KR101723434B1

KR101723434B1 - Novel human gene crucial for the replication of influenza virus and use thereof

Info

Publication number: KR101723434B1
Application number: KR1020160069604A
Authority: KR
Inventors: 민지영; 이지혜; 신동조
Original assignee: 재단법인 한국파스퇴르연구소
Priority date: 2016-06-03
Filing date: 2016-06-03
Publication date: 2017-04-05

Abstract

The present invention relates to a novel human gene involved in the replication of influenza virus and its use. More specifically, the present invention relates to an inhibitor of CTSC gene expression, an inhibitor of CTSC protein activity, or a mixture thereof, which is involved in replication of influenza virus A method for screening an antiviral agent against influenza virus and a method for screening an antiviral agent by measuring the level of the mRNA of the CTSC gene or a protein thereof, .

Description

New human genes involved in the replication of influenza viruses and their use {Novel human gene crucial for the replication of influenza virus and use thereof}

The present invention relates to a novel human gene involved in the replication of influenza virus and its use, and more particularly to a novel human gene involved in the replication of influenza virus, including a CTSC gene expression inhibitor, an inhibitor of CTSC protein activity, A method for screening an antiviral agent against influenza virus and a method for screening an antiviral agent by measuring the level of the mRNA of the CTSC gene or a protein thereof, .

Influenza drugs currently target only two proteins, the M2 ion channel and neuraminidase, both of which are viral gene products. Unfortunately, at present, resistance to both types of drugs is widespread, and there is an urgent need for novel antiviral drugs for the prevention of pandemic influenza and pandemic influenza worldwide. Host factors that regulate viral replication during infection have been considered attractive antiviral targets because they have distinctive features that lack the high mutation rate of influenza virus genes that enable the development of resistance to current drugs.

On the other hand, the CTSC (Cathepsin C) gene is a lysosomal cysteine proteinase and a peptidase, which are considered to be the central coordinators for the activation of many serine proteinases in the cells of the immune system And members of the C1 family (peptidiase C1 family). Selective splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is treated with protein hydrolysis to produce heavy and light chains that form disulfide-linked dimers . A portion of the propeptide acts as an intramolecular chaperone for the stabilization and folding of mature enzymes. This enzyme requires chlorine ions for its activity and can degrade glucagon. Defects in the encoded proteins have been shown to be causative of Papillon-Lefevre syndrome, an autosomal recessive disorder characterized by palmoplantar keratosis and periodontitis. However, the function of CTSC in relation to influenza virus is unknown.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication (Nature 463: 818-822, 2010) by Karlas A, et al., And presents various genes including GRIN2C gene as an important host factor for influenza virus replication There is, however, no mention of the fact that the CTSC gene plays an important role in influenza viruses.

Under these circumstances, the present inventors have made intensive efforts to search for a new target for development of an antiviral agent against influenza virus. As a result, the human CTSC gene acts as an important host factor required for replication of influenza virus, Or activity of the influenza virus is effective in preventing or treating influenza virus infection, the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating influenza virus infection comprising an inhibitor of CTSC (Cathepsin C) gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient.

It is another object of the present invention to provide a health functional food for preventing or ameliorating an influenza virus infection comprising an inhibitor of CTSC (Cathepsin C) gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient.

Yet another object of the present invention is to provide a method for detecting a CTSC gene, comprising the steps of: (a) treating an antiviral candidate substance to a separated sample expressing the CTSC gene, and then measuring the level of the mRNA of the CTSC gene or a protein thereof; And (b) selecting an antiviral agent for an influenza virus when the level of the mRNA or the protein of the gene is lower than that of a control sample not treated with the sample .

Another object of the present invention is to provide an anti-viral agent screening kit for an influenza virus comprising an agent for measuring the mRNA of the CTSC gene or a protein level thereof.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating an influenza virus infection comprising an inhibitor of CTSC (Cathepsin C) gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient.

According to a preferred embodiment of the present invention, the influenza virus may be influenza A virus.

According to another preferred embodiment of the present invention, the CTSC gene may be involved in replication of an influenza virus.

According to another preferred embodiment of the present invention, the CTSC gene expression inhibitor may be selected from the group consisting of antisense of CTSC gene, oligonucleotide, siRNA, shRNA and microRNA.

According to another preferred embodiment of the present invention, the siRNA may be any one selected from the group consisting of SEQ ID NOS: 1 to 4.

According to another preferred embodiment of the present invention, the activity inhibitor may be an antibody or an aptamer that specifically binds to a CTSC protein.

