KR20150130669A - Pharmaceutical composition for prevention and treatment for Hepatitis C containing Abi1 expression or activity inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating hepatitis C and, more specifically, to a composition containing an inhibitor suppressing the expression or activation of Abelson interactor 1 (Abi1). As a result of identifying cell proteins interacting with a HCV NS5A protein by using a protein microarray technique by inventors in the present invention, about 90 cell proteins were identified to interact with the HCV NS5A protein. Abi1 was selected from the partners of the NS5A. The binding between the HCV NS5A and Abi1 was confirmed by using an in vitro pull-down assay and a common precipitation analysis. Abi1 is essential for replication of HCV. In addition, EGF-induced Erk and Efr1 activation was inhibited by the NS5A, and the inhibitory effect was mediated by the Abi1 protein. Knockdown of the Abi1 expression impaired the replication of HCV, while overexpression of the Abi1 increased proliferation of HCV. In summary, these results support that HCV forces the Abi1 in host cells to modulate a MEK/ERK signaling pathway for proliferation. Therefore, a substance inhibiting the activation or expression of the Abi1 is useful as a prophylactic agent or a therapeutic agent for hepatitis C by inhibiting the proliferation of HCV.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, comprising an Abi1 expression or an activity inhibitor,

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, and more particularly to a composition comprising Abi1 (Abelson interactor 1) expression or activity inhibitor.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV is a transmembrane virus with a positive-sense, single-stranded RNA genome. HCV belongs to Flaviviridae family and genus Hepacivirus (1). The HCV genome is approximately 9.6 kb in length and encodes 3000 amino acid proteins from a single large open reading frame. This polyprotein precursor is cleaved by host cell proteases and viral proteins to produce three structural proteins (core, E1 and E2) and seven nonstructural proteins (p7, NS2 to NS5B) (2). While these structural proteins are components of virions, nonstructural proteins are involved in the replication of the viral genome. NS5A (Nonstructural 5A) is a multifunctional protein that interacts with many cellular proteins to regulate cell signaling pathways and virus proliferation. NS5A contains three matching proline-rich PXXP motifs that bind to the SH3 domain. These motifs are commonly found in many cell signaling molecules (3). NS5A modulates cell growth and activation by modulating the MAPK signaling pathway. NS5A specifically interacts with the two SH3 domains of growth factor receptor-bound protein 2 (Grb2) and inhibits the Erk MAPK pathway (4). The NS5A mutation in the C-terminal proline-rich motif did not interact with Grb2 nor blocked ERK phosphorylation stimulated by EGF (5-7). Furthermore, NS5A expressed in recombinant HSV-infected cells also interacted with Grb2 and inhibited the Erk1 / 2 signaling pathway (8). These data indicate that the NS5A regulates the MAPK path through Grb2. However, the exact mechanism by which NS5A inhibits Grb2 function has not been elucidated.

Abi1 (Abelson interactor 1), also known as E3B1, is an adapter protein in the receptor tyrosine kinase (RTK) signaling pathway. This protein contains three well-conserved domains: the homeo-domain homologous region (HHR), the proline-rich (PR) domain and SH3 (Src homology 3) .9 Abi1 binds to Eps8 and Sos1 It is also important to regulate actin rearrangement through RTKs to activate Ras and to form Rac-induced membrane wrinkles (10-13). Abi1 is also involved in cell proliferation, cell junctioning and cell migration regulation Abi1 is known to regulate EGF-mediated ERK pathway along with cytoskeletal rearrangement and lamellipodia formation during metastasis (16-18). Abi1 overexpression is mediated by EGF Induced Erk activation, but did not inhibit c-Jun N-terminal kinase or Akt activation. (14) The HCV NS5A protein interacts with Grb2 and does not inhibit Grb2-Sos interaction, Disturbing mediated Ras activation It should inhibit ERK1 / 2 activation and is further mediated by EGF already known (3,4).

