WO2022049814A1 - Growth inhibitor for herpes simplex virus - Google Patents
Growth inhibitor for herpes simplex virus Download PDFInfo
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- WO2022049814A1 WO2022049814A1 PCT/JP2021/011849 JP2021011849W WO2022049814A1 WO 2022049814 A1 WO2022049814 A1 WO 2022049814A1 JP 2021011849 W JP2021011849 W JP 2021011849W WO 2022049814 A1 WO2022049814 A1 WO 2022049814A1
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- hsv
- cells
- human
- infection
- inhibitor
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Definitions
- the present invention relates to an inhibitor for the growth of herpes simplex virus and a pharmaceutical or pharmaceutical composition containing the inhibitor. More specifically, it relates to an inhibitor for the growth of herpes simplex virus in photoreceptor cells and a drug or a pharmaceutical composition containing the inhibitor.
- lncRNA long non-coding RNA
- ARN Acute retinal necrosis
- HSV-1 herpesvirus type 1
- Non-Patent Document 2 steroids, antiviral agents, and antithrombotic agents are used for the treatment of ARN
- Non-Patent Document 2 the prognosis of visual acuity is very poor, which is considered to be mainly due to photoreceptor cell death caused by viral infection
- Document 3 the prognosis of visual acuity is very poor, which is considered to be mainly due to photoreceptor cell death caused by viral infection.
- an object of the present invention to provide a substance that inhibits the growth of herpes simplex virus in cells, and to provide a drug or a pharmaceutical composition for treating acute retinal necrosis containing the substance.
- Another object of the present invention is to provide a biomarker relating to a method for predicting visual acuity prognosis and an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis.
- the present inventors performed RNA sequencing analysis to search for lncRNA regulated under infection with herpes simplex virus type 1 (herpes simplex virus: HSV-1) against mouse retinal photoreceptor cells (661W cells). As a result, it was found that lncRNA U90926 was significantly upregulated by infection with HSV-1. To investigate the effect of lncRNA U90926 on HSV-1 proliferation, the inventors found that knocking down lncRNA U90926 markedly suppressed HSV-1 proliferation and increased host photoreceptor viability. It was revealed.
- Non-Patent Document 5 U90926 expression is only downregulated in adipose progenitor cell differentiation.
- Non-Patent Document 6 we identified the human homologue of U90926 and confirmed that the expression of human homologue RNA of U90926 was significantly increased in HSV-1 infected human umbilical cord blood venous endothelial cells.
- the present invention is the following (1) to (9).
- the inhibitor which is a growth inhibitor of herpes simplex virus (HSV) in cells and contains an expression inhibitor of U90926.
- a method for diagnosing HSV infection or a method for assisting diagnosis of HSV infection which comprises measuring the expression level of human U90926 in a sample derived from a subject.
- a method for predicting visual acuity prognosis or an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis which comprises measuring the expression level of human U90926 in a sample derived from a patient with acute retinal necrosis.
- the reference numeral “ ⁇ ” indicates a numerical range including the values on the left and right thereof.
- the present invention it is possible to suppress or inhibit the growth of herpes simplex virus, particularly HSV-1 in cells (particularly photoreceptor cells). Therefore, the present invention makes it possible to develop a therapeutic method and a therapeutic agent for ARN, which is an intractable disease.
- the drug of the present invention is unlikely to induce drug resistance and can exert an effect that enables long-term treatment of ARN.
- the present invention provides a method for predicting visual acuity prognosis in patients with acute retinal necrosis and an auxiliary method for predicting visual acuity prognosis. Therefore, the present invention may provide important judgment material in determining the treatment policy of a patient with acute retinal necrosis.
- RNA sequencing data for U90926 and ⁇ -actin in uninfected 661W cells (upper) and HSV-1 infected 661W cells (lower) are shown. Visualized using Integrative Genomics Viewer (registered trademark). The time course of the expression level of U90926 after HSV-1 infection is shown. Values are shown as mean ⁇ standard deviation.
- the results of measuring the relative expression levels of U90926, Neat1v2 and ⁇ -actin RNA in the whole cell, nucleus and cytoplasm in 661W cells 8 hours after HSV-1 infection are shown. Neat1v2 RNA and ⁇ -actin RNA were used as positive controls for RNA expression in the nucleus and cytoplasm, respectively. Values are shown as mean ⁇ standard deviation.
- * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 and **** p ⁇ 0.0001 are based on Student's t-test for control cells.
- the results of measuring the cell viability of control cells and U90926 knockdown cells after HSV-1 infection are shown.
- * p ⁇ 0.05, *** p ⁇ 0.001 and **** p ⁇ 0.0001 are based on Student's t-test for control cells.
- the measurement results of the relative ICP-0 (HSV-1 gene) RNA expression level and the relative ICP-4 (HSV-1 gene) RNA expression level in the control cell and the U90926 knockdown cell are shown.
- ICP-0 HSV-1 gene
- ICP-4 HSV-1 gene
- Correlation analysis between the expression level of human U90926 long transcript in vitreous humor derived from patients with acute retinal necrosis, viral load of HSV-1 in vitreous fluid derived from patients with acute retinal necrosis, and final corrected logMAR visual acuity in patients with acute retinal necrosis The result.
- A shows the correlation between the expression level of human U90926 long transcript in vitreous humor and the final corrected logMAR visual acuity.
- B shows the correlation between the viral load of HSV-1 in the vitreous humor and the final corrected logMAR visual acuity.
- C shows the correlation between the expression level of human U90926 long transcript and the amount of HSV-1 virus in the vitreous humor.
- the first embodiment is an inhibitor of the growth of herpes simplex virus (HSV) in cells, and the inhibitor containing a U90926 expression inhibitor (hereinafter, also referred to as "inhibitor of the present invention”).
- HSV herpes simplex virus
- U90926 expression inhibitor hereinafter, also referred to as "inhibitor of the present invention”
- Infectious varicella-zoster virus (VZV) which is an intraocular inflammatory disease, is included in the herpesvirus family, including HSV, cytomegalovirus (CMV), and varicella zoster virus (VZV). It is a disease that often develops due to the virus to which it belongs. In particular, when HSV or VZV infects the retina, severe uveitis called acute retinal necrosis occurs.
- U90926 (referring to the U90926 gene unless otherwise specified).
- U90926 is a gene identified in mice that is annotated and transcripts of the National Center for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/).
- lncRNA (NR_033483.1) is 522 bp long (Fig. 1 and SEQ ID NO: 17).
- human U90926 As a human homologue of U90926 (hereinafter also referred to as "human U90926”), the inventors have identified a gene displayed in the synteny region of the human genome with accession number AC110615.1.
- the gene has transcripts represented by AC110615.1-201 (human U90926 short transcript) (SEQ ID NO: 18) and AC110615.1-202 (human U90926 long transcript) (SEQ ID NO: 19).
- AC110615.1-201 human U90926 short transcript
- AC110615.1-202 human U90926 long transcript
- HSV in all embodiments of the present invention includes herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).
- HSV-1 and HSV-2 used in this embodiment are all strains classified into these (for example, KOS strain, F strain, 17 strain, VR3 strain, HF strain, HF10 classified into HSV-1). (Strains and SC16 strains, etc., as well as 186 strains, G strains, 333 strains, etc. classified as HSV-2) and all of those derived from these sub-strains are included, but the most preferred HSV is HSV-1.
- the "cell” in all the embodiments of the present invention is not particularly limited as long as it is a cell infected with HSV, and for example, vascular endothelial cells, oral mucosal epithelial cells, corneal epithelial cells, and corneal endothelium. Examples include cells, retinal ganglion cells, and photoreceptor cells.
- the inhibitor of the present invention has an action of inhibiting (or suppressing) the proliferation of HSV in cells, and increases the proliferation of HSV in the absence of the inhibitor by 50% or more, preferably 70% or more, particularly preferably. It can inhibit more than 80%.
- the U90926 expression inhibitor contained in the inhibitor of the present invention is not particularly limited as long as it is a substance that inhibits or suppresses the expression of U90926, which is a lncRNA, and examples thereof include antisense oligonucleotides and siRNA. Can be mentioned.
- antisense oligonucleotides, siRNAs and the like are not only nucleic acids, but also nucleic acid analogs such as LNA (locked nucleic acid), phosphorothioate (PS), morpholinooligo, borane phosphate, 2'-O-methyl. It may be composed of converted RNA (2'-OMe), 2'-O-methoxyethylated RNA (2'-MOE), or the like.
- the second embodiment is a pharmaceutical or pharmaceutical composition (hereinafter, also referred to as "pharmaceutical or the like of the present invention") containing a substance that inhibits the expression of U90926 (that is, the inhibitor of the present invention) as an active ingredient.
- the pharmaceutical product of the present invention has a medicinal effect as a therapeutic agent for a disease caused by infection with HSV (particularly HSV-1), that is, an HSV infection.
- HSV infection particularly HSV-1
- the pathophysiology of HSV infection for which the drug of the present invention has a therapeutic effect is not particularly limited, but if it is dared to give an example, stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis and meningitis You can mention flames.
- the drug of the present invention may be in the form of administering the expression-inhibiting substance itself of U90926, which is an active ingredient, but generally, a drug containing one or more pharmaceutical additives in addition to these active ingredients. It is desirable to administer in the form of a composition (hereinafter, also referred to as "the pharmaceutical composition of the present invention"). In addition, other known agents may be added to the pharmaceutical composition according to the embodiment of the present invention.
- the dosage form of the pharmaceutical or pharmaceutical composition of the present invention is not particularly limited, and examples thereof include liquid preparations, which may be dissolved or suspended in water or other suitable solvent at the time of use. good. Injectables are prepared by dissolving the antibody of the invention or a functional fragment thereof in water, but may be dissolved in saline or glucose solution as needed, as a buffer or storage. Agents may be added. Those skilled in the art will appropriately select the type of pharmaceutical additive used in the production of the pharmaceutical product of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical product or the pharmaceutical composition, depending on the form thereof. It is possible.
- an inorganic or organic substance, or a solid or liquid substance can be used, and generally, for example, 0.1% by weight to 99.9% by weight or 1% by weight to the weight of the active ingredient. It can be blended in an amount of 95.0% by weight, or between 1% by weight and 90.0% by weight.
- additives for preparation lactose, glucose, mannitt, dextrin, cyclodextrin, starch, fatty acid, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calciumcarboxymethylcellulose , Ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragant, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Examples thereof include sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white vaseline, fluorocarbon, nonionic surfactant, prop
- the active ingredient may be a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, etc., as required.
- a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, etc.
- reticine, polysorbate 80, polyoxyethylene hydrogenated castor oil or the like may be added to the active ingredient and emulsified in water to obtain an emulsion for injection.
- the dose and frequency of administration of the drug of the present invention are not particularly limited, and may be appropriately selected at the discretion of a doctor or pharmacist according to conditions such as the purpose of treatment of the disease to be treated, the type of disease, the weight and age of the patient. It is possible. In general, when used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (weight of active ingredient) continuously or intermittently to an adult.
- the pharmaceutical product and the like of the present invention may be provided in the form of a kit together with instructions such as an administration method.
- the medicines, etc. contained in the kit are supplied by a container made of a material that maintains the activity of the active ingredient effectively for a long period of time, does not adsorb the agent, etc. inside the container, and does not deteriorate the constituent ingredients. Will be done.
- the sealed glass ampoule may include a buffer encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
- the instruction manual of this kit may be printed on paper or the like, or may be stored and supplied in an electromagnetically readable medium such as a CD-ROM or a DVD-ROM.
- a third embodiment of the present invention is a method for treating a disease, particularly an HSV infection, which comprises administering the medicine of the present invention or the like to a patient.
- treatment means to prevent or alleviate the progression and aggravation of the disease in a patient who has already developed the disease, and the purpose thereof is to prevent or alleviate the progression and aggravation of the disease. It is a treatment.
- the administration route of the drug of the present invention or the like is not particularly limited, but intravitreal administration is preferable, for example.
- a fourth embodiment is a method for diagnosing HSV infection or an auxiliary diagnostic method for HSV infection (hereinafter, including measuring the expression level of human U90926, particularly human U90926 long transcript, in a sample derived from a subject. It is also described as "the diagnostic method and the diagnostic assist method of the present invention").
- the disease to be diagnosed in the present embodiment is an HSV infection, and examples of the pathological condition thereof include the above-mentioned stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis, and meningitis.
- the sample derived from the subject varies depending on the pathological condition of the HSV infection, and examples thereof include oral mucosal tissue, corneal epithelium, anterior aqueous humor, vitreous humor, and cerebrospinal fluid.
- the diagnostic method and diagnostic assist method of the present invention are human U90926 present in a sample derived from a subject and a control sample (for example, a sample derived from a healthy person (a person confirmed not to have a disease to be diagnosed)). Comparing the amounts of RNA, if the amount of human U90926 RNA in the sample from the subject is significantly higher, it is determined that the subject has or may have an infection with HSV. It is something to do.
- the fifth embodiment of the present invention is a candidate substance that inhibits HSV proliferation in HSV-infected cells using the expression level of U90926 (particularly, the expression level of human U90926 long transcript in the case of humans) as an index.
- U90926 particularly, the expression level of human U90926 long transcript in the case of humans
- the present inventors have found that infection of photoreceptor cells with HSV-1 increases the expression of U90926, and inhibition (or suppression) of the expression of U90926 inhibits (or suppresses) the proliferation of HSV-1. .. Based on this finding, substances that inhibit (or suppress) the expression of U90926 in HSV-infected cells, especially HSV-1-infected photoreceptor cells, are candidates for inhibiting (or suppressing) the growth of HSV in infected cells.
- the “candidate substance” is a substance having a function of inhibiting (or suppressing) the proliferation of HSV in cells at least in vitro, and can be used for further analysis in vivo.
- the “candidate substance” is not particularly limited, and may be a nucleic acid containing an antisense oligonucleotide and siRNA, an analog thereof, a protein, or the like.
- a sixth embodiment is a method for predicting visual acuity prognosis or visual acuity prognosis in a patient with acute retinal necrosis, which comprises measuring the expression level of human U90926, in particular, a human U90926 long transcript in a sample from a patient with acute retinal necrosis. It is an auxiliary method of prediction (hereinafter, also referred to as "a method for predicting visual acuity prognosis of the present invention or an auxiliary method for predicting visual acuity prognosis").
- examples of the sample derived from a patient with acute retinal necrosis include anterior aqueous humor and vitreous humor.
- the amount of human U90926 RNA (particularly human U90926 long transcript) present in a sample derived from a patient with acute retinal necrosis is derived from a healthy person.
- the "threshold value" is not particularly limited, but for example, data on the amount of human U90926 RNA in a sample obtained from a plurality of healthy subjects is obtained in advance, and the median value or the average value thereof is set as the threshold value. May be good.
- HSV-1 infection HSV-1 (KOS strain) was grown in monolayer Vero cells. For infection of 661W cells, the cells were seeded on culture plates one day before infection and then infected with HSV-1 with multiplicity of infection (MOI) 5. For infection of human umbilical vein endothelial cells, the cells were seeded on a culture plate one day before the infection, and then HSV-1 was infected with MOI 5.
- MOI multiplicity of infection
- RNA sequence analysis The RNA sequence library was constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the attached instruction manual. Based on standard protocols, 50-base pair single-end read RNA samples were prepared with the Illumina Hiseq 2500 sequencer. RNA expression was quantified based on RNA sequence data.
- the sequence data is HISAT2 for the mouse genome sequence and annotation data (mm10) obtained from the Center for Computational Biology at Johns Hopkins University (ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/). Alignment was performed with default parameters using v2.1.0 (Kim et al., Nat. Methods 12, 357-360 (2015).).
- Herpes simplex virus type 2 varicella / herpesvirus virus, Epstein bar virus, cytomegalovirus, human herpesvirus type 6, human herpesvirus type 7 and human herpesvirus type 8
- multiplex quantitative PCR method was used to quantify the amount of each virus in the vitreous fluid.
- RT-qPCR Reverse transcription-quantitative real-time polymerase chain reaction
- 661W cells HSV-1 infected 661W cells total RNA was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed into cDNA using Prime Script RT master Mix (Takara). .. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
- RNA of human umbilical vein endothelial cells and hyaline fluid HSV-1 infected human umbilical vein endothelial cells was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and Prime Script RT master Mix (Takara). It was reverse transcribed into cDNA using. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
- RNA in the vitreous humor was extracted using NucleoSpin miRNA plasma kit (Macherey-Nagel). The quality of total RNA was evaluated using the RNA6000 PicoKit (Agilent Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). A sample with an RNA integrity number of 7.0 or higher was used in the experiment.
- the cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara).
- qPCR was performed using the Thermal Cycler Dice Real Time System (Takara).
- GAPDH mRNA was used for transcript normalization. Relative RNA levels of AC 110615.1 transcripts in vitreous fluids from patients with acute retinal necrosis and sarcoidosis were calculated based on the amount of AC 110615.1 RNA in vitreous fluids from patients with intraocular malignant lymphoma.
- RNA in the nucleus and cytoplasm of 661W cells was extracted and the expression level of U90926 in the nucleus and cytoplasm was examined by RT-qPCR. Neat1 (v2) and ⁇ -actin were detected as indicators of fractionation.
- siRNA duplexes final concentration 10 nM
- SF solution SF solution
- silencer select-si control Sense sequence: 5'-GUACCUGACUAGUCGCAGA -3'(SEQ ID NO: 11) Antisense sequence: 5'-UCUGCGACUAGUCAGGUAC -3'(SEQ ID NO: 12) silencer select-si U90926 (1) Sense sequence: 5'-CCACUGAGCAGAAGAACUA -3'(SEQ ID NO: 13) Antisense sequence: 5'-UAGUUCUUCUGCUCAGUGG -3'(SEQ ID NO: 14) silencer select-si U90926 (2) Sense sequence: 5'-UGCUCAUACUGAUAAAGAA -3'(SEQ ID NO: 15) Antisense sequence: 5'-UUCUUUAUCAGUAUGACCA -3'(SEQ ID NO: 16)
- U90926 overexpression vector The U90926 overexpression vector was created by cloning the cDNA of U90926 directly under the CMV promoter of the pEGFP-C1 vector using the In-Fusion HD Cloning Kit (Takara). The U90926 overexpression vector was transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit (Lonza) according to the instructions for use. Briefly, the U90926 overexpression vector (final concentration 10 ng / ⁇ L) mixed with SF solution (Lonza) was transfected using program EN-138.
- HSV-1 DNA was quantified by qPCR using primers specific for the ICP-27 gene of HSV-1. The results were standardized for Gapdh.
- HSV-1 plaque assay A serially diluted HSV-1 sample was added to a single layer of Vero cells (12-well plate) and incubated at 37 ° C. for 1 hour to allow the virus to adhere to the cells and invade the interior. The supernatant was removed and the cells were washed twice with PBS (-). To form plaques, cells are placed in medium containing 0.2% ⁇ -globulin (Sigma-Aldrich) in 1% FBS-DMEM (high glucose) containing L-glutamine, phenol red and sodium pyruvate 5 Incubated at 37 ° C for 3 days under the condition of% CO 2 . The HSV-1 titer was calculated by counting the number of plaques and is shown as plaque forming units (PFU) / ml.
- PFU plaque forming units
- the evaluation of survival rate was calculated by the following formula: [(Absorbance of 450 nm of 661 W cells transfected with siRNA 3, 6, 9, 12 and 24 hours after HSV-1 infection)-(Asorbance of 450 nm of medium only)] / [(Non-infection at each time point) Sensation 661 W cell absorbance at 450 nm)-(absorbance of medium only at 450 nm)] Absorbance was measured with a Tecan Infinite F200 Microplate Reader (Tecan).
- the polyvinylidene fluoride membrane was blocked with 3% BSA / Tris buffered saline containing 0.1% Triton X-100 for 30 minutes at room temperature.
- the polyvinylidene fluoride membrane is 1 each of 300-fold diluted anti-mouse ICP-0 antibody (Abcam, ab6513), 5000-fold diluted anti-mouse ICP-4 antibody (ATCC, hb-8183), and 5000-fold diluted anti-mouse GAPDH antibody.
- the mixture was shaken at room temperature for 1 hour. All primary antibodies were diluted with IMMUNO SHOT reagent1 (Cosmobio).
- the polyvinylidene fluoride membrane was then shaken in a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (Dako, P0447) solution at room temperature for 1 hour.
- the secondary antibody was diluted with IMMUNO SHOT reagent2 (Cosmobio).
- each protein was visualized using a horseradish peroxidase substrate, and the chemiluminescent signal was detected using a Luminescent Image Analyzer LAS-4000 mini (Fujifilm).
- HSV-1 infected 661W cells To identify lncRNA whose expression was induced in HSV-1 infected 661W cells, HSV-1 infected 661W cells (cells infected for 2 hours) or non-infected cells. RNA selected by Poly A from infected cells was analyzed by a large-scale sequencing method. U90926 was identified as the second most upregulated lncRNA in infected cells (Table 1).
