Methods for treating hepatitis C virus infectious disease

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hepatitis C virus (HCV) infectious disease. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating HCV infectious disease or an antiviral composition for HCV, containing at least one selected from the group consisting of: GRIM19 protein or a fragment thereof; and a gene encoding the protein or a fragment of the protein.

Claims

1. A method for treating a hepatitis C virus infectious disease, the method comprising: selecting a subject in need of treatment for a hepatitis C virus infectious disease; and administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one selected from the group consisting of genes-associated with Retinoid-Interferon induced Morality 19 (GRIM19) protein or a fragment thereof, or a gene encoding the GRIM19 protein or the fragment thereof, wherein the GRIM19 protein consists of the amino acid sequence of SEQ ID NO: 2, wherein the gene encoding the GRIM19 protein consists of the base sequence of SEQ ID NO: 1, and wherein the fragment of GRIM19 protein is at least one selected from the group consisting of: a protein fragment comprising 1.sup.st to 36.sup.th amino acids of the amino acid sequence of SEQ ID NO: 2; a protein fragment comprising 37.sup.th to 72.sup.nd amino acids of the amino acid sequence of SEQ ID NO: 2; a protein fragment comprising 73.sup.rd to 101.sup.st amino acids of the amino acid sequence of SEQ ID NO: 2; and a protein fragment comprising 102.sup.nd to 144.sup.th amino acids of the amino acid sequence of SEQ ID NO: 2.

2. The method of claim 1, wherein the fragment of GRIM19 protein consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.

3. The method of claim 1, wherein the gene encoding the fragment of GRIM19 protein is at least one selected from the group consisting of: a gene comprising 3.sup.rd to 108.sup.th bases of the base sequence of SEQ ID NO: 1; a gene comprising 109.sup.th to 216.sup.th bases of the base sequence of SEQ ID NO: 1; a gene comprising 217.sup.th to 303.sup.rd bases of the base sequence of SEQ ID NO: 1; and a gene comprising 304.sup.th to 432.sup.nd bases of the base sequence of SEQ ID NO: 1.

4. The method of claim 1, wherein the gene encoding the fragment of GRIM19 protein consists of the base sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.

5. The method of claim 1, wherein the GRIM19 protein or the fragment thereof further comprises a cell penetrating peptide at an N-terminus, C-terminus, or both termini.

6. The method of claim 5, wherein the cell penetrating peptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

7. The method of claim 1, wherein the gene encoding the GRIM19protein or the fragment thereof is provided in a form included in a recombinant vector.

8. The method of claim 1, wherein the composition further comprises a substance inhibiting expression or activity of diacylglycerol acyltransferase-1 (DGAT-1).

9. The method of claim 8, wherein the substance inhibiting the expression or activity of DGAT-1 is siRNA, shRNA, or antisense oligonucleotide, specifically binding to the gene or mRNA of DGAT-1.

10. The method of claim 8, wherein the substance inhibiting the expression or activity of DGAT-1 is an antibody, an aptamer, or a compound of Formula 1: ##STR00007## or a salt thereof, specifically binding to DGAT-1.

11. The method of claim 1, wherein the composition further comprises a substance inhibiting expression or activity of RNA-dependent RNA polymerase.

12. The method of claim 11, wherein the substance inhibiting the expression or activity of RNA-dependent RNA polymerase is a compound of Formula 2: ##STR00008## or a salt thereof.

13. The method of claim 1, wherein the composition further comprises a substance activating AMP-activated protein kinase (AMPK).

14. The method of claim 13, wherein the substance activating AMP-activated protein kinase (AMPK) is metformin.

15. The method of claim 1, wherein the hepatitis C virus infectious disease is at least one of hepatitis C, liver fibrosis caused by hepatitis C virus, liver cirrhosis caused by hepatitis C virus, and liver cancer caused by hepatitis C virus.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a result of Western blot analysis of GRIM19 expression in liver tissues of patients with chronic liver disease caused by HCV. HCV-infected patients indicates patients infected with hepatitis C virus, HBV-infected patients indicates patients infected with hepatitis B virus, CHC indicates liver tissues of patients with chronic hepatitis C infection, CHB indicates liver tissues of patients with chronic hepatitis B infection. LC indicates liver tissues of patients with liver cirrhosis (LC), HCC indicates liver tissues of patients with liver cancer (HCC), Grim19 indicates the expression of GRIM19 protein, and -actin indicates the expression of -actin protein.

