(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`BS
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll lllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`( 43) International Publication Date
`21 February 2008 (21.02.2008)
`
`PCT
`
`(51) International Patent Classification:
`A61K 39142 (2006.01)
`
`(21) International Application Number:
`PCT/US2007/0l 7970
`
`(22) International Filing Date: 14 August 2007 (14.08.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/822,354
`
`14 August 2006 (14.08.2006) US
`
`(71) Applicant and
`(72) Inventor: LUO, Guang...:iang [-/US]; 2456 Olde Bridge
`Lane, Lexington, KY 40513 (US).
`
`(10) International Publication Number
`WO 2008/021353 A2
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, Fl, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(74) Agent: TANKHA, Ashok; Of Counsel, Lipton, Wein(cid:173)
`berger & Husick, 36 Greenleigh Drive, Sewell, NJ 08080
`(US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`with sequence listing part of description published sepa(cid:173)
`rately in electronic fonn and available upon request from
`the International Bureau
`
`-iiiiiiiiiiiiiii
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`~
`" ' (54) Title: CotvlPOSITION AND METHOD FOR CONTROLLING HEPATITIS C VIRUS INFECTION
`~
`'l"""""i M
`(57) Abstract: Disclosed herein are methods and compositions for the treatment and prevention of Hepatitis C Virus (HCV) in-
`0
`fection and methods of screening for antiviral agents against HCV infection and/or production. A method of using compositions
`Qo of certain apolipoprotein-specific monoclonal or polyclonal antibodies to inhibit HCV infectivity is disclosed. Further, methods of
`O using small interfering RN As (siRNAs) specific to apolipoproteins for treating and/or preventing HCV infection are disclosed. Also
`0
`disclosed are methods of using siRNAs specific and/or small molecule inhibitors to certain lipoprotein biosynthetic genes and of
`M using recombinant apolipoprotein E and/or their forms of lipoproteins to treat and/or prevent HCV infections. Screening methods
`0 for anti-HCV agents include assessing the effect of a candidate agent on apolipoprotein E and/or apolipoprotein C-I gene expression,
`
`:;;;..,.. assembly, and/or secretion and assessing the effect of a candidate agent on the blockage of the interaction and/or incorporation of
`~ HCV nonstructural proteins and/or their fusion forms with reporter proteins into HCV virions.
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`COMPOSITION AND METHOD FOR CONTROLLING HEPATITIS C VIRUS
`
`INFECTION
`This application claims the benefit of provisional application no. 60/822,354 titled
`
`"Composition And Methods For Treating And Preventing Hepatitis C Virus Infection and
`
`5
`
`Screening Methods For Identifying Anti-Hepatitis C Virus Agents" filed Aug. 14, 2006.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
`Research for this invention was made with support from the NCI, Grant Number
`
`CA093712, and the NIAID, Grant Number AI5 l 592.
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`10
`
`FIELD OF THE INVENTION
`The present invention relates to the treatment and prevention of Hepatitis C Virus (HCV)
`
`infection and screening for antiviral agents against HCV infection and/or production.
`
`15 BACKGROUND OF THE INVENTION
`The hepatitis C virus (HCV) was discovered in 1989 by molecular cloning and has since
`
`been recognized as a major cause of viral hepatitis in humans. HCV is a single-stranded positive(cid:173)
`sense RNA virus, which is about 9.6 kb in length. HCV belongs to the Hepacivirus genus of the
`family Flaviviridae. The viral RNA genome consists of the 5' untranslated region (5'UTR), a
`single open reading frame (ORF) encoding a viral polypeptide of3,010-3,040 amino acids, and
`the 3' untranslated region (3 'UTR) of variable length. Upon translation, the viral polyprotein is
`cleaved by cellular peptidases and viral proteases into core (C), envelope glycoproteins (El and
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`20
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`E2), P7, non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NSSB. Sequence
`
`analysis and comparison studies have revealed that both the 5'UTR and 3~UTR of the HCV
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`25
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`genome are highly conserved. In contrast, sequences of the ORF exhibit a variation among HCV
`
`. isolates. Based on the nucleotide sequence similarity, HCV has been further grouped into six
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`major genotypes and numerous subtypes.