- U90926 (NR_033483.1: SEQ ID NO: 17) is annotated at the National Center for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/) and is 522 bp long. It consists of five exons (Fig. 1). Analysis of the induction phenomenon of U90926 in 661W cells after HSV-1 infection revealed that the expression level of U90926 RNA gradually increased until 10 hours after HSV-1 infection, and then approximately 100 at 24 hours after HSV-1 infection. It increased dramatically by a factor of 2 (Fig. 2). However, transcription of the U90926 gene was scarcely observed in non-infected cells (Fig. 1). Analysis by cell fractionation revealed that U90926 RNA is localized to the nucleus in HSV-1 infected 661W cells (Fig. 3).
- U90926 RNA in knockdown cells (661W cells) of U90926 by introducing two different types of small interfering RNA (siRNA) is HSV-. At all time points measured after one infection, the expression level of control cells was less than 20% (Fig. 4A). HSV-1 genomic DNA levels and HSV-1 titers were measured to determine the extent of HSV-1 DNA replication and HSV-1 proliferation in U90926 knockdown cells. As a result, the amount of HSV-1 genomic DNA in U90926 knockdown cells was significantly lower after HSV-1 infection than in control cells (3 hours after infection (p ⁇ 0.05), 6 hours after infection and 6 hours after infection.
- the amount of HSV-1 genomic DNA in U90926 overexpressing cells was measured.
- the amount of HSV-1 genomic DNA in U90926 overexpressing cells (Fig. 5A) was significantly increased after HSV-1 infection compared with control cells (Fig. 5B, 3 hours and 6 hours after infection (Fig. 5B).
- p ⁇ 0.01), 9 hours and 12 hours after infection (p ⁇ 0.05)).
- 9 hours after infection the amount of HSV-1 DNA in U90926 overexpressing cells increased about 30-fold compared to the amount of DNA in control cells (Fig. 5).
- ICP-4 RNA 3 hours after infection and 6 hours after infection (p ⁇ 0.01) , 9 hours after infection and 12 hours after infection (p ⁇ 0.01)
- ICP-0 protein and ICP-4 protein in control cells and U90926 knockdown cells were detected by Western blotting.
- ICP-0 protein and ICP-4 protein were detected in control cells 6, 9, and 12 hours after infection, whereas ICP-0 protein and ICP-4 protein were HSV-1 infected in U90926 knockdown cells. It was not detected at any time after 3, 6, 9, or 12 hours (Fig. 8).
- HSV-1 infected human umbilical vein endothelial cells As mentioned above, U90926 dramatically responded to HSV-1 infection of the mouse photoreceptor line 661W cells. Expression increased. Similar to mouse U90926, a marked increase in the expression of human U90926 long transcript was observed in HUVEC cells 8 hours after infection with HSV-1 (about 7.5-fold, p ⁇ 0.0001) (Fig. 12A). On the other hand, no change was observed in the expression level of human U90926 short transcript (Fig. 12B).
- U90926 long transcripts in vitreous fluid from patients with acute retinal necrosis is a condition in which intraocular tissues such as the uveal tract and retina are inflamed, and infectious uveitis is based on the etiology. It is classified into three categories: inflammation, non-infectious uveitis and masked syndrome. In these classifications, acute retinal necrosis is classified as infectious uveitis.
- human U90926 transcripts in vitreous fluid from patients with acute retinal necrosis (infectious uveitis), sarcoidosis (non-infectious uveitis), and intraocular malignant lymphoma (mask syndrome). From qRT-PCR analysis, the expression of human U90926 long transcript was significantly increased in the vitreous humor derived from patients with acute retinal necrosis compared to the vitreous fluid derived from patients with sarcoidosis or intraocular malignant lymphoma.
- the pharmaceutical or pharmaceutical composition according to the present invention is unlikely to induce drug resistance and can be used for long-term treatment. Therefore, the present invention is highly expected to be used in the medical field.
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Abstract
The present invention addresses the problem of providing a substance that inhibits the growth of herpes simplex virus (HSV) in cells and also providing a medicine or a medicinal composition for treating acute retinal necrosis that comprises the aforesaid substance. More specifically, the present invention pertains to a growth inhibitor for herpes simplex virus, said inhibitor comprising a substance that inhibits the expression of U90926, and a medicine or a medicinal composition comprising the inhibitor.
Description
本発明は、単純ヘルペスウイルスの増殖阻害剤および該阻害剤を含む医薬または医薬組成物に関する。より具体的には、視細胞内における単純ヘルペスウイルスの増殖阻害剤および該阻害剤を含む医薬または医薬組成物に関する。
The present invention relates to an inhibitor for the growth of herpes simplex virus and a pharmaceutical or pharmaceutical composition containing the inhibitor. More specifically, it relates to an inhibitor for the growth of herpes simplex virus in photoreceptor cells and a drug or a pharmaceutical composition containing the inhibitor.
近年、次世代シーケンサーを用いた総トランスクリプトーム解析によって、長鎖非コードRNA（long non-coding RNA：lncRNA）と称されるタンパク質をコードしない転写産物が新たに同定された。これらのRNAは、ほ乳類ゲノムの多くの領域から転写されることが知られている。lncRNAは、転写調節、スプライシングおよびエピジェネティクスなどの様々な生命現象に関与することが示唆されている他、感染性因子への応答、ストレス状況への細胞応答において重要な役割を果たすと考えられている。そのため、lncRNAは、種々の疾患に対する新規の治療ターゲットとして注目されている。
In recent years, total transcriptome analysis using a next-generation sequencer has newly identified a transcript that does not encode a protein called long non-coding RNA (lncRNA). These RNAs are known to be transcribed from many regions of the mammalian genome. In addition to being suggested to be involved in various biological phenomena such as transcriptional regulation, splicing and epigenetics, lncRNA is thought to play an important role in response to infectious factors and cellular response to stress situations. ing. Therefore, lncRNA is attracting attention as a new therapeutic target for various diseases.
急性網膜壊死（acute retinal necrosis：ARN）は、ヘルペスウイルス1型（HSV-1）を含むヘルペスウイルス科に属するウイルスによって引き起こされるウイルス感染網膜炎の類型である（非特許文献１）。ステロイド剤、抗ウイルス剤、抗血栓剤がARNの治療に使用されるが（非特許文献２）、視力予後は非常に悪く、それは主としてウイルス感染によって引き起こされる視細胞死によると考えられる（非特許文献３）。3つのクラスの抗ウイルス剤（非環式グアノシンアナログ、非環式ヌクレオチドアナログおよびピロリン酸アナログ）がARN治療に対して承認されており、これら全ての剤はウイルスDNAポリメラーゼが標的である（非特許文献４）。しかしながら、これらの抗ウイルス剤による長期治療は、薬剤耐性を誘導するため、新たなARNに対する抗ウイルス薬が求められている。
Acute retinal necrosis (ARN) is a type of virus-infected retinitis caused by a virus belonging to the herpesvirus family, including herpesvirus type 1 (HSV-1) (Non-Patent Document 1). Although steroids, antiviral agents, and antithrombotic agents are used for the treatment of ARN (Non-Patent Document 2), the prognosis of visual acuity is very poor, which is considered to be mainly due to photoreceptor cell death caused by viral infection (Non-Patent Document 2). Document 3). Three classes of antiviral agents (acyclic guanosine analogs, acyclic nucleotide analogs and pyrophosphate analogs) have been approved for ARN treatment, all of which target viral DNA polymerases (non-patented). Document 4). However, long-term treatment with these antiviral agents induces drug resistance, so that new antiviral agents against ARN are required.
上記事情に鑑み、本発明は、細胞内での単純ヘルペスウイルスの増殖を阻害する物質の提供、および当該物質を含む急性網膜壊死の治療のための医薬または医薬組成物の提供を課題とする。
また、本発明は、急性網膜壊死患者の視力予後の予測方法および視力予後の予測の補助的方法に関するバイオマーカーの提供を課題とする。 In view of the above circumstances, it is an object of the present invention to provide a substance that inhibits the growth of herpes simplex virus in cells, and to provide a drug or a pharmaceutical composition for treating acute retinal necrosis containing the substance.
Another object of the present invention is to provide a biomarker relating to a method for predicting visual acuity prognosis and an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis.
また、本発明は、急性網膜壊死患者の視力予後の予測方法および視力予後の予測の補助的方法に関するバイオマーカーの提供を課題とする。 In view of the above circumstances, it is an object of the present invention to provide a substance that inhibits the growth of herpes simplex virus in cells, and to provide a drug or a pharmaceutical composition for treating acute retinal necrosis containing the substance.
Another object of the present invention is to provide a biomarker relating to a method for predicting visual acuity prognosis and an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis.
本発明者らは、マウス網膜視細胞（661W細胞）に対する単純ヘルペスウイルス1型（herpes simplex virus：HSV-1）の感染下において制御されるlncRNAを探索するためにRNAシーケンシング解析を行った。その結果、lncRNA U90926が、HSV-1の感染によって顕著に上方制御されることが分かった。発明者らは、HSV-1の増殖に対するlncRNA U90926の影響を検討するため、lncRNA U90926をノックダウンしたところ、HSV-1の増殖が顕著に抑制され、宿主視細胞の生存率が増大することが明らかになった。
The present inventors performed RNA sequencing analysis to search for lncRNA regulated under infection with herpes simplex virus type 1 (herpes simplex virus: HSV-1) against mouse retinal photoreceptor cells (661W cells). As a result, it was found that lncRNA U90926 was significantly upregulated by infection with HSV-1. To investigate the effect of lncRNA U90926 on HSV-1 proliferation, the inventors found that knocking down lncRNA U90926 markedly suppressed HSV-1 proliferation and increased host photoreceptor viability. It was revealed.
lncRNA U90926に関する報告は非常に少ない。例えば、マウスマクロファージにおいて、Toll-like receptorの刺激によってU90926の発現が上方制御され、インターロイキン-10の産生を正に制御し、CD80およびCD86の発現を負に制御すること（非特許文献５）、U90926の発現が脂肪前駆細胞分化においてダウンレギュレートされること（非特許文献６）が報告されているに過ぎない。
さらに、本発明者らは、U90926のヒトホモログを同定し、HSV-1が感染したヒト臍帯血静脈内皮細胞において、U90926のヒトホモログRNAの発現が有意に上昇していることを確認した。
以上の知見、すなわち、細胞内でのHSV-1の増殖にU90926の発現が影響を与え得るとの知見は、本発明者らによって初めて明らかにされたものである。 There are very few reports on lncRNA U90926. For example, in mouse macrophages, stimulation of Toll-like receptors upregulates the expression of U90926, positively regulates the production of interleukin-10, and negatively regulates the expression of CD80 and CD86 (Non-Patent Document 5). , U90926 expression is only downregulated in adipose progenitor cell differentiation (Non-Patent Document 6).
Furthermore, we identified the human homologue of U90926 and confirmed that the expression of human homologue RNA of U90926 was significantly increased in HSV-1 infected human umbilical cord blood venous endothelial cells.
The above findings, that is, the findings that the expression of U90926 can affect the proliferation of HSV-1 in cells, have been clarified for the first time by the present inventors.
さらに、本発明者らは、U90926のヒトホモログを同定し、HSV-1が感染したヒト臍帯血静脈内皮細胞において、U90926のヒトホモログRNAの発現が有意に上昇していることを確認した。
以上の知見、すなわち、細胞内でのHSV-1の増殖にU90926の発現が影響を与え得るとの知見は、本発明者らによって初めて明らかにされたものである。 There are very few reports on lncRNA U90926. For example, in mouse macrophages, stimulation of Toll-like receptors upregulates the expression of U90926, positively regulates the production of interleukin-10, and negatively regulates the expression of CD80 and CD86 (Non-Patent Document 5). , U90926 expression is only downregulated in adipose progenitor cell differentiation (Non-Patent Document 6).
Furthermore, we identified the human homologue of U90926 and confirmed that the expression of human homologue RNA of U90926 was significantly increased in HSV-1 infected human umbilical cord blood venous endothelial cells.
The above findings, that is, the findings that the expression of U90926 can affect the proliferation of HSV-1 in cells, have been clarified for the first time by the present inventors.
すなわち、本発明は、以下の（１）～（９）である。
（１）細胞内における単純ヘルペスウイルス（herpes simplex virus：HSV）の増殖阻害剤であって、U90926の発現阻害物質を含有する前記阻害剤。
（２）前記HSVがHSV-1であることを特徴とする上記（１）に記載の阻害剤。
（３）前記細胞がヒト視細胞であることを特徴とする上記（１）または（２）に記載の阻害剤。
（４）前記阻害物質がアンチセンスオリゴヌクレオチドまたはsiRNAであることを特徴とする上記（１）ないし（３）のいずれかに記載の阻害剤。
（５）上記（１）ないし（４）のいずれかに記載の阻害剤を有効成分として含有する医薬または医薬組成物。
（６）急性網膜壊死を治療対象とする上記（５）に記載の医薬または医薬組成物。
（７）HSV感染細胞内におけるU90926の発現量を指標として、当該細胞内におけるHSV増殖を阻害する候補物質をスクリーニングする方法。
（８）被験者由来のサンプル中のヒトU90926の発現量を測定することを含む、HSV感染症の診断方法またはHSV感染症の診断補助方法。
（９）急性網膜壊死患者由来のサンプル中のヒトU90926の発現量を測定することを含む、急性網膜壊死患者の視力予後の予測方法または視力予後の予測の補助的方法。
なお、本明細書において「～」の符号は、その左右の値を含む数値範囲を示す。 That is, the present invention is the following (1) to (9).
(1) The inhibitor which is a growth inhibitor of herpes simplex virus (HSV) in cells and contains an expression inhibitor of U90926.
(2) The inhibitor according to (1) above, wherein the HSV is HSV-1.
(3) The inhibitor according to (1) or (2) above, wherein the cell is a human photoreceptor cell.
(4) The inhibitor according to any one of (1) to (3) above, wherein the inhibitor is an antisense oligonucleotide or siRNA.
(5) A pharmaceutical or pharmaceutical composition containing the inhibitor according to any one of (1) to (4) above as an active ingredient.
(6) The pharmaceutical or pharmaceutical composition according to (5) above, which targets acute retinal necrosis.
(7) A method for screening a candidate substance that inhibits HSV proliferation in HSV-infected cells using the expression level of U90926 as an index.
(8) A method for diagnosing HSV infection or a method for assisting diagnosis of HSV infection, which comprises measuring the expression level of human U90926 in a sample derived from a subject.
(9) A method for predicting visual acuity prognosis or an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis, which comprises measuring the expression level of human U90926 in a sample derived from a patient with acute retinal necrosis.
In the present specification, the reference numeral “～” indicates a numerical range including the values on the left and right thereof.
（１）細胞内における単純ヘルペスウイルス（herpes simplex virus：HSV）の増殖阻害剤であって、U90926の発現阻害物質を含有する前記阻害剤。
（２）前記HSVがHSV-1であることを特徴とする上記（１）に記載の阻害剤。
（３）前記細胞がヒト視細胞であることを特徴とする上記（１）または（２）に記載の阻害剤。
（４）前記阻害物質がアンチセンスオリゴヌクレオチドまたはsiRNAであることを特徴とする上記（１）ないし（３）のいずれかに記載の阻害剤。
（５）上記（１）ないし（４）のいずれかに記載の阻害剤を有効成分として含有する医薬または医薬組成物。
（６）急性網膜壊死を治療対象とする上記（５）に記載の医薬または医薬組成物。
（７）HSV感染細胞内におけるU90926の発現量を指標として、当該細胞内におけるHSV増殖を阻害する候補物質をスクリーニングする方法。
（８）被験者由来のサンプル中のヒトU90926の発現量を測定することを含む、HSV感染症の診断方法またはHSV感染症の診断補助方法。
（９）急性網膜壊死患者由来のサンプル中のヒトU90926の発現量を測定することを含む、急性網膜壊死患者の視力予後の予測方法または視力予後の予測の補助的方法。
なお、本明細書において「～」の符号は、その左右の値を含む数値範囲を示す。 That is, the present invention is the following (1) to (9).
(1) The inhibitor which is a growth inhibitor of herpes simplex virus (HSV) in cells and contains an expression inhibitor of U90926.
(2) The inhibitor according to (1) above, wherein the HSV is HSV-1.
(3) The inhibitor according to (1) or (2) above, wherein the cell is a human photoreceptor cell.
(4) The inhibitor according to any one of (1) to (3) above, wherein the inhibitor is an antisense oligonucleotide or siRNA.
(5) A pharmaceutical or pharmaceutical composition containing the inhibitor according to any one of (1) to (4) above as an active ingredient.
(6) The pharmaceutical or pharmaceutical composition according to (5) above, which targets acute retinal necrosis.
(7) A method for screening a candidate substance that inhibits HSV proliferation in HSV-infected cells using the expression level of U90926 as an index.
(8) A method for diagnosing HSV infection or a method for assisting diagnosis of HSV infection, which comprises measuring the expression level of human U90926 in a sample derived from a subject.
(9) A method for predicting visual acuity prognosis or an auxiliary method for predicting visual acuity prognosis in patients with acute retinal necrosis, which comprises measuring the expression level of human U90926 in a sample derived from a patient with acute retinal necrosis.
In the present specification, the reference numeral “～” indicates a numerical range including the values on the left and right thereof.
本発明により、単純ヘルペスウイルス、特に、HSV-1の細胞（特に、視細胞）内での増殖の抑制または阻害が可能となる。従って、本発明により難治性疾患であるARNの治療方法および治療剤の開発が可能となる。
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress or inhibit the growth of herpes simplex virus, particularly HSV-1 in cells (particularly photoreceptor cells). Therefore, the present invention makes it possible to develop a therapeutic method and a therapeutic agent for ARN, which is an intractable disease.
本発明の医薬は、薬剤耐性を惹起する可能性が低く、ARNの長期治療を可能とする効果を発揮し得る。
The drug of the present invention is unlikely to induce drug resistance and can exert an effect that enables long-term treatment of ARN.
本発明は、急性網膜壊死患者の視力予後の予測方法および視力予後の予測の補助的方法を提供する。従って、急性網膜壊死患者の治療方針の決定における重要な判断材料が本発明によって提供され得る。
The present invention provides a method for predicting visual acuity prognosis in patients with acute retinal necrosis and an auxiliary method for predicting visual acuity prognosis. Therefore, the present invention may provide important judgment material in determining the treatment policy of a patient with acute retinal necrosis.
第１の実施形態は、細胞内における単純ヘルペスウイルス（herpes simplex virus：HSV）の増殖阻害剤であって、U90926の発現阻害物質を含有する前記阻害剤（以下「本発明の阻害剤」とも記載する）である。
眼内炎症性疾患であるぶどう膜炎のうち感染性ぶどう膜炎は、HSVの他、サイトメガロウイルス（cytomegalovirus：CMV）や水痘帯状疱疹ウイルス（varicella zoster virus：VZV）を含めたヘルペスウイルス科に属するウイルスが原因となって発症することが多い疾患である。特に、HSVやVZVが網膜に感染すると急性網膜壊死と称される重篤なぶどう膜炎が生じる。急性網膜壊死が発症すると高い確率で失明に至るが、失明を確実に防ぐための治療法は未だ確立されていない。
本発明の阻害剤のターゲットはU90926（特に付言しない限り、U90926遺伝子のことを指す）である。U90926は、マウスで同定された遺伝子で、National Centre for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/)においてアノテーションが付されており、その転写物であるlncRNA（NR_033483.1）は522 bp長である（図１および配列番号１７）。発明者らは、U90926のヒトホモログ（以下「ヒトU90926」とも記載する）として、ヒトゲノムのシンテニー領域にアクセッション番号AC110615.1で表示される遺伝子を同定している。同遺伝子はAC110615.1-201（ヒトU90926 short転写産物）（配列番号１８）および AC110615.1-202（ヒトU90926 long転写産物）（配列番号１９）で表示される転写産物を有している。このうち、マウスU90926と同様に、HSV-1が感染した細胞においてヒトU90926 long転写産物の発現量が上昇することを確認している。
なお、本明細書における「U90926」には、マウスU90926のみならず、他の動物種（例えば、ヒト）のホモログも含まれるものとする。 The first embodiment is an inhibitor of the growth of herpes simplex virus (HSV) in cells, and the inhibitor containing a U90926 expression inhibitor (hereinafter, also referred to as "inhibitor of the present invention"). To do).
Infectious varicella-zoster virus (VZV), which is an intraocular inflammatory disease, is included in the herpesvirus family, including HSV, cytomegalovirus (CMV), and varicella zoster virus (VZV). It is a disease that often develops due to the virus to which it belongs. In particular, when HSV or VZV infects the retina, severe uveitis called acute retinal necrosis occurs. Although there is a high probability that acute retinal necrosis will lead to blindness, no cure has yet been established to ensure that blindness is prevented.
