少なくとも１つの症状が改善することを意図する。サイトメガロウイルス関連疾患の治療の対象となるヒトは、ＣＭＶが感染しているヒトであれば特に限定されない。 As used herein, the term "treatment of a cytomegalovirus-related disease" means that the progression of symptoms of a cytomegalovirus-related disease is at least delayed as compared with the case where the agent according to one embodiment of the present invention is not administered. Preferably, at least one symptom is intended to be ameliorated. The human beings to be treated for cytomegalovirus-related diseases are not particularly limited as long as they are infected with CMV.
　なお、本発明の剤による効果は、例えば、ＣＭＶゲノムのリアルタイムＰＣＲなどの既存の方法によって評価され得る。 The effect of the agent of the present invention can be evaluated by an existing method such as real-time PCR of the CMV genome.
　（剤の有効成分）
　本発明の一実施形態に係るサイトメガロウイルス関連疾患の予防又は治療用の剤（以下、「本実施形態に係る剤」）は、（課題を解決するための手段）の欄に記載した１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制する成分（以下、「有効成分」）を含んでいる。これらの遺伝子は何れもヒト細胞に存在するターゲットである。そのため、本実施形態に係る剤は、従来の抗ウイルス薬等と比較して、薬剤耐性ＣＭＶが出現しにくいと期待される。また、本実施形態に係る剤は、従来の抗ウイルス薬等とは異なる、細胞因子を介した作用機序で抗ＣＭＶ効果を示す。そのため、本実施形態に係る剤は、薬剤耐性ＣＭＶに対しても補完的な治療効果を発揮する代替医薬品になることも期待される。 (Active ingredient of the agent)
Agents for the prevention or treatment of cytomegalovirus-related diseases according to one embodiment of the present invention (hereinafter, “agents according to the present embodiment”) are described in the column of (Means for Solving Problems). ~ 80. It contains a component (hereinafter, "active ingredient") that suppresses the expression or function of at least one gene selected from the group consisting of the genes of. All of these genes are targets present in human cells. Therefore, it is expected that the agent according to the present embodiment is less likely to develop drug-resistant CMV as compared with conventional antiviral agents and the like. In addition, the agent according to the present embodiment exhibits an anti-CMV effect by a mechanism of action mediated by a cell factor, which is different from conventional antiviral drugs and the like. Therefore, the agent according to the present embodiment is also expected to be an alternative drug that exerts a complementary therapeutic effect on drug-resistant CMV.
　本実施形態に係る剤は、有効成分として、上述の１．～８０．の遺伝子の中でも、以下１４個の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制する成分を含むことが好ましい場合がある。
５．　APC (adenomatous polyposis coli)
６．　C3orf74 (chromosome 3 open reading frame 74)
１１．　CREB1 (cAMP responsive element binding protein 1)
１６．　FAM160A1 (family with sequence similarity 160, member A1)
１９．　GPR126 (G protein-coupled receptor 126)
２７．　MUC5AC (mucin 5AC, oligomeric mucus/gel-forming)
２９．　NRP1 (neuropilin 1)
３６．　SCXA (scleraxis homolog A (mouse))
３７．　SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
４２．　WBP4 (WW domain binding protein 4 (formin binding protein 21))。
４４．　ATF1 (Activating Transcription Factor 1)
４５．　PGAM5 (PGAM Family Member 5, Mitochondrial Serine/Threonine Protein Phosphatase)
４６．　IPO8 (Importin 8)
４７．　OGA (O-GlcNAcase)
　なお、一例では、本実施形態に係る剤は、１．～８０．の遺伝子からなる群より選択される１０個以下、５個以下、４個以下、３個以下、２個以下、又は、１個の遺伝子の発現又は機能を抑制する有効成分（１種であっても、遺伝子の数に応じて複数種であってもよい）を含んでいる。典型的には、１つの有効成分は、対応する１個の遺伝子の発現又は機能を抑制するが、１つの有効成分が複数の遺伝子の発現又は機能を抑制してもよい。 The agent according to the present embodiment has the above-mentioned 1. ~ 80. Among the genes of, it may be preferable to contain a component that suppresses the expression or function of at least one gene selected from the group consisting of the following 14 genes.
5. APC (adenomatous polyposis coli)
6. C3orf74 (chromosome 3 open reading frame 74)
11. CREB1 (cAMP responsive element binding protein 1)
16. FAM160A1 (family with sequence similarity 160, member A1)
19. GPR126 (G protein-coupled receptor 126)
27. MUC5AC (mucin 5AC, oligomeric mucus / gel-forming)
29. NRP1 (neuropilin 1)
36. SCXA (scleraxis homolog A (mouse))
37. SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
42. WBP4 (WW domain binding protein 4 (formin binding protein 21)).
44. ATF1 (Activating Transcription Factor 1)
45. PGAM5 (PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase)
46. IPO8 (Importin 8)
47. OGA (O-GlcNA case)
In one example, the agent according to this embodiment is 1. ~ 80. An active ingredient (one type) that suppresses the expression or function of 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 gene selected from the group consisting of the genes of Also, there may be multiple species depending on the number of genes). Typically, one active ingredient suppresses the expression or function of one corresponding gene, but one active ingredient may suppress the expression or function of multiple genes.
　ここで、「遺伝子の発現又は機能を抑制する」とは、１）遺伝子からのタンパク質の合成（転写及び翻訳のプロセス）、２）転写された遺伝子（ｍＲＮＡ）の作用、及び、３）遺伝子から合成されたタンパク質の作用、の何れかを、直接的あるいは間接的に抑制することを指す。典型的な一例では、上記の有効成分は、ターゲットとなる遺伝子特異的に、その発現又は機能を抑制する。 Here, "suppressing gene expression or function" means 1) synthesis of protein from gene (transcription and translation process), 2) action of transcribed gene (mRNA), and 3) from gene. It refers to directly or indirectly suppressing any of the actions of synthesized proteins. In a typical example, the active ingredient described above suppresses its expression or function in a gene-specific manner as a target.
　このような有効成分としては、例えば、低分子化合物、オリゴヌクレオチドなどの核酸重合体、高分子化合物、及び、これらの組合せ等が挙げられるが、これらに限定されない。低分子化合物としては、例えば、上記の遺伝子がコードするタンパク質の阻害剤が挙げられる。オリゴヌクレオチドなどの核酸重合体としては、１）対象となる遺伝子のアンチセンスオリゴヌクレオチド、２）ＲＮＡｉ用の核酸重合体（オリゴヌクレオチド）、３）ヘテロ二本鎖核酸（ＤＮＡ/ＲＮＡ）、４）ｍｉＲＮＡ標的核酸、及び、５）上記の遺伝子がコードするタンパク質に対する核酸アプタマーなどが挙げられる。なお、ＣＭＶの感染を原因とする中枢神経系障害に対する予防又は治療に関しては、血液脳関門を通過可能な大きさの分子、並びに、物質送達手段が求められる場合もある。 Examples of such an active ingredient include, but are not limited to, low molecular weight compounds, nucleic acid polymers such as oligonucleotides, high molecular weight compounds, and combinations thereof. Examples of low molecular weight compounds include inhibitors of proteins encoded by the above genes. Nucleic acid polymers such as oligonucleotides include 1) antisense oligonucleotides of the target gene, 2) nucleic acid polymers for RNAi (oligonucleotides), 3) hetero double-stranded nucleic acids (DNA / RNA), 4). Examples thereof include miRNA target nucleic acids and 5) nucleic acid aptamers for proteins encoded by the above genes. For prevention or treatment of central nervous system disorders caused by CMV infection, a molecule having a size capable of crossing the blood-brain barrier and a substance delivery means may be required.
　以下、上記２）のＲＮＡｉ用の核酸重合体について説明する。ＲＮＡｉ用の核酸重合体は、典型的には、相補的な二本鎖部分を有するＲＮＡ分子である。相補的な二本鎖部分は、例えば１６ｐｂ以上であり、１７ｂｐ以上であることが好ましい場合があり、２０ｂｐ以上がより好ましい場合がある。この相補的な二本鎖部分は、例えば３０ｐｂ以下であり、２７又は２６ｂｐ以下であることが好ましい場合があり、２５ｂｐ以下がより好ましい場合がある。 Hereinafter, the nucleic acid polymer for RNAi in 2) above will be described. Nucleic acid polymers for RNAi are typically RNA molecules with complementary double-stranded moieties. The complementary double-stranded portion may be, for example, 16 bp or more, preferably 17 bp or more, and more preferably 20 bp or more. The complementary double-stranded portion may be, for example, 30 pb or less, preferably 27 or 26 bp or less, and more preferably 25 bp or less.