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`30
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`HCV infection is characterized by the establishment of chronic infection in up to about
`85% of individuals exposed to HCV. The chronic HCV infection carries an increased risk of
`developing fatal liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma.
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`HCV-associated end-stage liver disease is the leading cause of liver transplantation in the United
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`States (US). It is estimated that approximately 4 million people in the US and 170 million people
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`worldwide are persistently infected with HCV. Each year, HCV infection results in 8,000-10,000
`
`deaths in the US alone. HCV-related deaths are expected to triple within the next 10-20 years if
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`5
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`no effective intervention is made available. Currently, there is no specific and effective therapy
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`to treat HCV infection. Accordingly, there remains an urgent need in the art for specific antiviral
`
`targets and agents for effectively treating and preventing HCV infection.
`
`The structure and biochemical compositions of HCV virions have not been determined,
`although certain studies have found that HCV virions isolated from the plasma of hepatitis C
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`10
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`patients were associated with lipoproteins to form lipoviroparticles (LVPs). Apolipoproteins B
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`and E were detected in the low-density fractions of HCV RNA-containing particles, which could
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`also be captured by apolipoprotein-specific antibodies, suggesting an association of the low(cid:173)
`
`density HCV virions with human lipoproteins. However, the roles of apolipoproteins in HCV
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`15
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`assembly and production have not been defined.
`
`SUMMARY OF THE INVENTION
`
`The present invention addresses the above identified problems, and others, by pro~ding
`compositions and methods for treating and/or preventing hepatitis C virus infection in humans.
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`20
`
`The present invention further includes targets and methods for identification (screening) of
`effective anti-BCV agents.
`
`The present invention discloses a method of using compositions of apoE- and/or apoC-1-
`specific monoclonal or polyclonal antibodies to inhibit HCV infectivity. The method further
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`25
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`comprises the step of administering an effective amount of the composition to a patient.
`
`The present invention discloses methods of using siRNAs specific to apolipoproteins, for
`
`treating and/or preventing HCV infection. The present invention further includes siRNAs
`specific for certain lipoprotein biosynthetic genes for treating and/or preventing HCV infection.
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`Also disclosed is a method of using recombinant apoE (E2, E3, and E4) protein and/or their
`
`forms of lipoproteins to treat and/or prevent HCV infections.
`
`The present invention further discloses a method of screening for anti-HCV agents by
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`5
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`assessing the effect of a candidate agent on apoE and/or apoC-I gene expression, assembly,
`
`and/or secretion. The present invention also includes a method of screening for anti-HCV agents
`
`by assessing the effect of a candidate agent on the blockage of the interaction and/or
`
`incorporation of HCV nonstructural proteins and/or their fusion forms with reporter proteins into
`
`HCV virions.
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`10
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`The foregoing summary, as well as the following detailed description of the
`
`embodiments, is better understood when read in conjunction with the appended drawings. For
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`15
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`the purpose of illustrating the invention, exemplary illustrations of the invention are shown in the
`
`drawings. However, the invention is not limited to the specific methods disclosed herein.
`
`FIGURE 1 is a flow chart illustrating an exemplary method for preventing and/or treating
`Hepatitis C virus (HCV) infection in accordance with the present invention.
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`20
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`FIGURE 2 is a flow chart illustrating an exemplary method of screening for anti-HCV agents
`in accordance with the present invention.
`
`FIGURE 3A depicts the results of HCV virion RNA (vRNA) determined by a ribonuclease
`(RNase) protection assay (RPA) in a sucrose gradient sedimentation analysis ofHCV virions in
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`25
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`culture media and densities (g/ml) of each :fraction.
`
`FIGURE 3B is a Western blot analysis of HCV NS3 protein in cells infected with the different
`fractions identified in FIGURE 3A.
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`FIGURE 3C depicts the results ofRPA used to determine positive-strand HCV RNA in cells
`infected with the different :fractions identified in FIGURE 3A.
`
`FIGURE 3D is a Western.blot analysis ofapolipoproteins BIOO (apoBIOO), C-I (apoC-1), and
`
`S
`
`E (apoE), wherein a density gradient sedimentation analysis of HCV RNA-containing particles
`is performed as shown in FIGURE 3A, and apoBIOO, apoC-I, and apoE proteins were detected
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`by using apoB-, apoCI- and apoE-specific antibodies.