The present invention also provides a health functional food for preventing or ameliorating an influenza virus infection comprising an inhibitor of CTSC (Cathepsin C) gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient.

The present invention also provides a method for detecting a CTSC gene, comprising the steps of: (a) treating an antiviral candidate substance to a separated sample expressing the CTSC gene, and then measuring the level of the mRNA of the CTSC gene or a protein thereof; And (b) selecting an antiviral agent for an influenza virus when the level of mRNA of the gene or the protein thereof is lower than that of a control sample not treated with the sample.

According to a preferred embodiment of the present invention, the step of measuring the mRNA level of the CTSC gene may be performed using reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time reverse transcriptase polymerase Can be measured by real time quantitative RTPCR, RNase protection method, northern blotting or gene chip.

According to another preferred embodiment of the present invention, the step of measuring the mRNA level of the CTSC gene may use a primer or a probe specifically binding to the CTSC gene.

According to another preferred embodiment of the present invention, the step of measuring the level of the protein of the CTSC gene may use an antibody that specifically binds to the protein.

The present invention also provides a screening kit for an antiviral agent against influenza virus comprising an agent for measuring mRNA of CTSC gene or a protein level thereof.

According to a preferred embodiment of the present invention, the kit may be an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit or a protein chip kit.

The present invention has revealed that the CTSC gene acts as an important host factor for the replication of influenza virus. The present invention provides a pharmaceutical composition for preventing or treating an influenza virus infection using the CTSC gene of the present invention, a method for preventing or improving influenza virus infection A health functional food, a screening method of an antiviral agent against influenza virus, and a screening kit.

The CTSC gene used as a novel target in the present invention is a host factor that regulates viral replication during infection and has a distinctive feature that lacks the high mutation rate of the influenza virus gene that enables the development of resistance to current drugs Therefore, it can be usefully used for the development of a novel antiviral agent which can replace the antiviral agents reported in the prior art.

1 is a photograph showing human lung epithelial A549 cells infected with the rPR8 NS1-GFP virus, and the arrows show cells infected with the rPR8 NS1-GFP virus.
FIG. 2 is a photograph showing image-based assay results using recombinant influenza A virus expressing GFP-tagged NS1A protein, showing negative control (scramble) and positive control (siCSE1L and siNXF1), respectively .
Figure 3 is a schematic diagram outlining a wide range of human genome-wide high throughput siRNA screening of whole human genomes.
FIG. 4 is a photograph of a cell infectious ability measured using the analysis program of the present invention, the upper line is a photograph obtained from a confocal microscope, and the lower line is a photograph analyzed using an image analysis program of the present invention.
5 shows the infectivity of cells transfected with siRNA against the CTSC gene identified using the screening method of the present invention, using scrambled siRNA-transformed cells (negative control, infectivity 100%) and siCSE1L transformed cells Normalized to the control, infectious 0%), the red line represents the selection criteria (≥50% inhibition) and the bar represents the two-times mean ± SD.
6 is a graph showing the inhibitory effect of siCTSC on the replication of H7 avian influenza virus (H7N7).

Hereinafter, the present invention will be described in more detail.

As described above, there is an urgent need for new antiviral drugs for the prevention of influenza and pandemic influenza worldwide because of the widespread spread of antiviral drugs against influenza viruses.

Accordingly, the present invention confirms that the human CTSC gene is effective in preventing or treating influenza virus infection when acting as an important host factor required for replication of influenza virus and suppressing its expression or activity, and CTSC (Cathepsin C) A pharmaceutical composition for preventing or treating an influenza virus infection comprising an inhibitor of gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient, has been sought. Since the CTSC gene used as a new target in the present invention is a host factor that regulates viral replication during infection and has a distinctive feature that lacks the high mutation rate of the influenza virus gene that enables the development of resistance to drugs at present, May be useful for the development of novel antiviral agents that can replace the reported antiviral agents.

The present invention provides a pharmaceutical composition for preventing or treating influenza virus infection comprising an inhibitor of CTSC (Cathepsin C) gene expression, an inhibitor of CTSC protein activity, or a mixture thereof as an active ingredient.

The pharmaceutical composition of the present invention is effective for the prevention or treatment of an influenza virus, preferably an influenza A virus infection. Examples of the influenza A virus include A / Puerto Rico / 8/34 (H1N1) A / EM / A / Udorn / 307/72 (H3N2). However, the CTSC gene has not been identified in the present study. There is no particular limitation as long as the virus is a virus that inhibits expression or inhibits the activity of a protein to inhibit replication.