　ＲＮＡｉ用の核酸重合体は、ターゲットたるｍＲＮＡに対してＲＮＡ干渉を引き起こすものであれば、その構造等は特に限定されない。ＲＮＡｉ用の核酸重合体は、例えば、ｓｈＲＮＡ、ダンベル型、ｓｉＲＮＡ、又はｍｉＲＮＡ等であり得、一例ではｓｈＲＮＡである。ｓｈＲＮＡにおけるループ部分の塩基配列は特に限定されず、適宜設計することが可能である。例えば、ループ部分は「ＣＴＣＧＡＧ」とすることができる（５´末端から３´末端。鋳型となるＤＮＡの配列で示す。）。 The structure of the nucleic acid polymer for RNAi is not particularly limited as long as it causes RNA interference with the target mRNA. The nucleic acid polymer for RNAi can be, for example, shRNA, dumbbell type, siRNA, miRNA, or the like, and in one example, shRNA. The base sequence of the loop portion in the shRNA is not particularly limited and can be appropriately designed. For example, the loop portion can be "CTCGAG" (from 5'end to 3'end, indicated by the sequence of the template DNA).
　本実施形態における有効成分は、上述の核酸重合体を発現するベクターであってもよい。このようなベクターは、市販のキットを用いて作製することができる。ｓｈＲＮＡを発現するベクターは、転写によってｓｈＲＮＡを生成するＤＮＡを、市販のベクター（例えば、アデノウイルスベクター）に挿入することによって作製することができる。挿入するＤＮＡに含めるプロモーター及びターミネーターの塩基配列は特に限定されず、適宜設計することが可能である。例えば、プロモーター部分を「ＣＣＧＧ」、ターミネーター部分を「ＴＴＴＴＴＧ」とすることができる（いずれも５´末端から３´末端）。 The active ingredient in this embodiment may be a vector expressing the above-mentioned nucleic acid polymer. Such a vector can be prepared using a commercially available kit. A vector expressing shRNA can be prepared by inserting a DNA that produces shRNA by transcription into a commercially available vector (for example, an adenovirus vector). The base sequences of the promoter and terminator to be included in the DNA to be inserted are not particularly limited and can be appropriately designed. For example, the promoter portion can be "CCGG" and the terminator portion can be "TTTTTG" (both from the 5'end to the 3'end).
　後述するが、本実施形態に係る剤は、上述の有効成分の他に、その他の成分を含んでいてもよい。 As will be described later, the agent according to this embodiment may contain other ingredients in addition to the above-mentioned active ingredients.
　１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制するｓｈＲＮＡの具体的な配列としては、例えば、実施例に記載の表１に示した＃１～＃８０のＤＮＡ配列の発現産物（ＲＮＡ）が挙げられる。＃１～＃８０のＤＮＡ配列は何れも、５’側から順に（表中で左側から順に）、（１）プロモーター部分「ＣＣＧＧ」、（２）相補的な二本鎖部分を成す第一鎖（センス側）、（３）ループ部分「ＣＴＣＧＡＧ」、（４）相補的な二本鎖部分を成す第二鎖（アンチセンス側）、及び、（５）ターミネーター部分「ＴＴＴＴＴＧ」、を含んで構成されている。発現産物（ＲＮＡ）においては、上記（２）の第一鎖に対応する（第一鎖を鋳型とする）ＲＮＡと、上記（４）の第二鎖に対応するＲＮＡとが相補的な二本鎖部分を形成する。 1. ~ 80. As a specific sequence of shRNA that suppresses the expression or function of at least one gene selected from the group consisting of the genes of, for example, the DNA sequences of # 1 to # 80 shown in Table 1 described in Examples. Expression products (RNA) can be mentioned. The DNA sequences of # 1 to # 80 are all ordered from the 5'side (from the left side in the table), (1) the promoter portion "CCGG", and (2) the first strand forming a complementary double-stranded portion. Consists of (sense side), (3) loop portion "CTCGAG", (4) second strand (antisense side) forming a complementary double-stranded portion, and (5) terminator portion "TTTTTG". Has been done. In the expression product (RNA), the RNA corresponding to the first strand of (2) above (using the first strand as a template) and the RNA corresponding to the second strand of (4) above are complementary. Form a chain portion.
　表１に示した＃１～＃８０のＤＮＡ配列の発現産物（ＲＮＡ）は、上述の１．～８０．の遺伝子のうち対応する番号の遺伝子の発現又は機能をそれぞれ抑制することができる。 The expression products (RNA) of the DNA sequences # 1 to # 80 shown in Table 1 are described in 1. above. ~ 80. It is possible to suppress the expression or function of the gene having the corresponding number among the genes of.
　なお、表１に示したＤＮＡ配列の発現産物（ＲＮＡ）と同等の、相補的な二本鎖部分を有するＲＮＡ分子は、ＲＮＡｉ用のオリゴヌクレオチドとして機能する。配列表の配列番号１～８０は、表１に示した＃１～＃８０のＤＮＡ配列の、上記（２）の相補的な二本鎖部分を成す第一鎖（センス側）を示す。 Note that the RNA molecule having a complementary double-stranded portion equivalent to the expression product (RNA) of the DNA sequence shown in Table 1 functions as an oligonucleotide for RNAi. SEQ ID NOs: 1 to 80 in the sequence listing indicate the first strand (sense side) forming the complementary double-stranded portion of (2) above in the DNA sequences # 1 to # 80 shown in Table 1.
　ＲＮＡｉ用の核酸重合体として機能する上記ＲＮＡ分子の一例は、ａ）配列番号１～８０で示す塩基配列の何れかにおける連続する１６以上の塩基を含むＤＮＡを鋳型とするＲＮＡ、及び、ｂ）当該ＤＮＡに相補的なＤＮＡを鋳型とするＲＮＡ、の少なくとも一方を含んでなる。上記ＲＮＡ分子は、好ましくは、上記のａ）及びｂ）のＲＮＡｓが二本鎖構造を形成してなる核酸重合体である。このＲＮＡ分子は、ＤＮＡ（ベクター等）の形態で投与されうる。 Examples of the RNA molecule that functions as a nucleic acid polymer for RNAi include a) RNA using DNA containing 16 or more consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 80 as a template, and b). It contains at least one of RNA, which uses a DNA complementary to the DNA as a template. The RNA molecule is preferably a nucleic acid polymer in which the RNAs of a) and b) above form a double-stranded structure. This RNA molecule can be administered in the form of DNA (vector, etc.).
　上述の連続する塩基の数は、１７以上であることが好ましく、１８以上又は１９以上であることがより好ましく、２０以上であることがさらに好ましい場合がある。 The number of consecutive bases described above is preferably 17 or more, more preferably 18 or more or 19 or more, and even more preferably 20 or more.
　（有効成分としての核酸重合体の修飾）
　有効成分としての核酸重合体は、天然の核酸重合体であってもよく非天然の核酸重合体であってもよい。核酸重合体は修飾されたものであってもよく、例えば、ペプチド核酸（ＰＮＡ）、ロックド核酸（locked nucleic acids、ＬＮＡ）、ホスホロチオアートオリゴヌクレオチド、２’Ｏ－Ｍｅ修飾オリゴヌクレオチド、２’－Ｏ，４’－Ｃ－エチレン架橋型核酸種（ＥＮＡ）等が挙げられるがこれらに限定されない。このような修飾により生体内での核酸重合体の安定性を高めることができる。 (Modification of nucleic acid polymer as an active ingredient)
The nucleic acid polymer as an active ingredient may be a natural nucleic acid polymer or a non-natural nucleic acid polymer. Nucleic acid polymers may be modified, eg, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate oligonucleotides, 2'O-Me modified oligonucleotides, 2'. Examples include, but are not limited to, -O, 4'-C-ethylene cross-linked nucleic acid species (ENA). Such modification can enhance the stability of the nucleic acid polymer in vivo.
　（その他の成分）
　本実施形態に係る剤を構成する有効成分以外の成分は特に限定されず、例えば、薬学的に許容される担体、潤滑剤、保存剤、安定剤、湿潤剤、乳化剤、浸透圧調整用の塩類、緩衝剤、着色剤、香味料、甘味料、抗酸化剤、及び粘度調整剤などと混合することができる。 (Other ingredients)
Ingredients other than the active ingredient constituting the agent according to the present embodiment are not particularly limited, and for example, pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmoregulation, etc. , Buffers, colorants, flavors, sweeteners, antioxidants, viscosity modifiers and the like.