`
`FIGURE 4A depicts the results of a study carried out to determine the HCV-neutralizing
`activity of apoE-specific monoclonal antibodies, where HCV positive-strand RNA levels in the
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`10
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`Huh? .5 cells infected with HCV in the presence of apoE-specific monoclonal antibodies were
`determined by RP A.
`
`FIGURE 4B is a graph showing the correlation between HCV-neutralizing activity and
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`15
`
`concentrations of apoE-specific monoclonal antibodies using the quantitative data derived from
`FIGURE4A.
`
`FIGURE 4C shows the reduction ofinfectious HCV titers in the cell culture supernatant of
`
`Huh? .5 cells that were infected with HCV in the presence of apoE-specific monoclonat
`antibodies. The infectious HCV titers were determined by immunofluorescence assay (IF A) as
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`20
`
`foci-forming units per milliliter (ffu/ml) of cell culture supernatant.
`
`FIGURE 4D depicts the results of an analysis of the inhibition of HCV infectivity in the
`infectious fractions 3 to 5 of the sedimentation analysis as shown in FIGURES 3B and JC by
`25 HCV E2- and ApoE-specific monoclonal antibodies.
`
`FIGURE 5 shows the reduction ofHCV infectivity by an apoC-I polyclonal antibody, as
`determined by Western blot analysis ofHCV NS3 protein in cells infected with HCV in the
`
`presence of increasing concentrations of apoC-I polyclonal antibody.
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`FIGURE 6 indicates human apolipoprotein E gene sequences. The protein coding sequences
`are highlighted in bold letters and 50% gray in color, and the small interfering RNA (siRNA)(cid:173)
`
`targeting sequences are shown in black color.
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`5
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`FIGURE 7 A depicts the results of a study conducted to determine the effects of ApoE-specific
`siRNA and a non-specific control (NSC) siRNA on suppression of HCV virion assembly and
`production, where the levels of ApoE and apoB proteins in the cell culture supematants of the
`BCV-infected and siRNA-transfected cells were determined by Western blot analysis.
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`10
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`FIGURE 7B depicts the correlation between siRNA concentrations and apoE levels in the cell
`
`culture supernatants using the quantified data derived from FIGURE 7A.
`
`FIGURE 7C depicts the correlation between siRNA concentrations and apoB levels in the cell
`culture supematants using the quantified data derived from FIGURE 7 A.
`
`FIGURE SA depicts the results of a study conducted to determine the effects of ApoE-speci:fic
`
`and NSC siRNAs on suppression of HCV virion assembly and production, where cell culture
`
`supernatant was used to infect naive Huh7.5 cells; At 3 days post-infection, the levels ofHCV
`NS3 protein and positive-strand RNA were determined by Western blot analysis and RPA,
`respectively.
`
`FIGURE SB is a graph showing the correlation between siRNA concentrations and HCV
`
`positive-strand RNA levels relative to the control (without siRNA transfection) in Huh7.5 cells
`infected with the supernatants described in FIGURE SA. The data plotted in the graph are
`derived from the quantification of H<:;V positive-strand RNA levels in FIGURE SA.
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`15
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`20
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`25
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`FIGURE SC depicts the results of infectious HCV titers (ffu/ml) in the supematants of the
`
`BCV-infected and apoE and NSC siRNA-transfected Huh7 .5 cells.
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`30
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`FIGURE 9A depicts the results of a study conducted to determine the effects of ApoE-specific
`and NSC siRNAs on suppression of HCV production, where the virion RNA (vRNA) levels of
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`BCV RNA-containing particles secreted into the culture supematants of the BCV-infected and
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`apoE and NSC siRNA-transfected cells were determined by RP A.
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`FIGURE 9B depicts the results of a study conducted to determine the effects of ApoE-specific
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`5
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`and NSC siRNAs on suppression of BCV production, where BCV vRNA levels(%) are plotted
`
`against NSC and apoE siRNA concentrations (nM). The vRNA levels relative to control
`
`(without siRNA) are calculated as percentage of control(%) using the quantified data derived
`
`from FIGURE 9A.