1. Giannini C, Brechot C. 2003. Hepatitis C virus biology. Cell Death Differ. 10 (Suppl 1): S27-S38. 2. Moradpour D, Penin F, Rice CM. 2007. Replication of hepatitis C virus. Nat. Rev. Microbiol. 5: 453-463. 3. Tan SL, Nakao H, He Y, Vijaysri S, Neddermann P, Jacobs BL, Mayer BJ, Katze MG. 1999. NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2, a Src homology 3 domain / ligand-dependent manner and perturbs mitogenic signaling. Proc. Natl. Acad. Sci. U.S.A. 96: 5533-5538. 4. He Y, Nakao H, Tan SL, Polyak SJ, Neddermann P, Vijaysri S, Jacobs BL, Katze MG. 2002. Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase. J. Virol. 76: 9207-9217. 5. Macdonald, Crowder K, Street A, McCormick C, Saksela K, Harris M. 2003. Hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-ERK pathway signaling. J. Biol. Chem. 278: 17775-17784. 6. Macdonald A, Chan JKY, Harris M. 2005. Perturbation of epidermal growth factor receptor complex formation and Ras signaling in cells harboring the hepatitis C virus subgenomic replicon. J. Gen. Virol. 86: 1027-1033. 7. Mankouri J, Griffin S, Harris M. 2008. Hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor. Traffic 9: 1497-1509. 8. Georgopouloui, Caravokiri K, Mavromara P. 2003. Suppression of the ERK1 / 2 signaling pathway from HCV NS5A protein expressed by herpes simplex recombinant viruses. Arch. Virol. 148: 237-251. 9. Jenei V, Andersson T, Jakus J, Dib K. 2005. E3B1, a human homologue of the mouse gene product Abi-1, sensitizing activation of Rap1 in response to epidermal growth factor. Exp. Cell. Res. 310: 463-473. 10. Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C, Di Fiore PP. 1999. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401: 290-293. 11. Lanzetti L, Rybin V, Malabarba MG, Christoforidis S, Scita G, Zerial M, Di Fiore PP. 2000. Eps8 protein coordinates EGF receptor signaling through Rac and trafficking through Rab5. Nature 408: 374-377. 12. Innocenti M, Tenca P, Frittoli E, Faretta M, Tocchettia, Di Fiore PP, Scita G. 2002. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J. Cell. Biol. 156: 125-136. 13. Chen H, Wu X, Pan ZK, Huang S. 2010. Integrity of SOS1 / EPS8 / ABI1 tri-complex determined ovarian cancer metastasis. Cancer. Res. 70: 9979-9990. 14. Fan PD, Goff SP. 2000. Abl interactor 1 binds to sos and inhibits epidermal growth factor- and v-Abl-induced activation of extracellular signal-regulated kinases. Mol. Cell. Biol. 20: 7591-7601. 15. Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, Di Fiore PP, Scita G. 2003. Phosphoinositide 3-kinase activates Eps8, Abi1, and Sos-1. J. Cell. Biol. 160: 17-23. 16. Innocenti M, Zucconia, Disanza A, Frittoli E, Areces LB, Steffen A, Stradal TE, Di Fiore PP, Carlier MF, Scita G. 2004. Abi1 is essential for the formation and activation of a WAVE2 signaling complex. Nat. Cell. Biol. 6: 319-327. 17. Cui M, Yu W, Dong J, Chen J, Zhang X, Liu Y. 2010. Downregulation of ABI 1 expression affects the progression and prognosis of human gastric carcinoma. Med. Oncol. 27: 632-639. 18. Kotula L. 2012. Abi1, a critical molecule coordinating actin cytoskeleton reorganization with PI-3 kinase and growth signaling. FEBS Lett. 586: 2790-2794. 19. Choi SH, Hwang SB. 2006. Modulation of the transforming growth factor-beta signal transduction pathway by hepatitis C virus nonstructural 5A protein. J. Biol. Chem. 281: 7468-7478. 20. Park CY, Choi SH, Kang SM, Kang JI, Ahn BY, Kim H, Jung G, Choi KY, Hwang SB. 2009. Nonstructural 5A protein activates beta-catenin signaling cascades: implication of hepatitis C virus-induced liver pathogenesis. J. Hepatol. 51: 853-864. 21. Lim YS, Tran HT, Park SJ, Yim SA, Hwang SB. 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation. J. Virol. 85: 8777-8788. 22. Ngo TTH, Pham LV, Kim JW, Lim YS, Hwang SB. 2013. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein. J. Virol. 87: 5718-5731. 23. Park KJ, Choi SH, Choi DH, Park JM, Yie SW, Lee SY, Hwang SB. 2003. Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2. J. Biol. Chem. 278: 30711-30718. 24. Nanda SK, Herion D, Liang TJ. 2006. The SH3 binding motif of HCV NS5A protein interacts with Bin1 and apoptosis and infectivity. Gastroenterology 130: 794-809. 25. Penin F, Brass V, Appel N, Ramboarina S, Montserret R, Ficheux D, Blum HE, Bartenschlager R, Moradpour D. 2004. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 279: 40835-40843. 26. Biesova Z, Piccoli C, Wong WT. 1997. Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogen 14: 233-241. 27. NiggEA. 1993. Cellular substrates of p34 (cdc2) and its companion cyclin-dependent kinases. Trends Cell Biol. 3: 296-301. 28. Clark-Lewis I, Sanghera JS, and Pelechnll SL. 1991. Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. J. Biol. Chem. 266: 15180-15184. 29. Gretton S, Hughes M, Harris M. 2010. Hepatitis C virus RNA replication is regulated by Ras-Erk signalalling. J. Gen. Virol. 91: 671-680. 30. Ndjomou J, Park IW, Liu Y, Mayo LD, He JJ. 2009. Up-regulation of hepatitis C virus replication and production by inhibition of MEK / ERK signaling. PLoS One 4: e7498

The present invention aims at solving the problem that there is no effective therapeutic agent for hepatitis C and providing an effective preventive and therapeutic agent for hepatitis C infection.

The HCV life cycle is highly dependent on host cell machinery. HCV NS5A interacts with many cellular proteins that regulate cell signaling pathways and virus proliferation. In the present invention, the inventors described intracellular Abi1 as a protein that interacts with NS5A. NS5A regulates MEK / ERK signaling mediated by epithelial growth factor through Abi1 protein. Abi1 expression knockdown impaired HCV replication, whereas Abi1 overexpression increased HCV proliferation. These data suggest that HCV may illegally use intracellular Abi1 to modulate the MEK / ERK signaling pathway for its own proliferation and thus the MEK / ERK pathway may be a potential target for anti-HCV therapy .

The inventors of the present invention have identified cell proteins that interact with the HCV NS5A protein using protein microarray technology and have identified about 90 cell proteins that interact with HCV NS5A. Of these NS5A partners, Abi1 was chosen. The binding of HCV NS5A to Abi1 was demonstrated using in vitro pull down and common precipitation assays. Abi1 is essential for HCV replication. In addition, EGF-stimulated Erk and Egr1 activation was inhibited by NS5A, and this inhibitory effect was mediated by the Abi1 protein. Taken together, these results support the hypothesis that HCV inhibits the MEK / ERK signaling pathway for its own proliferation by forcing Abi1 in host cells.

The HCV NS5A protein is a membrane-associated protein that is an essential component of the viral replication complex. NS5A is known to interact with a variety of host proteins that regulate cell growth and cell signaling pathways. To identify cellular factors essential for HCV proliferation, we performed protein microarray screening using the NS5A protein as a probe. Of the approximately 9,000 human proteins, about 90 cell proteins have been identified as new NS5A interactors. Because Abi1 is an adapter protein in the RTK signaling pathway and plays a crucial role in actin rearrangement and cell proliferation (10,14,15), we have investigated the possible involvement of the NS5A protein in the EGF-mediated Abi1 signaling pathway.

We first identified protein interactions between NS5A and Abi1 by in vitro binding and common immunoprecipitation assays. HCV NS5A interacted with endogenous Abi1 in the context of HCV replication. Further, we have demonstrated that NS5A and Abi1 proteins are located in the same cell in the cytoplasm of Jc1-infected cells, and NS5A interacts with Abi1 by having an N-terminal amphipathic spiral through part II of Abi1 and NS5A domain I . Domain I of NS5A contains not only membrane localization but also a portion of the membrane that is essential for HCV replication (25). Membrane targeting of NS5A has been reported to be essential for inhibition of the EGF-mediated Ras-Erk signaling pathway (7). Deletion of N-terminal 32 residues in NS5A failed to block the expression of AP-1 induced luciferase, which is stimulated by EGF (7). On the other hand, Abi1 moiety II is rich in serine and threonine and thus targets serine / threonine kinase (26). There are three putative phosphorylation sites (S / T-P-X-K / R) in cyclin-dependent kinases (27) and seven putative phosphorylation sites (S / T-P) in MAP kinases. Therefore, the present inventors have assumed that the protein interaction between Abi1 and NS5A is involved in the MAPK / ERK signaling pathway.