　上記薬学的に許容される担体は、特に限定されないが、担体であって、上記有効成分と同時投与された場合に有効成分の効能を阻害せず、かつ、投与対象となるヒトに対して実質的な悪影響を及ぼさないという性質を備えることが好ましい。このような担体としては、この分野で従来公知のものを広く使用でき、具体的には、例えば、水、各種塩溶液、アルコール、植物油、ポリエチレングリコール、ゼラチン、ラクトース、アミロース、ステアリン酸マグネシウム、タルク、ケイ酸、パラフィン、脂肪酸モノグリセリド、脂肪酸ジグリセリド、ヒドロキシメチルセルロース、及びポリビニルピロリドンなどが挙げられるが、特にこれらに限定されない。担体の種類は、本実施形態に係る剤の剤型、投与方法などに応じて、適宜選択すればよい。 The pharmaceutically acceptable carrier is not particularly limited, but is a carrier that does not inhibit the efficacy of the active ingredient when co-administered with the active ingredient, and is substantially the same for a human to be administered. It is preferable to have the property of not having an adverse effect. As such a carrier, those conventionally known in this field can be widely used, and specifically, for example, water, various salt solutions, alcohol, vegetable oil, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, and talc. , Silica, paraffin, fatty acid monoglyceride, fatty acid diglyceride, hydroxymethyl cellulose, polyvinylpyrrolidone and the like, but are not particularly limited thereto. The type of carrier may be appropriately selected according to the dosage form, administration method, etc. of the agent according to the present embodiment.
　（既存の抗サイトメガロウイルス剤との併用）
　本実施形態に係る剤は、既存の抗サイトメガロウイルス剤と併用することも出来る。併用の一形態は、本実施形態に係る剤が、既存の抗サイトメガロウイルス剤を含んで製剤化されている、いわゆる配合剤の形態である。併用の他の形態は、本実施形態に係る剤と、抗サイトメガロウイルス剤とが別々に製剤化されていて、これらの剤を組合せて使用する（別々の剤を同時投与又は逐次投与する）形態である。 (Combination with existing anti-cytomegalovirus agents)
The agent according to this embodiment can also be used in combination with an existing anti-cytomegalovirus agent. One form of the combination is a so-called combination drug in which the agent according to the present embodiment is formulated containing an existing anti-cytomegalovirus agent. In another form of the combination, the agent according to the present embodiment and the anti-cytomegalovirus agent are separately formulated, and these agents are used in combination (separate agents are administered simultaneously or sequentially). It is a form.
　既存の抗サイトメガロウイルス剤としては、例えば、ガンシクロビル及びその塩、ホスカルネット及びその塩（ホスカルネットナトリウム水和物など）、ホスフェニトインナトリウム及びその塩（水和物など）、バルガンシクロビル及びその塩（バルガンシクロビル塩酸塩など）、レテルモビル、シドフォビル及びその塩、ならびに、ホミビルセンからなる群を挙げることができる。 Existing anti-cytomegalovirus agents include, for example, ganciclovir and its salts, foscarnet and its salts (such as foscarnet sodium hydrate), sodium phosphenitoin and its salts (such as hydrate), valganciclovir and Examples include the group consisting of its salts (such as valganciclovir hydrochloride), letermovir, cidofovir and its salts, and fomivirsen.
　〔２．剤の投与〕
　本実施形態に係る剤は、サイトメガロウイルス関連疾患の予防又は治療を目的として、ヒトの個体に投与することができる。 [2. Administration of drug]
The agent according to the present embodiment can be administered to an individual human for the purpose of preventing or treating a cytomegalovirus-related disease.
　ヒトの個体を投与対象とする場合、ヒトに対して、サイトメガロウイルス関連疾患の予防又は治療に有効な量の有効成分を投与する。ここで、有効成分は、単独で投与してもよく、又は、投与の目的に適した剤の一構成成分として投与してもよい。 When a human individual is targeted for administration, an amount of the active ingredient effective for the prevention or treatment of cytomegalovirus-related diseases is administered to the human. Here, the active ingredient may be administered alone or as a component of an agent suitable for the purpose of administration.
　本実施形態に係る剤に含まれる有効成分の投与量は特に限定されず、投与対象の疾患状態、体重、治療に対する反応、剤の投与形態、疾患の経過の段階、又は投与の間隔に依存して適宜調整され得る。なお、投与は、１回～数回に分けておこなわれ得、例えば、１日あたり１～数回投与され得る。 The dose of the active ingredient contained in the agent according to the present embodiment is not particularly limited, and depends on the disease state, body weight, response to treatment, administration form of the agent, stage of disease course, or administration interval. Can be adjusted as appropriate. The administration may be divided into one to several times, for example, one to several times per day.
　本実施形態に係る剤の投与経路は特に限定されず、例えば、経口投与、静脈内又は動脈内への血管内投与、並びに腸内投与といった手法により全身投与されてもよい。また、本実施形態に係る剤は、注入（注射器又は注入ポンプなどを利用）、経皮投与、舌下投与、及び臓器表面投与といった手法により局所投与されてもよい。投与量及び投与方法は、治療目的、患者の体重、年齢、及び症状などに応じて、当業者が適宜選択すればよい。本実施形態に係る剤の剤型は特に限定されず、例えば、錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、坐剤、貼り薬、塗り薬、及び注射剤などが挙げられる。 The administration route of the agent according to this embodiment is not particularly limited, and may be systemically administered by a method such as oral administration, intravenous or intraarterial administration, and intestinal administration. In addition, the agent according to the present embodiment may be locally administered by a method such as injection (using a syringe or an infusion pump), transdermal administration, sublingual administration, and organ surface administration. The dosage and administration method may be appropriately selected by those skilled in the art according to the purpose of treatment, the weight, age, symptoms and the like of the patient. The dosage form of the agent according to the present embodiment is not particularly limited, and for example, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, patches, ointments, injections and the like. Can be mentioned.
　本実施形態における有効成分が核酸重合体である場合、本実施形態に係る剤は、遺伝子治療剤であり得る。当該遺伝子治療剤は、有効成分を、注射によりヒトに直接投与する形態のものであってもよく、あるいは、上記核酸重合体が組み込まれたベクターを、注射によりヒトに直接投与する形態のものであってもよい。また、上記ベクターは、特に限定されないが、アデノウイルスベクター、アデノ関連ウイルスベクター、アデノ随伴ウイルスベクター、ヘルペスウイルスベクター、ワクシニアウイルスベクター、及びレトロウイルスベクターなど、遺伝子治療に適用可能なベクターが挙げられる。なお、上記遺伝子治療剤はリポソーム製剤であってもよい。 When the active ingredient in this embodiment is a nucleic acid polymer, the agent according to this embodiment can be a gene therapy agent. The gene therapy agent may be in the form of directly administering the active ingredient to humans by injection, or in the form of directly administering the vector incorporating the nucleic acid polymer to humans by injection. There may be. The vector is not particularly limited, and examples thereof include vectors applicable to gene therapy, such as an adenovirus vector, an adeno-related virus vector, an adeno-associated virus vector, a herpesvirus vector, a vaccinia virus vector, and a retrovirus vector. The gene therapy agent may be a liposome preparation.
　上記遺伝子治療剤を構成する上記ベクターには、標的部位において特異的に上記有効成分を発現させる発現調節配列が組み込まれていることが好ましい。ここで、発現調節配列とは、例えば、プロモーター又はエンハンサーである。 It is preferable that the vector constituting the gene therapy agent incorporates an expression regulatory sequence that specifically expresses the active ingredient at the target site. Here, the expression regulatory sequence is, for example, a promoter or an enhancer.
　〔３．本発明に係るスクリーニング方法〕
　本実施の形態のスクリーニング方法は、サイトメガロウイルス関連疾患の予防又は治療用の候補物質のスクリーニング方法であって、１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制するかを一つの指標とするスクリーニング方法である。本発明の一態様のスクリーニング方法は、細胞試料と被験物質とを接触させる工程（接触工程）、該細胞試料における１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を評価する工程（評価工程）、並びに、対照と比較して上記１．～８０．の遺伝子の発現又は機能が抑制された場合に、該被検物質を、候補物質として選択する工程（選択工程）を包含する。 [3. Screening method according to the present invention]
The screening method of the present embodiment is a screening method for a candidate substance for prevention or treatment of cytomegalovirus-related diseases, and 1. ~ 80. This is a screening method in which one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the above genes. One aspect of the screening method of the present invention is a step of contacting a cell sample with a test substance (contact step), 1. ~ 80. A step of evaluating the expression or function of at least one gene selected from the group consisting of the above genes (evaluation step), and the above 1. ~ 80. This includes a step of selecting a test substance as a candidate substance (selection step) when the expression or function of the gene is suppressed.