The target of the inhibitor of the present invention is U90926 (referring to the U90926 gene unless otherwise specified). U90926 is a gene identified in mice that is annotated and transcripts of the National Center for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/). lncRNA (NR_033483.1) is 522 bp long (Fig. 1 and SEQ ID NO: 17). As a human homologue of U90926 (hereinafter also referred to as "human U90926"), the inventors have identified a gene displayed in the synteny region of the human genome with accession number AC110615.1. The gene has transcripts represented by AC110615.1-201 (human U90926 short transcript) (SEQ ID NO: 18) and AC110615.1-202 (human U90926 long transcript) (SEQ ID NO: 19). Among these, it has been confirmed that the expression level of human U90926 long transcript is increased in cells infected with HSV-1, similar to mouse U90926.
In addition, "U90926" in the present specification includes not only mouse U90926 but also homologs of other animal species (for example, human).
眼内炎症性疾患であるぶどう膜炎のうち感染性ぶどう膜炎は、HSVの他、サイトメガロウイルス（cytomegalovirus：CMV）や水痘帯状疱疹ウイルス（varicella zoster virus：VZV）を含めたヘルペスウイルス科に属するウイルスが原因となって発症することが多い疾患である。特に、HSVやVZVが網膜に感染すると急性網膜壊死と称される重篤なぶどう膜炎が生じる。急性網膜壊死が発症すると高い確率で失明に至るが、失明を確実に防ぐための治療法は未だ確立されていない。
本発明の阻害剤のターゲットはU90926（特に付言しない限り、U90926遺伝子のことを指す）である。U90926は、マウスで同定された遺伝子で、National Centre for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/)においてアノテーションが付されており、その転写物であるlncRNA（NR_033483.1）は522 bp長である（図１および配列番号１７）。発明者らは、U90926のヒトホモログ（以下「ヒトU90926」とも記載する）として、ヒトゲノムのシンテニー領域にアクセッション番号AC110615.1で表示される遺伝子を同定している。同遺伝子はAC110615.1-201（ヒトU90926 short転写産物）（配列番号１８）および AC110615.1-202（ヒトU90926 long転写産物）（配列番号１９）で表示される転写産物を有している。このうち、マウスU90926と同様に、HSV-1が感染した細胞においてヒトU90926 long転写産物の発現量が上昇することを確認している。
なお、本明細書における「U90926」には、マウスU90926のみならず、他の動物種（例えば、ヒト）のホモログも含まれるものとする。 The first embodiment is an inhibitor of the growth of herpes simplex virus (HSV) in cells, and the inhibitor containing a U90926 expression inhibitor (hereinafter, also referred to as "inhibitor of the present invention"). To do).
Infectious varicella-zoster virus (VZV), which is an intraocular inflammatory disease, is included in the herpesvirus family, including HSV, cytomegalovirus (CMV), and varicella zoster virus (VZV). It is a disease that often develops due to the virus to which it belongs. In particular, when HSV or VZV infects the retina, severe uveitis called acute retinal necrosis occurs. Although there is a high probability that acute retinal necrosis will lead to blindness, no cure has yet been established to ensure that blindness is prevented.
The target of the inhibitor of the present invention is U90926 (referring to the U90926 gene unless otherwise specified). U90926 is a gene identified in mice that is annotated and transcripts of the National Center for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/). lncRNA (NR_033483.1) is 522 bp long (Fig. 1 and SEQ ID NO: 17). As a human homologue of U90926 (hereinafter also referred to as "human U90926"), the inventors have identified a gene displayed in the synteny region of the human genome with accession number AC110615.1. The gene has transcripts represented by AC110615.1-201 (human U90926 short transcript) (SEQ ID NO: 18) and AC110615.1-202 (human U90926 long transcript) (SEQ ID NO: 19). Among these, it has been confirmed that the expression level of human U90926 long transcript is increased in cells infected with HSV-1, similar to mouse U90926.
In addition, "U90926" in the present specification includes not only mouse U90926 but also homologs of other animal species (for example, human).
本発明の全実施形態におけるHSVには、単純ヘルペスウイルス1型（HSV-1）および単純ヘルペスウイルス2型（HSV-2）が含まれる。また、本実施形態で用いられるHSV-1およびHSV-2は、これらに分類されるあらゆる株（例えば、HSV-1に分類されるKOS株、F株、17株、VR3株、HF株、HF10株およびSC16 株など、ならびに、HSV-2に分類される186株、G株および333株など）およびこれらの亜株に由来するものの全てが含まれるが、特に好ましいHSVはHSV-1である。
また、本発明の全実施形態における「細胞」は、HSVが感染する細胞であれば特に限定されず、敢えて例を挙げるとすれば、血管内皮細胞、口腔粘膜上皮細胞、角膜上皮細胞、角膜内皮細胞、網膜神経節細胞、および視細胞などを挙げることができる。 HSV in all embodiments of the present invention includes herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). In addition, HSV-1 and HSV-2 used in this embodiment are all strains classified into these (for example, KOS strain, F strain, 17 strain, VR3 strain, HF strain, HF10 classified into HSV-1). (Strains and SC16 strains, etc., as well as 186 strains, G strains, 333 strains, etc. classified as HSV-2) and all of those derived from these sub-strains are included, but the most preferred HSV is HSV-1.
Further, the "cell" in all the embodiments of the present invention is not particularly limited as long as it is a cell infected with HSV, and for example, vascular endothelial cells, oral mucosal epithelial cells, corneal epithelial cells, and corneal endothelium. Examples include cells, retinal ganglion cells, and photoreceptor cells.
また、本発明の全実施形態における「細胞」は、HSVが感染する細胞であれば特に限定されず、敢えて例を挙げるとすれば、血管内皮細胞、口腔粘膜上皮細胞、角膜上皮細胞、角膜内皮細胞、網膜神経節細胞、および視細胞などを挙げることができる。 HSV in all embodiments of the present invention includes herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). In addition, HSV-1 and HSV-2 used in this embodiment are all strains classified into these (for example, KOS strain, F strain, 17 strain, VR3 strain, HF strain, HF10 classified into HSV-1). (Strains and SC16 strains, etc., as well as 186 strains, G strains, 333 strains, etc. classified as HSV-2) and all of those derived from these sub-strains are included, but the most preferred HSV is HSV-1.
Further, the "cell" in all the embodiments of the present invention is not particularly limited as long as it is a cell infected with HSV, and for example, vascular endothelial cells, oral mucosal epithelial cells, corneal epithelial cells, and corneal endothelium. Examples include cells, retinal ganglion cells, and photoreceptor cells.
本発明の阻害剤は、HSVの細胞内における増殖を阻害（または抑制）する作用を有し、本阻害剤非存在下におけるHSVの増殖を、50%以上、好ましくは70%以上、特に好ましくは80%以上阻害することができる。
また、本発明の阻害剤に含まれるU90926の発現阻害物質としては、lncRNAであるU90926の発現を阻害または抑制する物質であれば、特に限定はされず、例えば、アンチセンスオリゴヌクレオチド、siRNAなどを挙げることができる。
ここで、アンチセンスオリゴヌクレオチド、siRNAなどは、核酸のみならず、核酸アナログ、例えば、LNA（locked nucleic acid）、ホスホロチオエート（phosphorothioate：PS）、モルフォリノオリゴ、ボラノホスフェート、2’-O-メチル化RNA（2’-OMe）、2’-O-メトキシエチル化RNA（2’-MOE）などからなるものであってもよい。 The inhibitor of the present invention has an action of inhibiting (or suppressing) the proliferation of HSV in cells, and increases the proliferation of HSV in the absence of the inhibitor by 50% or more, preferably 70% or more, particularly preferably. It can inhibit more than 80%.
The U90926 expression inhibitor contained in the inhibitor of the present invention is not particularly limited as long as it is a substance that inhibits or suppresses the expression of U90926, which is a lncRNA, and examples thereof include antisense oligonucleotides and siRNA. Can be mentioned.
Here, antisense oligonucleotides, siRNAs and the like are not only nucleic acids, but also nucleic acid analogs such as LNA (locked nucleic acid), phosphorothioate (PS), morpholinooligo, borane phosphate, 2'-O-methyl. It may be composed of converted RNA (2'-OMe), 2'-O-methoxyethylated RNA (2'-MOE), or the like.
また、本発明の阻害剤に含まれるU90926の発現阻害物質としては、lncRNAであるU90926の発現を阻害または抑制する物質であれば、特に限定はされず、例えば、アンチセンスオリゴヌクレオチド、siRNAなどを挙げることができる。
ここで、アンチセンスオリゴヌクレオチド、siRNAなどは、核酸のみならず、核酸アナログ、例えば、LNA（locked nucleic acid）、ホスホロチオエート（phosphorothioate：PS）、モルフォリノオリゴ、ボラノホスフェート、2’-O-メチル化RNA（2’-OMe）、2’-O-メトキシエチル化RNA（2’-MOE）などからなるものであってもよい。 The inhibitor of the present invention has an action of inhibiting (or suppressing) the proliferation of HSV in cells, and increases the proliferation of HSV in the absence of the inhibitor by 50% or more, preferably 70% or more, particularly preferably. It can inhibit more than 80%.
The U90926 expression inhibitor contained in the inhibitor of the present invention is not particularly limited as long as it is a substance that inhibits or suppresses the expression of U90926, which is a lncRNA, and examples thereof include antisense oligonucleotides and siRNA. Can be mentioned.
Here, antisense oligonucleotides, siRNAs and the like are not only nucleic acids, but also nucleic acid analogs such as LNA (locked nucleic acid), phosphorothioate (PS), morpholinooligo, borane phosphate, 2'-O-methyl. It may be composed of converted RNA (2'-OMe), 2'-O-methoxyethylated RNA (2'-MOE), or the like.
第２の実施形態は、U90926の発現阻害物質（すなわち、本発明の阻害剤）を有効成分として含む、医薬または医薬組成物（以下「本発明の医薬等」とも記載する。）である。
本発明の医薬等は、HSV（特にHSV-1）が感染することで惹起される疾患、すなわち、HSV感染症の治療薬としての薬効を有する。本発明の医薬等が治療効果を有するHSV感染症の病態は、特に限定されるものではないが、敢えて例示すれば、口内炎、口唇炎、角膜炎、虹彩炎、急性網膜壊死、脳炎および髄膜炎などを挙げることができる。 The second embodiment is a pharmaceutical or pharmaceutical composition (hereinafter, also referred to as "pharmaceutical or the like of the present invention") containing a substance that inhibits the expression of U90926 (that is, the inhibitor of the present invention) as an active ingredient.
The pharmaceutical product of the present invention has a medicinal effect as a therapeutic agent for a disease caused by infection with HSV (particularly HSV-1), that is, an HSV infection. The pathophysiology of HSV infection for which the drug of the present invention has a therapeutic effect is not particularly limited, but if it is dared to give an example, stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis and meningitis You can mention flames.
本発明の医薬等は、HSV（特にHSV-1）が感染することで惹起される疾患、すなわち、HSV感染症の治療薬としての薬効を有する。本発明の医薬等が治療効果を有するHSV感染症の病態は、特に限定されるものではないが、敢えて例示すれば、口内炎、口唇炎、角膜炎、虹彩炎、急性網膜壊死、脳炎および髄膜炎などを挙げることができる。 The second embodiment is a pharmaceutical or pharmaceutical composition (hereinafter, also referred to as "pharmaceutical or the like of the present invention") containing a substance that inhibits the expression of U90926 (that is, the inhibitor of the present invention) as an active ingredient.
The pharmaceutical product of the present invention has a medicinal effect as a therapeutic agent for a disease caused by infection with HSV (particularly HSV-1), that is, an HSV infection. The pathophysiology of HSV infection for which the drug of the present invention has a therapeutic effect is not particularly limited, but if it is dared to give an example, stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis and meningitis You can mention flames.
本発明の医薬は、有効成分であるU90926の発現阻害物質自体を投与するための形態でもよいが、一般的には、これらの有効成分の他、1または2以上の製剤用添加物を含む医薬組成物（以下「本発明の医薬組成物」とも記載する）の形態で投与することが望ましい。また、本発明の実施形態にかかる医薬組成物中には、公知の他の薬剤を併せて配合してもよい。
The drug of the present invention may be in the form of administering the expression-inhibiting substance itself of U90926, which is an active ingredient, but generally, a drug containing one or more pharmaceutical additives in addition to these active ingredients. It is desirable to administer in the form of a composition (hereinafter, also referred to as "the pharmaceutical composition of the present invention"). In addition, other known agents may be added to the pharmaceutical composition according to the embodiment of the present invention.
本発明の医薬または医薬組成物の剤形は、特に限定されず、例えば、液体製剤などを挙げることができ、用時、水または他の適当な溶媒に溶解または懸濁するものであってもよい。注射剤の場合には、本発明の抗体またはその機能的断片を水に溶解させて調製されるが、必要に応じて生理食塩水あるいはブドウ糖溶液に溶解させてもよく、また、緩衝剤や保存剤を添加してもよい。
本発明の医薬等の製造に用いられる製剤用添加物の種類、有効成分に対する製剤用添加物の割合、あるいは、医薬または医薬組成物の製造方法は、その形態に応じて当業者が適宜選択することが可能である。製剤用添加物としては無機または有機物質、あるいは、固体または液体の物質を用いることができ、一般的には、有効成分重量に対して、例えば、0.1重量％～99.9重量％、1重量％～95.0重量％、または1重量％～90.0重量％の間で配合することができる。具体的には、製剤用添加物の例として乳糖、ブドウ糖、マンニット、デキストリン、シクロデキストリン、デンプン、蔗糖、メタケイ酸アルミン酸マグネシウム、合成ケイ酸アルミニウム、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルデンプン、カルボキシメチルセルロースカルシウム、イオン交換樹脂、メチルセルロース、ゼラチン、アラビアゴム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、ポリビニルアルコール、軽質無水ケイ酸、ステアリン酸マグネシウム、タルク、トラガント、ベントナイト、ビーガム、酸化チタン、ソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、グリセリン、脂肪酸グリセリンエステル、精製ラノリン、グリセロゼラチン、ポリソルベート、マクロゴール、植物油、ロウ、流動パラフィン、白色ワセリン、フルオロカーボン、非イオン性界面活性剤、プロピレングルコールまたは水等が挙げられる。 The dosage form of the pharmaceutical or pharmaceutical composition of the present invention is not particularly limited, and examples thereof include liquid preparations, which may be dissolved or suspended in water or other suitable solvent at the time of use. good. Injectables are prepared by dissolving the antibody of the invention or a functional fragment thereof in water, but may be dissolved in saline or glucose solution as needed, as a buffer or storage. Agents may be added.
Those skilled in the art will appropriately select the type of pharmaceutical additive used in the production of the pharmaceutical product of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical product or the pharmaceutical composition, depending on the form thereof. It is possible. As the additive for the pharmaceutical product, an inorganic or organic substance, or a solid or liquid substance can be used, and generally, for example, 0.1% by weight to 99.9% by weight or 1% by weight to the weight of the active ingredient. It can be blended in an amount of 95.0% by weight, or between 1% by weight and 90.0% by weight. Specifically, as examples of additives for preparation, lactose, glucose, mannitt, dextrin, cyclodextrin, starch, fatty acid, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calciumcarboxymethylcellulose , Ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragant, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Examples thereof include sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white vaseline, fluorocarbon, nonionic surfactant, propylene glycol or water.
本発明の医薬等の製造に用いられる製剤用添加物の種類、有効成分に対する製剤用添加物の割合、あるいは、医薬または医薬組成物の製造方法は、その形態に応じて当業者が適宜選択することが可能である。製剤用添加物としては無機または有機物質、あるいは、固体または液体の物質を用いることができ、一般的には、有効成分重量に対して、例えば、0.1重量％～99.9重量％、1重量％～95.0重量％、または1重量％～90.0重量％の間で配合することができる。具体的には、製剤用添加物の例として乳糖、ブドウ糖、マンニット、デキストリン、シクロデキストリン、デンプン、蔗糖、メタケイ酸アルミン酸マグネシウム、合成ケイ酸アルミニウム、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルデンプン、カルボキシメチルセルロースカルシウム、イオン交換樹脂、メチルセルロース、ゼラチン、アラビアゴム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、ポリビニルアルコール、軽質無水ケイ酸、ステアリン酸マグネシウム、タルク、トラガント、ベントナイト、ビーガム、酸化チタン、ソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、グリセリン、脂肪酸グリセリンエステル、精製ラノリン、グリセロゼラチン、ポリソルベート、マクロゴール、植物油、ロウ、流動パラフィン、白色ワセリン、フルオロカーボン、非イオン性界面活性剤、プロピレングルコールまたは水等が挙げられる。 The dosage form of the pharmaceutical or pharmaceutical composition of the present invention is not particularly limited, and examples thereof include liquid preparations, which may be dissolved or suspended in water or other suitable solvent at the time of use. good. Injectables are prepared by dissolving the antibody of the invention or a functional fragment thereof in water, but may be dissolved in saline or glucose solution as needed, as a buffer or storage. Agents may be added.
Those skilled in the art will appropriately select the type of pharmaceutical additive used in the production of the pharmaceutical product of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical product or the pharmaceutical composition, depending on the form thereof. It is possible. As the additive for the pharmaceutical product, an inorganic or organic substance, or a solid or liquid substance can be used, and generally, for example, 0.1% by weight to 99.9% by weight or 1% by weight to the weight of the active ingredient. It can be blended in an amount of 95.0% by weight, or between 1% by weight and 90.0% by weight. Specifically, as examples of additives for preparation, lactose, glucose, mannitt, dextrin, cyclodextrin, starch, fatty acid, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calciumcarboxymethylcellulose , Ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragant, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Examples thereof include sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white vaseline, fluorocarbon, nonionic surfactant, propylene glycol or water.
注射剤を製造するには、有効成分を必要に応じて、塩酸、水酸化ナトリウム、乳糖、乳酸、ナトリウム、リン酸一水素ナトリウムまたはリン酸二水素ナトリウムなどのｐＨ調整剤、塩化ナトリウムまたはブドウ糖などの等張化剤と共に注射用蒸留水に溶解し、無菌濾過してアンプルに充填するか、さらに、マンニトール、デキストリン、シクロデキストリンまたはゼラチンなどを加えて真空凍結乾燥し、用事溶解型の注射剤としてもよい。また、有効成分にレチシン、ポリソルベート80またはポリオキシエチレン硬化ヒマシ油などを加えて水中で乳化させ、注射剤用乳剤とすることもできる。
To produce injectables, the active ingredient may be a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, etc., as required. Dissolve in distilled water for injection together with the tonicity agent, filter sterile and fill in an ampol, or add mannitol, dextrin, cyclodextrin, gelatin, etc. and freeze-dry in vacuum to make a solution-type injection. May be good. Further, reticine, polysorbate 80, polyoxyethylene hydrogenated castor oil or the like may be added to the active ingredient and emulsified in water to obtain an emulsion for injection.
本発明の医薬等の投与量および投与回数は特に限定されず、治療対象疾患の治療の目的、疾患の種類、患者の体重や年齢などの条件に応じて、医師または薬剤師の判断により適宜選択することが可能である。
一般的には、注射剤として用いる場合には、成人に対して1日量0.001～100ｍｇ（有効成分重量）を連続投与または間欠投与することが望ましい。 The dose and frequency of administration of the drug of the present invention are not particularly limited, and may be appropriately selected at the discretion of a doctor or pharmacist according to conditions such as the purpose of treatment of the disease to be treated, the type of disease, the weight and age of the patient. It is possible.
In general, when used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (weight of active ingredient) continuously or intermittently to an adult.
一般的には、注射剤として用いる場合には、成人に対して1日量0.001～100ｍｇ（有効成分重量）を連続投与または間欠投与することが望ましい。 The dose and frequency of administration of the drug of the present invention are not particularly limited, and may be appropriately selected at the discretion of a doctor or pharmacist according to conditions such as the purpose of treatment of the disease to be treated, the type of disease, the weight and age of the patient. It is possible.
In general, when used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (weight of active ingredient) continuously or intermittently to an adult.
本発明の医薬等は、投与方法等の説明書と共にキットの形態で提供してもよい。キット中に含まれる医薬等は、有効成分の活性を長期間有効に持続し、剤等が容器内側に吸着することなく、また、構成成分を変質させることのない材質で製造された容器により供給される。例えば、封着されたガラスアンプルは、窒素ガスのような中性で不反応性を示すガスの存在下で封入されたバッファーなどを含んでもよい。
また、本キットの使用説明は、紙などに印刷されたものであっても、CD-ROMまたはDVD-ROMなどの電磁的に読み取り可能な媒体に保存され、供給されてもよい。 The pharmaceutical product and the like of the present invention may be provided in the form of a kit together with instructions such as an administration method. The medicines, etc. contained in the kit are supplied by a container made of a material that maintains the activity of the active ingredient effectively for a long period of time, does not adsorb the agent, etc. inside the container, and does not deteriorate the constituent ingredients. Will be done. For example, the sealed glass ampoule may include a buffer encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
Further, the instruction manual of this kit may be printed on paper or the like, or may be stored and supplied in an electromagnetically readable medium such as a CD-ROM or a DVD-ROM.