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`IO
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`FIGURE lOA depicts the results of a study conducted to determine the effects of ApoE(cid:173)
`
`speci:fic and NSC siRNAs on suppression of HCV assembly and production, where intracellular
`
`HCV virions were used to infect naive Huh7.5 cells. At 3 days post-infection, the levels of
`
`HCV positive-strand RNA in the infected cells were determined by RP A.
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`FIGURE lOB depicts the correlation between siRNA concentrations and HCV positive-strand
`
`RNA levels relative to the control (without siRNA transfection) in Huh7.5 cells infected with
`
`intracellular HCV virions described in FIGURE lOA. The data plotted in the graph are derived
`
`from the quantification of BCV positive-strand RNA levels in FIGURE lOA.
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`FIGURE lOC depicts the results of infectious BCV titers (ffu/ml) of the intracellular BCV
`
`virions isolated from the BCV-infected and NSC and apoE siRNA-transfected Buh7.5 cells.
`
`FIGURE 11 depicts the results of a study conducted to determine.the effects ofNSC and
`ApoE-specific siRNAs on BCV RNA replication, where subgenomic BCV RNA-harboring
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`25
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`cells were transfected with NSC and apoE siRNAs; and, at 3 days post-transfection, BCV
`
`positive-strand RNA levels were determined by RP A.
`
`FIGURE 12 depicts the results of a study to determine the effects of Sterol 0-acyltransferase
`
`(SOAT or ACAT) and microsomal triglyceride transfer protein (MTP)-specific siRNAs on
`
`30 HCV production, wherein the BCV-infected Buh7.5 cells were transfected with increasing
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`concentrations ofNSC, ACAT, and MTP siRNAs, respectively, and, the cell culture
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`supernatants were used to infect naive Huh7.5 cells. At 3 days post-infection~ HCV RNA was
`extracted from cells with Trizol reagent, and HCV positive-strand RNA levels were determined
`
`byRPA.
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`FIGURE 13 depicts the structures of CP-346086, glybenclamide, hesperetin, quercetin, and
`
`naringenin.
`
`FIGURE 14A depicts the results ofa study conducted to determine the effects ofCP-346086, a
`
`microsomal triglyceride transfer protein (MTP) inhibitor, on suppression of HCV production,
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`10 where apoBlOO and apoE levels in the supernatants ofCP-346086-treated cells were
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`determined by Western blot analysis.
`
`FIGURE 14B depicts the results of a study conducted to determine the effects of CP-346086
`
`on suppression ofHCV production, where the supernatants ofCP-346086-treated and HCV-
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`15
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`infected cells were used to infect naive Huh7.5 cells. At 3 days post-infection, the levels of
`
`HCV NS3 protein was determined by Western blot analysis.
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`FIGURE 14C depicts the results ofa study conducted to determine the effects ofCP-346086
`
`on suppression of HCV production, where apoB, apoE, and HCV NS3 protein levels derived
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`20
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`from quantification of FIGURES 13A and 138 are used to plot against CP-346086
`
`concentrations (µM).
`
`FIGURE 14D depicts the results ofa study conducted to determine the effects ofCP-346086
`
`on suppression ofHCV production, where the supematants of CP-346086-treated and HCV-
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`25
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`infected cells were used to infect naive Huh7.5 cells. At 3 days post-infection, the levels of
`
`HCV positive-strand RNA were determined by RP A.
`
`FIGURE 14E depicts the results of a study conducted to determine the effects of CP-346086
`
`on suppression ofHCV production, where infectious HCV titers (ffu/ml) in the cell culture
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`supematants of the CP-346086-treated cells were determined by IFA and are used to plot
`against CP346086 concentrations.
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`FIGURE 14F depicts the results of a study conducted to determine the effects of CP-346086
`on HCV RNA replication, where subgenomic HCV RNA-harboring cells were treated with CP-
`
`346086, and HCV positive-strand RNA levels in the cell were then determined by RP A.
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`FIGURE 15 depicts the results of an RP A used to determine the effects of glybenclamide,
`hesperetin, and quercetin on HCV production; where the HCV-infected Huh7.5 cells were
`
`treated with increasing concentrations of glybenclamide, hesperetin, and quercetin,
`
`respectively; and, the cell ~ulture supernatants were used to infect naive Huh7.5 cells. At 3 days
`post-infection, HCV RNA was extracted with Trizol reagent, and HCV positive-strand RNA
`levels were determined by RP A.