The term "CTSC" is abbreviation of " Cathepsic C ", defects of the protein encoded by the CTSC gene include autosomal recessive disorder characterized by palmoplantar keratosis and periodontitis, Papillon- Lefevre syndrome, but the function of CTSC in relation to influenza virus is unknown.

The CTSC gene and protein sequence can be obtained from known databases such as NCBI. Specifically, the CTSC gene sequence may be one disclosed in Gene Accession NM_001814 of the NCBI database, and the CTSC protein sequence may be one disclosed in the NCBI database NP_001805.3, but is not limited thereto.

In the present invention, it was first confirmed that the CTSC gene acts as an important host factor for influenza virus replication, thereby confirming that influenza virus infection can be effectively treated by suppressing the expression of the gene or the activity of the protein.

In one embodiment of the present invention, recombinant influenza A virus (rPR8 NS1-GFP) expressing GFP-tagged NS1A protein (rPR8 NS1-GFP) was prepared (Fig. 1) and then subjected to an image-based assay Wide human genome-wide high throughput siRNA screening of whole human genetic material (Figure 3).

In order to compare the inhibitory effects of selected genes through siRNA high-speed screening (HTS), non-specific scrambled siRNAs (scrambles) were used as negative controls and human CSE1L (chromosome segregation 1-like), which has been identified as an important host factor controlling influenza, And siRNA targeting human NXF1 (nuclear RNA export factor 1) were used as positive controls to optimize image-based assays (FIG. 2).

The knockdown effect of each individual human gene was measured by calculating the infectivity (number of cells expressing GFP / number of total cells) using an in-house image analysis program (FIG. 4) A human gene CTSC (Cytoskeleton), which causes a 50% inhibitory effect on replication of influenza A virus through comparison of Z-score analysis and positive control (siCSE1L, 0% infection rate) and negative control (Scramble, 100% infection rate) And the results are shown in FIG.

As shown in FIG. 5, it was confirmed that the siRNA targeting the CTSC gene of the present invention exhibited superior infection inhibition rates than the siRNA targeting GRIN2C, which is conventionally known as a human gene important for influenza virus replication. Thus, inhibition of the expression of the CTSC gene of the present invention is considered to be more effective for treating influenza virus infection than inhibiting the expression of the GRIN2C gene.

Furthermore, as shown in FIG. 6, it was confirmed that the siRNA targeting the CTSC gene of the present invention also inhibited the replication of H7 avian influenza virus (H7N7). Thus, inhibiting the expression of the CTSC gene can be used to prevent or treat symptoms of avian influenza virus infection.

Based on the sequence of CTSC, an inhibitor of CTSC expression or an activity inhibitor can be designed, and the sequence can be modified to some extent in this design. Those skilled in the art will appreciate that sequences that retain more than 80%, in particular more than 90%, more specifically more than 95%, and more particularly 98% Do.

As used herein, the term "inhibitor of CTSC gene expression" refers collectively to a substance that decreases the expression or activity of CTSC. More specifically, the term " agent that decreases CTSC expression at a transcription level or a protein level " . The substance that inhibits CTSC expression can be used without limitation in the form of a compound, a nucleic acid, a peptide, a virus, or a vector containing the nucleic acid, which can inhibit CTSC expression or activity by targeting CTSC.

Specifically, the CTSC gene expression inhibitor may be any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the CTSC gene.

The term "antisense oligonucleotide " in the present invention means DNA, RNA or derivatives thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein .

The term "small interfering RNA (siRNA)" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method.

Specifically, the siRNA may be any one selected from the group consisting of SEQ ID NOS: 1 to 4.

The term "short hairpin RNA (shRNA)" in the present invention refers to an oligodeoxynucleotide synthesizing oligonucleotide linking 3-10 base linkers between the sense of the siRNA sequence for the target gene and the complementary nonsense, (ShRNA) (short hairpin RNA), which is loop-shaped, is produced and converted into siRNA by intracellular Dicer to produce RNAi Effect. The shRNA shows a relatively long-term RNAi effect as compared to siRNA.

Such an expression inhibitor can be easily designed by those skilled in the art to induce the suppression of the expression of the CTSC gene according to techniques commonly used in the art.

In addition, the activity inhibitor of CTSC protein may be an antibody or an aptamer that specifically binds to a protein expressed from CTSC gene, but is not limited thereto.