(2) FIG. 2 is a graph showing relative comparison of GRIM19 expression in liver tissues of patients with chronic liver disease caused by HCV. Normal indicates normal comparative group, HCV-CLD indicates patients with chronic hepatitis C infected with hepatitis C virus, and HBV-CLD indicates patients with chronic hepatitis B infected with hepatitis B virus.

(3) FIG. 3 shows a result of Western blot analysis of GRIM19 expression in in vitro HCV infection model. Huh7 indicates a result before infection with HCV, and HCVcc (Days) indicates the number of days of infection with HCV.

(4) FIG. 4 shows a result of Western blot confirming GRIM19 overexpression after transfection of a vector expressing GRIM19. Mock indicates cells with no treatment, pcDNA3 indicates cells transfected with a vector expressing no GRIM19, and pcDNA3_GRIM19 indicates the result of protein expression in cells transfected with a vector expressing GRIM19.

(5) FIG. 5a shows a result confirming a change in level of HCV RNA 48 hours after transfection of Huh7 cells, which were infected with HCVcc, with pcDNA3_GRIM19.

(6) FIG. 5b shows a change in level of HCV RNA according to GRIM19 overexpression. Mock indicates cells with no treatment, and pcDNA3_GRIM19 indicates the result in cells transfected with a vector expressing GRIM19.

(7) FIG. 6a shows a change in degree of lipid accumulation in Huh7 cells when a vector expressing GRIM19 was transfected and GRIM19 was overexpressed. BSA indicates the case of addition of bovine serum albumin as control group, and OA indicates the case of addition of oleic acid.

(8) FIG. 6b shows that decreased level of HCV RNA in Huh7 cells due to transfection of a vector expressing GRIM19 to overexpress GRIM19 was restored through treatment with lipid.

(9) FIG. 7a shows a change in expression of proteins involved in fatty acid synthesis in the case of transfection with pcDNA3_GRIM19 to overexpress GRIM19. SIRT indicates sirtuin protein, p-Acc indicates phosphor-acetyl-CoA carboxylase, Acc indicates acetyl-CoA carboxylase, p-AMPK indicates phosphor-AMP-activated protein kinase, AMPK indicates AMP-activated protein kinase, and Actin indicates actin protein.

(10) FIG. 7b shows a change in expression of acetyl-CoA carbosylase (Acc) in the case of transfection with pcDNA3_GRIM19 to overexpress GRIM19.

(11) FIG. 8 shows a change in level of HCV RNA in the case of transfection with pcDNA3_GRIM19 to overexpress GRIM19 and treatment with metformin. Mock indicates cells with no treatment, and pcDNA3_GRIM19 indicates the result in cells transfected with a vector expressing GRIM19.

(12) FIG. 9 shows a change in level of HCV RNA in the case of treatment of pcDNA3_GRIM19 transfected cells with a compound (DGAT-1 inhibitor) of Formula 1 in combination. DMSO indicates the control group treated with dimethyl sulfoxide (DMSO), instead of the compound of Formula 1, Mock indicates cells with no treatment, and pcDNA3_GRIM19 indicates the result in cells transfected with a vector expressing GRIM19.

(13) FIG. 10 shows a change in level of HCV RNA in the case of treatment of pcDNA3_GRIM19 transfected cells with a compound (activity inhibitor of RNA-dependent RNA polymerase) of Formula 2. Sofosbuvir indicates the compound of Formula 2.

(14) FIG. 11a is a schematic view fragmenting GRIM19 protein into four domains according to the present invention.

(15) FIG. 11b shows a change in level of HCV RNA in the case of transfection with GRIM19 protein and four domains of GRIM19 protein fragmented according to the present invention in in vitro HCV infection model.

(16) FIG. 12 shows a change in level of HCV RNA in the case of direct treatment of in vitro HCV infection model with peptides where cell permeable peptide sequences (CP) bind to four domains of GRIM19 protein fragmented according to the present invention.

(17) FIG. 13 shows a cleavage map that shows the structure of pJFH-1.