Signaling from activated EGFR activates a number of downstream targets, including Grb2 and Eps8. It has been reported that the HCV NS5A protein interacts with Grb2 and inhibits the Ras-Erk pathway stimulated by EGF without disturbing the Grb2-Sos interaction (6). In the present invention, the present inventors have found that NS5A interacts with Abi1 and inhibits EGF-stimulated Egr1 promoter activity. This inhibition disappeared without Abi1, which means that NS5A regulates EGF signaling through the Abi1 protein. It is noteworthy that NS5A inhibits EGF-mediated signal transduction without disturbing Abi1-Sos1 interactions. We have shown that the NS5A protein modulates EGF-mediated signal transduction by the NS5A protein by forming a complex of three with Abi1 and Sos1.

How can NS5A disrupt the EGF signaling pathway in two different ways? In fact, Abi1 and Grb2 compete for the same binding site VPVPPPVPPRRR on Sos1 (12). Sos1 can act as a guanine nucleotide exchange factor (GEF) for Ras (by binding to Grb2) and Rac (as part of the Eps8 / Abi1 / PI3K complex). When Abi1 was overexpressed, Sos1 did not bind to Grb2 and could not activate Ras, thus inhibiting the Ras / Raf / MAPK / ERK signaling pathway involved in GEF-Ras stimulated cell proliferation. Instead, Sos1 complexes with Eps8 / Abi1, and GEF-Rac can activate Rac to activate actin polymerization (12,15). Thus, as a component of the Abi1 complex consisting of four, NS5A appears to be able to down-regulate EGF-induced ERK activation. The present inventors predicted that NS5A at different stages of the HCV life cycle would differently modulate EGF signaling by unauthorized use of Grb2 or Abi1. However, further study is needed to elucidate the detailed mechanism of the different regulation of NS5A in EGF signaling.

Ras-Erk signaling pathway disruption appears to be a mechanism to regulate the level of HCV RNA replication. Although low Ras-Erk signaling activity is essential for HCV RNA replication, complete inhibition of Ras-Erk signaling has been reported to inhibit replication (29). Other researchers have also reported that MEK / ERK signaling inhibition augments HCV proliferation (30). The present inventors have found that Abi1 silencing results in enhanced ERK activation. HCV NS5A could not inhibit the EGF signaling pathway in the absence of Abi1. This implies that protein interactions between Abi1 and NS5A are essential for inhibiting MEK / ERK signaling. Finally, the present inventors have examined the possibility of Abi1 association in HCV proliferation. We have shown that siRNA-mediated Abi1 knockdown reduces intracellular HCV RNA levels, HCV protein levels and HCV infectivity. In addition, exogenous expression of Abi1 in Abi1 knockdown cells restored HCV protein levels. In fact, Abi1 overexpression in HCV-infected cells markedly increased JFH1-luc luciferase activity and HCV protein levels. Taken together, HCV has used Abi1 in host cells for its replication, and thus MEK / ERK signaling modulation by NS5A appears to contribute to HCV infection.

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C comprising an Abi1 (Abelson interactor 1) expression or activity inhibitor.

The present invention also relates to a method for producing a C-type antibody, which is characterized in that the Abi1 expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementary to mRNA of the Abi1 gene, short RNA (shRNA) and short interfering RNA To a pharmaceutical composition for preventing and treating hepatitis.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating hepatitis C, wherein the siRNA is SEQ ID NO: 1 to 4. However, since it is easy for a person skilled in the art to produce siRNA targeting Abi1 in addition to SEQ ID NOS: 1, 2, 3 and 4, the siRNAs of SEQ ID NOS: 1, 2, 3 and 4 It is obvious that it is not limited to the number of times.

In addition, the present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, wherein the Abi1 activity inhibitor is an anti-Abi1 antibody binding to Abi1 protein.

The composition for the prevention and treatment of hepatitis C infection comprising the Abi1 protein expression or activity inhibitor as an active ingredient of the present invention contains the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the composition.

The composition of the present invention may contain at least one active ingredient which exhibits the same or similar function to the Abi1 protein expression or activity inhibitor. The composition of the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.

In the present invention, the inhibitor for Abi1 may be an antisense oligonucleotide for the mRNA of the gene.

Antisense oligonucleotides have been used successfully to achieve gene-specific inhibition both in vivo as well as in vitro. Antisense nucleotides are short length DNA synthesis strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target. Antisense oligonucleotides have been proposed to inhibit the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping expression at the transcription, translation or splicing step. Antisense oligonucleotides have also been successfully used in animal models of cell culture and disease (Hogrefe, 1999). Other variations of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. The antisense oligonucleotides used herein include double stranded or single stranded DNA, double stranded or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and oligonucleotides with base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of oligonucleotide backbones known in the art, modifications of sugar moieties, or modifications of bases.

In addition, the inhibitor for Abi1 may be siRNA (Small Interfering RNA) of the gene.

That is, the nucleotide sequence of the siRNA may be a sequence of 19 to 23 contiguous nucleotides of the nucleotide sequence of the Abi1 gene (mRNA).

The sequence of the siRNA represents a sense strand for convenience, and constitutes a double ribonucleic acid chain together with an antisense strand indicating an actual gene suppression effect.

siRNA has the potential to become a very potent drug against the inhibition of the expression of specific genes in vivo in terms of long-lasting effects in cell culture and in vivo, the ability to transfect cells in vivo, and resistance to serum degradation (Bertrand et al., 2002). Expression constructs / vectors including siRNA delivery and siRNA are known to those skilled in the art. For example, U.S. Patent Application Nos. 2004/106567 and 2004/0086884 disclose delivery mechanisms including viral vectors, non-viral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, / Vector.

siRNAs have been proposed as an alternative to antisense oligonucleotides because they can achieve equivalent or even higher effects than those obtained by antisense oligonucleotides at relatively low concentrations (Thompson, 2002). The use of siRNAs has been shown to inhibit gene expression in animal models of disease. Those skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs as desired using methods known in the art (Andreas Henschel, Frank Buchholzl and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32 (Web Server Issue): W113-W120 Reference). Also, those skilled in the art are well aware of regulatory sequences (e.g., constitutive promoters, inducible promoters, tissue-specific promoters, or combinations thereof) useful in expression constructs / vectors with antisense oligonucleotides or siRNA.

The antisense oligonucleotides or siRNA of the present invention used for the treatment of hepatitis C can be administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, sustained or delayed release of the active ingredient after administration.

The Abi1 gene inhibitor may be any substance that inhibits the expression of the gene in addition to the antisense oligonucleotide or siRNA. Therefore, compounds known as inhibitors of the genes in the art are also available.