　上記接触工程に用いられる細胞試料は、例えばＣＭＶの感染対象となる細胞を含む試料である。細胞試料としては、非ヒト生物、各種の細胞株の培養細胞、及び生物から分離された生物学的試料等が挙げられる。培養細胞は、例えばヒト及び非ヒト生物の生体細胞、ＥＳ細胞、及びｉＰＳ細胞等であり、より具体的にはヒト神経系細胞株（U373MG細胞株）、及びヒト線維芽細胞株等が挙げられる。 The cell sample used in the above contact step is, for example, a sample containing cells to be infected with CMV. Examples of the cell sample include non-human organisms, cultured cells of various cell lines, and biological samples isolated from the organism. The cultured cells are, for example, living cells of human and non-human organisms, ES cells, iPS cells and the like, and more specifically, a human nervous system cell line (U373MG cell line), a human fibroblast cell line and the like can be mentioned. ..
　被検物質としては、低分子化合物、オリゴヌクレオチドなどの核酸重合体、ペプチド、タンパク質、高分子化合物、及び、これらの組合せ等が挙げられるが、これらに限定されない。被験物質は天然に存在するものでもよく、化学合成されたものでもよい。候補物質は、好ましくは、生体内で無害な物質であり得る。被験物質の一例はｓｈＲＮＡである。 Examples of the test substance include, but are not limited to, low molecular weight compounds, nucleic acid polymers such as oligonucleotides, peptides, proteins, high molecular weight compounds, and combinations thereof. The test substance may be naturally occurring or chemically synthesized. The candidate substance can preferably be a substance that is harmless in vivo. An example of a test substance is shRNA.
　細胞試料と被験物質とを接触させる具体的な方法は特に限定されない。例えば、細胞試料が培養細胞である場合、細胞を培養している培養液へ、候補物質を直接投与することが可能である。特に限定されないが、被験物質がオリゴヌクレオチドである場合には、リポフェクション法又はエレクトロポレーション法などを用いることによって、より確実に被験物質を細胞内へ導入することができる。また、被験物質がポリペプチド又は高分子化合物である場合には、インジェクション法又はリポソーム法などを用いることによって、より確実に被験物質を細胞内へ導入することができる。被検物質の添加濃度は、その種類に応じて、細胞の生育に悪影響を及ぼさない範囲で適宜選択され得る。接触期間、および、接触期間中の温度なども適宜選択され得る。 The specific method of contacting the cell sample with the test substance is not particularly limited. For example, when the cell sample is a cultured cell, the candidate substance can be directly administered to the culture medium in which the cell is cultured. Although not particularly limited, when the test substance is an oligonucleotide, the test substance can be more reliably introduced into the cell by using a lipofection method, an electroporation method, or the like. When the test substance is a polypeptide or a polymer compound, the test substance can be more reliably introduced into the cell by using an injection method, a liposome method, or the like. The concentration of the test substance added can be appropriately selected according to the type of the substance as long as it does not adversely affect the growth of cells. The contact period, the temperature during the contact period, and the like can also be appropriately selected.
　評価工程における遺伝子の発現又は機能の評価は、例えば、遺伝子発現量をリアルタイムＰＣＲ、デジタルＰＣＲ等の定量ＰＣＲ、又はマイクロアレイ解析などの解析により評価することができる。あるいは、該遺伝子から転写されるタンパクを標的にしてＥＬＩＳＡ又はウエスタンブロッディング法、サザンブロッディング法及び免疫蛍光染色などを用いて評価してもよいし、細胞死が見られるかどうかで評価してもよい。これらの操作は、常法に従って行うことができる。 The evaluation of gene expression or function in the evaluation step can be evaluated by, for example, real-time PCR, quantitative PCR such as digital PCR, or analysis such as microarray analysis. Alternatively, the protein transcribed from the gene may be targeted and evaluated using ELISA or Western blotting method, Southern blotting method, immunofluorescent staining, etc., or evaluated based on whether cell death is observed. May be good. These operations can be performed according to a conventional method.
　指標とする遺伝子は、１．～８０．の遺伝子からなる群より選択される、少なくとも１つの遺伝子であり、好ましくは２つ以上、より好ましくは３つ以上、さらにより好ましくは４つ以上、特に好ましくは５つ以上、最も好ましくは１０個以上であることが好ましい。 The genes used as indicators are 1. ~ 80. It is at least one gene selected from the group consisting of genes of, preferably two or more, more preferably three or more, even more preferably four or more, particularly preferably five or more, most preferably ten. The above is preferable.
　上記選択工程において、遺伝子の発現又は機能活性の程度を、対照（例えば被検物質を投与しなかった細胞試料における遺伝子の発現又は機能活性の程度）と比較する。比較は、好ましくは有意差の有無を考慮して行われる。その結果、対照と比較して、遺伝子の発現又は機能が抑制されたときに、該被検物質を、ＣＭＶ関連疾患の予防又は治療用の候補物質として選択する。 In the above selection step, the degree of gene expression or functional activity is compared with a control (for example, the degree of gene expression or functional activity in a cell sample to which the test substance was not administered). The comparison is preferably made in consideration of the presence or absence of a significant difference. As a result, when the expression or function of the gene is suppressed as compared with the control, the test substance is selected as a candidate substance for prevention or treatment of CMV-related diseases.
　以上のようなスクリーニング方法は、ＣＭＶ関連疾患の予防又は治療用の候補物質を効率よく得る方法として非常に有用である。 The screening method as described above is very useful as a method for efficiently obtaining a candidate substance for prevention or treatment of CMV-related diseases.
　以上に示したように、本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 As shown above, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments can be used. Embodiments obtained by appropriately combining them are also included in the technical scope of the present invention.
　〔４．まとめ〕
　上述の通り、本発明は、例えば、以下のような態様を包含している。 [4. Summary]
As described above, the present invention includes, for example, the following aspects.
　（Ａ）　（課題を解決するための手段）の欄に記載した１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制する成分を含む、サイトメガロウイルス関連疾患の予防又は治療用の剤。 1. Described in the column of (A) (Means for solving the problem). ~ 80. An agent for the prevention or treatment of cytomegalovirus-related diseases, which comprises a component that suppresses the expression or function of at least one gene selected from the group consisting of the genes of.
　（Ｂ）　上記成分が、核酸重合体である、（Ａ）の剤。 (B) The agent of (A), wherein the above component is a nucleic acid polymer.
　（Ｃ）　上記核酸重合体が、１）配列番号１～８０で示す塩基配列の何れかにおける連続する１６以上の塩基を含むＤＮＡを鋳型とするＲＮＡ、及び、当該ＤＮＡに相補的なＤＮＡを鋳型とするＲＮＡ、の少なくとも一方を含むか、２）上記１）のＲＮＡを含む核酸重合体を転写により生成するＤＮＡを含んでいる、（Ｂ）の剤。 (C) The nucleic acid polymer 1) uses RNA as a template for DNA containing 16 or more consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 80, and DNA complementary to the DNA as a template. The agent (B), which contains at least one of the RNAs to be used, or 2) a DNA produced by transcription of a nucleic acid polymer containing the RNA of 1) above.
　（Ｄ）　上記核酸重合体が、ｓｉＲＮＡである、（Ｂ）又は（Ｃ）の剤。 (D) The agent of (B) or (C), wherein the nucleic acid polymer is siRNA.
　（Ｅ）　５．、６．、１１．、１６．、１９．、２７．、２９．、３６．、３７．、４２．及び４４．～４７．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現を抑制する成分を含む、（Ａ）～（Ｄ）のいずれかの剤。 (E) 5. , 6. , 11. , 16. , 19. , 27. , 29. , 36. , 37. , 42. And 44. ~ 47. The agent according to any one of (A) to (D), which comprises a component that suppresses the expression of at least one gene selected from the group consisting of the genes of.
　（Ｆ）　抗サイトメガロウイルス剤を含むか、抗サイトメガロウイルス剤と組合せられている、（Ａ）～（Ｅ）のいずれかの剤。 (F) Any of (A) to (E) containing an anti-cytomegalovirus agent or combined with an anti-cytomegalovirus agent.