(18) FIG. 14 shows a structure of GRIM19 overexpression vector prepared by using pcDNA3.

(19) FIG. 15 shows a cleavage map that shows the structure of pCMV-3Tag-3A vector.

BEST MODE FOR CARRYING OUT INVENTION

(20) Hereinafter, the present invention is described in detail with reference to examples. The following examples, however, are only to illustrate the present invention and the present invention is not limited to the following examples.

EXAMPLE 1

Confirmation of Decrease in GRIM19 Expression in Liver Tissue of Patients with Chronic Liver Disease Caused by HCV

(21) In order to evaluate the expression level of GRIM19 in tissue of patients with chronic liver disease (CLD) caused by infection with HBV and HCV, which are main viruses causing chronic hepatitis, Western blot analysis was performed using anti-GRIM19 antibody.

(22) Patients with chronic hepatitis (CHB, CHC), liver cirrhosis (LC), and liver cancer (HCC) caused by infection with HBV or HCV were selected from patients approved by Institutional Review Board (IRB) among patients of Korea Seoul St. Mary's Hospital, and liver tissues subjected to biopsy was obtained therefrom. After lysing 20 mg of the obtained liver tissues (CHC n=4, LC n=3, HCC n=2, for hepatitis C patients; CHB n=3, LC n=3, HCC n=3 for hepatitis B patients) using PROPREP reagent (INTRON BIOTECHNOLOGY), Western blot analysis was performed using 20 g of lysates. As anti-GRIM19 monoclonal antibody, a product from Abcam was used, and as anti--actin monoclonal antibody, a product from Sigma was used.

(23) As a result of experiment, it was confirmed that unlike liver tissues of HBV infected patients, in tissues of patients with chronic liver disease caused by HCV, the expression of GRIM19 was specifically decreased (FIG. 1 and FIG. 2).

EXAMPLE 2

Confirmation of Decrease in GRIM19 Expression in In Vitro HCV Infection Model

(24) HCV does not infect small animals such as mice, and the sole animal model is chimpanzee. Thus, most of recent studies on HCV are conducted through in vitro infection model.

(25) Hepatitis C Virus cell culture (HCV cc) system was constructed with pJFH-1 (FIG. 13), a vector comprising infectious clone JFH-1 clone provided by the research team led by professor Takaji Wakita of Japan National Institute of Infectious Diseases which constructed HCVcc using JFH-1 strain.

(26) In order to construct a recombinant infectious HCV model, infectious HCV genome which was synthesized through in vitro transcription was introduced into Huh7 cells (Korean Cell Line Bank) through electroporation, followed by passage every three days, to culture cells.

(27) HCV RNA in Huh7 cells after electroporation was evaluated through quantitative realtime RT PCR. The total RNA was extracted using Trizol LS reagents (Invitrogen), and cDNA was synthesized using 2 g of the extracted RNA. Quantitative realtime RT PCR was carried out using the synthesized cDNA, Lightcycler 480 probe master (Roche), HCV 5-UTR specific fluorescent probes (SEQ ID NO: 3), and primers (SEQ ID Nos: 4 and 5).

(28) TABLE-US-00002 TABLE2 Gene Sequence(5.fwdarw.3) SEQIDNO:3 HCV5UTR CTGCGGAACCGGTGAGTACAC probe SEQIDNO:4 HCV5UTR GCGCCTAGCCATGGCGTTAGT ForwardPrimer ATGAGTGTC SEQIDNO:5 HCV5UTR ACCACAAGGCCTTTCGCAACC ReversePrimer CAACGCTAC

(29) Three days after electroporation, about 110.sup.6 copies were detected per 1 g of the total RNA. As a result of examination of the amount of HCVcc discharged into the cell culture medium through the level of HCV RNA, it was confirmed that the level of HCV RNA was retained at about 110.sup.7 per 1 g of RNA in 1 ml of the culture medium. From this, it was confirmed that in vitro HCV infectious system which is the experimental basis of HCV research was constructed.

(30) A change in GRIM19 expression derived from HCV infection was examined using in vitro HCV infection system. After seeding 1.510.sup.6 Huh7 cells in 10-cm culture dish, the cells were infected with HCVcc next day. Thereafter, every three days, cells and cell media were subcultured at a ratio of 1:4.