The present invention also provides a composition for treating hepatitis C comprising an inhibitor against a protein expressed from Abi1 gene. When the gene of the present invention is inhibited, expression of the protein is inhibited and HCV proliferation is inhibited. Therefore, inhibition of the protein of the gene can inhibit HCV proliferation.

Accordingly, the present invention provides a therapeutic use of an inhibitor of the above-mentioned protein for hepatitis C virus. In the present invention, treatment of hepatitis C includes prevention and inhibition of hepatitis C virus.

The inhibitor for the protein may be an antibody to the Abi1 protein expressed from the gene of the present invention. The monoclonal antibody to the protein may be prepared by using a monoclonal antibody production method as is customary in the art, or a commercially available monoclonal antibody may be used. In addition, a polyclonal antibody recognizing the protein may be used in place of the monoclonal antibody, or it may be produced and used through an antiserum preparation method customary in the art.

The composition for treating hepatitis C of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. When the inhibitor for the protein of the present invention is an antibody, the pharmaceutically acceptable carrier may be composed of a minimum amount of auxiliary substance such as a wetting agent or an emulsifying agent, an antiseptic or a buffer to increase the shelf life or effectiveness of the binding protein.

In addition, the composition for treating hepatitis C of the present invention can be used together with one or more therapeutic agents for hepatitis C virus. The composition for treating hepatitis C of the present invention may further comprise a chemotherapeutic agent well known to those skilled in the art.

The composition for treating hepatitis C of the present invention may include pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective components. Examples of such adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a solubilizing agent.

In addition, the composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. 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. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical preparation form of the hepatitis C preventive and therapeutic composition of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, Release formulations, and the like.

The composition for the prevention and treatment of hepatitis C of the present invention can be administered orally or parenterally by intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, Lt; RTI ID = 0.0 > route. ≪ / RTI >

In the treatment method of the present invention, "effective amount" means an amount required for achieving the effect of inhibiting hepatitis C infection. Thus, the "effective amount" of the active ingredient of the present invention will vary depending upon factors such as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, The dosage, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like. In the case of an adult, 0.01 ng / kg to 10 mg / kg in the case of siRNA and 0.01 ng / kg to 10 mg / kg in the case of antisense oligonucleotides to mRNA of the gene when the gene or protein inhibitor is administered once to several times a day, Kg to 10 mg / kg in the case of a compound, and 0.1 ng / kg to 10 mg / kg in the case of a monoclonal antibody against the protein.

In the present invention, the Abi1 gene may be a base sequence in which one or more bases of a known base sequence are deleted, substituted or inserted.

It is apparent to those skilled in the art that the siRNA sequence of the Abi1 gene is not limited to the examples. Sequences having substantial sequence identity or substantial sequence homology to the sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence identity" is used to denote that the sequence represents substantial structural or functional identity with another sequence. These differences are due, for example, to inherent variations in the codon usage between different species. If there is a significant amount of sequence redundancy or similarity between two or more different sequences, the structural differences will be negligible if they have similar physical properties even if the length or structure of these sequences is different.

(2001)) and Frederick et al. (Frederick et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (Ausubel et al., Current Protocols in Molecular Biology Volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

The composition of the present invention may be used alone or in combination with radiation therapy, chemotherapy, and a method using a biological response modifier.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

According to the present invention, Abi1 knockdown impairs HCV proliferation, whereas overexpression of Abi1 promotes HCV proliferation.

Accordingly, the Abi1 activity or expression inhibiting substance or method of the present invention is useful as a preventive and therapeutic agent for hepatitis C, or a method for preventing and treating hepatitis by inhibiting the proliferation of HCV.