また、本キットの使用説明は、紙などに印刷されたものであっても、CD-ROMまたはDVD-ROMなどの電磁的に読み取り可能な媒体に保存され、供給されてもよい。 The pharmaceutical product and the like of the present invention may be provided in the form of a kit together with instructions such as an administration method. The medicines, etc. contained in the kit are supplied by a container made of a material that maintains the activity of the active ingredient effectively for a long period of time, does not adsorb the agent, etc. inside the container, and does not deteriorate the constituent ingredients. Will be done. For example, the sealed glass ampoule may include a buffer encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
Further, the instruction manual of this kit may be printed on paper or the like, or may be stored and supplied in an electromagnetically readable medium such as a CD-ROM or a DVD-ROM.
本発明の第３の実施形態は、本発明の医薬等を患者に投与することを含む、疾患、特に、HSV感染症の治療方法である。
ここで「治療」とは、すでに疾患を発症した患者において、その病態の進行および悪化を阻止または緩和することを意味し、これによって当該疾患の進行および悪化を阻止または緩和することを目的とする処置のことである。
第３の実施形態の治療方法において、HSV感染症の病態が急性網膜壊死である場合、本発明の医薬等の投与経路は、特に限定はしないが、例えば、硝子体内投与が好ましい。 A third embodiment of the present invention is a method for treating a disease, particularly an HSV infection, which comprises administering the medicine of the present invention or the like to a patient.
The term "treatment" as used herein means to prevent or alleviate the progression and aggravation of the disease in a patient who has already developed the disease, and the purpose thereof is to prevent or alleviate the progression and aggravation of the disease. It is a treatment.
In the treatment method of the third embodiment, when the pathological condition of the HSV infection is acute retinal necrosis, the administration route of the drug of the present invention or the like is not particularly limited, but intravitreal administration is preferable, for example.
ここで「治療」とは、すでに疾患を発症した患者において、その病態の進行および悪化を阻止または緩和することを意味し、これによって当該疾患の進行および悪化を阻止または緩和することを目的とする処置のことである。
第３の実施形態の治療方法において、HSV感染症の病態が急性網膜壊死である場合、本発明の医薬等の投与経路は、特に限定はしないが、例えば、硝子体内投与が好ましい。 A third embodiment of the present invention is a method for treating a disease, particularly an HSV infection, which comprises administering the medicine of the present invention or the like to a patient.
The term "treatment" as used herein means to prevent or alleviate the progression and aggravation of the disease in a patient who has already developed the disease, and the purpose thereof is to prevent or alleviate the progression and aggravation of the disease. It is a treatment.
In the treatment method of the third embodiment, when the pathological condition of the HSV infection is acute retinal necrosis, the administration route of the drug of the present invention or the like is not particularly limited, but intravitreal administration is preferable, for example.
第４の実施形態は、被験者由来のサンプル中のヒトU90926、特に、ヒトU90926 long 転写産物の発現量を測定することを含む、HSV感染症の診断方法またはHSV感染症の補助的診断方法（以下「本発明の診断方法および診断補助方法」とも記載する）である。本実施形態における診断対象疾患は、HSV感染症であり、その病態として、例えば、上述の口内炎、口唇炎、角膜炎、虹彩炎、急性網膜壊死、脳炎および髄膜炎などを挙げることができる。また、被験者由来のサンプルは、HSV感染症の病態によって異なるが、例えば、口腔内粘膜組織、角膜上皮、前房水、硝子体液、髄液などを挙げることができる。
本発明の診断方法および診断補助方法は、被験者由来のサンプルと対照サンプル（例えば、健常者（診断対象疾患に罹患していないことが確認されている者）由来のサンプル）中に存在するヒトU90926 RNAの量を比較し、被験者由来のサンプル中のヒトU90926 RNAの量の方が有意に多ければ、当該被験者はHSVに感染症に罹患している、または罹患している可能性があると判断するものである。 A fourth embodiment is a method for diagnosing HSV infection or an auxiliary diagnostic method for HSV infection (hereinafter, including measuring the expression level of human U90926, particularly human U90926 long transcript, in a sample derived from a subject. It is also described as "the diagnostic method and the diagnostic assist method of the present invention"). The disease to be diagnosed in the present embodiment is an HSV infection, and examples of the pathological condition thereof include the above-mentioned stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis, and meningitis. Further, the sample derived from the subject varies depending on the pathological condition of the HSV infection, and examples thereof include oral mucosal tissue, corneal epithelium, anterior aqueous humor, vitreous humor, and cerebrospinal fluid.
The diagnostic method and diagnostic assist method of the present invention are human U90926 present in a sample derived from a subject and a control sample (for example, a sample derived from a healthy person (a person confirmed not to have a disease to be diagnosed)). Comparing the amounts of RNA, if the amount of human U90926 RNA in the sample from the subject is significantly higher, it is determined that the subject has or may have an infection with HSV. It is something to do.
本発明の診断方法および診断補助方法は、被験者由来のサンプルと対照サンプル（例えば、健常者（診断対象疾患に罹患していないことが確認されている者）由来のサンプル）中に存在するヒトU90926 RNAの量を比較し、被験者由来のサンプル中のヒトU90926 RNAの量の方が有意に多ければ、当該被験者はHSVに感染症に罹患している、または罹患している可能性があると判断するものである。 A fourth embodiment is a method for diagnosing HSV infection or an auxiliary diagnostic method for HSV infection (hereinafter, including measuring the expression level of human U90926, particularly human U90926 long transcript, in a sample derived from a subject. It is also described as "the diagnostic method and the diagnostic assist method of the present invention"). The disease to be diagnosed in the present embodiment is an HSV infection, and examples of the pathological condition thereof include the above-mentioned stomatitis, cheilitis, keratitis, irisitis, acute retinal necrosis, encephalitis, and meningitis. Further, the sample derived from the subject varies depending on the pathological condition of the HSV infection, and examples thereof include oral mucosal tissue, corneal epithelium, anterior aqueous humor, vitreous humor, and cerebrospinal fluid.
The diagnostic method and diagnostic assist method of the present invention are human U90926 present in a sample derived from a subject and a control sample (for example, a sample derived from a healthy person (a person confirmed not to have a disease to be diagnosed)). Comparing the amounts of RNA, if the amount of human U90926 RNA in the sample from the subject is significantly higher, it is determined that the subject has or may have an infection with HSV. It is something to do.
本発明の第５の実施形態は、HSV感染細胞内におけるU90926の発現量（特に、ヒトの場合はヒトU90926 long転写産物の発現量）を指標として、当該細胞内におけるHSV増殖を阻害する候補物質をスクリーニングする方法である。
本発明者らは、視細胞にHSV-1が感染するとU90926の発現が上昇すること、U90926の発現を阻害（または抑制）するとHSV-1の増殖が阻害（または抑制）されることを見出した。この知見に基づくと、HSV感染細胞、特に、HSV-1感染視細胞内におけるU90926の発現を阻害（または抑制）する物質は、感染細胞内におけるHSVの増殖を阻害（または抑制）する候補物質として、その後の解析に使用することができる。ここで「候補物質」とは、少なくともin vitroにおいて、細胞内でのHSVの増殖を阻害（または抑制）する機能を有する物質のことで、in vivoにおけるさらなる解析に使用することができる。また、「候補物質」は、特に限定されるものではなく、アンチセンスオリゴヌクレオチドおよびsiRNAなどを含む核酸およびそのアナログ、あるいはタンパク質などであってもよい。 The fifth embodiment of the present invention is a candidate substance that inhibits HSV proliferation in HSV-infected cells using the expression level of U90926 (particularly, the expression level of human U90926 long transcript in the case of humans) as an index. Is a method of screening.
The present inventors have found that infection of photoreceptor cells with HSV-1 increases the expression of U90926, and inhibition (or suppression) of the expression of U90926 inhibits (or suppresses) the proliferation of HSV-1. .. Based on this finding, substances that inhibit (or suppress) the expression of U90926 in HSV-infected cells, especially HSV-1-infected photoreceptor cells, are candidates for inhibiting (or suppressing) the growth of HSV in infected cells. , Can be used for subsequent analysis. Here, the "candidate substance" is a substance having a function of inhibiting (or suppressing) the proliferation of HSV in cells at least in vitro, and can be used for further analysis in vivo. Further, the "candidate substance" is not particularly limited, and may be a nucleic acid containing an antisense oligonucleotide and siRNA, an analog thereof, a protein, or the like.
本発明者らは、視細胞にHSV-1が感染するとU90926の発現が上昇すること、U90926の発現を阻害（または抑制）するとHSV-1の増殖が阻害（または抑制）されることを見出した。この知見に基づくと、HSV感染細胞、特に、HSV-1感染視細胞内におけるU90926の発現を阻害（または抑制）する物質は、感染細胞内におけるHSVの増殖を阻害（または抑制）する候補物質として、その後の解析に使用することができる。ここで「候補物質」とは、少なくともin vitroにおいて、細胞内でのHSVの増殖を阻害（または抑制）する機能を有する物質のことで、in vivoにおけるさらなる解析に使用することができる。また、「候補物質」は、特に限定されるものではなく、アンチセンスオリゴヌクレオチドおよびsiRNAなどを含む核酸およびそのアナログ、あるいはタンパク質などであってもよい。 The fifth embodiment of the present invention is a candidate substance that inhibits HSV proliferation in HSV-infected cells using the expression level of U90926 (particularly, the expression level of human U90926 long transcript in the case of humans) as an index. Is a method of screening.
The present inventors have found that infection of photoreceptor cells with HSV-1 increases the expression of U90926, and inhibition (or suppression) of the expression of U90926 inhibits (or suppresses) the proliferation of HSV-1. .. Based on this finding, substances that inhibit (or suppress) the expression of U90926 in HSV-infected cells, especially HSV-1-infected photoreceptor cells, are candidates for inhibiting (or suppressing) the growth of HSV in infected cells. , Can be used for subsequent analysis. Here, the "candidate substance" is a substance having a function of inhibiting (or suppressing) the proliferation of HSV in cells at least in vitro, and can be used for further analysis in vivo. Further, the "candidate substance" is not particularly limited, and may be a nucleic acid containing an antisense oligonucleotide and siRNA, an analog thereof, a protein, or the like.
第６の実施形態は、急性網膜壊死患者由来のサンプル中のヒトU90926、特に、ヒトU90926 long 転写産物の発現量を測定することを含む、急性網膜壊死患者の視力予後の予測方法または視力予後の予測の補助的方法（以下「本発明の視力予後の予測方法または視力予後の予測の補助的方法」とも記載する）である。
本実施形態において、急性網膜壊死患者由来のサンプルは、例えば、前房水、硝子体液などを挙げることができる。
本発明の視力予後を予測する方法および視力予後の予測の補助的方法は、急性網膜壊死患者由来のサンプル中に存在するヒトU90926 RNA（特に、ヒトU90926 long 転写産物）の量が、健常者由来のサンプルから導きだされる閾値よりも有意に多く、さらにそのRNA量が多ければ多いほど、当該患者の視力予後は不良であると判断するものである。ここで「閾値」として、特に限定はしないが、例えば、複数の健常者から得たサンプル中のヒトU90926 RNAの量に関するデータを予め取得し、その中央値または平均値などを当該閾値として定めてもよい。 A sixth embodiment is a method for predicting visual acuity prognosis or visual acuity prognosis in a patient with acute retinal necrosis, which comprises measuring the expression level of human U90926, in particular, a human U90926 long transcript in a sample from a patient with acute retinal necrosis. It is an auxiliary method of prediction (hereinafter, also referred to as "a method for predicting visual acuity prognosis of the present invention or an auxiliary method for predicting visual acuity prognosis").
In the present embodiment, examples of the sample derived from a patient with acute retinal necrosis include anterior aqueous humor and vitreous humor.
In the method for predicting visual acuity prognosis and the auxiliary method for predicting visual acuity prognosis of the present invention, the amount of human U90926 RNA (particularly human U90926 long transcript) present in a sample derived from a patient with acute retinal necrosis is derived from a healthy person. The greater the amount of RNA, which is significantly higher than the threshold derived from the sample, the poorer the visual acuity prognosis of the patient. Here, the "threshold value" is not particularly limited, but for example, data on the amount of human U90926 RNA in a sample obtained from a plurality of healthy subjects is obtained in advance, and the median value or the average value thereof is set as the threshold value. May be good.
本実施形態において、急性網膜壊死患者由来のサンプルは、例えば、前房水、硝子体液などを挙げることができる。
本発明の視力予後を予測する方法および視力予後の予測の補助的方法は、急性網膜壊死患者由来のサンプル中に存在するヒトU90926 RNA（特に、ヒトU90926 long 転写産物）の量が、健常者由来のサンプルから導きだされる閾値よりも有意に多く、さらにそのRNA量が多ければ多いほど、当該患者の視力予後は不良であると判断するものである。ここで「閾値」として、特に限定はしないが、例えば、複数の健常者から得たサンプル中のヒトU90926 RNAの量に関するデータを予め取得し、その中央値または平均値などを当該閾値として定めてもよい。 A sixth embodiment is a method for predicting visual acuity prognosis or visual acuity prognosis in a patient with acute retinal necrosis, which comprises measuring the expression level of human U90926, in particular, a human U90926 long transcript in a sample from a patient with acute retinal necrosis. It is an auxiliary method of prediction (hereinafter, also referred to as "a method for predicting visual acuity prognosis of the present invention or an auxiliary method for predicting visual acuity prognosis").
In the present embodiment, examples of the sample derived from a patient with acute retinal necrosis include anterior aqueous humor and vitreous humor.
In the method for predicting visual acuity prognosis and the auxiliary method for predicting visual acuity prognosis of the present invention, the amount of human U90926 RNA (particularly human U90926 long transcript) present in a sample derived from a patient with acute retinal necrosis is derived from a healthy person. The greater the amount of RNA, which is significantly higher than the threshold derived from the sample, the poorer the visual acuity prognosis of the patient. Here, the "threshold value" is not particularly limited, but for example, data on the amount of human U90926 RNA in a sample obtained from a plurality of healthy subjects is obtained in advance, and the median value or the average value thereof is set as the threshold value. May be good.
本明細書が英語に翻訳されて、単数形の「a」、「an」、および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものも含むものとする。
以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 If the specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include multiple items.
Hereinafter, the present invention will be described with reference to examples, but the present embodiment is merely an example of an embodiment of the present invention and does not limit the scope of the present invention.
以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 If the specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include multiple items.
Hereinafter, the present invention will be described with reference to examples, but the present embodiment is merely an example of an embodiment of the present invention and does not limit the scope of the present invention.
１．実験方法
１－１．細胞培養
マウス視細胞株661WおよびVero細胞は大阪バイオサイエンス研究所から入手した。661W細胞およびVero細胞は、L-グルタミン、フェノールレッド、HEPESおよびピルビン酸ナトリウムを含む、ダルベッコ改変イーグル培地（Dulbecco’s modified Eagle’s medium：DMEM）／Ham’s F12（661W）または高グルコースDMEM（Vero）（以上、富士フイルム和光純薬）に10% の熱不活性化ウシ胎児血清（GIBCO）を添加した培地中にて、5% CO2の加湿インキュベーターで培養した。
ヒト臍帯静脈内皮細胞は、ATCCから入手した。ヒト臍帯静脈内皮細胞はEGM-2 Bullet Kit（Lonza）に10% の熱不活性化ウシ胎児血清（GIBCO）を添加した培地中にて、5% CO2の加湿インキュベーターで培養した。 1. 1. Experimental method 1-1. Cell culture mouse photoreceptor line 661W and Vero cells were obtained from Osaka Bioscience Institute. 661W cells and Vero cells are Dulbecco's modified Eagle's medium (DMEM) / Ham's F12 (661W) or high glucose DMEM (Vero) containing L-glutamine, phenol red, HEPES and sodium pyruvate (above, It was cultured in a medium containing 10% heat-inactivated fetal bovine serum (GIBCO) in Fujifilm Wako Pure Medicine) in a humidified incubator with 5% CO 2 .
Human umbilical vein endothelial cells were obtained from ATCC. Human umbilical vein endothelial cells were cultured in a medium containing 10% heat-inactivated fetal bovine serum (GIBCO) in EGM-2 Bullet Kit (Lonza) in a humidified incubator with 5% CO 2 .
１－１．細胞培養
マウス視細胞株661WおよびVero細胞は大阪バイオサイエンス研究所から入手した。661W細胞およびVero細胞は、L-グルタミン、フェノールレッド、HEPESおよびピルビン酸ナトリウムを含む、ダルベッコ改変イーグル培地（Dulbecco’s modified Eagle’s medium：DMEM）／Ham’s F12（661W）または高グルコースDMEM（Vero）（以上、富士フイルム和光純薬）に10% の熱不活性化ウシ胎児血清（GIBCO）を添加した培地中にて、5% CO2の加湿インキュベーターで培養した。
ヒト臍帯静脈内皮細胞は、ATCCから入手した。ヒト臍帯静脈内皮細胞はEGM-2 Bullet Kit（Lonza）に10% の熱不活性化ウシ胎児血清（GIBCO）を添加した培地中にて、5% CO2の加湿インキュベーターで培養した。 1. 1. Experimental method 1-1. Cell culture mouse photoreceptor line 661W and Vero cells were obtained from Osaka Bioscience Institute. 661W cells and Vero cells are Dulbecco's modified Eagle's medium (DMEM) / Ham's F12 (661W) or high glucose DMEM (Vero) containing L-glutamine, phenol red, HEPES and sodium pyruvate (above, It was cultured in a medium containing 10% heat-inactivated fetal bovine serum (GIBCO) in Fujifilm Wako Pure Medicine) in a humidified incubator with 5% CO 2 .
Human umbilical vein endothelial cells were obtained from ATCC. Human umbilical vein endothelial cells were cultured in a medium containing 10% heat-inactivated fetal bovine serum (GIBCO) in EGM-2 Bullet Kit (Lonza) in a humidified incubator with 5% CO 2 .
１－２．患者
本研究は、東京大学大学院医学系研究科、東京大学医学部、防衛医科大学校病院の倫理委員会の承認を受けて実施した。全ての患者から書面によるインフォームド・コンセントを得た上で、全ての手順はヘルシンキ宣言の原則に基づいて行われた。患者は、2013年12月から2018年12月の間に、東京大学医学部附属病院および防衛医科大学校病院において急性網膜壊死、サルコイドーシスおよび眼内悪性リンパ腫の診断および／または治療のために経毛様体扁平部硝子体切除術を受けた21人21眼（男性12名、女性9名；平均年齢＝59.0±18.1歳）を対象にした。8名の患者（11眼）は、急性網膜壊死と診断され（男性7名、女性4名；53.5±16.7歳）、5名の患者（5眼）は、サルコイドーシスと診断され（男性2名、女性3名；57.0±20.0歳）、5名の患者（5眼）は、眼内悪性リンパ腫と診断された（男性3名、女性2名；73.2±14.0歳）。急性網膜壊死、サルコイドーシスおよび眼内悪性リンパ腫は、下記の診断基準に従って診断した。 1-2. Patients This study was conducted with the approval of the Institutional Review Board of the Graduate School of Medicine, the University of Tokyo, the Faculty of Medicine, the University of Tokyo, and the National Defense Medical College Hospital. All procedures were carried out in accordance with the principles of the Declaration of Helsinki, with written informed consent from all patients. Patients were ciliary for the diagnosis and / or treatment of acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma at the University of Tokyo Hospital and National Defense Medical College Hospital between December 2013 and December 2018. The subjects were 21 eyes (12 males and 9 females; average age = 59.0 ± 18.1 years) who underwent pars plana vitrectomy. Eight patients (11 eyes) were diagnosed with acute retinal necrosis (7 men, 4 women; 53.5 ± 16.7 years), and 5 patients (5 eyes) were diagnosed with sarcoidosis (2 men, 2 men). Three females; 57.0 ± 20.0 years, and five patients (5 eyes) were diagnosed with intraocular malignant lymphoma (3 males, 2 females; 73.2 ± 14.0 years). Acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma were diagnosed according to the following diagnostic criteria.