(31) HCVcc infected Huh7 cells were harvested on 3.sup.rd, 6.sup.th, 9.sup.th, and 12.sup.th days, and the expression level of GRIM19 was confirmed through Western blot analysis. After lysing cells using PROPREP reagent (INTRON BIOTECHNOLOGY), Western blot analysis was performed using 20 g of lysates. As anti-GRIM19 monoclonal antibody, a product from Abcam was used, and as anti--actin monoclonal antibody, a product from Sigma was used.

(32) As in HCV-CLD tissues (chronic hepatitis C patients infected with hepatitis C virus), it was confirmed that in HCVcc infected Huh7, the level of GRIM19 was decreased. The level was decreased by about 80% three days after infection, and by about 60% six days after infection (FIG. 3).

EXAMPLE 3

Decrease in HCV RNA Level by GRIM19 Overexpression

(33) From Examples 1 and 2, it was confirmed that artificial increase in the expression of GRIM19 produces the effect of inhibiting HCV replication based on the deduction that the GRIM19 expression in HCV infected tissues and cells was regulated by virus in order to form environment favorable to HCV replication.

(34) GRIM19 overexpression vector was prepared to evaluate the effect of inhibiting HCV replication through GRIM19 overexpression. The gene of GRIM19 was obtained from mRNA of Huh7 cells through RT PCR and cloned to pcDNA3 vector (Invitrogen) (FIG. 14) using restriction enzymes BglII and HindIII. The overexpression of GRIM19 was evaluated through Western blot analysis (FIG. 4).

(35) After seeding 1.510.sup.6 Huh7 cells in 10-cm culture dish, the cells were infected with HCVcc next day. Thereafter, every three days, cells and cell media were subcultured at a ratio of 1:4. The cells which were subcultured on 1.sup.st, 4.sup.th, 7.sup.th and 10.sup.th days of infection were transfected with 5 g of pcDNA3_GRIM1 9 using FUGENE HD (Promega). The cells were harvested 48 hours after transfection, and the level of HCV RNA was evaluated through quantitative realtime RT PCR.

(36) FIG. 5a, which shows the level of HCV RNA 48 hours after transfection of Huh7 cells, which were infected with HCVcc, with pcDNA3_GRIM19, confirmed that HCV RNA was decreased when GRIM19 protein is expressed even though Huh7 cells were infected with HCVcc.

(37) Also, with reference to FIG. 5b, in the case of HCVcc infected Huh7 cells, about 9.510.sup.5 HCV RNA copies per 1 g of the total RNA on 3.sup.rd day, about 6.9.510.sup.6 HCV RNA copies per 1 g of the total RNA on 6.sup.th day, about 5.010.sup.7 HCV RNA copies per 1 g of the total RNA on 9.sup.th day, and about 1.910.sup.8 HCV RNA copies per 1 g of the total RNA on 12.sup.th day were detected. As a result of GRIM19 overexpression due to transfection with pcDNA3_GRIM19, the level of HCV RNA was decreased down to about 5.710.sup.5 HCV RNA copies, 3.510.sup.6 HCV RNA copies, 1.810.sup.7 HCV RNA copies, and 3.810.sup.7 HCV RNA copies, respectively. From this, it was confirmed that as a result of GRIM19 overexpression in HCV infected Huh7 cells, the level of HCV RNA was decreased by about 40 to 80%.

(38) Therefore, it was clearly proved that the expression of GRIM19 protein in HCV infected cells has anti-HCV activity.

EXAMPLE 4

Confirmation of Association Between HCV RNA Level and Lipid Accumulation in Cells

(39) The level of HCV RNA was evaluated after treating oleic acid (OA) to Huh7 cells transfected with pcDNA3_GRIM19, in order to confirm whether the effect of decrease in HCV RNA by GRIM19 overexpression is associated with lipid accumulation in cells, based on the fact that HCV replication is known to be much affected by lipid in cells.