1A is a schematic diagram showing the construction of an NS5A expression plasmid as a V5-His fusion protein.
The left panel of FIG. 1B shows the NS5A-V5-His fusion protein expressed in E. coli was purified and stained with Coomassie blue. The right panel was obtained by immunoblotting purified NS5A with NS5A antibody.
Figure 1C shows the purified NS5A protein verified with anti-V5 and anti-NS5A antibodies. The purified NS5A protein was also immunoprecipitated with the anti-V5 antibody and immunoblotted with the NS5A antibody.
Figure 1D shows the results of applying the purified NS5A protein onto the spot of a human protein microarray kit. NS5A interactors were determined by measuring the average spot intensity with a Z-score of 3 or greater as the cutoff value.
2A through 2E show that Abi1 interacts with NS5A in both in vitro and in vivo.
2A: HEK293T cells were transiently transfected with Flag-labeled Abi1 plasmid. Total cell lysates were collected 48 hours later and incubated with GST or GST-NS5A protein. The bound protein was precipitated with glutathione sepharose beads and detected by immunoblotting with anti-Flag monoclonal antibody. Protein expression of GST and GST-NS5A fusion proteins was confirmed by immunoblot analysis using anti-GST antibodies.
Figure 2B: HEK293T cells were transfected with Myc-tagged NS5A or Flag-labeled Abi1 expression plasmids or co-transfected with two plasmids. After 48 hours, the cell lysate was immunoprecipitated with anti-Myc monoclonal antibody and the bound protein was detected by immunoblot analysis with anti-Flag monoclonal antibody (middle panel). Immunoprecipitation efficiency was confirmed by immunoblot analysis using anti-Myc antibodies (bottom panel).
Figure 2C: Total cell lysates collected from HCV replicon cells were immunoprecipitated with IgG or anti-NS5A antibodies. Binding proteins were detected by immunoblotting with anti-AbI antibodies.
2D: Huh7.5 cells were electroporated with 10 [mu] g Jc1 RNA and incubated for 3 days. Cell lysates were immunoprecipitated with IgG or anti-NS5A antibodies and the bound proteins were detected by immunoblotting with anti-AbI1 antibody (top panel). Immunoprecipitation efficiency was confirmed by immunoblot analysis using NS5A antibody (bottom panel).
2E: Huh7.5 cells were infected with Jc1 for four hours. On the second day, the cells were transfected with a plasmid expressing Flag-labeled Abi1. After 24 hours, the cells were fixed with 4% paraformaldehyde and immunofluorescent staining was performed to detect Abi1 (green) using anti-Flag monoclonal antibody and FITC-conjugated goat anti-mouse IgG, Immunofluorescent staining was performed to detect NS5A (red) using an anti-NS5A antibody and a TRITC-conjugated donkey anti-rabbit IgG antibody. Double staining showed yellow fluorescence in the overlapping image, showing Abi1 and NS5A in the same position in the cytoplasm. Cells were back-stained with DAPI (4,6-diamidino-2-phenylindole) to label the nucleus (blue). In the overlapping image, the rectangle is cropped to the right.
3A-3D show that Abi1 interacts with domain I of NS5A through aa 107-253 of Abi1.
Figure 3A: Schematic representation of wild-type and mutant NS5A proteins.
Figure 3B: HEK293T cells were cotransfected with a mutagenic construct of Flag-labeled Abi1 and Myc-labeled NS5A. Total cell lysate was collected after 48 hours and immunoprecipitated with anti-Flag antibody, and the bound protein was immunoblotted with anti-Myc antibody.
Figure 3C: A schematic diagram of wild type and mutant Abi1 proteins.
Figure 3D: HEK293T cells were cotransfected with Myc-labeled NS5A and Flag-labeled Abi1 mutant constructs. NS5A was immunoprecipitated with anti-Myc antibody and the bound protein was immunoblotted with anti-Flag antibody. Protein expression of Myc-tagged NS5A and Flag-labeled Abi1 was confirmed by immunoblotting with anti-Myc and anti-Flag antibodies, respectively, using the same cell lysate.
Figures 4A-4E show that NS5A down-regulates EGF signaling through regulation of Abi1.
4A: Huh7 cells were transfected with negative control siRNA (-) or Abi1 siRNA (+). After 24 hours, the cells were transfected with 2 [mu] g of Myc-labeled NS5A for 24 hours. Cells were treated with 100 ng / ml EGF for 30 min after blocking the feeding for 24 h. After incubation for 8 hours, total cell lysate was immunoblotted with the indicated antibody.
4B: (upper panel) Huh7 cells were transfected with negative control siRNA or Abi1 siRNA and co-transfected 24 h later with 2 ug of Myc-labeled NS5A and 1 ng of Egrl-luc promoter and 0.5 ug of pCH110 . Cells were treated with 100 ng / ml EGF for 30 min after blocking the feeding for 24 h. Egrl promoter activity was measured 8 hours after treatment (bottom panel). Protein expression was measured with the indicated antibodies using the same cell lysate.
Figure 4C: (Left panel) Huh7 cells containing HCV subgenomic replicons were transfected with negative control siRNA (-) or Abi1 siRNA (+). After 48 hours, the cells were treated for 24 hours with no treatment or with 50 ng / ml EGF for 30 minutes. After incubation for 8 hours, the cell lysate was immunoblotted with the indicated antibody. (Middle panel) Huh7 cells containing HCV subgenomic replicon were treated in the same manner as the left panel. Intracellular Abi1 and Egr1 mRNA levels were quantitated by qRT-PCR. (Right panel) Huh7 cells were treated in the same manner as the left panel. Twenty-four hours after transfection, cells were infected with Jcl for four hours. After 48 hours of infection, the cells were blocked for 24 hours and treated with EGF as described above. After 4 hours, intracellular Abi1 and Egrl mRNA levels were quantitated by qRT-PCR. The asterisk indicates a significant difference from the negative control (*, p <0.05; **, p <0.01; ***, p <0.001). The experiment was performed twice. The error bars represent the standard deviation.
Figure 4D: Huh7 cells containing (top panel) HCV subgenomic replicons were transfected with negative control siRNA (-) or Abi1 siRNA (+). After 24 hours, the cells were co-transfected with the Egr1-luc promoter and pCH110. Cells were blocked for 24 h with feeding and treated with 50 ng / ml EGF for 30 min. Egrl promoter activity was measured 8 hours after treatment. (Bottom panel) Protein expression was measured with the indicated antibodies using the same cell lysate.
Figure 4E: Huh7 cells containing the HCV subgenomic replicon were transfected with the indicated siRNA. After 24 hours, the cells were transfected with an empty vector or Flag-labeled Abi1 plasmid for 48 hours. Total cell lysates were collected and immunoblotted with the indicated antibodies. Abi1 siRNA # 1 targets the 3'UTR of Abi1, while Abi1 siRNA # 2 binds to the inner sequence of the Abi1 coding region.
Figure 5 shows the possibility that HCV NS5A forms a complex of three with Abi1 and Sos1. HEK293T cells were co-transfected with Flag-labeled Abi1, Myc-labeled NS5A and HA-labeled Sos1. After 48 hours, the cell lysate was immunoprecipitated with anti-HA antibody, and the bound protein was immunoblotted with anti-Myc antibody and anti-Flag antibody.
6A to 6G show that Abi1 knockdown suppresses HCV proliferation.
6A: Huh7 cells containing HCV subgenomic replicon (genotype 1b) were transfected with 40 nM of indicated siRNA constructs. Total cell lysate was collected after 96 hours and immunoblotted with the indicated antibody. Negative: unrelated siRNA, positive: HCV-specific siRNA, siRNA # 1 and # 2 refer to siRNA targeting different parts of Abi1.
Figure 6B: Total RNA was extracted from siRNA-transfected HCV replicon cells and the levels of intracellular HCV RNA and Abi1 mRNA were quantitated by qRT-PCR. The asterisk indicates a significant difference from the negative control (*, #, p &lt;0.05); (**, ##, p < 0.01). The experiment was performed three times, and the error bars represent the standard deviation from the mean.
6C: Huh7.5 cells were transfected with 40 nM of indicated siRNA constructs. After 48 hours, the cells were infected with Jc1 for four hours. 48 hours after infection with HCV, total cell lysates were collected and immunoblotted with the indicated antibodies.
6D: Huh7.5 cells were transfected with indicated siRNA. After 96 hours, cell viability was measured by MTT assay.
6E: Huh7.5 cells were transfected with indicated siRNA and infected with Jc1 after 48 hours. After 96 hours of siRNA transfection, intracellular HCV RNA and Abi1 mRNA levels were quantitated by qRT-PCR. The asterisk indicates a significant difference from the negative control (*, #, p &lt;0.05); (**, ##, p < 0.01). The experiment was carried out three times, and the error bars represent the standard deviation from the mean.
6F: Huh7.5 cells were transfected with indicated siRNA for 48 hours and then infected with Jc1 for four hours. On the second day, culture supernatants were collected and used to infect uninfected Huh7.5 cells. HCV infectivity was measured by FFU / ml. The experiment was performed three times. The error bars represent the standard deviation from the mean.
Figure 6G: Huh7.5 cells were transfected with indicated siRNAs for 24 hours. Cells were transfected with empty vector or Flag-labeled Abi1 plasmid for 24 h and then infected with Jc1 for 48 h. Total cell lysates were collected and immunoblotted with the indicated antibodies. Abi1 siRNA # 1 targets the 3'UTR of Abi1, while Abi1 siRNA # 2 binds to the inner sequence of the Abi1 coding region.
Figures 7A-7C show that Abi1 overexpression increases HCV proliferation.
7A: Huh7.5 cells were electroporated with 10 [mu] g JFH1-luc RNA. After 48 hours, the cells were transfected with an increasing amount of Flag-labeled Abi1. Luciferase activity was measured 48 hours after transfection. Protein expression of Flag-labeled Abi1 and GAPDH was confirmed with the indicated antibodies using the same cell culture medium (lower panel).
7B: Huh7.5 cells were electroporated with JFHl-luc RNA and co-transfected with the same expression plasmids as described in Figure 7A above. Luciferase activity was measured at the indicated time.
Figure 7C: Huh7.5 cells were transfected with 2 [mu] g of control vector or Flag-labeled Abi1 for 24 hours and then infected with either additive or Jc1 for four hours. On the second day, total cell lysate was immunoblotted with the indicated antibody.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