本研究は、東京大学大学院医学系研究科、東京大学医学部、防衛医科大学校病院の倫理委員会の承認を受けて実施した。全ての患者から書面によるインフォームド・コンセントを得た上で、全ての手順はヘルシンキ宣言の原則に基づいて行われた。患者は、2013年12月から2018年12月の間に、東京大学医学部附属病院および防衛医科大学校病院において急性網膜壊死、サルコイドーシスおよび眼内悪性リンパ腫の診断および／または治療のために経毛様体扁平部硝子体切除術を受けた21人21眼（男性12名、女性9名；平均年齢＝59.0±18.1歳）を対象にした。8名の患者（11眼）は、急性網膜壊死と診断され（男性7名、女性4名；53.5±16.7歳）、5名の患者（5眼）は、サルコイドーシスと診断され（男性2名、女性3名；57.0±20.0歳）、5名の患者（5眼）は、眼内悪性リンパ腫と診断された（男性3名、女性2名；73.2±14.0歳）。急性網膜壊死、サルコイドーシスおよび眼内悪性リンパ腫は、下記の診断基準に従って診断した。 1-2. Patients This study was conducted with the approval of the Institutional Review Board of the Graduate School of Medicine, the University of Tokyo, the Faculty of Medicine, the University of Tokyo, and the National Defense Medical College Hospital. All procedures were carried out in accordance with the principles of the Declaration of Helsinki, with written informed consent from all patients. Patients were ciliary for the diagnosis and / or treatment of acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma at the University of Tokyo Hospital and National Defense Medical College Hospital between December 2013 and December 2018. The subjects were 21 eyes (12 males and 9 females; average age = 59.0 ± 18.1 years) who underwent pars plana vitrectomy. Eight patients (11 eyes) were diagnosed with acute retinal necrosis (7 men, 4 women; 53.5 ± 16.7 years), and 5 patients (5 eyes) were diagnosed with sarcoidosis (2 men, 2 men). Three females; 57.0 ± 20.0 years, and five patients (5 eyes) were diagnosed with intraocular malignant lymphoma (3 males, 2 females; 73.2 ± 14.0 years). Acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma were diagnosed according to the following diagnostic criteria.
急性網膜壊死の診断は、以前に報告された日本における急性網膜壊死の診断基準（Takaseら, Jpn. J. Ophtalmol. 59, 14-20 (2015) https://doi.org/10.1007/s10384-014-0362-0）に従った。サルコイドーシスの診断は、眼サルコイドーシスの国際ワークショップによって確立された診断基準を使用した（Mochizukiら, Br. J. Ophthalmol. 103, 1418-1422 (2019) https://doi.org/10.1136/bjophthalmol-2018-313356.）。眼内悪性リンパ腫に関しては、以下の4基準のうち少なくとも2つを満たす場合に確定診断とした。（１）ライトギムザ染色またはパパニコロウ染色を行った細胞診標本がパパニコロウ分類でclass III以上（Kaburakiら, Br. J. Haematol. 179, 246-255 (2017) https://doi.org/10.1111/bjh.14848.）、（２）眼内液中のIL10/IL6比>1またはIL10>50 pg/mL（Cassouxら, Invest. Ophthalmol. Vis. Sci. 48, 3253-3259 (2007) https://doi.org/10.1167/iovs.06-0031.：Whitcupら, Arch. Ophthalmol. 115, 1157-1160 (1997)https://doi.org/10.1001/archopht.1997.01100160327010.）、（３）フローサイトメーター解析における免疫グロブリン軽鎖のκ/λ比の強い偏移（Davisら, Am. J. Ophthalmol. 140, 822-829 (2005) https://doi.org/10.1016/j.ajo.2005.05.032.）、（４）免疫グロブリン重鎖遺伝子の再構成の有無（Baehringら, Cancer. 104, 591-597 (2005) https://doi.org/10.1002/cncr.21191.：Sugitaら, Jpn. J. Ophthalmol. 53, 209-214 (2009) https://doi.org/10.1007/s10384-009-0662-y.：Ohtaら, Jpn. J. Ophthalmol. 51, 147-149 (2007). https://doi.org/10.1007/s10384-006-0406-1.）。
The diagnosis of acute retinal necrosis is the previously reported diagnostic criteria for acute retinal necrosis in Japan (Takase et al., Jpn. J. Optalmol. 59, 14-20 (2015) https://doi.org/10.1007/s10384- 014-0362-0) was followed. The diagnosis of sarcoidosis used the diagnostic criteria established by the International Workshop on Ophthalmic Sarcoidosis (Mochizuki et al., Br. J. Ophthalmol. 103, 1418-1422 (2019) https://doi.org/10.1136/bjophthalmol- 2018-313356.). For intraocular malignant lymphoma, a definitive diagnosis was made if at least two of the following four criteria were met. (1) Cytopathological specimens stained with light gimza or Papanicolaou are classified as class III or higher in the Papanicolaou classification (Kaburaki et al., Br. J. Haematol. 179, 246-255 (2017) https://doi.org/10.1111/bjh .14848.), (2) IL10 / IL6 ratio in intraocular fluid> 1 or IL10> 50 pg / mL (Cassoux et al., Invest. Ophthalmol. Vis. Sci. 48, 3253-3259 (2007) https: // doi.org/10.1167/iovs.06-0031 .: Whitcup et al., Arch. Ophthalmol. 115, 1157-1160 (1997) https://doi.org/10.1001/archopht.1997.01100160327010.), (3) Flow cytometer Strong shift of κ / λ ratio of immunoglobulin light chain in analysis (Davis et al., Am. J. Ophthalmol. 140, 822-829 (2005) https://doi.org/10.1016/j.ajo.2005.05.032 .), (4) Presence or absence of rearrangement of immunoglobulin heavy chain gene (Baehring et al., Cancer. 104, 591-597 (2005) https://doi.org/10.1002/cncr.21191 .: Sugita et al., Jpn. J. Ophthalmol. 53, 209-214 (2009) https://doi.org/10.1007/s10384-009-0662-y .: Ohta et al., Jpn. //doi.org/10.1007/s10384-006-0406-1.).
１－３．HSV-1の感染
HSV-1（KOS株）は単層のVero細胞中で増殖させた。
661W細胞への感染ついては、感染の1日前に培養プレートに播種し、その後、multiplicity of infection （MOI）5でHSV-1を感染させた。
また、ヒト臍帯静脈内皮細胞への感染についても、感染の1日前に培養プレートに播種し、その後、MOI 5でHSV-1を感染させた。 1-3. HSV-1 infection HSV-1 (KOS strain) was grown in monolayer Vero cells.
For infection of 661W cells, the cells were seeded on culture plates one day before infection and then infected with HSV-1 with multiplicity of infection (MOI) 5.
For infection of human umbilical vein endothelial cells, the cells were seeded on a culture plate one day before the infection, and then HSV-1 was infected withMOI 5.
HSV-1（KOS株）は単層のVero細胞中で増殖させた。
661W細胞への感染ついては、感染の1日前に培養プレートに播種し、その後、multiplicity of infection （MOI）5でHSV-1を感染させた。
また、ヒト臍帯静脈内皮細胞への感染についても、感染の1日前に培養プレートに播種し、その後、MOI 5でHSV-1を感染させた。 1-3. HSV-1 infection HSV-1 (KOS strain) was grown in monolayer Vero cells.
For infection of 661W cells, the cells were seeded on culture plates one day before infection and then infected with HSV-1 with multiplicity of infection (MOI) 5.
For infection of human umbilical vein endothelial cells, the cells were seeded on a culture plate one day before the infection, and then HSV-1 was infected with
１－４．RNAシーケンス解析
RNA配列ライブラリは、TruSeq Stranded mRNA Sample Prep Kit（Illumina）を用いて、添付の使用説明書に従って構築した。標準的なプロトコールに基づき、50-ベースペアsingle-endリードRNAサンプルをIllumina Hiseq2500 sequencerで調製した。RNAの発現はRNA配列データに基づいて定量化した。配列データは、Centre for Computational Biology at Johns Hopkins University（ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/）から取得したマウスゲノム配列およびアノテーションデータ（mm10）に対し、HISAT2 v2.1.0（Kimら, Nat. Methods 12, 357-360 (2015).）を用い、デフォルトパラメーターでアライメントを行った。アライメントデータは、デフォルトパラメーターでStringTie v1.3.4d （Perteaら,Nat. Biotechnol. 33, 290-295 (2015)：Perteaら, Nat. Protoc. 11, 1650-1667 (2016).）にインプットし、個々の転写物の発現をFPKM（fragments per kilobase of transcript per million mapped reads）として評価した。 1-4. RNA sequence analysis The RNA sequence library was constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the attached instruction manual. Based on standard protocols, 50-base pair single-end read RNA samples were prepared with the Illumina Hiseq 2500 sequencer. RNA expression was quantified based on RNA sequence data. The sequence data is HISAT2 for the mouse genome sequence and annotation data (mm10) obtained from the Center for Computational Biology at Johns Hopkins University (ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/). Alignment was performed with default parameters using v2.1.0 (Kim et al., Nat.Methods 12, 357-360 (2015).). Alignment data is input to StringTie v1.3.4d (Pertea et al., Nat. Biotechnol. 33, 290-295 (2015): Pertea et al., Nat. Protoc. 11, 1650-1667 (2016).) With default parameters. The expression of individual transcripts was evaluated as FPKM (fragments per kilobase of transcript per million mapped reads).
RNA配列ライブラリは、TruSeq Stranded mRNA Sample Prep Kit（Illumina）を用いて、添付の使用説明書に従って構築した。標準的なプロトコールに基づき、50-ベースペアsingle-endリードRNAサンプルをIllumina Hiseq2500 sequencerで調製した。RNAの発現はRNA配列データに基づいて定量化した。配列データは、Centre for Computational Biology at Johns Hopkins University（ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/）から取得したマウスゲノム配列およびアノテーションデータ（mm10）に対し、HISAT2 v2.1.0（Kimら, Nat. Methods 12, 357-360 (2015).）を用い、デフォルトパラメーターでアライメントを行った。アライメントデータは、デフォルトパラメーターでStringTie v1.3.4d （Perteaら,Nat. Biotechnol. 33, 290-295 (2015)：Perteaら, Nat. Protoc. 11, 1650-1667 (2016).）にインプットし、個々の転写物の発現をFPKM（fragments per kilobase of transcript per million mapped reads）として評価した。 1-4. RNA sequence analysis The RNA sequence library was constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the attached instruction manual. Based on standard protocols, 50-base pair single-end read RNA samples were prepared with the Illumina Hiseq 2500 sequencer. RNA expression was quantified based on RNA sequence data. The sequence data is HISAT2 for the mouse genome sequence and annotation data (mm10) obtained from the Center for Computational Biology at Johns Hopkins University (ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/). Alignment was performed with default parameters using v2.1.0 (Kim et al., Nat.
１－５．硝子体液の採取および硝子体液中のウイルス量の測定
23ゲージまたは25ゲージのトロカールを用いた標準的な3ポート硝子体茎離断術を行い、希釈することなく無菌的に硝子体液を採取した。採取した硝子体液は、すぐに滅菌済みのクライオチューブ中に入れて、-80℃で保存した。既報（Sugitaら, Br J Ophthalmol. 92, 928-32. (2008) https://doi.org/10.1136/bjo.2007.133967）に従い、硝子体液からDNAを抽出した上で、8種のウイルス（HSV-1、単純ヘルペスウイルス2型、水痘・帯状疱疹ウイルス、エプスタイン・バール・ウイルス、サイトメガロウイルス、ヒトヘルペスウイルス6型、ヒトヘルペスウイルス7型およびヒトヘルペスウイルス8型）について、マルチプレックス定量PCR法を用いて、硝子体液中の各ウイルス量を定量した。 1-5. Collection of vitreous fluid and measurement of viral load in vitreous fluid A standard 3-port vitreous stalk transection using a 23-gauge or 25-gauge trocar was performed, and the vitreous fluid was collected aseptically without dilution. The collected vitreous fluid was immediately placed in a sterile cryotube and stored at -80 ° C. According to the previous report (Sugita et al., Br J Ophthalmol. 92, 928-32. (2008) https://doi.org/10.1136/bjo.2007.133967), DNA was extracted from the vitreous fluid, and then eight viruses (HSV) were extracted. -1, Herpessimplex virus type 2, varicella / herpesvirus virus, Epstein bar virus, cytomegalovirus, human herpesvirus type 6, human herpesvirus type 7 and human herpesvirus type 8), multiplex quantitative PCR method Was used to quantify the amount of each virus in the vitreous fluid.
23ゲージまたは25ゲージのトロカールを用いた標準的な3ポート硝子体茎離断術を行い、希釈することなく無菌的に硝子体液を採取した。採取した硝子体液は、すぐに滅菌済みのクライオチューブ中に入れて、-80℃で保存した。既報（Sugitaら, Br J Ophthalmol. 92, 928-32. (2008) https://doi.org/10.1136/bjo.2007.133967）に従い、硝子体液からDNAを抽出した上で、8種のウイルス（HSV-1、単純ヘルペスウイルス2型、水痘・帯状疱疹ウイルス、エプスタイン・バール・ウイルス、サイトメガロウイルス、ヒトヘルペスウイルス6型、ヒトヘルペスウイルス7型およびヒトヘルペスウイルス8型）について、マルチプレックス定量PCR法を用いて、硝子体液中の各ウイルス量を定量した。 1-5. Collection of vitreous fluid and measurement of viral load in vitreous fluid A standard 3-port vitreous stalk transection using a 23-gauge or 25-gauge trocar was performed, and the vitreous fluid was collected aseptically without dilution. The collected vitreous fluid was immediately placed in a sterile cryotube and stored at -80 ° C. According to the previous report (Sugita et al., Br J Ophthalmol. 92, 928-32. (2008) https://doi.org/10.1136/bjo.2007.133967), DNA was extracted from the vitreous fluid, and then eight viruses (HSV) were extracted. -1, Herpes
１－６．RT-qPCR（Reverse transcription-quantitative real-time polymerase chain reaction）
１－６－１．661W細胞
HSV-1感染661W細胞の総RNAは、NucleoSpin RNA kit（Macherey-Nagel）を使用して調製し、Prime Script RT master Mix（Takara）を用いてcDNAに逆転写した。cDNAは、SYBR Premix Ex Taq II（Takara）を用いて、以下に示すプライマーセットで増幅した。qPCRは、Thermal Cycler Dice Real Time System（Takara）を用いて行った。Gapdh mRNAは転写物の標準化のために使用した。
U90926遺伝子
フォワードプライマー：5’- GTGATTCTGATGGCCCTTCT -3’（配列番号１）
リバースプライマー：5’- ATCTTGCCAGGGAATCTTGA -3’（配列番号２）
β-アクチン遺伝子
フォワードプライマー：5’- GTACCCAGGCATTGCTGACA -3’（配列番号３）
リバースプライマー：5’- CGCAGCTCAGTAACAGTCCG -3’（配列番号４）
Neat1v2遺伝子
フォワードプライマー：5’- CTTGCCACACCTTGTCTTGC -3’（配列番号５）
リバースプライマー：5’- TAGCTGGTGCATCCTGTGTG -3’（配列番号６）
ICP-27遺伝子
フォワードプライマー：5’- TCCGACAGCGATCTGGAC -3’（配列番号７）
リバースプライマー：5’- TCCGACGAGGAACACTCC -3’（配列番号８）
GAPDH遺伝子
フォワードプライマー：5’- GGTCCCAGCTTAGGTTCATCA -3’（配列番号９）
リバースプライマー：5’- CCAATACGGCCAAATCCGTT -3’（配列番号１０） 1-6. RT-qPCR (Reverse transcription-quantitative real-time polymerase chain reaction)
1-6-1. 661W cells HSV-1 infected 661W cells total RNA was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed into cDNA using Prime Script RT master Mix (Takara). .. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
U90926 gene <br /> Forward primer: 5'-GT GATTCTGATGGCCCTTCT -3'(SEQ ID NO: 1)
Reverse primer: 5'-ATCTTGCCAGGGAATCTTGA -3'(SEQ ID NO: 2)
β-actin gene <br /> Forward primer: 5'-GTACCCAGGCATTGCTGACA -3'(SEQ ID NO: 3)
Reverse primer: 5'-CGCAGCTCAGTAACAGTCCG -3'(SEQ ID NO: 4)
Neat1v2 gene <br /> Forward primer: 5'-CTTGCCACACCTTGTCTTGC -3'(SEQ ID NO: 5)
Reverse primer: 5'-TAGCTGGTGCATCCTGTGTG -3'(SEQ ID NO: 6)
ICP-27 gene <br /> Forward primer: 5'-TCCGACAGCGATCTGGAC -3'(SEQ ID NO: 7)
Reverse primer: 5'-TCCGAGGAGAACACTCC -3'(SEQ ID NO: 8)
GAPDH gene <br /> Forward primer: 5'-GGTCCCAGCTTAGGTTCATCA -3'(SEQ ID NO: 9)
Reverse primer: 5'-CCAATACGGCCAAATCCGTT -3'(SEQ ID NO: 10)
１－６－１．661W細胞
HSV-1感染661W細胞の総RNAは、NucleoSpin RNA kit（Macherey-Nagel）を使用して調製し、Prime Script RT master Mix（Takara）を用いてcDNAに逆転写した。cDNAは、SYBR Premix Ex Taq II（Takara）を用いて、以下に示すプライマーセットで増幅した。qPCRは、Thermal Cycler Dice Real Time System（Takara）を用いて行った。Gapdh mRNAは転写物の標準化のために使用した。
U90926遺伝子
フォワードプライマー：5’- GTGATTCTGATGGCCCTTCT -3’（配列番号１）
リバースプライマー：5’- ATCTTGCCAGGGAATCTTGA -3’（配列番号２）
β-アクチン遺伝子
フォワードプライマー：5’- GTACCCAGGCATTGCTGACA -3’（配列番号３）
リバースプライマー：5’- CGCAGCTCAGTAACAGTCCG -3’（配列番号４）
Neat1v2遺伝子
フォワードプライマー：5’- CTTGCCACACCTTGTCTTGC -3’（配列番号５）
リバースプライマー：5’- TAGCTGGTGCATCCTGTGTG -3’（配列番号６）
ICP-27遺伝子
フォワードプライマー：5’- TCCGACAGCGATCTGGAC -3’（配列番号７）
リバースプライマー：5’- TCCGACGAGGAACACTCC -3’（配列番号８）
GAPDH遺伝子
フォワードプライマー：5’- GGTCCCAGCTTAGGTTCATCA -3’（配列番号９）
リバースプライマー：5’- CCAATACGGCCAAATCCGTT -3’（配列番号１０） 1-6. RT-qPCR (Reverse transcription-quantitative real-time polymerase chain reaction)
1-6-1. 661W cells HSV-1 infected 661W cells total RNA was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed into cDNA using Prime Script RT master Mix (Takara). .. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
U90926 gene <br /> Forward primer: 5'-GT GATTCTGATGGCCCTTCT -3'(SEQ ID NO: 1)
Reverse primer: 5'-ATCTTGCCAGGGAATCTTGA -3'(SEQ ID NO: 2)
β-actin gene <br /> Forward primer: 5'-GTACCCAGGCATTGCTGACA -3'(SEQ ID NO: 3)
Reverse primer: 5'-CGCAGCTCAGTAACAGTCCG -3'(SEQ ID NO: 4)
Neat1v2 gene <br /> Forward primer: 5'-CTTGCCACACCTTGTCTTGC -3'(SEQ ID NO: 5)
Reverse primer: 5'-TAGCTGGTGCATCCTGTGTG -3'(SEQ ID NO: 6)
ICP-27 gene <br /> Forward primer: 5'-TCCGACAGCGATCTGGAC -3'(SEQ ID NO: 7)
Reverse primer: 5'-TCCGAGGAGAACACTCC -3'(SEQ ID NO: 8)
GAPDH gene <br /> Forward primer: 5'-GGTCCCAGCTTAGGTTCATCA -3'(SEQ ID NO: 9)
Reverse primer: 5'-CCAATACGGCCAAATCCGTT -3'(SEQ ID NO: 10)
１－６－２．ヒト臍帯静脈内皮細胞および硝子体液
HSV-1感染ヒト臍帯静脈内皮細胞（HUVEC細胞）の総RNAは、NucleoSpin RNA kit（Macherey-Nagel）を使用して調製し、Prime Script RT master Mix（Takara）を用いてcDNAに逆転写した。cDNAは、SYBR Premix Ex Taq II（Takara）を用いて、以下に示すプライマーセットで増幅した。qPCRは、Thermal Cycler Dice Real Time System（Takara）を用いて行った。Gapdh mRNAは転写物の標準化のために使用した。 1-6-2. Total RNA of human umbilical vein endothelial cells and hyaline fluid HSV-1 infected human umbilical vein endothelial cells (HUVEC cells) was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and Prime Script RT master Mix (Takara). It was reverse transcribed into cDNA using. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
HSV-1感染ヒト臍帯静脈内皮細胞（HUVEC細胞）の総RNAは、NucleoSpin RNA kit（Macherey-Nagel）を使用して調製し、Prime Script RT master Mix（Takara）を用いてcDNAに逆転写した。cDNAは、SYBR Premix Ex Taq II（Takara）を用いて、以下に示すプライマーセットで増幅した。qPCRは、Thermal Cycler Dice Real Time System（Takara）を用いて行った。Gapdh mRNAは転写物の標準化のために使用した。 1-6-2. Total RNA of human umbilical vein endothelial cells and hyaline fluid HSV-1 infected human umbilical vein endothelial cells (HUVEC cells) was prepared using the NucleoSpin RNA kit (Macherey-Nagel) and Prime Script RT master Mix (Takara). It was reverse transcribed into cDNA using. The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). Gapdh mRNA was used for transcript standardization.