(40) The degree of lipid accumulated in cells was evaluated by measuring the degree of fluorescence after Nile Red staining. Specifically, after seeding 210.sup.5 Huh7 cells in 6 well plates, the cells were transfected with pcDNA3_GRIM19 after 24 hours. After transfection with pcDNA3_GRIM19, the cells were cultured using serum free DMEM media. After 24 hours, the cells transfected with pcDNA3_GRIM19 were treated with 100 M of oleic acid (OA) using serum free DMEM media supplemented with 1% BSA. After 24 hours, the amount of lipid in cells, increased by OA treatment, was evaluated through Nile Red staining. The cells in which pcDNA3_GRIM19 was overexpressed and OA treatment was completed were fixed using 3.7% paraformaldehyde, and the lipid in cells was subjected to fluorescence staining using PBS solution including 1 g/ml of Nile Red. The nucleus was stained using 0.2 g/ml of DAPI in order to predict the degree of lipid accumulation for the number of cells. The degree of lipid accumulation in cells was evaluated through the intensity of fluorescence measured by a microplate reader, and the degree of lipid accumulation was relatively compared and analyzed through the degree of DAPI.

(41) As a result, with reference to FIG. 6a, it was confirmed that in the case of GRIM19 overexpression due to transfection with pcDNA3_GRIM19, the degree of lipid accumulated in Huh7 cells was decreased.

(42) Also, with reference to FIG. 6b, it was confirmed that the level of HCV RNA decreased by GRIM19 overexpression due to transfection with pcDNA3_GRIM19 (Example 3) was restored through treatment with oleic acid (OA). From this, it was proved that decrease in the level of HCV RNA by GRIM19 overexpression is associated with lipid accumulation in cells.

EXAMPLE 5

Confirmation of Mechanism of Lipid Decrease in Cells by GRIM19 Overexpression

(43) With reference to Example 6 above, in the case of GRIM19 overexpression due to transfection with pcDNA3_GRIM19, the degree of lipid accumulated in Huh7 cells was decreased. In order to confirm such mechanism, a change in expression of proteins involved in fatty acid synthesis was examined including acetyl-CoA carbosylase (Acc), an enzyme promoting fatty acid synthesis in a fatty acid synthesis process.

(44) After seeding 1.510.sup.6 Huh7 cells in 10-cm culture dish, the cells were cultured using serum free DMEM. After 24 hours, the cells were transfected with pcDNA3_GRIM19, and after 48 hours, the cells were harvested, followed by Western blot analysis. The harvested cells were treated with PRO-PREP Protein Extraction Solution of INTRON BIOTECHNOLOGY, to obtain protein lysates. 30 g of protein lysates was separated according to size through 8% SDS-PAGE and then transferred to nitrocellulose membrane, and the expression level of ACC was evaluated using ACC specific antibody. As an anti-ACC antibody, a product purchased from Cell Signaling was used. The expression level of protein was visualized through chemiluminescence system (Amersham Pharmacia Biotech). The expression level of ACC was relatively compared by analyzing the density between the visualized ACC proteins bands and loading control -actin band.

(45) With reference to FIG. 7a and FIG. 7b, it was confirmed that the GRIM19 overexpression due to transfection with pcDNA3_GRIM19 decreases the expression of acetyl-CoA carbosylase.

(46) It was reported that when infected with hepatitis C virus, lipid accumulation in cells was observed, and that in such a case, the activity of SIRT1 is decreased, and accordingly, the activity of AMPK is also decreased, which results in an increase of the activity of ACC and promotion of production of lipid in cells. With reference to FIG. 7a, it was confirmed that the activity of ACC was decreased, whereas the activities of SIRT1 and AMPK were also decreased, which is due to GRIM19 overexpression.

(47) From this, it was proved that the expression level of ACC, among main enzymes involved in lipid metabolism in cells by HCV, is decreased by GRIM19 overexpression, and accordingly, lipid in cells is decreased.

EXAMPLE 6

Synergistic Effect of Inhibiting HCV Through GRIM19 Overexpression and a Combination Treatment of Acc Activity Inhibitor

(48) From Example 5 above, it was confirmed that the expression of acetyl-CoA carbosylase (Acc) was decreased when GRIM19 was overexpressed. In this regard, a change in HCV RNA copies was examined in the case of administration of metformin (Sigama Aldrich, Cat. D150959), an activator of AMP-activated protein kinase (AMPK) inhibiting the activity of ACC, in combination.