Plasmid construction

Plasmids expressing the Myc-labeled wild-type NS5A and plasmids expressing the mutants were constructed according to the prior art (19,20). Abi1 coding region was amplified by PCR using total RNA extracted from HEK293T cells. The PCR product was inserted into the Eco RI and Kpn I restriction sites of p3XFlag-CMV10 vector (Sigma Aldrich). The Abi1 mutation was constructed using the same restriction enzyme sites of the p3XFlag-CMV10 plasmid. The Egr1 promoter (-688 to +1) linked to the luciferase gene was provided by Dr. Kang-Wook Kang of Korea University and pMT2-HA-hSOS1 Pier Paolo Di Fiore (Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy).

Cell culture

All cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 5% CO ₂ , 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C. Both interferon-treated cells and HCV subgenomic replicon cells were cultured as in the prior art (21).

Antibody

The Abi1 antibody is Giorgio Scita (Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy). Flag antibodies are from Sigma-Aldrich, GAPDH, Erk1 and Myc antibodies are from Santa Cruz, and p-Erk1 antibodies are from Cell Signaling. The respective antibody receptors for HCV core, NS3 and NS5A were described in the Examples (21).

Microarray  Probe Manufacturing

The HCV NS5A protein expressed in E. coli was purified using Invitrogen Ni-NTA (nitrilotriacetic acid) -agarose beads as directed by the manufacturer. The protein concentration was determined by the Bradford method (Bio-Rad) and the protein was stored at -70 ° C.

Protein Array Screening

Protein microarray screening was performed as in the prior art using version 5.0 of Invitrogen Protoarray (22). Important responses were identified based on the Z-score cut-off value of 3.0.

Immunoprecipitation method

HEK293T cells were cotransfected with 2 [mu] g of Myc-labeled NS5A and 2 [mu] g of Flag-labeled Abi1. The total amount of DNA was adjusted by adding an empty vector. Forty-eight hours later, cells were collected and analyzed for immunoprecipitation (21).

Invitro  Pulldown analysis

About 1 μg of the purified NS5A protein was incubated with 50 μl of glutathion sepharose beads at 4 ° C for 1 hour with gentle stirring. The beads were washed three times with buffer (50 mM Na ₂ HPO ₄ (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1% protein inhibitor cocktail) and incubated at 4 ° C with the cell lysate expressing Flag- Lt; / RTI &gt; Samples were washed five times with lysis buffer and the bound proteins were detected by immunoblot analysis with anti-Flag monoclonal antibody.

HCV Jc1  Generation and virus infection

Infectious HCV was produced by slightly modifying the prior art (22). Briefly, the pFK-Jc1 plasmid was linearized with Mlu I digest cloth and purified with phenol-chloroform extraction. RNA transcripts were prepared using the T7 RiboMAX express large scale RNA production system (Promega) and purified according to the manufacturer's protocol using Ribo EX (Gene All). Ten μg Invitrogen-genomic Jc1 RNA was mixed with 7.5 x ¹⁰⁶ Huh7.5 cells in a 0.4 cm gap cuvette and electroporated at 270 V and 950 μF using a Bio-Rad GenePulser II electroporator. Cells were carefully transferred with complete medium (10% fetal calf serum, 100 U / ml penicillin, 100 ug / ml streptomycin, 2 mM L-glutamine, 1 mM nonessential amino acid and low glucose DMEM containing 10 mM HEPES) Placed on a 150 mm dish. Four days after electroporation, the culture medium was collected and filtered through a 0.45 mm syringe-top filter unit (Millipore) and stored as a virus stock solution. Cells were washed twice with PBS (phosphate-buffered saline) and incubated with HCVcc (cell culture-grown HCV) for 4 hours for virus infection. HCVcc-infected cells were washed again with PBS and cultured in complete medium.

RNA  Interference

(# 1 sense, 5'-CUGUUGUACACUGGUUCAA-3 ', antisense, 5'-UUGAACCAGUGUACAACAG-3'), (# 2 sense, 5'- UCUCUAGCUAGUGUUGCUU-3 ', antisense, 5'- AAGCAACACUAGCUAGAGA- 3 &apos;) and seven generalized negative control siRNAs were purchased from Bioneer. SiRNA (5'-CCUCAAAGAAAAACCAAACUU-3 ') targeting the 5' NTR (nontranslated region) of Jc1 was used as a positive control (21, 22). siRNA transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. siRNA # 1 targets the 3 'UTR of Abi1 and siRNA # 2 targets the coding region of Abi1.

HCV RNA  dose

Both intracellular and extracellular RNA were isolated from HCVcc-infected cells, culture medium or replicon cells using Trizol LS reagent (Invitrogen, Carlsbad, Calif.) And the SuperScript Vilo cDNA synthesis kit (Invitrogen, Carlsbad, Respectively. Quantitative real-time PCR (qRT-PCR) was performed using an iQ5 multicolor real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) under the following conditions: 15 min at 95 ° C, 55 cycles of 20 seconds, and 72 cycles of 20 seconds. The melting curve was drawn by raising the temperature from 60 ° C to 95 ° C by 0.5 ° C every 10 seconds.

virus Potency  decision

Huh7.5 cells that had not been exposed to the virus were seeded at 2 x 10 ⁴ / well with 200 μl of culture medium in 8-well chamber culture slides (Becton Dickinson / Falcon, Franklin Lakes, NJ). After 24 hours of incubation, Huh7.5 cells were inoculated with cell culture medium collected in HCVcc-infected cells, which were sequentially diluted. Two days after inoculation, indirect immunofluorescence analysis was performed on the presence of intracellular core antibodies to measure the number of FFUs (focus-forming units). Each cluster of infected cells identified by staining for NS5A was considered as an infection focus. Virus titers were calculated as FFU per ml.