　（Ｇ）　上記抗サイトメガロウイルス剤が、ガンシクロビル及びその塩、ホスカルネット及びその塩（ホスカルネットナトリウム水和物など）、ホスフェニトインナトリウム及びその塩（水和物など）、バルガンシクロビル及びその塩（バルガンシクロビル塩酸塩など）、レテルモビル、シドフォビル及びその塩、ならびに、ホミビルセンからなる群から選択される少なくとも１つである、（Ｆ）の剤。 (G) The anti-cytomegalovirus agent is ganciclovir and its salt, foscarnet and its salt (foscarnet sodium hydrate, etc.), phosphenitoin sodium and its salt (hydrate, etc.), valganciclovir and its salt. The agent of (F), which is at least one selected from the group consisting of salts (such as valganciclovir hydrochloride), letermovir, cidofovir and salts thereof, and homivirsen.
　（Ｈ）　（課題を解決するための手段）の欄に記載した１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制するかを一つの指標とする、サイトメガロウイルス関連疾患の予防又は治療用の候補物質のスクリーニング方法。 1. Described in the column of (H) (Means for solving problems). ~ 80. A method for screening candidate substances for the prevention or treatment of cytomegalovirus-related diseases, wherein one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the genes of.
　以下に、実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
　以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施の形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施の形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples are shown below, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects can be used for details. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments obtained by appropriately combining the disclosed technical means Is also included in the technical scope of the present invention. In addition, all the documents described in the present specification are incorporated by reference.
　〔実施例〕
　１．shRNAスクリーニング及び標的遺伝子の推定
　以下の手順でshRNAの塩基配列を決定して標的遺伝子を推定した。 〔Example〕
1. 1. shRNA screening and estimation of target gene The target gene was estimated by determining the base sequence of shRNA by the following procedure.
　標的細胞として、ヒト神経系細胞（U373MG細胞株）を用いた。U373MG細胞は、培地として１０％ウシ胎児血清（BioWest社）及び抗生物質‐抗真菌剤混合溶液（ペニシリン１００ｕ／ｍＬ、ストレプトマイシン１００μｇ／ｍＬ及びアムホテリシンＢ２５０ｎｇ／ｍＬ）を含むダルベッコ変法イーグル培地（ナカライテスク）を用い、３７℃、５％ＣＯ２のインキュベーターで培養した。 Human nervous system cells (U373MG cell line) were used as target cells. U373MG cells contain 10% fetal bovine serum (BioWest) and an antibiotic-antifungal mixed solution (penicillin 100 u / mL, streptomycin 100 μg / mL and amphotericin B 250 ng / mL) as a medium. ) Was used, and the cells were cultured in an incubator at 37 ° C. and 5% CO 2.
　市販のキットに含まれる、レンチウイルスに組み込まれたshRNAのライブラリーを、U373MG細胞に導入した。shRNAが導入されたU373MG細胞を、１０μｇ／ｍＬ　Puromycin（Invivogen）培地を用いて３７℃、５％ＣＯ２のインキュベーターで１４日間培養し、Puromycin耐性を示す細胞集団を選択した。これにより、U373MG細胞の生存及び増殖を妨げないshRNAが導入されたU373MG細胞集団（以下、shRNA導入U373MG細胞集団）を獲得した。 A library of lentivirus-incorporated shRNAs included in a commercially available kit was introduced into U373MG cells. U373MG cells into which shRNA was introduced were cultured in a 10 μg / mL Puromycin (Invivogen) medium at 37 ° C. in a 5% CO 2 incubator for 14 days, and a cell population showing Puromycin resistance was selected. As a result, a U373MG cell population into which shRNAs that do not interfere with the survival and proliferation of U373MG cells were introduced (hereinafter referred to as shRNA-introduced U373MG cell population) was acquired.
　shRNA導入U373MG細胞集団にヒトサイトメガロウイルスTowne株（ATCC、製品番号VR-977）を感染させ、培養を続けて細胞死への抵抗性を観察した。また、対照としてshRNAライブラリーを導入していないU373MG細胞に対しても同様にヒトサイトメガロウイルスTowne株を感染させた。結果を図１に示す。図１中で、細胞集団Ａは強度耐性クローンを、細胞集団Ｂは中等度耐性クローンを示す。 The shRNA-introduced U373MG cell population was infected with the human cytomegalovirus Towne strain (ATCC, product number VR-977), and the culture was continued to observe resistance to cell death. In addition, as a control, U373MG cells into which the shRNA library had not been introduced were similarly infected with the human cytomegalovirus Towne strain. The results are shown in FIG. In FIG. 1, cell population A shows a strength-tolerant clone and cell population B shows a moderately resistant clone.
　ヒトサイトメガロウイルスTowne株に感染しているshRNA導入U373MG細胞集団のうち、約３週間経過後に生存している細胞集団を、ヒトサイトメガロウイルスTowne株に耐性を示す細胞集団（以下、ヒトCMV耐性細胞集団）とした。なお、対照実験に用いたU373MG細胞は、感染後約３週間でほとんどの細胞が死滅した。 Among the shRNA-introduced U373MG cell populations infected with the human cytomegalovirus Towne strain, the cell populations that survive after about 3 weeks are the cell populations that are resistant to the human cytomegalovirus Towne strain (hereinafter referred to as human CMV resistance). Cell population). Most of the U373MG cells used in the control experiment died about 3 weeks after infection.
　９６ウェルプレートを用いた限界希釈法を用いて、ヒトCMV耐性細胞集団に対してシングルセルクローニングを行った。続いて、単一細胞クローンそれぞれから常法に従ってゲノムDNAを抽出した。抽出したゲノムDNAのshRNA領域をPolymerase Chain Reaction（PCR）法で増幅した。続いて、PCR産物をプラスミドへそれぞれ導入し、shRNAの塩基配列をジデオキシ法により決定した。さらに、キットの販売者が提供するshRNAライブラリーの情報に基づいて、決定したshRNAの塩基配列から各shRNAが標的とする細胞遺伝子を特定した。shRNAの塩基配列、及び各shRNAが標的とする細胞遺伝子を以下の表１及び表２に示す。 Single-cell cloning was performed on a human CMV-resistant cell population using the limiting dilution method using a 96-well plate. Subsequently, genomic DNA was extracted from each single cell clone according to a conventional method. The shRNA region of the extracted genomic DNA was amplified by the Polymerase Chain Reaction (PCR) method. Subsequently, each PCR product was introduced into a plasmid, and the nucleotide sequence of shRNA was determined by the dideoxy method. Furthermore, based on the information in the shRNA library provided by the seller of the kit, the cell gene targeted by each shRNA was identified from the determined nucleotide sequence of shRNA. The nucleotide sequences of shRNA and the cell genes targeted by each shRNA are shown in Tables 1 and 2 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-I000004
　２．効果の検証
　（HCMV遺伝子の発現抑制）
　上述のヒトCMV耐性細胞集団からシングルセルクローニングを行って得た単一細胞クローンのうちの６クローン（サンプルＡ～Ｆとする）と、陰性対照shRNAを導入したU373MG細胞（対照）とを用い、各細胞におけるHCMV遺伝子の発現を比較した。より具体的には、HCMV遺伝子の発現量は、HCMV遺伝子から転写されるIE2タンパク質の発現量を指標にして比較した。IE2タンパク質の検出にはウエスタンブロッディング法及び免疫蛍光染色を用いた。ウエスタンブロッディング法では６クローンのうちの３クローンを用い、ローディングコントロールとしてβ‐アクチンを用いた。免疫蛍光染色では残りの3クローンを用いた。また、細胞核はDAPI染色を行った。その結果、特定のshRNAが導入された、ヒトサイトメガロウイルスに耐性を有するU373MG細胞では、IE2タンパク質の発現抑制がみられることから、HCMV遺伝子の発現の抑制が示唆された（結果は図示せず）。 2. Verification of effect (suppression of HCMV gene expression)
Six clones (referred to as Samples A to F) of the single cell clones obtained by single cell cloning from the above-mentioned human CMV resistant cell population and U373MG cells (control) into which negative control shRNA was introduced were used. The expression of the HCMV gene in each cell was compared. More specifically, the expression level of the HCMV gene was compared using the expression level of the IE2 protein transcribed from the HCMV gene as an index. Western blotting and immunofluorescence staining were used to detect the IE2 protein. In the western blotting method, 3 out of 6 clones were used, and β-actin was used as a loading control. The remaining 3 clones were used for immunofluorescence staining. The cell nuclei were stained with DAPI. As a result, suppression of IE2 protein expression was observed in U373MG cells resistant to human cytomegalovirus into which a specific shRNA was introduced, suggesting suppression of HCMV gene expression (results not shown). ).