硝子体液中の総RNAは、NucleoSpin miRNA plasma kit（Macherey-Nagel）を使用して抽出した。総RNAの質の評価は、RNA 6000 Pico Kit（Agilent Technologies）を用いて、Agilent 2100 Bioanalyzer（Agilent Technologies）で行った。RNA integrity numberが7.0以上のサンプルを実験に使用した。
Total RNA in the vitreous humor was extracted using NucleoSpin miRNA plasma kit (Macherey-Nagel). The quality of total RNA was evaluated using the RNA6000 PicoKit (Agilent Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). A sample with an RNA integrity number of 7.0 or higher was used in the experiment.
Prime Script RT master Mix（Takara）を用いて、総RNAをcDNAに逆転写した。cDNAは、SYBR Premix Ex Taq II（Takara）を用いて、以下に示すプライマーセットで増幅した。qPCRは、Thermal Cycler Dice Real Time System（Takara）を用いて行った。GAPDH mRNAは、転写物の正規化に使用した。急性網膜壊死およびサルコイドーシス患者由来の硝子体液中のAC 110615.1転写産物の相対的RNA量は、眼内悪性リンパ腫患者由来の硝子体液中のAC 110615.1RNA量に基づいて算出した。
AC-110615.1-201（ヒトU90926 short転写産物）
フォワードプライマー：5’-TGGCTTTGGGCAAGTTATTT-3’（配列番号２０）
リバースプライマー：5’-ACAGCTCTCAGCCCACATCT-3’（ 配列番号２１）
AC-110615.1-202（ヒトU90926 long転写産物）
フォワードプライマー：5’-GGCTGCTATGGTTTGGATGT-3’（配列番号２２）
リバースプライマー：5’-CTCCCATGACCCAAACACTT-3’（ 配列番号２３）
GAPDH mRNA
フォワードプライマー：5’-GGCCTCCAAGGAGTAAGACC-3’（配列番号２４）
リバースプライマー：5’-AGGGGTCTACATGGCAACTG-3’（ 配列番号２５） Total RNA was reverse transcribed into cDNA using Prime Script RT master Mix (Takara). The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). GAPDH mRNA was used for transcript normalization. Relative RNA levels of AC 110615.1 transcripts in vitreous fluids from patients with acute retinal necrosis and sarcoidosis were calculated based on the amount of AC 110615.1 RNA in vitreous fluids from patients with intraocular malignant lymphoma.
AC-110615.1-201 (human U90926 short transcript)
Forward primer: 5'-TGGCTTTGGGCAAGTTATTT-3'(SEQ ID NO: 20)
Reverse primer: 5'-ACAGCTCTCAGCCCACATCT-3'(SEQ ID NO: 21)
AC-110615.1-202 (Human U90926 long transcript)
Forward primer: 5'-GGCTGCTATGGTTTGGATGT-3'(SEQ ID NO: 22)
Reverse primer: 5'-CTCCCATGACCCAAACACTT-3'(SEQ ID NO: 23)
GAPDH mRNA
Forward primer: 5'-GGCCTCCAAGGAGTAAGACC-3'(SEQ ID NO: 24)
Reverse primer: 5'-AGGGGTCTACATGGCAACTG-3'(SEQ ID NO: 25)
AC-110615.1-201（ヒトU90926 short転写産物）
フォワードプライマー：5’-TGGCTTTGGGCAAGTTATTT-3’（配列番号２０）
リバースプライマー：5’-ACAGCTCTCAGCCCACATCT-3’（ 配列番号２１）
AC-110615.1-202（ヒトU90926 long転写産物）
フォワードプライマー：5’-GGCTGCTATGGTTTGGATGT-3’（配列番号２２）
リバースプライマー：5’-CTCCCATGACCCAAACACTT-3’（ 配列番号２３）
GAPDH mRNA
フォワードプライマー：5’-GGCCTCCAAGGAGTAAGACC-3’（配列番号２４）
リバースプライマー：5’-AGGGGTCTACATGGCAACTG-3’（ 配列番号２５） Total RNA was reverse transcribed into cDNA using Prime Script RT master Mix (Takara). The cDNA was amplified with the primer set shown below using SYBR Premix Ex Taq II (Takara). qPCR was performed using the Thermal Cycler Dice Real Time System (Takara). GAPDH mRNA was used for transcript normalization. Relative RNA levels of AC 110615.1 transcripts in vitreous fluids from patients with acute retinal necrosis and sarcoidosis were calculated based on the amount of AC 110615.1 RNA in vitreous fluids from patients with intraocular malignant lymphoma.
AC-110615.1-201 (human U90926 short transcript)
Forward primer: 5'-TGGCTTTGGGCAAGTTATTT-3'(SEQ ID NO: 20)
Reverse primer: 5'-ACAGCTCTCAGCCCACATCT-3'(SEQ ID NO: 21)
AC-110615.1-202 (Human U90926 long transcript)
Forward primer: 5'-GGCTGCTATGGTTTGGATGT-3'(SEQ ID NO: 22)
Reverse primer: 5'-CTCCCATGACCCAAACACTT-3'(SEQ ID NO: 23)
GAPDH mRNA
Forward primer: 5'-GGCCTCCAAGGAGTAAGACC-3'(SEQ ID NO: 24)
Reverse primer: 5'-AGGGGTCTACATGGCAACTG-3'(SEQ ID NO: 25)
１－７．配列アライメント
各配列のアライメントは、Clustal Omega（Sieversら, Mol. Syst. Biol. 7, 539. (2011) https://doi.org/10.1038/msb.2011.75.）を用いて行った。 1-7. Sequence Alignment Alignment of each sequence was performed using Clustal Omega (Sievers et al., Mol. Syst. Biol. 7, 539. (2011) https://doi.org/10.1038/msb.2011.75.).
各配列のアライメントは、Clustal Omega（Sieversら, Mol. Syst. Biol. 7, 539. (2011) https://doi.org/10.1038/msb.2011.75.）を用いて行った。 1-7. Sequence Alignment Alignment of each sequence was performed using Clustal Omega (Sievers et al., Mol. Syst. Biol. 7, 539. (2011) https://doi.org/10.1038/msb.2011.75.).
１－８．細胞分画
核画分および細胞質画分は、PARIS kit（Invitrogen）を用い、使用説明書に従って分離した。661W細胞の核および細胞質のRNAを抽出し、核および細胞質におけるU90926の発現レベルを、RT-qPCR法で調べた。Neat1（v2）およびβ－アクチンは分画の指標として検出した。 1-8. Cell fractions The nuclear fractions and cytoplasmic fractions were separated using the PARIS kit (Invitrogen) according to the instructions for use. RNA in the nucleus and cytoplasm of 661W cells was extracted and the expression level of U90926 in the nucleus and cytoplasm was examined by RT-qPCR. Neat1 (v2) and β-actin were detected as indicators of fractionation.
核画分および細胞質画分は、PARIS kit（Invitrogen）を用い、使用説明書に従って分離した。661W細胞の核および細胞質のRNAを抽出し、核および細胞質におけるU90926の発現レベルを、RT-qPCR法で調べた。Neat1（v2）およびβ－アクチンは分画の指標として検出した。 1-8. Cell fractions The nuclear fractions and cytoplasmic fractions were separated using the PARIS kit (Invitrogen) according to the instructions for use. RNA in the nucleus and cytoplasm of 661W cells was extracted and the expression level of U90926 in the nucleus and cytoplasm was examined by RT-qPCR. Neat1 (v2) and β-actin were detected as indicators of fractionation.
１－９．トランスフェクション
１－９－１．siRNA
全てのsiRNAはAmbio（登録商標）（Thermo Fisher Scientific Inc）から購入した。siRNA配列を以下に示す。siRNAを、4D-Nucleofector X Unit（Lonza）を用い使用説明書に従い、エレクトロポレーション法で661W細胞にトランスフェクションした。簡単に説明すると、SF solution（Lonza）と混合したsiRNA二重鎖（最終濃度10 nM）を、プログラムEN-138を使用してトランスフェクションした。
silencer select-si control
センス配列：5’- GUACCUGACUAGUCGCAGA -3’（配列番号１１）
アンチセンス配列：5’- UCUGCGACUAGUCAGGUAC -3’（配列番号１２）
silencer select-si U90926 (1)
センス配列：5’- CCACUGAGCAGAAGAACUA -3’（配列番号１３）
アンチセンス配列：5’- UAGUUCUUCUGCUCAGUGG -3’（配列番号１４）
silencer select-si U90926 (2)
センス配列：5’- UGCUCAUACUGAUAAAGAA -3’（配列番号１５）
アンチセンス配列：5’- UUCUUUAUCAGUAUGACCA -3’（配列番号１６） 1-9. Transfection 1-9-1. siRNA
All siRNAs were purchased from Ambio® (Thermo Fisher Scientific Inc). The siRNA sequence is shown below. The siRNA was transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit (Lonza) according to the instructions for use. Briefly, siRNA duplexes (final concentration 10 nM) mixed with SF solution (Lonza) were transfected using program EN-138.
silencer select-si control
Sense sequence: 5'-GUACCUGACUAGUCGCAGA -3'(SEQ ID NO: 11)
Antisense sequence: 5'-UCUGCGACUAGUCAGGUAC -3'(SEQ ID NO: 12)
silencer select-si U90926 (1)
Sense sequence: 5'-CCACUGAGCAGAAGAACUA -3'(SEQ ID NO: 13)
Antisense sequence: 5'-UAGUUCUUCUGCUCAGUGG -3'(SEQ ID NO: 14)
silencer select-si U90926 (2)
Sense sequence: 5'-UGCUCAUACUGAUAAAGAA -3'(SEQ ID NO: 15)
Antisense sequence: 5'-UUCUUUAUCAGUAUGACCA -3'(SEQ ID NO: 16)
１－９－１．siRNA
全てのsiRNAはAmbio（登録商標）（Thermo Fisher Scientific Inc）から購入した。siRNA配列を以下に示す。siRNAを、4D-Nucleofector X Unit（Lonza）を用い使用説明書に従い、エレクトロポレーション法で661W細胞にトランスフェクションした。簡単に説明すると、SF solution（Lonza）と混合したsiRNA二重鎖（最終濃度10 nM）を、プログラムEN-138を使用してトランスフェクションした。
silencer select-si control
センス配列：5’- GUACCUGACUAGUCGCAGA -3’（配列番号１１）
アンチセンス配列：5’- UCUGCGACUAGUCAGGUAC -3’（配列番号１２）
silencer select-si U90926 (1)
センス配列：5’- CCACUGAGCAGAAGAACUA -3’（配列番号１３）
アンチセンス配列：5’- UAGUUCUUCUGCUCAGUGG -3’（配列番号１４）
silencer select-si U90926 (2)
センス配列：5’- UGCUCAUACUGAUAAAGAA -3’（配列番号１５）
アンチセンス配列：5’- UUCUUUAUCAGUAUGACCA -3’（配列番号１６） 1-9. Transfection 1-9-1. siRNA
All siRNAs were purchased from Ambio® (Thermo Fisher Scientific Inc). The siRNA sequence is shown below. The siRNA was transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit (Lonza) according to the instructions for use. Briefly, siRNA duplexes (
silencer select-si control
Sense sequence: 5'-GUACCUGACUAGUCGCAGA -3'(SEQ ID NO: 11)
Antisense sequence: 5'-UCUGCGACUAGUCAGGUAC -3'(SEQ ID NO: 12)
silencer select-si U90926 (1)
Sense sequence: 5'-CCACUGAGCAGAAGAACUA -3'(SEQ ID NO: 13)
Antisense sequence: 5'-UAGUUCUUCUGCUCAGUGG -3'(SEQ ID NO: 14)
silencer select-si U90926 (2)
Sense sequence: 5'-UGCUCAUACUGAUAAAGAA -3'(SEQ ID NO: 15)
Antisense sequence: 5'-UUCUUUAUCAGUAUGACCA -3'(SEQ ID NO: 16)
１－９－２．U90926過剰発現ベクター
U90926過剰発現ベクターはU90926のcDNAをpEGFP-C1ベクターのCMVプロモーター直下にIn-Fusion HD Cloning Kit (Takara) を用いてクローニングすることで作成した。U90926過剰発現ベクターは4D-Nucleofector X Unit（Lonza）を用い使用説明書に従い、エレクトロポレーション法で661W細胞にトランスフェクションした。簡単に説明すると、SF solution（Lonza）と混合したU90926過剰発現ベクター（最終濃度10 ng/μL）を、プログラムEN-138を使用してトランスフェクションした。 1-9-2. U90926 overexpression vector The U90926 overexpression vector was created by cloning the cDNA of U90926 directly under the CMV promoter of the pEGFP-C1 vector using the In-Fusion HD Cloning Kit (Takara). The U90926 overexpression vector was transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit (Lonza) according to the instructions for use. Briefly, the U90926 overexpression vector (final concentration 10 ng / μL) mixed with SF solution (Lonza) was transfected using program EN-138.
U90926過剰発現ベクターはU90926のcDNAをpEGFP-C1ベクターのCMVプロモーター直下にIn-Fusion HD Cloning Kit (Takara) を用いてクローニングすることで作成した。U90926過剰発現ベクターは4D-Nucleofector X Unit（Lonza）を用い使用説明書に従い、エレクトロポレーション法で661W細胞にトランスフェクションした。簡単に説明すると、SF solution（Lonza）と混合したU90926過剰発現ベクター（最終濃度10 ng/μL）を、プログラムEN-138を使用してトランスフェクションした。 1-9-2. U90926 overexpression vector The U90926 overexpression vector was created by cloning the cDNA of U90926 directly under the CMV promoter of the pEGFP-C1 vector using the In-Fusion HD Cloning Kit (Takara). The U90926 overexpression vector was transfected into 661W cells by electroporation using a 4D-Nucleofector X Unit (Lonza) according to the instructions for use. Briefly, the U90926 overexpression vector (
１－１０．HSV-1 DNAのqPCR解析
NucleoSpin RNA kitおよびNucleoSpin RNA/DNA buffer set（Macherey-Nagel）を用いて、HSV-1感染661W細胞から総DNAを抽出した。HSV-1 DNAは、HSV-1のICP-27遺伝子に特異的なプライマーを用いてqPCRを行い定量化した。結果は、Gapdhに対して標準化した。 1-10. QPCR analysis of HSV-1 DNA Total DNA was extracted from HSV-1 infected 661W cells using the NucleoSpin RNA kit and NucleoSpin RNA / DNA buffer set (Macherey-Nagel). HSV-1 DNA was quantified by qPCR using primers specific for the ICP-27 gene of HSV-1. The results were standardized for Gapdh.
NucleoSpin RNA kitおよびNucleoSpin RNA/DNA buffer set（Macherey-Nagel）を用いて、HSV-1感染661W細胞から総DNAを抽出した。HSV-1 DNAは、HSV-1のICP-27遺伝子に特異的なプライマーを用いてqPCRを行い定量化した。結果は、Gapdhに対して標準化した。 1-10. QPCR analysis of HSV-1 DNA Total DNA was extracted from HSV-1 infected 661W cells using the NucleoSpin RNA kit and NucleoSpin RNA / DNA buffer set (Macherey-Nagel). HSV-1 DNA was quantified by qPCR using primers specific for the ICP-27 gene of HSV-1. The results were standardized for Gapdh.
１－１１．HSV-1プラークアッセイ
段階希釈したHSV-1サンプルを単層のVero細胞（12ウェルプレート）に添加し、37℃、1時間インキュベートして、ウイルスを細胞に接着させ、内部に侵入させた。上清を除去し、細胞をPBS(-)で２回洗浄した。プラークを形成させるために、細胞をL-グルタミン、フェノールレッドおよびピルビン酸ナトリウムを含む1% FBS-DMEM（高グルコース）に0.2% γ-グロブリン（Sigma-Aldrich）を添加した培地中にて、5%CO2の条件で、37℃、3日間インキュベートした。HSV-1力価はプラーク数をカウントすることにより算出し、プラーク形成ユニット（plaque forming units：PFU）／mlとして示した。 1-11. HSV-1 plaque assay A serially diluted HSV-1 sample was added to a single layer of Vero cells (12-well plate) and incubated at 37 ° C. for 1 hour to allow the virus to adhere to the cells and invade the interior. The supernatant was removed and the cells were washed twice with PBS (-). To form plaques, cells are placed in medium containing 0.2% γ-globulin (Sigma-Aldrich) in 1% FBS-DMEM (high glucose) containing L-glutamine, phenol red andsodium pyruvate 5 Incubated at 37 ° C for 3 days under the condition of% CO 2 . The HSV-1 titer was calculated by counting the number of plaques and is shown as plaque forming units (PFU) / ml.
段階希釈したHSV-1サンプルを単層のVero細胞（12ウェルプレート）に添加し、37℃、1時間インキュベートして、ウイルスを細胞に接着させ、内部に侵入させた。上清を除去し、細胞をPBS(-)で２回洗浄した。プラークを形成させるために、細胞をL-グルタミン、フェノールレッドおよびピルビン酸ナトリウムを含む1% FBS-DMEM（高グルコース）に0.2% γ-グロブリン（Sigma-Aldrich）を添加した培地中にて、5%CO2の条件で、37℃、3日間インキュベートした。HSV-1力価はプラーク数をカウントすることにより算出し、プラーク形成ユニット（plaque forming units：PFU）／mlとして示した。 1-11. HSV-1 plaque assay A serially diluted HSV-1 sample was added to a single layer of Vero cells (12-well plate) and incubated at 37 ° C. for 1 hour to allow the virus to adhere to the cells and invade the interior. The supernatant was removed and the cells were washed twice with PBS (-). To form plaques, cells are placed in medium containing 0.2% γ-globulin (Sigma-Aldrich) in 1% FBS-DMEM (high glucose) containing L-glutamine, phenol red and
１－１２．HSV-1が感染した661W細胞の生存率の測定
96ウェルプレートに播種した661W細胞の生存率は、HSV-1の感染後3、6、9、12および24時間後の時点で、Cell Counting Kit-8（Dojindo）を使用して評価した。生存率の評価は以下の計算式により算出した：
［（HSV-1感染後3、6、9、12および24時間後にsiRNAをトランスフェクトした661W細胞の450 nmの吸光度）－（培地のみの450 nmの吸光度）］／［（各時点における非感染感661W細胞の450 nmの吸光度）－（培地のみの450 nmの吸光度）］
吸光度は、Tecan Infinite F200 Microplate Reader（Tecan）で測定した。 1-12. Measuring the viability of HSV-1 infected 661W cells The viability of 661W cells seeded in 96-well plates is 3, 6, 9, 12 and 24 hours after HSV-1 infection, Cell Counting Kit. Evaluated using -8 (Dojindo). The evaluation of survival rate was calculated by the following formula:
[(Absorbance of 450 nm of 661 W cells transfected with siRNA 3, 6, 9, 12 and 24 hours after HSV-1 infection)-(Asorbance of 450 nm of medium only)] / [(Non-infection at each time point) Sensation 661 W cell absorbance at 450 nm)-(absorbance of medium only at 450 nm)]
Absorbance was measured with a Tecan Infinite F200 Microplate Reader (Tecan).
96ウェルプレートに播種した661W細胞の生存率は、HSV-1の感染後3、6、9、12および24時間後の時点で、Cell Counting Kit-8（Dojindo）を使用して評価した。生存率の評価は以下の計算式により算出した：
［（HSV-1感染後3、6、9、12および24時間後にsiRNAをトランスフェクトした661W細胞の450 nmの吸光度）－（培地のみの450 nmの吸光度）］／［（各時点における非感染感661W細胞の450 nmの吸光度）－（培地のみの450 nmの吸光度）］
吸光度は、Tecan Infinite F200 Microplate Reader（Tecan）で測定した。 1-12. Measuring the viability of HSV-1 infected 661W cells The viability of 661W cells seeded in 96-well plates is 3, 6, 9, 12 and 24 hours after HSV-1 infection, Cell Counting Kit. Evaluated using -8 (Dojindo). The evaluation of survival rate was calculated by the following formula:
[(Absorbance of 450 nm of 661 W cells transfected with
Absorbance was measured with a Tecan Infinite F200 Microplate Reader (Tecan).