Such an antibody may be a polyclonal antibody, a monoclonal antibody or fragments of the antibody as long as the antibody has antigen binding ability. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, and a human antibody. In addition to the novel antibody, antibodies that are already known in the art may also be included. The antibody comprises a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, so long as the antibody has the property of binding specifically recognizing the protein expressed from the CTSC gene. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes, but is not limited to, Fab, F (ab ') 2, F (ab') 2 and Fv.

In the present invention, the term "Aptamer" is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity. Aptamers are comparable to monoclonal antibodies due to their inherent high affinity (usually pM levels) and their ability to bind to target molecules with specificity, and there is a high likelihood of being an alternative antibody, especially as a "chemoantibody".

The term "prevention" as used in the present invention means all the actions of inhibiting or delaying the infection of influenza virus by administering the composition to an individual. The term "treatment" as used in the present invention means all the acts of administering the composition to influenza virus infected individuals so as to improve or improve the symptoms of infection.

The pharmaceutical composition of the present invention may be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, and examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or a solubilizing agent such as a flavoring agent can be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical preparation form of the pharmaceutical composition of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drips or injectable solutions, . The pharmaceutical composition of the present invention may be administered orally or parenterally through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, &Lt; / RTI > An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating infection. Thus, the type of infection, the severity of the infection, the type and amount of active and other ingredients contained in the composition, the type of formulation and the age, body weight, general health status, sex and diet, time of administration, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, the inhibitor of the present invention may be used at a dose of 0.1 ng / kg to 10 g / kg when the active ingredient is a compound once or several times a day, 0.1 ng / kg to 10 g / kg for peptides, proteins or antibodies, and 0.01 ng / kg to 10 g / kg for antisense oligonucleotides, siRNA, shRNAi and miRNA.

The kind of the food is not particularly limited, and includes food in a conventional sense. Non-limiting examples of foods to which the material can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice creams, , A drink, an alcoholic beverage, and a vitamin complex.

When the health functional food of the present invention is a beverage composition, it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Non-limiting examples of such natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweetening agents such as dextrin, cyclodextrin; Synthetic sweetening agents such as saccharin and aspartame, and the like. The proportion of the additional component added may be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit drink or vegetable drink. These components may be used independently or in combination of two or more. The ratios of these additives can also be suitably selected by those skilled in the art.

The level of CTSC of the present invention is measured in a cell expressing the CTSC gene in the absence of a candidate substance capable of preventing or treating an influenza virus infection and the level of the CTSC of the present invention is measured in the presence of the candidate substance, , A substance which reduces the expression level of CTSC when the candidate substance is present in the absence of the candidate substance can be predicted as an antiviral agent for preventing or treating an influenza virus infection.

The step of measuring the level of the mRNA of the CTSC gene or the protein thereof may be measured by an agent for measuring the level of the CTSC gene mRNA. The agent may be a preparation used for determining the expression of CTSC contained in the sample, (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting a primer or a probe capable of specifically binding to a target gene used in a method such as blotting and gene chip analysis, but is not particularly limited thereto.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but the present invention is not limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

As used herein, the term " measuring the level of a protein "refers to a process for determining the presence and the degree of expression of a protein expressed from a CTSC gene, specifically, an antibody that specifically binds to the protein of the gene Can be used to confirm the amount of protein.

In the present invention, "antibody" means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein produced by expression of a CTSC gene, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The production of an antibody that specifically binds to a CTSC protein can be easily carried out using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting CTSC protein antigens into animals and obtaining blood from animals to obtain sera containing antibodies. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 1992, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991). The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

Methods for assaying the amount of the target protein bound to the antibody using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip Protein (FACS), Immunoprecipitation Assay, Immunoprecipitation Assay, Ouchterlony Immunoprecipitation, Rocket Immunoelectrophoresis, chip, but are not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Cell culture

Human lung epithelial A549 cells used in the present invention were purchased from the American Type Culture Collection (ATCC, Catalog No. CCL-185 ^TM ), and cultured in Dulbecco's Modified Eagle Medium - high glucose (DMEM-HG) supplemented with 10% fetal bovine serum FBS) and 1% penicillin & streptomycin.