　（標的細胞の遺伝子の発現抑制）
　上述のヒトCMV耐性細胞集団のうちの＃５のshRNAが導入されていた単一細胞クローンにおいてReverse Transcription-PCR (RT-PCR)法を用い、それぞれのshRNAが標的とする細胞遺伝子の発現を調べた。RT-PCR法のプライマーはshRNAの塩基配列に基づいて設計した。逆転写反応試薬はPrimeScript II 1st strand cDNA Synthesis Kit（タカラバイオ）を、RT-PCR用試薬は、Tks GflexTM DNA Polymerase（タカラバイオ）を用い、解析装置としてVeritiサーマルサイクラー(Applied Biosystems)を使用した。得られたPCR産物を電気泳動によって確認した。ローディングコントロールにはTBPを用いた。図２に示すように、導入したshRNAは、標的とする細胞遺伝子の発現を抑制していた。 (Suppression of gene expression in target cells)
Using the Reverse Transcription-PCR (RT-PCR) method in single cell clones into which # 5 shRNA was introduced in the above-mentioned human CMV-resistant cell population, the expression of cell genes targeted by each shRNA was examined. It was. The primers for the RT-PCR method were designed based on the nucleotide sequence of shRNA. PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio) was used as the reverse transcription reaction reagent, Tks Gflex TM DNA Polymerase (Takara Bio) was used as the reagent for RT-PCR, and Veriti Thermal Cycler (Applied Biosystems) was used as the analyzer. .. The obtained PCR product was confirmed by electrophoresis. TBP was used as the loading control. As shown in FIG. 2, the introduced shRNA suppressed the expression of the target cellular gene.
　（shRNAの単独導入）
　新たにU373MG細胞を用意し、上述の条件（１０％ウシ胎児血清及び抗生物質‐抗真菌剤混合溶液を含むダルベッコ変法イーグル培地、３７℃、５％ＣＯ２インキュベーター）で培養した。新たに＃５および＃１１のshRNAをそれぞれ導入したU373MG細胞と、陰性対照shRNAを導入したU373MG細胞（対照）とを１０μｇ／ｍＬ　Puromycin培地を用いて３７℃、５％ＣＯ２のインキュベーターで１４日間培養した後、Puromycin耐性を示す細胞集団を選択した。選択した細胞集団にヒトサイトメガロウイルスTowne株を感染させ、細胞死への抵抗性を比較した。図２に示すように、特定のshRNAを導入したU373MG細胞が、CMV感染にともなう細胞死に対して抵抗性を示すことを確認した。 (Introduction of shRNA alone)
U373MG cells were newly prepared and cultured under the above conditions (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and an antibiotic-antifungal mixed solution, 37 ° C., 5% CO 2 incubator). U373MG cells newly introduced with # 5 and # 11 shRNA and U373MG cells (control) introduced with negative control shRNA were used in a 10 μg / mL Puromycin medium at 37 ° C. in a 5% CO 2 incubator for 14 days. After culturing, a cell population showing Puromycin resistance was selected. Selected cell populations were infected with the human cytomegalovirus Towne strain and their resistance to cell death was compared. As shown in FIG. 2, it was confirmed that U373MG cells into which a specific shRNA was introduced showed resistance to cell death associated with CMV infection.

Claims (8)

  1. 　以下の１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制する成分を含む、サイトメガロウイルス関連疾患の予防又は治療用の剤。
    １．　AHNAK2 (AHNAK nucleoprotein 2)
    ２．　AIDA (axin interactor, dorsalization associated)
    ３．　AKAP5 (A kinase (PRKA) anchor protein 5)
    ４．　AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
    ５．　APC (adenomatous polyposis coli)
    ６．　C3orf74 (chromosome 3 open reading frame 74)
    ７．　C9orf9 (chromosome 9 open reading frame 9)
    ８．　C11orf67 (chromosome 11 open reading frame 67)
    ９．　C19orf33 (chromosome 19 open reading frame 33)
    １０．　CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
    １１．　CREB1 (cAMP responsive element binding protein 1)
    １２．　CYTH1 (cytohesin 1)
    １３．　EML1 (echinoderm microtubule associated protein like 1)
    １４．　EMP2 (epithelial membrane protein 2)
    １５．　EPHB6 (EPH receptor B6)
    １６．　FAM160A1 (family with sequence similarity 160, member A1)
    １７．　FAM176A (family with sequence similarity 176, member A)
    １８．　FXR2 (fragile X mental retardation, autosomal homolog 2)
    １９．　GPR126 (G protein-coupled receptor 126)
    ２０．　HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
    ２１．　HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
    ２２．　TESPA1 (Thymocyte Expressed, Positive Selection Associated 1) 
    ２３．　PALD1 (Phosphatase Domain Containing Paladin 1) 
    ２４．　KRTAP12-1 (keratin associated protein 12-1)
    ２５．　MBD1 (methyl-CpG binding domain protein 1)
    ２６．　MESDC2 (mesoderm development candidate 2)
    ２７．　MUC5AC (mucin 5AC, oligomeric mucus/gel-forming)
    ２８．　NOL12 (nucleolar protein 12)
    ２９．　NRP1 (neuropilin 1)
    ３０．　NUP153 (nucleoporin 153kDa)
    ３１．　PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
    ３２.　PCBD1 (pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha)
    ３３．　PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
    ３４．　RBM26 (RNA binding motif protein 26)
    ３５．　RSBN1 (round spermatid basic protein 1)
    ３６．　SCXA (scleraxis homolog A (mouse))
    ３７．　SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
    ３８．　TATDN3 (TatD DNase domain containing 3)
    ３９．　TBC1D3G (TBC1 domain family, member 3G)
    ４０．　TSC22D1 (TSC22 domain family, member 1)
    ４１．　UNC119B (unc-119 homolog B (C. elegans))
    ４２．　WBP4 (WW domain binding protein 4 (formin binding protein 21))
    ４３．　ZNF354A (zinc finger protein 354A)
    ４４．　ATF1 (Activating Transcription Factor 1)
    ４５．　PGAM5 (PGAM Family Member 5, Mitochondrial Serine/Threonine Protein Phosphatase)
    ４６．　IPO8 (Importin 8)
    ４７．　OGA (O-GlcNAcase)
    ４８．　PROX1 (Prospero Homeobox 1)
    ４９．　CIDEC (Cell Death Inducing DFFA Like Effector C)
    ５０．　JPH2 (Junctophilin 2)
    ５１．　RNF208 (Ring Finger Protein 208)
    ５２．　STEAP1B (STEAP Family Member 1B)
    ５３．　TENM4 (Teneurin Transmembrane Protein 4)
    ５４．　GCDH (Glutaryl-CoA Dehydrogenase)
    ５５．　INTS6L (Integrator Complex Subunit 6 Like)
    ５６．　MAN1A2 (Mannosidase Alpha Class 1A Member 2)
    ５７．　RBM5 (RNA Binding Motif Protein 5)
    ５８．　PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
    ５９．　SMARCA2 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
    ６０．　MYCBP2 (MYC Binding Protein 2)
    ６１．　SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
    ６２．　RNF13 (Ring Finger Protein 13)
    ６３．　DUSP22 (Dual Specificity Phosphatase 22)
    ６４．　SMNDC1 (Survival Motor Neuron Domain Containing 1)
    ６５．　SYF2 (SYF2 Pre-MRNA Splicing Factor)
    ６６．　SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
    ６７．　RASA1 (RAS P21 Protein Activator 1)
    ６８．　ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
    ６９．　CEP85 (Centrosomal Protein 85)
    ７０．　PXN (Paxillin)
    ７１．　PEX12 (Peroxisomal Biogenesis Factor 12)
    ７２．　COMMD8 (COMM Domain Containing 8)
    ７３．　RHOQ (Ras Homolog Family Member Q)
    ７４．　SIL1 (SIL1 Nucleotide Exchange Factor)
    ７５．　F8 (Coagulation Factor VIII)
    ７６．　TCHP (Trichoplein Keratin Filament Binding)
    ７７．　NUP210 (Nucleoporin 210)
    ７８．　MIR7-3HG (MIR7-3 Host Gene)
    ７９．　MFAP5 (Microfibril Associated Protein 5)
    ８０．　TUG1 (Taurine Up-Regulated 1)     The following 1. ~ 80. An agent for the prevention or treatment of cytomegalovirus-related diseases, which comprises a component that suppresses the expression or function of at least one gene selected from the group consisting of the genes of.