(49) With reference to FIG. 8, the case of transfection with pcDNA3_GRIM19 to overexpress GRIM19 and treatment with metformin leads to a considerably lower increase in HCV RNA than the case of treatment with metformin alone. Thus, it was confirmed that the combination thereof has a synergistic effect of inhibiting HCV.

(50) From this, it was proved that the regulation of fatty acid synthesis including AMPK and Acc protein is involved in the mechanism of decrease in the level of HCV RNA by GRIM19 overexpression.

EXAMPLE 7

Synergistic Effect of Inhibiting HCV Through GRIM19 Overexpression and a Combination Treatment of DGAT-1 Inhibitor

(51) As a result of simultaneous treatment of 40 M of a compound (A922500, MERK) of Formula 1:

(52) ##STR00005##
an inhibitor of DGAT-1, with cells where GRIM19 is overexpressed in HCVcc infected Huh7 cells, based on the fact that HCV replication complex is formed dependent on lipid membrane, and that lipid droplets play an important role in formation of HCV particles, it was confirmed that a synergistic effect of inhibiting HCV replication was shown (FIG. 9).

EXAMPLE 8

Synergistic Effect of Inhibiting HCV Through Treatment of Compound Inhibiting RNA-Dependent RNA Polymerase Activity in In Vitro HCV Infection Model

(53) HCVcc infected Huh7 cells were transfected with pcDNA3_GRIM19 using FUGENE HD, as in Example 3 above, the cells were treated with sofosbuvir (PharmaBLOCK R&D Co. Ltd.) (Formula 2):

(54) ##STR00006##
a compound inhibiting RNA-dependent RNA polymerase activity of HCV, or the cells were transfected with pcDNA3_GRIM19 and treated with sofosbuvir. Then, a change in HCV RNA copies was evaluated.

(55) With reference to FIG. 10, it was confirmed that for all the cases excluding the control group treated with dimethyl sulfoxide (DMSO), the level of HCV RNA copies was decreased till 3.sup.rd day, and then increased again. Further, it was confirmed that the case of transfection with pcDNA3_GRIM19 and treatment with sofosbuvir leads to a lower increase in HCV RNA than the case of treatment with sofosbuvir alone, and thus the combination thereof has a synergistic effect of inhibiting HCV, and that a considerably lower increase of about 45% or more was observed.

EXAMPLE 9

Confirmation of Domains Having Anti-HCV Effect in GRIM19 Protein

(56) GRIM19 protein consists of 144 amino acids. The protein was fragmented into four domains as shown in Table 1 below to confirm domains having anti-HCV effect (FIG. 11a).

(57) TABLE-US-00003 TABLE3 Domain Aminoacidsequence Genesequence(5.fwdarw.3) GRIM19-D1 MAASKVKQDMPPPGG atggcggcgtcaaaggtgaagcaggacatgcctccgccg (aa1-36) YGPIDYKRNLPRRGLS gggggctatgggcccatcgactacaaacggaacttgccg GYSML cgtcgaggactgtcgggctacagcatgctg (SEQIDNO:6) (SEQIDNO:7) GRIM19-D2 MAIGIGTLIYGHWSIM atggccatagggattggaaccctgatctacgggcactgga (aa37-72) KWNRERRRLQIEDFEA gcataatgaagtggaaccgtgagcgcaggcgcctacaaa RIALL tcgaggacttcgaggctcgcatcgcgctgttg (SEQIDNO:8) (SEQIDNO:9) GRIM19-D3 MPLLQAETDRRTLQM atgccactgttacaggcagaaaccgaccggaggaccttg (aa73-101) LRENLEEEAIIMKDV cagatgcttcgggagaacctggaggaggaggccatcatc (SEQIDNO:10) atgaaggacgtg(SEQIDNO:11) GRIM19-D4 MDWKVGESVFHTTRW atgcccgactggaaggtgggggagtctgtgttccacacaa (aa102-144) VPPLIGELYGLRTTEEA cccgctgggtgccccccttgatcggggagctgtacgggct LHASHGFMWYT gcgcaccacagaggaggctctccatgccagccacggctt (SEQIDNO:12) catgtggtacacg(SEQIDNO:13)

(58) An expression vector which can express the GRIM19-D2 (aa 1-36), GRIM19-D2 (aa 37-72), GRIM19-D3 (aa 73-101), and GRIM19-D4 (aa 102-144) above was prepared, and the effect of each domain was evaluated in in vitro HCV infection model in Example 2.