MTT  analysis

Huh7.5 cells transfected with the indicated siRNAs. After 96 hours, MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide} preparation (Sigma) was added to the cells and cultured at 37 ° C for two hours. Cell viability was determined as in the prior art (22).

Lucifer Reyes  Reporter gene analysis

Huh7.5 cells were transfected with Egrl-Luc and pCH110 plasmid for 24 hours. Cells were blocked for 24 h with nutrient feed and then treated with 100 ng / ml EGF for 30 min. After 8 hours, the cells were collected and subjected to Luciferase analysis as in the prior art (22). The JFH-Luc reporter assay is described in the prior art (21).

Result 1: Using protein arrays HCV NS5A Cell proteins that interact with

To identify cellular proteins that interact with the HCV NS5A protein, an NS5A expression plasmid was constructed as a V5-His fusion protein (Figure 1A). The NS5A protein expressed in E. coli was purified using Ni-NTA agarose beads and identified as NS5A antibody (Fig. 1B). The purified NS5A protein was again confirmed by immunoblot analysis using anti-V5 and anti-NS5A antibodies or immunoprecipitation using anti-V5 antibody (FIG. 1C). The NS5A protein was applied to a spot of human protein microarray kit (Invitrogen) containing approximately 9,000 rodml human protein. Statistically significant candidate interactors were determined by measuring the average spot intensity with Z-score 3 and above as cut-off values (Fig. 1D). Analysis of the scanned array data shows that approximately 90 cellular proteins have been identified as HCV NS5A interactors (Tables 1-5). Abi1 was identified as one of the candidate hits. Both TRAF2 (23) and B1N1 (24) proteins were shown as positive control hits.

Result 2: Abi1 In In Vitro and In Vivo HCV NS5A Lt; / RTI &gt;

Abi1 plays a crucial role by regulating actin polymerization (16-18) and signaling Ras to Rac (10-13) in regulating EGF signaling. Abi1 overexpression inhibits EGF-induced ERK activation (14). NS5A has also been reported to interfere with the MEK / ERK signaling pathway under EGF stimulation through interaction with Grb2 (3,4), but this mechanism is not clear. Thus, the inventors selected Abi1 to demonstrate the possibility that Abi1 may be involved in EGF signal modulation that regulates HCV proliferation. To identify the protein array data, first insert E. coli And an in vitro GST pulldown assay using the GST-NS5A protein purified from the cell lysate expressing Flag-labeled Abi1 was performed. Figure 2A shows that Abi1 selectively binds to the GST-NS5A protein but not the GST protein (middle panel). In order to confirm the binding result with in-vitro, a co-immunoprecipitation assay was performed. HEK293T cells were co-transfected with Myc-tagged NS5A and Flag labeled Abi1, the cell lysate was immunoprecipitated with anti-Myc antibody, and the co-precipitated protein was detected by immunoblot analysis using anti-Flag antibody . Common immunoprecipitation data again confirmed that Abi1 specifically interacts with the NS5A protein (Fig. 2B). Next, we examined whether the HCV NS5A protein interacts with endogenous Abi1 in the context of HCV replication. The cell lysate collected from Huh7 cells containing HCV replicon was immunoprecipitated with anti-NS5A antibody or control IgG, and the bound protein was immunoblotted with anti-AbI antibody. As a result, HCV NS5A interacted with the endogenous Abi1 protein (Fig. 2C). We reaffirmed whether interfering Abi1 and NS5A interact in HCV-infected cells (Figure 2D). These data suggest that NS5A and Abi1 are in the same position in HCV-infected cells. To investigate this possibility, Huh7.5 cells were either mock-infected or infected with Jc1. Two days after infection, cells were transfected with Flag-labeled Abi1 expression plasmid and immunofluorescence analysis was performed. As shown in FIG. 2E, Abi1 and HCV NS5A were in the same position in the cytoplasm as yellow fluorescence in both overlapping images in Jc1 infected cells, but not in the additive cells (data not shown). Taken together, this data suggests that Abi1 specifically interacts with HCV NS5A in both in vitro and in vivo.

Result 3: HCV NS5A The NS5A  Domain I and Abi1 Through Area II of Abi1 Lt; / RTI &gt;

Interaction between Abi1 and the various deletion mutants of NS5A (Figure 3A) was assayed by transfection-based common immunoprecipitation to measure the domains in NS5A binding to Abi1. As shown in Figure 3B, Abi1 interacted with domain I mutations but did not interact with mutations with domains II and III of NS5A. This result clearly indicates that NS5A domain I is responsible for binding to Abi1. Next, the portion of Abi1 binding to NS5A was measured. To this end, the inventors mutated Abi1 into three well-conserved regions (Figure 3C). Zone I contains a syntaxin-binding region (SNARE) and a WAVE binding domain. Zone II includes a homeome-domain-like zone enriched in serine and threonine. Zone III consists of proline-rich zones. Zone IV contains Src-like 3 domains. Using these various mutations of Abi1, the binding site was determined as described above. As in Figure 3D, NS5A interacted with mutations containing region I + II and mutants containing region II + III + IV. However, NS5A did not interact with a mutation containing region III + IV, indicating that NS5A interacts with Abi1 through a portion of Abi1 containing region II (aa 107-253) (Fig. 3D ).

Result 4: NS5A The Abi1 Through EGF  Down regulation of signaling.