    1. 1. AHNAK2 (AHNAK nucleoprotein 2)
    2. AIDA (axin interactor, dorsalization associated)
    3. 3. AKAP5 (A kinase (PRKA) anchor protein 5)
    4. AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
    5. APC (adenomatous polyposis coli)
    6. C3orf74 (chromosome 3 open reading frame 74)
    7. C9orf9 (chromosome 9 open reading frame 9)
    8. C11orf67 (chromosome 11 open reading frame 67)
    9. C19orf33 (chromosome 19 open reading frame 33)
    10. CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
    11. CREB1 (cAMP responsive element binding protein 1)
    12. CYTH1 (cytohesin 1)
    13. EML1 (echinoderm microtubule associated protein like 1)
    14. EMP2 (epithelial membrane protein 2)
    15. EPHB6 (EPH receptor B6)
    16. FAM160A1 (family with sequence similarity 160, member A1)
    17. FAM176A (family with sequence similarity 176, member A)
    18. FXR2 (fragile X mental retardation, autosomal homolog 2)
    19. GPR126 (G protein-coupled receptor 126)
    20. HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
    21. HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
    22. TESPA1 (Thymocyte Expressed, Positive Selection Associated 1)
    23. PALD1 (Phosphatase Domain Containing Paladin 1)
    24. KRTAP12-1 (keratin associated protein 12-1)
    25. MBD1 (methyl-CpG binding domain protein 1)
    26. MESDC2 (mesoderm development candidate 2)
    27. MUC5AC (mucin 5AC, oligomeric mucus / gel-forming)
    28. NOL12 (nucleolar protein 12)
    29. NRP1 (neuropilin 1)
    30. NUP153 (nucleoporin 153kDa)
    31. PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
    32. PCBD1 (pterin-4 alpha-carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha)
    33. PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
    34. RBM26 (RNA binding motif protein 26)
    35. RSBN1 (round spermatid basic protein 1)
    36. SCXA (scleraxis homolog A (mouse))
    37. SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
    38. TATDN3 (TatD DNase domain containing 3)
    39. TBC1D3G (TBC1 domain family, member 3G)
    40. TSC22D1 (TSC22 domain family, member 1)
    41. UNC119B (unc-119 homolog B (C. elegans))
    42. WBP4 (WW domain binding protein 4 (formin binding protein 21))
    43. ZNF354A (zinc finger protein 354A)
    44. ATF1 (Activating Transcription Factor 1)
    45. PGAM5 (PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase)
    46. IPO8 (Importin 8)
    47. OGA (O-GlcNA case)
    48. PROX1 (Prospero Homeobox 1)
    49. CIDEC (Cell Death Inducing DFFA Like Effector C)
    50. JPH2 (Junctophilin 2)
    51. RNF208 (Ring Finger Protein 208)
    52. STEAP1B (STEAP Family Member 1B)
    53. TENM4 (Teneurin Transmembrane Protein 4)
    54. GCDH (Glutaryl-CoA Dehydrogenase)
    55. INTS6L (Integrator Complex Subunit 6 Like)
    56. MAN1A2 (Mannosidase Alpha Class 1A Member 2)
    57. RBM5 (RNA Binding Motif Protein 5)
    58. PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
    59. SMARCA2 (SWI / SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
    60. MYCBP2 (MYC Binding Protein 2)
    61. SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
    62. RNF13 (Ring Finger Protein 13)
    63. DUSP22 (Dual Specificity Phosphatase 22)
    64. SMNDC1 (Survival Motor Neuron Domain Containing 1)
    65. SYF2 (SYF2 Pre-MRNA Splicing Factor)
    66. SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
    67. RASA1 (RAS P21 Protein Activator 1)
    68. ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
    69. CEP85 (Centrosomal Protein 85)
    70. PXN (Paxillin)
    71. PEX12 (Peroxisomal Biogenesis Factor 12)
    72. COMMD8 (COMM Domain Containing 8)
    73. RHOQ (Ras Homolog Family Member Q)
    74. SIL1 (SIL1 Nucleotide Exchange Factor)
    75. F8 (Coagulation Factor VIII)
    76. TCHP (Trichoplein Keratin Filament Binding)
    77. NUP210 (Nucleoporin 210)
    78. MIR7-3HG (MIR7-3 Host Gene)
    79. MFAP5 (Microfibril Associated Protein 5)
    80. TUG1 (Taurine Up-Regulated 1)
  2. 　上記成分は、核酸重合体である、請求項１に記載の剤。 The agent according to claim 1, wherein the above component is a nucleic acid polymer.
  3. 　上記核酸重合体は、
    １）配列番号１～８０で示す塩基配列の何れかにおける連続する１６以上の塩基を含むＤＮＡを鋳型とするＲＮＡ、及び、当該ＤＮＡに相補的なＤＮＡを鋳型とするＲＮＡ、の少なくとも一方を含むか、
    ２）上記１）のＲＮＡを含む核酸重合体を転写により生成するＤＮＡを含んでいる、
    請求項２に記載の剤。     The above nucleic acid polymer is
    1) Includes at least one of an RNA using a DNA containing 16 or more consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 80 as a template, and an RNA using a DNA complementary to the DNA as a template. Or
    2) Contains DNA that is produced by transcription of the nucleic acid polymer containing the RNA of 1) above.
    The agent according to claim 2.
  4. 　上記核酸重合体は、ｓｉＲＮＡである、請求項２又は３に記載の剤。 The agent according to claim 2 or 3, wherein the nucleic acid polymer is siRNA.
  5. 　上記５．、６．、１１．、１６．、１９．、２７．、２９．、３６．、３７．、４２．及び４４．～４７．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現を抑制する成分を含む、請求項１～４のいずれか１項に記載の剤。 Above 5. , 6. , 11. , 16. , 19. , 27. , 29. , 36. , 37. , 42. And 44. ~ 47. The agent according to any one of claims 1 to 4, which comprises a component that suppresses the expression of at least one gene selected from the group consisting of the genes of.
  6. 　抗サイトメガロウイルス剤を含むか、抗サイトメガロウイルス剤と組合せられている、請求項１～５のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 5, which contains an anti-cytomegalovirus agent or is combined with an anti-cytomegalovirus agent.
  7. 　上記抗サイトメガロウイルス剤は、ガンシクロビル及びその塩、ホスカルネット及びその塩（ホスカルネットナトリウム水和物など）、ホスフェニトインナトリウム及びその塩（水和物など）、バルガンシクロビル及びその塩（バルガンシクロビル塩酸塩など）、レテルモビル、シドフォビル及びその塩、ならびに、ホミビルセンからなる群から選択される少なくとも１つである、請求項６に記載の剤。 The anti-cytomegalovirus agent includes ganciclovir and its salt, foscarnet and its salt (foscarnet sodium hydrate, etc.), phosphenitoin sodium and its salt (hydrate, etc.), valganciclovir and its salt (val). The agent according to claim 6, which is at least one selected from the group consisting of ganciclovir hydrochloride, etc.), letermovir, cidofovir and salts thereof, and fomivirsen.