Preparation of Recombinant Influenza A Virus Expressing Green Fluorescent Protein

The recombinant influenza virus (A / Puerto Rico / 8/34) expressing the green fluorescent protein used in the present invention is commercially available from Adolfo Garcia-Sastre (Professor in the Department of Microbiology and Director of the Institute of Mount Sinai School of Health and Emerging Pathogens of Medicine in New York), and the approximate manufacturing method is as follows. The open reading frame (ORF) of the NS1 gene encoding the nonstructural protein 1 (NS1 protein) of the influenza gene is fused with the GFP gene encoding the green fluorescent protein to generate the NS-GFP gene fragment . This gene fragment is inserted into a DNA plasmid that can be expressed in cells. The DNA plasmid containing the NS-GFP gene fragment was transfected with each of seven plasmids containing seven genes (influenza viruses other than NS gene) (PB2, PB1, PA, HA, NP, Into the cell via the method. Within the cell, these plasmids produce recombinant influenza virus through reverse genetics based technology.

rPR8 NS1- GFP Image based essay using virus

Human lung epithelial A549 cells were infected with the rPR8 NS1-GFP virus prepared in Example 1 for 10 hours. Cells were fixed and cell nuclei were stained. The infected cells were imaged on a confocal fluorescence imaging system (Evotec Technologies High-Throughput Cell Analyzer Opera, Perkin Elmer, USA) at 20x magnification and analyzed with an in-house image mining (IM) platform.

As shown in Fig. 1, NS1-GFP and nuclei were visualized in green and red, respectively, and the arrows indicate rPR8 NS1-GFP virus infected cells.

In the present invention, a non-specific scramble siRNA (Scramble, Dharmacon) was used as a negative control to compare the inhibitory effect of the selected gene through the high-speed screening of siRNA (HTS), and the human CSE1L (Dharmacon) targeting segregation 1-like and human nuclear export factor 1 (NXF1) as a positive control to optimize image-based assays.

Cells were then fixed and stained after infection with the rPR8 NS1-GFP virus as described above, imaged on a 20X magnification confocal fluorescence imaging system and analyzed with an internal IM platform. As a result, knockdown of CSE1L and NXF1 as shown in Fig. 2 effectively blocked the expression of GFP-tagged virus NS1A protein.

Wide range of human genome siRNA Screening

RNA interference (RNAi) technology is a powerful tool for understanding host pathogen relationships. In this example, extensive siRNA high-speed screening (HTS) of the genome was used as shown in the schematic diagram of FIG. 3 to identify host factors required for replication of influenza virus in human cells.

For the reading on HTS, the recombinant influenza A virus (rPR8 NS1-GFP) prepared in Example 1 above was used and the image-based assay of Example 2 was used to detect 18,055 labeled genes in human lung epithelial A549 cells (Dharmacon, # GU-105005, Human ON-TARGETplus siRNA Library - Whole Genome) of an entire human genome consisting of approximately 72,000 siRNAs targeted to the target gene (4 siRNA pools per gene).

Add 10 μl of siRNA to a 384-well plate and dispense 5 μl each of the solution containing the transfection reagent into each well. After allowing to stand at room temperature for 20 minutes, A549 cells are dispensed into 384-well plates in 30 쨉 l increments. After 48 hours, the transfected A549 cells are infected with rPR8 NS1-GFP virus for 10 hours. A549 cells transfected with individual siRNAs are fixed with paraformaldehyde and stained with Hoechst 33342 (Thermo Fiusher Scientific), a nuclear dyeing reagent. 48 hours before infection with the rPR8 NS1-GFP virus, A549 cells transfected with individual siRNAs were fixed and stained 10 hours after infection to monitor viral infectivity.

The knockdown effect of each individual human gene was determined by calculating the infectivity (number of cells expressing GFP / number of total cells) using an in-house image analysis program (FIG. 4).

A genomic human gene that causes more than 50% inhibition of replication of influenza A virus through a strong Z-score analysis and comparison of positive control (siCSE1L, 0% infection rate) and negative control (Scramble, 100% infection rate) The gene was identified, and the result is as shown in Fig.

In addition, it was confirmed that the siRNA targeting the CTSC gene of the present invention exhibited superior infection inhibition rates than siRNA targeting GRIN2C, which is conventionally known as a human gene important for influenza virus replication. Thus, inhibition of the expression of the CTSC gene of the present invention is considered to be more effective for treating influenza virus infection than inhibiting the expression of the GRIN2C gene.

The sequences of the siRNA targeting CTSC and GRIN2C used in this example are shown in Table 1 below.