It has been previously reported that the HCV NS5A protein inhibits EGF-stimulated activation of the protein kinase pathway that is activated by Ras-ERK cell division promoters (6). Because NS5A interacts with the Abi1 protein, Abi1 was chosen to explain whether it is likely to be involved in the regulation of the EGF signaling pathway. We have shown that overexpression of NS5A inhibits EGF-stimulated ERK activation (Fig. 4A, lane 3). However, NS5A did not inhibit EGF-stimulated ERK activation in Abi1 knockdown cells (Fig. 4A, lane 4). In the EGF signaling pathway, EGF-stimulated phosphorylated-ERK migrated to the nucleus and then activated Egrl (early growth response protein 1) promoter activity. Egr1 promoter activity was analyzed in Abi1 knockdown cells to reaffirm Abi1's association with the inhibitory activity of NS5A on EGF-stimulated ERK activation. As a result, the activation of Egr1 promoter mediated by EGF was inhibited by NS5A (Fig. 4B, lane 3) and the inhibitory activity of NS5A disappeared without Abi1 (Fig. 4B, lane 4). We have also shown that negative regulation of EGF-stimulated ERK activation in HCV replicon cells is also impaired in Abi1 knockdown cells (Fig. 4C, lane 4 in the left panel). NS5A expression It is also noteworthy that ERK is minimally activated in the absence of EGF stimulation in damaged Abi1 knockdown cells (Fig. 4C, lane 3 on the left panel). In replicon cells, Egrl mRNA levels were significantly higher in EGF stimulated cells than in control cells, and the level of Egr1 mRNA stimulated by this EGF was significantly increased in Abi1 knockdown cells (Figure 4C, middle panel). Similar results were obtained using Jc1 infected cells (Figure 4C, right panel). Similarly, Egr1 promoter activation mediated by EGF in HCV replicon cells is no longer inhibited in the absence of Abi1, suggesting that Abi1 is essential for the negative regulation of the EGF signaling pathway in HCV replicon cells (Figure 4D, lane 4 ). To exclude the off-target effect of Abi1 siRNA, EGF mediated by overexpression of Abi1 using siRNA targeting Abi1's 3'UTR (# 1) or Abi1 encoding portion (# 2) The ability to restore p-ERK activation was measured. For this, a Flag-labeled Abi1 plasmid containing only the coding portion of Abi1 and no 3'UTR portion was used. Figure 4E shows that EGF-stimulated ERK activation in HCV replicon cells is increased by knockdown of Abi1 (lane 3 to lane 5). However, exogenous expression of Abi1 in Abi # 1 siRNA knockdown cells inhibited EGF mediated p-ERK activation (Fig. 4E, lane 4) but not Abi # 2 siRNA knockdown cells (Fig. 4E, lane 6). Taken together, these data suggest that protein interactions between Abi1 and NS5A are essential for the regulation of the EGF signaling pathway.

Result 5: HCV NS5A The Abi1  And Sos1 And a complex of three.

To investigate how NS5A modulates EGF signaling, we examined whether NS5A and Abi1 binding are interfered with by Abi1-Sos1 interactions. To prove this, HEK293T cells were cotransfected with Flag-Abi1, Myc-NS5A and HA-Sos1. Forty-eight hours later, the cell suspension was immunoprecipitated with HA antibody and immunoprecipitated with Myc antibody or HA antibody to detect co-precipitated proteins. As in Figure 5, Abi1 precipitated with NS5A and Sos1. In vivo noninvasive data suggest that NS5A and Abi1 can form a complex composed of Sos1 and a set of three. It is clear that NS5A can disrupt the EGF signaling transduction without interfering with the Abi1-Sos1 interaction. These results suggest that NS5A regulates EGF signaling through Abi1, which contributes to HCV-induced pathogenesis.

Result 6: Abi1  Knockdown HCV  It damages the proliferation.

We have shown that Abi1 knockdown downregulates EGF-mediated signaling transduction as well as reduces HCV replication (Fig. 4C). To further investigate the involvement of Abi1 in HCV replication, HCV subgenomic replicon cells were transfected with siRNA targeted at two different parts of Abi1 or with control siRNA, and then HCV RNA and protein levels were measured . Figure 6A shows that Abi1 knockdown in the replicon cells decreases the level of HCV protein. Similarly, Abi1 knockdown significantly reduced HCV RNA levels (40% reduction with Abi1 # 1 siRNA and 60% decrease with Abi1 # 2 siRNA) as measured by qRT-PCR (Figure 6B). These results suggest that Abi1 is involved in HCV replication. To further investigate the effect of Abi1 on HCV proliferation, Huh7.5 cells transfected with Abi1-targeted siRNA or indicated control siRNA constructs were infected with Jc1. HCV protein levels were measured after 48 hours. Abi1 expression silencing led to a strong decrease in HCV protein levels (Fig. 6C) without cytotoxicity (Fig. 6D). Abi1 knockdown caused a dramatic decrease in intracellular HCV RNA levels (Figure 6E) and HCV infectivity (Figure 6F). To exclude the effect of Abi1 siRNA, siRNA (# 1) targeting Abi1 3'UTR or siRNA (# 2) targeting Abi1 coding region in Abi1 knockdown cells was labeled with Flag Abi1 was overexpressed and recovery experiments were performed. As shown in Figure 6G, exogenous expression of Abi1 restored HCV protein expression in Abi # 1 siRNA knockdown cells (lane 4), but not Abi # 2 siRNA knockdown cells (lane 6) Because it contains only the encrypted part of Abi1. Taken together, these data suggest that Abi1 is essential for HCV proliferation.

Result 7: Abi1  Overexpression HCV  Promotes proliferation.

Because Abi1 silencing inhibits HCV replication, we predicted that Abi1 overexpression would have a positive effect on HCV replication. To determine this possibility, Huh7.5 cells were electroporated with JFHl-luc RNA for 48 hours and transiently transfected with Flag labeled Abi1. As a result, Abi1 overexpression significantly increased luciferase activity in a dose- and time-dependent manner compared to the vector transfected with the vector (Fig. 7A and Fig. 7B). Further, the present inventors have found that HCV protein levels are increased in Jc1-infected cells compared to Jc1-infected cells in which Abi1 overexpression transfected (Fig. 7C). Taken together, these data suggest that Abi1 is essential for HCV proliferation.

<110> Industry Academic Cooperateion Foundation, Hallym University <120> Pharmaceutical composition for prevention and treatment for          Hepatitis C containing Abi1 expression or activity inhibitor <130> HallymU-SBHwang-Abi1 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targeing Abi1 <400> 1 cuguuguaca cugguucaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targeing Abi1 <400> 2 uugaaccagu guacaacag 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targeing Abi1 <400> 3 ucucuagcua guguugcuu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targeing Abi1 <400> 4 aagcaacacu agcuagaga 19 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA targeing Jc1 5'NTR <400> 5 ccucaaagaa aaaccaaacu u 21

Claims (4)

A pharmaceutical composition for the prevention and treatment of hepatitis C comprising Abi1 (Abelson interactor 1) expression or activity inhibitor.
The method according to claim 1,
Wherein the Abi1 expression inhibitor is any one selected from the group consisting of antisense nucleotides, shRNA (short hairpin RNA) and siRNA (short interfering RNA) that complementarily bind to mRNA of Abi1 gene. A pharmaceutical composition.
3. The method of claim 2,
Wherein said siRNA is SEQ ID NOS: 1 to 4.
The method according to claim 1,
Wherein the Abi1 activity inhibitor is an anti-Abi1 antibody binding to Abi1 protein.

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