１－１３．ウエスタンブロッティング法によるウイルスタンパク質の検出
HSV-1感染させたコントロ－ル細胞、U90926ノックダウン細胞から細胞溶解液（50 mmol/L Tris-HCl [pH 7.4]、1 mmol/L EDTA、1% Triton X-100、1% SDS、5% Glycerol）を用いて総タンパク質を抽出した。タンパク質抽出液は4-15% Mini-PROTECAN TGX Precast Gels（Bio-Rad）を用いて、電気泳動を行った後に、ポリフッ化ビニリデン膜（Millipore）に転写した。ポリフッ化ビニリデン膜は0.1% Triton X-100を含有する3% BSA/Tris緩衝生理食塩水を用いて、室温で30分間ブロッキングを行った。次に、ポリフッ化ビニリデン膜は300倍希釈抗マウスICP-0抗体（Abcam、ab6513）、5000倍希釈抗マウスICP-4抗体（ATCC、hb-8183）、5000倍希釈抗マウスGAPDH抗体の各１次抗体溶液の中で、室温で１時間振盪した。全ての１次抗体はIMMUNO SHOT reagent1（Cosmobio）を用いて希釈した。その後、ポリフッ化ビニリデン膜は5000倍希釈ホースラディッシュ・ペルオキシダーゼ標識ヤギ抗マウス免疫グロブリン抗体（Dako、P0447）溶液の中で、室温で１時間振盪した。２次抗体はIMMUNO SHOT reagent2（Cosmobio）を用いて希釈した。最後に各タンパク質はホースラディッシュ・ペルオキシダーゼ基質を用いて視覚化し、化学発光シグナルはLuminescent Image Analyzer LAS-4000 mini（Fujifilm）を用いて検出した。 1-13. Detection of viral proteins by Western blotting HSV-1 Infected control cells, U90926 Knockdown cells from cell lysates (50 mmol / L Tris-HCl [pH 7.4], 1 mmol / L EDTA, 1% Triton X Total protein was extracted using -100, 1% SDS, 5% Glycerol). The protein extract was electrophoresed using 4-15% Mini-PROTECAN TGX Precast Gels (Bio-Rad) and then transferred to a polyvinylidene fluoride membrane (Millipore). The polyvinylidene fluoride membrane was blocked with 3% BSA / Tris buffered saline containing 0.1% Triton X-100 for 30 minutes at room temperature. Next, the polyvinylidene fluoride membrane is 1 each of 300-fold diluted anti-mouse ICP-0 antibody (Abcam, ab6513), 5000-fold diluted anti-mouse ICP-4 antibody (ATCC, hb-8183), and 5000-fold diluted anti-mouse GAPDH antibody. In the next antibody solution, the mixture was shaken at room temperature for 1 hour. All primary antibodies were diluted with IMMUNO SHOT reagent1 (Cosmobio). The polyvinylidene fluoride membrane was then shaken in a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (Dako, P0447) solution at room temperature for 1 hour. The secondary antibody was diluted with IMMUNO SHOT reagent2 (Cosmobio). Finally, each protein was visualized using a horseradish peroxidase substrate, and the chemiluminescent signal was detected using a Luminescent Image Analyzer LAS-4000 mini (Fujifilm).
HSV-1感染させたコントロ－ル細胞、U90926ノックダウン細胞から細胞溶解液（50 mmol/L Tris-HCl [pH 7.4]、1 mmol/L EDTA、1% Triton X-100、1% SDS、5% Glycerol）を用いて総タンパク質を抽出した。タンパク質抽出液は4-15% Mini-PROTECAN TGX Precast Gels（Bio-Rad）を用いて、電気泳動を行った後に、ポリフッ化ビニリデン膜（Millipore）に転写した。ポリフッ化ビニリデン膜は0.1% Triton X-100を含有する3% BSA/Tris緩衝生理食塩水を用いて、室温で30分間ブロッキングを行った。次に、ポリフッ化ビニリデン膜は300倍希釈抗マウスICP-0抗体（Abcam、ab6513）、5000倍希釈抗マウスICP-4抗体（ATCC、hb-8183）、5000倍希釈抗マウスGAPDH抗体の各１次抗体溶液の中で、室温で１時間振盪した。全ての１次抗体はIMMUNO SHOT reagent1（Cosmobio）を用いて希釈した。その後、ポリフッ化ビニリデン膜は5000倍希釈ホースラディッシュ・ペルオキシダーゼ標識ヤギ抗マウス免疫グロブリン抗体（Dako、P0447）溶液の中で、室温で１時間振盪した。２次抗体はIMMUNO SHOT reagent2（Cosmobio）を用いて希釈した。最後に各タンパク質はホースラディッシュ・ペルオキシダーゼ基質を用いて視覚化し、化学発光シグナルはLuminescent Image Analyzer LAS-4000 mini（Fujifilm）を用いて検出した。 1-13. Detection of viral proteins by Western blotting HSV-1 Infected control cells, U90926 Knockdown cells from cell lysates (50 mmol / L Tris-HCl [pH 7.4], 1 mmol / L EDTA, 1% Triton X Total protein was extracted using -100, 1% SDS, 5% Glycerol). The protein extract was electrophoresed using 4-15% Mini-PROTECAN TGX Precast Gels (Bio-Rad) and then transferred to a polyvinylidene fluoride membrane (Millipore). The polyvinylidene fluoride membrane was blocked with 3% BSA / Tris buffered saline containing 0.1% Triton X-100 for 30 minutes at room temperature. Next, the polyvinylidene fluoride membrane is 1 each of 300-fold diluted anti-mouse ICP-0 antibody (Abcam, ab6513), 5000-fold diluted anti-mouse ICP-4 antibody (ATCC, hb-8183), and 5000-fold diluted anti-mouse GAPDH antibody. In the next antibody solution, the mixture was shaken at room temperature for 1 hour. All primary antibodies were diluted with IMMUNO SHOT reagent1 (Cosmobio). The polyvinylidene fluoride membrane was then shaken in a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (Dako, P0447) solution at room temperature for 1 hour. The secondary antibody was diluted with IMMUNO SHOT reagent2 (Cosmobio). Finally, each protein was visualized using a horseradish peroxidase substrate, and the chemiluminescent signal was detected using a Luminescent Image Analyzer LAS-4000 mini (Fujifilm).
１－１４．視力測定
少数視力は、ランドルト環を用いて測定し、次式を用いてlogMAR視力に変換した：logMAR 視力 = log (1 / 少数視力)。既報（Oshika, Small incision cataract surgery (In Japanese), IGAKU-SHOIN Ltd., Tokyo, 1994.）に従い、指数弁、手動弁および光覚弁のlogMAR 視力は、各々、2.4、2.7および3.0と定義した。logMAR値が高い程、視力は低いことを意味している。 1-14. Visual acuity measurement Minority visual acuity was measured using the Randold ring and converted to logMAR visual acuity using the following equation: logMAR visual acuity = log (1 / minority visual acuity). According to the previous report (Oshika, Small incision cataract surgery (In Japanese), IGAKU-SHOIN Ltd., Tokyo, 1994.), The logMAR visual acuity of the exponential valve, manual valve and photosensitivity valve was defined as 2.4, 2.7 and 3.0, respectively. .. The higher the logMAR value, the lower the visual acuity.
少数視力は、ランドルト環を用いて測定し、次式を用いてlogMAR視力に変換した：logMAR 視力 = log (1 / 少数視力)。既報（Oshika, Small incision cataract surgery (In Japanese), IGAKU-SHOIN Ltd., Tokyo, 1994.）に従い、指数弁、手動弁および光覚弁のlogMAR 視力は、各々、2.4、2.7および3.0と定義した。logMAR値が高い程、視力は低いことを意味している。 1-14. Visual acuity measurement Minority visual acuity was measured using the Randold ring and converted to logMAR visual acuity using the following equation: logMAR visual acuity = log (1 / minority visual acuity). According to the previous report (Oshika, Small incision cataract surgery (In Japanese), IGAKU-SHOIN Ltd., Tokyo, 1994.), The logMAR visual acuity of the exponential valve, manual valve and photosensitivity valve was defined as 2.4, 2.7 and 3.0, respectively. .. The higher the logMAR value, the lower the visual acuity.
１－１５．統計学的解析
データ値は、平均値±標準偏差で示した。2グループ間の連続変数の比較は、スチューデントのt検定で行った。0.05未満のp値を有意差有りとした。スチューデントのt検定はGraphPad Prism software version 7（GraphPad Software、San Diego、CA、USA）を用いて行った。 1-15. Statistical analysis data values are shown as mean ± standard deviation. The comparison of continuous variables between the two groups was done by Student's t-test. A p-value less than 0.05 was considered to have a significant difference. Student's t-test was performed using GraphPad Prism software version 7 (GraphPad Software, San Diego, CA, USA).
データ値は、平均値±標準偏差で示した。2グループ間の連続変数の比較は、スチューデントのt検定で行った。0.05未満のp値を有意差有りとした。スチューデントのt検定はGraphPad Prism software version 7（GraphPad Software、San Diego、CA、USA）を用いて行った。 1-15. Statistical analysis data values are shown as mean ± standard deviation. The comparison of continuous variables between the two groups was done by Student's t-test. A p-value less than 0.05 was considered to have a significant difference. Student's t-test was performed using GraphPad Prism software version 7 (GraphPad Software, San Diego, CA, USA).
２．結果
２－１．HSV-1感染661W細胞において最も発現が上昇したlncRNAの同定
HSV-1感染661W細胞内で発現誘導されたlncRNAを同定するために、HSV-1感染661W細胞（2時間感染させた細胞）または非感染細胞からポリAで選択したRNAを大規模シークエンス法で解析した。U90926は、感染細胞において、2番目に発現が上昇したlncRNAとして同定した（表１）。U90926（NR_033483.1：配列番号１７）は、National Centre for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/)においてアノテーションが付されており、522 bp長で5つのエクソンからなる（図１）。HSV-1感染後の661W細胞におけるU90926の誘導現象を解析したところ、U90926 RNAの発現レベルは、HSV-1感染10時間後まで徐々に増大し、その後、HSV-1感染後24時間ではおよそ100倍にまで劇的に増大した（図２）。しかし、非感染細胞では、U90926遺伝子の転写はほとんど認められなかった（図１）。細胞分画による解析により、HSV-1感染661W細胞において、U90926 RNAは核に局在することが明らかとなった（図３）。
2. 2. Result 2-1. Identification of the most upregulated lncRNA in HSV-1 infected 661W cells To identify lncRNA whose expression was induced in HSV-1 infected 661W cells, HSV-1 infected 661W cells (cells infected for 2 hours) or non-infected cells. RNA selected by Poly A from infected cells was analyzed by a large-scale sequencing method. U90926 was identified as the second most upregulated lncRNA in infected cells (Table 1). U90926 (NR_033483.1: SEQ ID NO: 17) is annotated at the National Center for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/) and is 522 bp long. It consists of five exons (Fig. 1). Analysis of the induction phenomenon of U90926 in 661W cells after HSV-1 infection revealed that the expression level of U90926 RNA gradually increased until 10 hours after HSV-1 infection, and then approximately 100 at 24 hours after HSV-1 infection. It increased dramatically by a factor of 2 (Fig. 2). However, transcription of the U90926 gene was scarcely observed in non-infected cells (Fig. 1). Analysis by cell fractionation revealed that U90926 RNA is localized to the nucleus in HSV-1 infected 661W cells (Fig. 3).
２－１．HSV-1感染661W細胞において最も発現が上昇したlncRNAの同定
HSV-1感染661W細胞内で発現誘導されたlncRNAを同定するために、HSV-1感染661W細胞（2時間感染させた細胞）または非感染細胞からポリAで選択したRNAを大規模シークエンス法で解析した。U90926は、感染細胞において、2番目に発現が上昇したlncRNAとして同定した（表１）。U90926（NR_033483.1：配列番号１７）は、National Centre for Biotechnology Information Reference Sequence database (http://www.ncbi.nlm.nih.gov/RefSeq/)においてアノテーションが付されており、522 bp長で5つのエクソンからなる（図１）。HSV-1感染後の661W細胞におけるU90926の誘導現象を解析したところ、U90926 RNAの発現レベルは、HSV-1感染10時間後まで徐々に増大し、その後、HSV-1感染後24時間ではおよそ100倍にまで劇的に増大した（図２）。しかし、非感染細胞では、U90926遺伝子の転写はほとんど認められなかった（図１）。細胞分画による解析により、HSV-1感染661W細胞において、U90926 RNAは核に局在することが明らかとなった（図３）。
２．HSV-1の複製、増殖に対するU90926の影響
2種類の異なる低分子干渉RNA（small interfering RNA：siRNA）を導入してU90926のノックダウン細胞（661W細胞）における、U90926 RNAの発現レベルは、HSV-1感染後に測定した全ての時点において、コントロール細胞の発現レベルの20%未満であった（図４Ａ）。
U90926ノックダウン細胞におけるHSV-1のDNA複製とHSV-1の増殖の程度を調べるために、HSV-1ゲノムDNA量およびHSV-1力価を測定した。その結果、U90926ノックダウン細胞におけるHSV-1 ゲノムDNA量は、コントロール細胞に比べて、HSV-1感染後、有意に低下していた（感染後3時間（p<0.05）、感染後6時間および9時間（p<0.01）および感染後12時間（p<0.001））。特に、HSV-1感染から12時間後では、U90926ノックダウン細胞におけるHSV-1 ゲノムDNA量はコントロール細胞のDNA量より、約80%低下していた（図４Ｂ）。さらに、U90926ノックダウン細胞のHSV-1力価は、感染後3時間、6時間、9時間および12時間の時点で有意（p<0.0001）に減少していた（図４Ｃ）。 2. 2. Effect of U90926 on HSV-1 replication and proliferation The expression level of U90926 RNA in knockdown cells (661W cells) of U90926 by introducing two different types of small interfering RNA (siRNA) is HSV-. At all time points measured after one infection, the expression level of control cells was less than 20% (Fig. 4A).
HSV-1 genomic DNA levels and HSV-1 titers were measured to determine the extent of HSV-1 DNA replication and HSV-1 proliferation in U90926 knockdown cells. As a result, the amount of HSV-1 genomic DNA in U90926 knockdown cells was significantly lower after HSV-1 infection than in control cells (3 hours after infection (p <0.05), 6 hours after infection and 6 hours after infection. 9 hours (p <0.01) and 12 hours after infection (p <0.001)). In particular, 12 hours after HSV-1 infection, the amount of HSV-1 genomic DNA in U90926 knockdown cells was about 80% lower than that in control cells (Fig. 4B). In addition, the HSV-1 titers of U90926 knockdown cells were significantly (p <0.0001) reduced at 3, 6, 9 and 12 hours post-infection (FIG. 4C).
2種類の異なる低分子干渉RNA（small interfering RNA：siRNA）を導入してU90926のノックダウン細胞（661W細胞）における、U90926 RNAの発現レベルは、HSV-1感染後に測定した全ての時点において、コントロール細胞の発現レベルの20%未満であった（図４Ａ）。
U90926ノックダウン細胞におけるHSV-1のDNA複製とHSV-1の増殖の程度を調べるために、HSV-1ゲノムDNA量およびHSV-1力価を測定した。その結果、U90926ノックダウン細胞におけるHSV-1 ゲノムDNA量は、コントロール細胞に比べて、HSV-1感染後、有意に低下していた（感染後3時間（p<0.05）、感染後6時間および9時間（p<0.01）および感染後12時間（p<0.001））。特に、HSV-1感染から12時間後では、U90926ノックダウン細胞におけるHSV-1 ゲノムDNA量はコントロール細胞のDNA量より、約80%低下していた（図４Ｂ）。さらに、U90926ノックダウン細胞のHSV-1力価は、感染後3時間、6時間、9時間および12時間の時点で有意（p<0.0001）に減少していた（図４Ｃ）。 2. 2. Effect of U90926 on HSV-1 replication and proliferation The expression level of U90926 RNA in knockdown cells (661W cells) of U90926 by introducing two different types of small interfering RNA (siRNA) is HSV-. At all time points measured after one infection, the expression level of control cells was less than 20% (Fig. 4A).
HSV-1 genomic DNA levels and HSV-1 titers were measured to determine the extent of HSV-1 DNA replication and HSV-1 proliferation in U90926 knockdown cells. As a result, the amount of HSV-1 genomic DNA in U90926 knockdown cells was significantly lower after HSV-1 infection than in control cells (3 hours after infection (p <0.05), 6 hours after infection and 6 hours after infection. 9 hours (p <0.01) and 12 hours after infection (p <0.001)). In particular, 12 hours after HSV-1 infection, the amount of HSV-1 genomic DNA in U90926 knockdown cells was about 80% lower than that in control cells (Fig. 4B). In addition, the HSV-1 titers of U90926 knockdown cells were significantly (p <0.0001) reduced at 3, 6, 9 and 12 hours post-infection (FIG. 4C).
次に、U90926過剰発現細胞におけるHSV-1ゲノムDNA量を測定した。その結果、U90926過剰発現細胞（図５Ａ）におけるHSV-1 ゲノムDNA量は、コントロール細胞に比べて、HSV-1感染後、有意に上昇していた（図５Ｂ、感染後3時間および6時間（p<0.01）、感染後9時間および12時間（p<0.05））。特に、感染後9時間では、U90926過剰発現細胞におけるHSV-1 DNA量はコントロール細胞のDNA量と比較して、約30倍上昇していた（図５）。
最後に、HSV-1感染細胞の生存率を調べたところ、HSV-1感染後24時間において、U90926ノックダウン細胞の生存率は、80.2%（siU90926(1)）および82.6%（siU90926(2)）であったのに対し、コントロール細胞の生存率は21.3%であった（図６）。 Next, the amount of HSV-1 genomic DNA in U90926 overexpressing cells was measured. As a result, the amount of HSV-1 genomic DNA in U90926 overexpressing cells (Fig. 5A) was significantly increased after HSV-1 infection compared with control cells (Fig. 5B, 3 hours and 6 hours after infection (Fig. 5B). p <0.01), 9 hours and 12 hours after infection (p <0.05)). In particular, 9 hours after infection, the amount of HSV-1 DNA in U90926 overexpressing cells increased about 30-fold compared to the amount of DNA in control cells (Fig. 5).
Finally, when the viability of HSV-1 infected cells was examined, the viability of U90926 knockdown cells was 80.2% (siU90926 (1)) and 82.6% (siU90926 (2)) 24 hours after HSV-1 infection. ), While the survival rate of control cells was 21.3% (Fig. 6).
最後に、HSV-1感染細胞の生存率を調べたところ、HSV-1感染後24時間において、U90926ノックダウン細胞の生存率は、80.2%（siU90926(1)）および82.6%（siU90926(2)）であったのに対し、コントロール細胞の生存率は21.3%であった（図６）。 Next, the amount of HSV-1 genomic DNA in U90926 overexpressing cells was measured. As a result, the amount of HSV-1 genomic DNA in U90926 overexpressing cells (Fig. 5A) was significantly increased after HSV-1 infection compared with control cells (Fig. 5B, 3 hours and 6 hours after infection (Fig. 5B). p <0.01), 9 hours and 12 hours after infection (p <0.05)). In particular, 9 hours after infection, the amount of HSV-1 DNA in U90926 overexpressing cells increased about 30-fold compared to the amount of DNA in control cells (Fig. 5).
Finally, when the viability of HSV-1 infected cells was examined, the viability of U90926 knockdown cells was 80.2% (siU90926 (1)) and 82.6% (siU90926 (2)) 24 hours after HSV-1 infection. ), While the survival rate of control cells was 21.3% (Fig. 6).
３．HSV-1遺伝子発現に対するU90926の影響
U90926ノックダウン細胞（661W細胞）におけるHSV-1遺伝子の発現を調べるために、ICP-0（HSV-１遺伝子）ならびにICP-4（HSV-1遺伝子）のRNA量を測定した。その結果、U90926ノックダウン細胞におけるICP-0 RNA量ならびにICP-4 RNA量は、コントロール細胞に比べて、HSV-1感染後、有意に低下していた（ICP-0 RNA：感染後3時間（p<0.05）、感染後6時間（p<0.001）、感染後9時間および感染後12時間（p<0.05））（ICP-4 RNA: 感染後3時間および感染後6時間（p＜0.01）、感染後9時間および感染後12時間（p＜0.01））（図７）。さらにコントロール細胞、U90926ノックダウン細胞におけるICP-0タンパク質、ICP-4タンパク質をウエスタンブロッティング法で検出した。その結果、コントロール細胞では感染後6、9、12時間後でICP-0タンパク質ならびにICP-4タンパク質が検出されたが、U90926ノックダウン細胞ではICP-0タンパク質ならびにICP-4タンパク質はHSV-1感染3、6、9、12時間後のいずれの時点でも検出されなかった（図８）。 3. 3. Effect of U90926 on HSV-1 gene expression To investigate the expression of HSV-1 gene in U90926 knockdown cells (661W cells), RNA of ICP-0 (HSV-1 gene) and ICP-4 (HSV-1 gene) The amount was measured. As a result, the amount of ICP-0 RNA and ICP-4 RNA in U90926 knockdown cells were significantly lower after HSV-1 infection than in control cells (ICP-0 RNA: 3 hours after infection (ICP-0 RNA: 3 hours after infection). p <0.05), 6 hours after infection (p <0.001), 9 hours after infection and 12 hours after infection (p <0.05)) (ICP-4 RNA: 3 hours after infection and 6 hours after infection (p <0.01) , 9 hours after infection and 12 hours after infection (p <0.01)) (Fig. 7). Furthermore, ICP-0 protein and ICP-4 protein in control cells and U90926 knockdown cells were detected by Western blotting. As a result, ICP-0 protein and ICP-4 protein were detected in control cells 6, 9, and 12 hours after infection, whereas ICP-0 protein and ICP-4 protein were HSV-1 infected in U90926 knockdown cells. It was not detected at any time after 3, 6, 9, or 12 hours (Fig. 8).