Gene Symbol Sequence CTSC GCUUUGAGAUUGUGUUGAA (SEQ ID NO: 1) GCACCUAUCUUGACCUGCU (SEQ ID NO: 2) CAACUGCUCGGUUAUGGGA (SEQ ID NO: 3) GUAGUGGUGUACCUUCAGA (SEQ ID NO: 4) GRIN2C GAGCAUGGCGUCCUAUACA (SEQ ID NO: 5) UCAGAAGUGUGAGUUAUCA (SEQ ID NO: 6) CGCAGUAACUACCGUGACA (SEQ ID NO: 7) CAAGAGCAAUACAUCGACA (SEQ ID NO: 8)

Inhibitory effect of A type influenza virus on subtype

In order to confirm whether the suppression of the gene expression by the siRNA targeting the CTSC gene showed an inhibitory effect on other subtype virus replication in addition to the rPR8 NS1-GFP virus of the type A influenza virus used for screening, Additional experiments were performed using influenza virus (A / EM / Korea / W152 / 2006, H7N7).

The degree of infection of H7 avian influenza virus was imaged through immunofluorescence assay using an antibody that specifically binds to the viral protein nucleoprotein (NP). Sensitivity (number of cells expressing NP / number of total cells) was measured using an internal image analysis program. The measured infectious capacity was normalized by a negative control (scramble, 100% infection rate) and displayed as a bar graph.

As a result, as shown in Fig. 6, it was confirmed that the replication of the H7N7 subtype of influenza A virus was suppressed by about 20% by inhibiting the expression of the CTSC gene. Therefore, it was confirmed that infection of H7 avian influenza virus can be significantly prevented or treated through inhibition of CTSC gene expression.

<110> Institut Pasteur Korea <120> Novel human gene crucial for the replication of influenza virus and use thereof <130> 1060769 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CTSC siRNA <400> 1 gcuuugagau uguguugaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CTSC siRNA <400> 2 gcaccuaucu ugaccugcu 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CTSC siRNA <400> 3 caacugcucg guuauggga 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CTSC siRNA <400> 4 guaguggugu accuucaga 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> GRIN2C siRNA <400> 5 gagcauggcg uccuauaca 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> GRIN2C siRNA <400> 6 ucagaagugu gaguuauca 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> GRIN2C siRNA <400> 7 cgcaguaacu accgugaca 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> GRIN2C siRNA <400> 8 caagagcaau acaucgaca 19

Claims

A / Puerto Rico / 8/34 (H1N1) and A / EM (H1N1) strains containing as an active ingredient siRNA of any one or more CTSC genes selected from the group consisting of SEQ ID NOS: 1 to 4 as inhibitors of CTSC (Cathepsin C) / Korea / W152 / 2006 (H7N7). The present invention also relates to a pharmaceutical composition for preventing or treating infection with influenza A virus.

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The pharmaceutical composition according to claim 1, wherein the CTSC gene is involved in replication of the influenza virus.

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A / Puerto Rico / 8/34 (H1N1) and A / EM (H1N1) strains containing as an active ingredient siRNA of any one or more CTSC genes selected from the group consisting of SEQ ID NOS: 1 to 4 as inhibitors of CTSC (Cathepsin C) / Korea / W152 / 2006 (H7N7) for the prevention or amelioration of infection by any one or more influenza A viruses selected from the group consisting of:

(a) treating an antiviral candidate substance to a separated sample expressing the CTSC gene, and then measuring the level of the mRNA of the CTSC gene or a protein thereof; And
(b) A / Puerto Rico / 8/34 (H1N1) and A / EM / Korea / W152 / 2006 (H7N7) when the level of the mRNA of the gene or the protein thereof is lower than that of the control sample And a step of selecting an antiviral agent against any one or more types of influenza A viruses selected from the group consisting of:

The method according to claim 8, wherein the step of measuring the mRNA level of the CTSC gene comprises the steps of RT-PCR, competitive RT-PCR, real-time RT quantitative RT-PCR, RNase protection method, northern blotting or gene chip.

9. The screening method according to claim 8, wherein the step of measuring the mRNA level of the CTSC gene comprises using a primer or a probe specifically binding to the CTSC gene.

9. The screening method according to claim 8, wherein the level of the protein of the CTSC gene is measured using an antibody that specifically binds to the protein.

A / EM / Korea / W152 / 2006 (H7N7) selected from the group consisting of A / Puerto Rico / 8/34 (H1N1) and A / EM / Korea / W152 / 2006 (H7N7), which comprises an agent for measuring mRNA of CTSC gene or its protein level Screening kit for antiviral agents against influenza virus.

13. The screening kit according to claim 12, wherein the kit is an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit or a protein chip kit.