  8. 　以下の１．～８０．の遺伝子からなる群より選択される少なくとも１つの遺伝子の発現又は機能を抑制するかを一つの指標とする、サイトメガロウイルス関連疾患の予防又は治療用の候補物質のスクリーニング方法。
    １．　AHNAK2 (AHNAK nucleoprotein 2)
    ２．　AIDA (axin interactor, dorsalization associated)
    ３．　AKAP5 (A kinase (PRKA) anchor protein 5)
    ４．　AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
    ５．　APC (adenomatous polyposis coli)
    ６．　C3orf74 (chromosome 3 open reading frame 74)
    ７．　C9orf9 (chromosome 9 open reading frame 9)
    ８．　C11orf67 (chromosome 11 open reading frame 67)
    ９．　C19orf33 (chromosome 19 open reading frame 33)
    １０．　CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
    １１．　CREB1 (cAMP responsive element binding protein 1)
    １２．　CYTH1 (cytohesin 1)
    １３．　EML1 (echinoderm microtubule associated protein like 1)
    １４．　EMP2 (epithelial membrane protein 2)
    １５．　EPHB6 (EPH receptor B6)
    １６．　FAM160A1 (family with sequence similarity 160, member A1)
    １７．　FAM176A (family with sequence similarity 176, member A)
    １８．　FXR2 (fragile X mental retardation, autosomal homolog 2)
    １９．　GPR126 (G protein-coupled receptor 126)
    ２０．　HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
    ２１．　HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
    ２２．　TESPA1 (Thymocyte Expressed, Positive Selection Associated 1) 
    ２３．　PALD1 (Phosphatase Domain Containing Paladin 1) 
    ２４．　KRTAP12-1 (keratin associated protein 12-1)
    ２５．　MBD1 (methyl-CpG binding domain protein 1)
    ２６．　MESDC2 (mesoderm development candidate 2)
    ２７．　MUC5AC (mucin 5AC, oligomeric mucus/gel-forming)
    ２８．　NOL12 (nucleolar protein 12)
    ２９．　NRP1 (neuropilin 1)
    ３０．　NUP153 (nucleoporin 153kDa)
    ３１．　PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
    ３２.　PCBD1 (pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha)
    ３３．　PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
    ３４．　RBM26 (RNA binding motif protein 26)
    ３５．　RSBN1 (round spermatid basic protein 1)
    ３６．　SCXA (scleraxis homolog A (mouse))
    ３７．　SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
    ３８．　TATDN3 (TatD DNase domain containing 3)
    ３９．　TBC1D3G (TBC1 domain family, member 3G)
    ４０．　TSC22D1 (TSC22 domain family, member 1)
    ４１．　UNC119B (unc-119 homolog B (C. elegans))
    ４２．　WBP4 (WW domain binding protein 4 (formin binding protein 21))
    ４３．　ZNF354A (zinc finger protein 354A)
    ４４．　ATF1 (Activating Transcription Factor 1)
    ４５．　PGAM5 (PGAM Family Member 5, Mitochondrial Serine/Threonine Protein Phosphatase)
    ４６．　IPO8 (Importin 8)
    ４７．　OGA (O-GlcNAcase)
    ４８．　PROX1 (Prospero Homeobox 1)
    ４９．　CIDEC (Cell Death Inducing DFFA Like Effector C)
    ５０．　JPH2 (Junctophilin 2)
    ５１．　RNF208 (Ring Finger Protein 208)
    ５２．　STEAP1B (STEAP Family Member 1B)
    ５３．　TENM4 (Teneurin Transmembrane Protein 4)
    ５４．　GCDH (Glutaryl-CoA Dehydrogenase)
    ５５．　INTS6L (Integrator Complex Subunit 6 Like)
    ５６．　MAN1A2 (Mannosidase Alpha Class 1A Member 2)
    ５７．　RBM5 (RNA Binding Motif Protein 5)
    ５８．　PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
    ５９．　SMARCA2 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
    ６０．　MYCBP2 (MYC Binding Protein 2)
    ６１．　SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
    ６２．　RNF13 (Ring Finger Protein 13)
    ６３．　DUSP22 (Dual Specificity Phosphatase 22)
    ６４．　SMNDC1 (Survival Motor Neuron Domain Containing 1)
    ６５．　SYF2 (SYF2 Pre-MRNA Splicing Factor)
    ６６．　SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
    ６７．　RASA1 (RAS P21 Protein Activator 1)
    ６８．　ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
    ６９．　CEP85 (Centrosomal Protein 85)
    ７０．　PXN (Paxillin)
    ７１．　PEX12 (Peroxisomal Biogenesis Factor 12)
    ７２．　COMMD8 (COMM Domain Containing 8)
    ７３．　RHOQ (Ras Homolog Family Member Q)
    ７４．　SIL1 (SIL1 Nucleotide Exchange Factor)
    ７５．　F8 (Coagulation Factor VIII)
    ７６．　TCHP (Trichoplein Keratin Filament Binding)
    ７７．　NUP210 (Nucleoporin 210)
    ７８．　MIR7-3HG (MIR7-3 Host Gene)
    ７９．　MFAP5 (Microfibril Associated Protein 5)
    ８０．　TUG1 (Taurine Up-Regulated 1)     The following 1. ~ 80. A method for screening candidate substances for the prevention or treatment of cytomegalovirus-related diseases, wherein one index is whether to suppress the expression or function of at least one gene selected from the group consisting of the genes of.
    1. 1. AHNAK2 (AHNAK nucleoprotein 2)
    2. AIDA (axin interactor, dorsalization associated)
    3. 3. AKAP5 (A kinase (PRKA) anchor protein 5)
    4. AP1S2 (adaptor-related protein complex 1, sigma 2 subunit)
    5. APC (adenomatous polyposis coli)
    6. C3orf74 (chromosome 3 open reading frame 74)
    7. C9orf9 (chromosome 9 open reading frame 9)
    8. C11orf67 (chromosome 11 open reading frame 67)
    9. C19orf33 (chromosome 19 open reading frame 33)
    10. CHTF18 (CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae))
    11. CREB1 (cAMP responsive element binding protein 1)
    12. CYTH1 (cytohesin 1)
    13. EML1 (echinoderm microtubule associated protein like 1)
    14. EMP2 (epithelial membrane protein 2)
    15. EPHB6 (EPH receptor B6)
    16. FAM160A1 (family with sequence similarity 160, member A1)
    17. FAM176A (family with sequence similarity 176, member A)
    18. FXR2 (fragile X mental retardation, autosomal homolog 2)
    19. GPR126 (G protein-coupled receptor 126)
    20. HDHD1A (haloacid dehalogenase-like hydrolase domain containing 1A)
    21. HNRNPH3 (heterogeneous nuclear ribonucleoprotein H3 (2H9))
    22. TESPA1 (Thymocyte Expressed, Positive Selection Associated 1)
    23. PALD1 (Phosphatase Domain Containing Paladin 1)
    24. KRTAP12-1 (keratin associated protein 12-1)
    25. MBD1 (methyl-CpG binding domain protein 1)
    26. MESDC2 (mesoderm development candidate 2)
    27. MUC5AC (mucin 5AC, oligomeric mucus / gel-forming)
    28. NOL12 (nucleolar protein 12)
    29. NRP1 (neuropilin 1)
    30. NUP153 (nucleoporin 153kDa)
    31. PATL2 (protein associated with topoisomerase II homolog 2 (yeast))
    32. PCBD1 (pterin-4 alpha-carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha)
    33. PPKRIR (protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor))
    34. RBM26 (RNA binding motif protein 26)
    35. RSBN1 (round spermatid basic protein 1)
    36. SCXA (scleraxis homolog A (mouse))
    37. SPINT3 (serine peptidase inhibitor, Kunitz type, 3)
    38. TATDN3 (TatD DNase domain containing 3)
    39. TBC1D3G (TBC1 domain family, member 3G)
    40. TSC22D1 (TSC22 domain family, member 1)
    41. UNC119B (unc-119 homolog B (C. elegans))
    42. WBP4 (WW domain binding protein 4 (formin binding protein 21))
    43. ZNF354A (zinc finger protein 354A)
    44. ATF1 (Activating Transcription Factor 1)
    45. PGAM5 (PGAM Family Member 5, Mitochondrial Serine / Threonine Protein Phosphatase)
    46. IPO8 (Importin 8)
    47. OGA (O-GlcNA case)
    48. PROX1 (Prospero Homeobox 1)
    49. CIDEC (Cell Death Inducing DFFA Like Effector C)
    50. JPH2 (Junctophilin 2)
    51. RNF208 (Ring Finger Protein 208)
    52. STEAP1B (STEAP Family Member 1B)
    53. TENM4 (Teneurin Transmembrane Protein 4)
    54. GCDH (Glutaryl-CoA Dehydrogenase)
    55. INTS6L (Integrator Complex Subunit 6 Like)
    56. MAN1A2 (Mannosidase Alpha Class 1A Member 2)
    57. RBM5 (RNA Binding Motif Protein 5)
    58. PIGW (Phosphatidylinositol Glycan Anchor Biosynthesis Class W)
    59. SMARCA2 (SWI / SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 2)
    60. MYCBP2 (MYC Binding Protein 2)
    61. SEC24B (SEC24 Homolog B, COPII Coat Complex Component)
    62. RNF13 (Ring Finger Protein 13)
    63. DUSP22 (Dual Specificity Phosphatase 22)
    64. SMNDC1 (Survival Motor Neuron Domain Containing 1)
    65. SYF2 (SYF2 Pre-MRNA Splicing Factor)
    66. SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B)
    67. RASA1 (RAS P21 Protein Activator 1)
    68. ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase)
    69. CEP85 (Centrosomal Protein 85)
    70. PXN (Paxillin)
    71. PEX12 (Peroxisomal Biogenesis Factor 12)
    72. COMMD8 (COMM Domain Containing 8)
    73. RHOQ (Ras Homolog Family Member Q)
    74. SIL1 (SIL1 Nucleotide Exchange Factor)
    75. F8 (Coagulation Factor VIII)
    76. TCHP (Trichoplein Keratin Filament Binding)
    77. NUP210 (Nucleoporin 210)
    78. MIR7-3HG (MIR7-3 Host Gene)
    79. MFAP5 (Microfibril Associated Protein 5)
    80. TUG1 (Taurine Up-Regulated 1)
PCT/JP2020/044004 2019-11-27 2020-11-26 Prophylactic or therapeutic agent for cytomegalovirus-related diseases WO2021107005A1 (en)

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