U90926ノックダウン細胞（661W細胞）におけるHSV-1遺伝子の発現を調べるために、ICP-0（HSV-１遺伝子）ならびにICP-4（HSV-1遺伝子）のRNA量を測定した。その結果、U90926ノックダウン細胞におけるICP-0 RNA量ならびにICP-4 RNA量は、コントロール細胞に比べて、HSV-1感染後、有意に低下していた（ICP-0 RNA：感染後3時間（p<0.05）、感染後6時間（p<0.001）、感染後9時間および感染後12時間（p<0.05））（ICP-4 RNA: 感染後3時間および感染後6時間（p＜0.01）、感染後9時間および感染後12時間（p＜0.01））（図７）。さらにコントロール細胞、U90926ノックダウン細胞におけるICP-0タンパク質、ICP-4タンパク質をウエスタンブロッティング法で検出した。その結果、コントロール細胞では感染後6、9、12時間後でICP-0タンパク質ならびにICP-4タンパク質が検出されたが、U90926ノックダウン細胞ではICP-0タンパク質ならびにICP-4タンパク質はHSV-1感染3、6、9、12時間後のいずれの時点でも検出されなかった（図８）。 3. 3. Effect of U90926 on HSV-1 gene expression To investigate the expression of HSV-1 gene in U90926 knockdown cells (661W cells), RNA of ICP-0 (HSV-1 gene) and ICP-4 (HSV-1 gene) The amount was measured. As a result, the amount of ICP-0 RNA and ICP-4 RNA in U90926 knockdown cells were significantly lower after HSV-1 infection than in control cells (ICP-0 RNA: 3 hours after infection (ICP-0 RNA: 3 hours after infection). p <0.05), 6 hours after infection (p <0.001), 9 hours after infection and 12 hours after infection (p <0.05)) (ICP-4 RNA: 3 hours after infection and 6 hours after infection (p <0.01) , 9 hours after infection and 12 hours after infection (p <0.01)) (Fig. 7). Furthermore, ICP-0 protein and ICP-4 protein in control cells and U90926 knockdown cells were detected by Western blotting. As a result, ICP-0 protein and ICP-4 protein were detected in
４．ヒトU90926ホモログ転写産物の同定
Ensembl genome browser（www.ensembl.org）上で、マウスU90926遺伝子は、5番染色体上のUso1とPpef2遺伝子の間に位置することを確認した（図９上）。さらにヒトゲノムにおいて、AC110615.1遺伝子は、4番染色体上のヒトとマウスで進化的に保存されている領域であるUSO1遺伝子とPPEF2遺伝子の間に存在することを確認した（図９中）。また、AC110615.1遺伝子は、AC110615.1-201（592 塩基、6エクソン）（配列番号１８）およびAC110615.1-202（1,955塩基、1エクソン）（配列番号１９）と称される2つの転写産物を持つことを見出した。 4. Identification of Human U90926 Homolog Transcript Product On the Ensembl genome browser (www.ensembl.org), it was confirmed that the mouse U90926 gene is located between the Uso1 and Ppef2 genes on chromosome 5 (Fig. 9, top). Furthermore, in the human genome, it was confirmed that the AC110615.1 gene exists between the USO1 gene and the PPEF2 gene, which are evolutionarily conserved regions in humans and mice on chromosome 4 (in FIG. 9). In addition, the AC110615.1 gene has two transcripts called AC110615.1-201 (592 bases, 6 exons) (SEQ ID NO: 18) and AC110615.1-202 (1,955 bases, 1 exon) (SEQ ID NO: 19). Found to have a product.
Ensembl genome browser（www.ensembl.org）上で、マウスU90926遺伝子は、5番染色体上のUso1とPpef2遺伝子の間に位置することを確認した（図９上）。さらにヒトゲノムにおいて、AC110615.1遺伝子は、4番染色体上のヒトとマウスで進化的に保存されている領域であるUSO1遺伝子とPPEF2遺伝子の間に存在することを確認した（図９中）。また、AC110615.1遺伝子は、AC110615.1-201（592 塩基、6エクソン）（配列番号１８）およびAC110615.1-202（1,955塩基、1エクソン）（配列番号１９）と称される2つの転写産物を持つことを見出した。 4. Identification of Human U90926 Homolog Transcript Product On the Ensembl genome browser (www.ensembl.org), it was confirmed that the mouse U90926 gene is located between the Uso1 and Ppef2 genes on chromosome 5 (Fig. 9, top). Furthermore, in the human genome, it was confirmed that the AC110615.1 gene exists between the USO1 gene and the PPEF2 gene, which are evolutionarily conserved regions in humans and mice on chromosome 4 (in FIG. 9). In addition, the AC110615.1 gene has two transcripts called AC110615.1-201 (592 bases, 6 exons) (SEQ ID NO: 18) and AC110615.1-202 (1,955 bases, 1 exon) (SEQ ID NO: 19). Found to have a product.
各配列のアライメントにより、マウスU90926転写産物とヒトAC110615.1-201転写産物間には、45.3%の類似性が（図１０）、マウスU90926転写産物とヒトAC110615.1-202転写産物間には、38.5%の類似性が（図１１）認められた。そこで、AC110615.1-201およびAC110615.1-202を、各々、ヒトU90926 short転写産物およびヒトU90926 long転写産物と名付けた。
Due to the alignment of each sequence, there was a 45.3% similarity between the mouse U90926 transcript and the human AC110615.1-201 transcript (Fig. 10), and between the mouse U90926 transcript and the human AC110615.1-202 transcript. , 38.5% similarity was observed (Fig. 11). Therefore, AC110615.1-201 and AC110615.1-202 were named human U90926 short transcript and human U90926 long transcript, respectively.
５．HSV-1感染ヒト臍帯静脈内皮細胞（HUVEC細胞）におけるヒトU90926 long 転写産物の発現上昇
上述のとおり、マウス視細胞株である661W細胞に対するHSV-1の感染に応答して、U90926は劇的に発現が上昇した。マウスU90926同様に、HSV-1の感染から8時間後のHUVEC細胞において、ヒトU90926 long転写産物の著しい発現上昇が認められた（約7.5倍、p<0.0001）（図１２Ａ）。他方、ヒトU90926 short転写産物の発現量の変化はみられなかった（図１２Ｂ）。 5. Increased expression of human U90926 long transcripts in HSV-1 infected human umbilical vein endothelial cells (HUVEC cells) As mentioned above, U90926 dramatically responded to HSV-1 infection of the mouse photoreceptor line 661W cells. Expression increased. Similar to mouse U90926, a marked increase in the expression of human U90926 long transcript was observed in HUVEC cells 8 hours after infection with HSV-1 (about 7.5-fold, p <0.0001) (Fig. 12A). On the other hand, no change was observed in the expression level of human U90926 short transcript (Fig. 12B).
上述のとおり、マウス視細胞株である661W細胞に対するHSV-1の感染に応答して、U90926は劇的に発現が上昇した。マウスU90926同様に、HSV-1の感染から8時間後のHUVEC細胞において、ヒトU90926 long転写産物の著しい発現上昇が認められた（約7.5倍、p<0.0001）（図１２Ａ）。他方、ヒトU90926 short転写産物の発現量の変化はみられなかった（図１２Ｂ）。 5. Increased expression of human U90926 long transcripts in HSV-1 infected human umbilical vein endothelial cells (HUVEC cells) As mentioned above, U90926 dramatically responded to HSV-1 infection of the mouse photoreceptor line 661W cells. Expression increased. Similar to mouse U90926, a marked increase in the expression of human U90926 long transcript was observed in HUVEC cells 8 hours after infection with HSV-1 (about 7.5-fold, p <0.0001) (Fig. 12A). On the other hand, no change was observed in the expression level of human U90926 short transcript (Fig. 12B).
６．急性網膜壊死患者由来の硝子体液中におけるヒトU90926 long 転写産物の発現上昇
ぶどう膜炎は、ぶどう膜や網膜などの眼内組織が炎症を起こした状態であり、病因に基づいて、感染性ぶどう膜炎、非感染性ぶどう膜炎および仮面症候群の3つに分類される。これらの分類において、急性網膜壊死は感染性ぶどう膜炎に分類される。そこで、急性網膜壊死（感染性ぶどう膜炎）患者、サルコイドーシス（非感染性ぶどう膜炎）患者および眼内悪性リンパ腫（仮面症候群）患者由来の硝子体液中のヒトU90926転写産物の発現を調べた。qRT-PCR解析から、ヒトU90926 long 転写産物の発現は、サルコイドーシスまたは眼内悪性リンパ腫患者由来の硝子体液中と比較して、急性網膜壊死患者由来の硝子体液中で著しくその発現量が上昇していた（サルコイドーシス患者由来の硝子体液に対して約35倍、p=0.0017：眼内悪性リンパ腫患者由来の硝子体液に対して約38倍、p=0.0017）（図１３Ａ）。一方で、ヒトU90926 short 転写産物の発現は、急性網膜壊死患者、サルコイドーシス患者および眼内悪性リンパ腫患者間で、ほぼ一定であった（図１３Ｂ）。 6. Increased expression of human U90926 long transcripts in vitreous fluid from patients with acute retinal necrosis Uveitis is a condition in which intraocular tissues such as the uveal tract and retina are inflamed, and infectious uveitis is based on the etiology. It is classified into three categories: inflammation, non-infectious uveitis and masked syndrome. In these classifications, acute retinal necrosis is classified as infectious uveitis. Therefore, we investigated the expression of human U90926 transcripts in vitreous fluid from patients with acute retinal necrosis (infectious uveitis), sarcoidosis (non-infectious uveitis), and intraocular malignant lymphoma (mask syndrome). From qRT-PCR analysis, the expression of human U90926 long transcript was significantly increased in the vitreous humor derived from patients with acute retinal necrosis compared to the vitreous fluid derived from patients with sarcoidosis or intraocular malignant lymphoma. (Approximately 35 times compared to vitreous fluid derived from sarcoidosis patients, p = 0.0017: Approximately 38 times compared to vitreous fluid derived from patients with intraocular malignant lymphoma, p = 0.0017) (Fig. 13A). On the other hand, the expression of human U90926 short transcript was almost constant among patients with acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma (Fig. 13B).
ぶどう膜炎は、ぶどう膜や網膜などの眼内組織が炎症を起こした状態であり、病因に基づいて、感染性ぶどう膜炎、非感染性ぶどう膜炎および仮面症候群の3つに分類される。これらの分類において、急性網膜壊死は感染性ぶどう膜炎に分類される。そこで、急性網膜壊死（感染性ぶどう膜炎）患者、サルコイドーシス（非感染性ぶどう膜炎）患者および眼内悪性リンパ腫（仮面症候群）患者由来の硝子体液中のヒトU90926転写産物の発現を調べた。qRT-PCR解析から、ヒトU90926 long 転写産物の発現は、サルコイドーシスまたは眼内悪性リンパ腫患者由来の硝子体液中と比較して、急性網膜壊死患者由来の硝子体液中で著しくその発現量が上昇していた（サルコイドーシス患者由来の硝子体液に対して約35倍、p=0.0017：眼内悪性リンパ腫患者由来の硝子体液に対して約38倍、p=0.0017）（図１３Ａ）。一方で、ヒトU90926 short 転写産物の発現は、急性網膜壊死患者、サルコイドーシス患者および眼内悪性リンパ腫患者間で、ほぼ一定であった（図１３Ｂ）。 6. Increased expression of human U90926 long transcripts in vitreous fluid from patients with acute retinal necrosis Uveitis is a condition in which intraocular tissues such as the uveal tract and retina are inflamed, and infectious uveitis is based on the etiology. It is classified into three categories: inflammation, non-infectious uveitis and masked syndrome. In these classifications, acute retinal necrosis is classified as infectious uveitis. Therefore, we investigated the expression of human U90926 transcripts in vitreous fluid from patients with acute retinal necrosis (infectious uveitis), sarcoidosis (non-infectious uveitis), and intraocular malignant lymphoma (mask syndrome). From qRT-PCR analysis, the expression of human U90926 long transcript was significantly increased in the vitreous humor derived from patients with acute retinal necrosis compared to the vitreous fluid derived from patients with sarcoidosis or intraocular malignant lymphoma. (Approximately 35 times compared to vitreous fluid derived from sarcoidosis patients, p = 0.0017: Approximately 38 times compared to vitreous fluid derived from patients with intraocular malignant lymphoma, p = 0.0017) (Fig. 13A). On the other hand, the expression of human U90926 short transcript was almost constant among patients with acute retinal necrosis, sarcoidosis and intraocular malignant lymphoma (Fig. 13B).
７．急性網膜壊死患者における硝子体液中のヒトU90926 long転写産物の発現量、硝子体液中のウイルス量、最終矯正logMAR視力間の関係性
急性網膜壊死患者における硝子体液中のヒトU90926 long転写産物の発現量、硝子体液中のウイルス量、硝子体液中の最終矯正logMAR視力について各パラメーター間の相関関係を調べた。その結果、硝子体液中のヒトU90926 long転写産物の発現量は、最終矯正logMAR視力と強く相関していた（r = 0.7414、p = 0.0090）（図１４Ａおよび図１５）。これに対して、硝子体液中のウイルス量と最終矯正logMAR視力（p = 0.4333）、硝子体液中のヒトU90926 long転写産物の発現量と硝子体液中のウイルス量との間には相関がみられなかった（p = 0.0785）（図１４Ｂおよび図１４C）。 7. Expression of human U90926 long transcript in vitreous humor in patients with acute retinal necrosis, viral load in vitreous fluid, relationship between final corrected logMAR visual acuity Expression of human U90926 long transcript in vitreous fluid in patients with acute retinal necrosis , The viral load in the vitreous humor, and the final corrected logMAR visual acuity in the vitreous humor were investigated for the correlation between each parameter. As a result, the expression level of human U90926 long transcript in vitreous humor was strongly correlated with the final corrected logMAR visual acuity (r = 0.7414, p = 0.0090) (FIGS. 14A and 15). In contrast, there was a correlation between the viral load in the vitreous humor and the final corrected logMAR visual acuity (p = 0.4333), and the expression level of the human U90926 long transcript in the vitreous humor and the viral load in the vitreous humor. Not (p = 0.0785) (FIGS. 14B and 14C).
急性網膜壊死患者における硝子体液中のヒトU90926 long転写産物の発現量、硝子体液中のウイルス量、硝子体液中の最終矯正logMAR視力について各パラメーター間の相関関係を調べた。その結果、硝子体液中のヒトU90926 long転写産物の発現量は、最終矯正logMAR視力と強く相関していた（r = 0.7414、p = 0.0090）（図１４Ａおよび図１５）。これに対して、硝子体液中のウイルス量と最終矯正logMAR視力（p = 0.4333）、硝子体液中のヒトU90926 long転写産物の発現量と硝子体液中のウイルス量との間には相関がみられなかった（p = 0.0785）（図１４Ｂおよび図１４C）。 7. Expression of human U90926 long transcript in vitreous humor in patients with acute retinal necrosis, viral load in vitreous fluid, relationship between final corrected logMAR visual acuity Expression of human U90926 long transcript in vitreous fluid in patients with acute retinal necrosis , The viral load in the vitreous humor, and the final corrected logMAR visual acuity in the vitreous humor were investigated for the correlation between each parameter. As a result, the expression level of human U90926 long transcript in vitreous humor was strongly correlated with the final corrected logMAR visual acuity (r = 0.7414, p = 0.0090) (FIGS. 14A and 15). In contrast, there was a correlation between the viral load in the vitreous humor and the final corrected logMAR visual acuity (p = 0.4333), and the expression level of the human U90926 long transcript in the vitreous humor and the viral load in the vitreous humor. Not (p = 0.0785) (FIGS. 14B and 14C).
本発明にかかる医薬または医薬組成物は薬剤抵抗性を惹起する可能性が低く長期治療に使用することが可能である。従って、本発明は、医療分野における利用が大いに期待される。
The pharmaceutical or pharmaceutical composition according to the present invention is unlikely to induce drug resistance and can be used for long-term treatment. Therefore, the present invention is highly expected to be used in the medical field.
Claims (9)
- 細胞内における単純ヘルペスウイルス（herpes simplex virus：HSV）の増殖阻害剤であって、U90926の発現阻害物質を含有する前記阻害剤。 The inhibitor that is an inhibitor of the growth of herpes simplex virus (HSV) in cells and contains an inhibitor of the expression of U90926.
- 前記HSVがHSV-1であることを特徴とする請求項１に記載の阻害剤。 The inhibitor according to claim 1, wherein the HSV is HSV-1.
- 前記細胞がヒト視細胞であることを特徴とする請求項１または２に記載の阻害剤。 The inhibitor according to claim 1 or 2, wherein the cell is a human photoreceptor.
- 前記阻害物質がアンチセンスオリゴヌクレオチドまたはsiRNAであることを特徴とする請求項１ないし３のいずれかに記載の阻害剤。 The inhibitor according to any one of claims 1 to 3, wherein the inhibitor is an antisense oligonucleotide or siRNA.
- 請求項１ないし４のいずれかに記載の阻害剤を有効成分として含有する医薬または医薬組成物。 A pharmaceutical or pharmaceutical composition containing the inhibitor according to any one of claims 1 to 4 as an active ingredient.
- 急性網膜壊死を治療対象とする請求項５に記載の医薬または医薬組成物。 The pharmaceutical or pharmaceutical composition according to claim 5, wherein acute retinal necrosis is treated.
- HSV感染細胞内におけるU90926の発現量を指標として、当該細胞内におけるHSV増殖を阻害する候補物質をスクリーニングする方法。 A method for screening candidate substances that inhibit HSV proliferation in HSV-infected cells using the expression level of U90926 as an index.
- 被験者由来のサンプル中のヒトU90926の発現量を測定することを含む、HSV感染症の診断方法またはHSV感染症の診断補助方法。 A method for diagnosing HSV infection or a method for assisting diagnosis of HSV infection, which comprises measuring the expression level of human U90926 in a sample derived from a subject.
- 急性網膜壊死患者由来のサンプル中のヒトU90926の発現量を測定することを含む、急性網膜壊死患者の視力予後の予測方法または視力予後の予測の補助的方法。
A method for predicting visual acuity prognosis in patients with acute retinal necrosis or an auxiliary method for predicting visual acuity prognosis, which comprises measuring the expression level of human U90926 in a sample derived from a patient with acute retinal necrosis.
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Non-Patent Citations (4)
| Title |
|---|
| CHEN J, LIU Y, LU S, YIN L, ZONG C, CUI S, QIN D, YANG Y, GUAN Q, LI X, WANG X: "The role and possible mechanism of lncRNA U90926 in modulating 3T3-L1 preadipocyte differentiation", INTERNATIONAL JOURNAL OF OBESITY, NATURE PUBLISHING GROUP UK, LONDON, vol. 41, no. 2, 1 February 2017 (2017-02-01), London, pages 299 - 308, XP055913727, ISSN: 0307-0565, DOI: 10.1038/ijo.2016.189 * |
| SABIKUNNAHAR SABIKUNNAHAR, VARNUM STELLA, BIVONA J J, COOPER RACHEL, KREMENTSOV DIMITRY, MAY J IMMUNOL: "Functional characterization of a novel myeloid cell-specific lncRNA U90926", J IMMUNOL, vol. 187, no. 1 Supp., 1 May 2019 (2019-05-01), pages 26, XP055913754 * |
| SHEN, R. ET AL.: "Identification of long non-coding RNAs expressed during early adipogenesis", J. BIOL. SCI., vol. 19, no. 4, 2019, pages 245 - 259, XP055913737, DOI: doi.org/10.3844/ojbsci.2019.245.259 * |
| SHIRAHAMA SHINTARO, SHIRAHAMA SHINTARO, ONOGUCHI MIZUTANI RENA, KAWATA KENTARO, TANIUE KENZUI, MIKI ATSUKO, KATO AKIHISA, KAWAGUCH: "Long noncoding RNA U90926 is crucial for herpes simplex virus type 1 proliferation in murine retinal photoreceptor cells", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, vol. 10, no. 1, 1 December 2020 (2020-12-01), pages 19406, XP055913755, DOI: 10.1038/s41598-020-76450-2 * |
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