(59) In order to prepare the expression vector, the gene for each domain of GRIM19 was obtained through PCR with template of the vector expressing pcDNA3_GRIM19, and each domain was subcloned into pCMV-3Tag-3A vector (Agilent Technologies, Inc.) using restriction sites of restriction enzymes EcoRI and SalI. For GRIM19-D2, GRIM19-D3, and GRIM19-D4 to be expressed in the vector, a start codon was added prior to 5 of the actually corresponding gene sequence of GRIM19. Accordingly, for each protein fragment, methionine was added prior to the N terminus of the actually corresponding amino acid sequence of GRIM19.

(60) With reference to FIG. 11b, it was confirmed that in the case of overexpression of GRIM19-D1, GRIM19-D2, GRIM19-D3, and GRIM19-D4 domains due to transfection with expression vector that can express each of the prepared domains in in vitro HCV infection model, the level of HCV RNA was significantly decreased in all the domains. Further, it was confirmed that in the case of overexpression of each domain, the level of HCV RNA was significantly decreased, as compared with the case of expression of GRIM19 protein.

(61) Accordingly, it was proved that in the case of the expression of each of four domains of GRIM19, as well as GRIM19 protein, excellent anti-HCV activity was achieved.

EXAMPLE 10

Confirmation of Anti-HCV Effect of Each Domain of GRIM19 Protein

(62) In order to confirm whether an anti-HCV effect is shown when penetrating each domain itself of GRIM19 protein into cells, the effect was evaluated in in vitro HCV infection model by synthesizing peptides in which cell permeable peptide sequences (CP) bind to N-terminal of each domain of GRIM19 of Example 8. The sequences of each domain of GRIM19 comprising the synthesized cell permeable peptide sequences (CP) were analyzed in Table 4 below.

(63) TABLE-US-00004 TABLE4 Aminoacidsequence CP-GRIM19-D1 RRRRRRRRRMAASKVKQDMPPPGGYGPIDYKRNL PRRGLSGYSML(SEQIDNO:14) CP-GRIM19-D2 RRRRRRRRRAIGIGTLIYGHWSIMKWNRERRRLQ IEDFEARIALL(SEQIDNO:15) CP-GRIM19-D3 RRRRRRRRRPLLQAETDRRTLQMLRENLEEEAII MKDV(SEQIDNO:16)

(64) After treating Huh7 cells infected with HCVcc through in vitro HCV infection model of Example 2 with 10 M of CP-GRIM19-D1, CP-GRIM19-D2, and CP-GRIM19-D3 for 48 hours, the level of RNA in HCV was examined.

(65) With reference to FIG. 12, it was confirmed that the level of HCV RNA was significantly decreased in HCV infected cells when treating the cells with CP-GRIM19-D1, CP-GRIM19-D2, and CP-GRIM19-D3.

(66) Therefore, it was proved that in addition to the case of transfection with GRIM19 protein or domains of GRIM19 protein and expression of them directly in cells, in the case of treatment with GRIM19 protein or domains of GRIM19 protein directly out of cells, an excellent anti-HCV effect is achieved.

(67) The present invention was conducted by the following research and development program support.

(68) 1) Support institute: Korea Seoul St. Mary's Hospital

(69) 2) Project: Preparation of corresponding project such as clinics research funds, excellent researcher's project

(70) 3) Task: Research on inhibition of hepatitis C virus replication using STAT3 activity inhibitor

(71) 4) Chief of research: YOON, Seung Kew

(72) 5) Research period: Nov. 1, 2013 to Oct. 31, 2014

INDUSTRIAL AVAILABILITY

(73) The pharmaceutical composition for preventing or treating a hepatitis C virus infectious disease or the antiviral composition against hepatitis C virus of the present invention, comprising GRIM19 protein or a fragment thereof, and a gene encoding the protein or the fragment thereof, can treat patients on whom the standard-of-care treatment for hepatitis C virus does not work, and treat or prevent a hepatitis C virus infectious diseases by inhibiting replication of hepatitis C virus, irrespective of genotype, thereby being useable in the field of treatment of hepatitis C.