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`FIGURE 16A depicts the results of a Western blot analysis ofHCV NS3 protein in a study to
`determine the inhibition of HCV infection by a recombinant apoE3 protein.
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`15
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`FIGURE i6B depicts the results of a study to determine the effect of a purified apoC-1 protein
`on the inhibition ofHCV infection, where the levels ofHCV positive-strand RNA in Huh7.5
`
`cells infected with HCV in the presence ofapoC-1 protein were determined by RPA.
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`20
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`FIGURE 17A is a Western blot analysis used to assess the inhibition ofHCV infection by
`human high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density
`lipoproteins {VLDL ).
`
`FIGURE 17B depicts the results of an RP A used to assess the inhibition of HCV infection by
`human HDL, LDL, and VLDL.
`
`FIGURE 18 is a Western blot analysis ofHCV Core (C), NS3, and NSSA proteins, wherein a
`
`density gradient sedimentation analysis ofHCV RNA-containing particles is performed as in
`FIGURE 3A; The C, NS3, and NSSA were detected by Western blot analysis using C-, NS3-,
`and NS5A-specific monoclonal antibodies.
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`FIGURE 19A depicts the construction ofinfectious HCV expressing an NS5A-GFP fusion
`protein and fluorescence photographs showing green fluorescence protein (GFP) expression in
`
`Huh7.5 cells infected with NS5A-GFP-expressing HCVs. GFP is inserted into NS5A (466
`
`amino acids) at different positions indicated by amino acid numbers (247, 363, and 429).
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`5
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`FIGURE 19B is a Western blot analysis of HCV NS5A or NS5A-GFP fusion protein
`
`expression in Huh7.5 cells infected with wild type HCV or mutant HCVs expressing NS5A(cid:173)
`GFP fusion protein using NS5A- and GFP-specific monoclonal antibodies.
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`10
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`FIGURES 19C is a Western blot analysis of the NS5A-GFP fusion protein present in HCV
`RNA-containing particles of the sucrose-gradient fractions using a GFP-specific monoclonal
`
`antibody.
`
`DESCRIPTION OF EMBODIMENTS OF THE INVENTION
`
`15
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`20
`
`25
`
`The present invention includes compositions and methods for treating and/or preventing
`
`hepatitis C virus infection in humans. The present invention further includes targets and methods
`
`for identification (screening) of effective anti-HCV agents.
`
`In accordance with the present invention, HCV assembly and/or HCV infectivity may be
`controlled by suppressing the levels of certain apolipoproteins (e.g., apoE and apoC-I) in
`patients, thereby treating and/or preventing hepatitis C virus infection. In accordance with the
`
`present invention, HCV may also be treated by targeting certain lipid and lipoprotein
`
`biosynthesis genes and/or pathways.
`
`The present invention further includes methods of using monoclonal and/or polyclonal
`
`antibodies specific to apolipoprotein E and/or apolipoprotein C-I to treat and/or prevent HCV
`infection.
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`30
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`The present invention also includes methods of using siRNAs specific to apolipoproteins,
`including but not limited to apoE, for treating and/or preventing HCV infection. The present
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`invention further includes siRNAs specific for certain lipoprotein biosynthetic genes, including:
`acyl coenzyme A:cholesterol acyltransferase (ACAT; also known as sterol 0-acyltransferase
`(SOA n), and microsomal triglyceride transfer protein {MTP), for treating and/or preventing
`HCV infection. Such siRNAs may be obtained, for example, from Dharmacon Company
`(Lafayette, CO).
`
`5
`
`The present invention also includes a method of using small molecular inhibitors of
`lipoprotein biosynthesis to treat and/or prevent HCV infections, for example, inhibitors of ACAT
`
`and MTP may be used. Examples of ACAT and MTP inhibitors include, but are not limited to:
`CP-346086, glybenclamide, hesperetin, quercetin, and naringenin.
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`10
`
`The present invention additionally includes a method of using recombinant apoE (E2, E3,
`and E4) protein and/or their forms oflipoproteins to treat and/or prevent HCV infections,
`including high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-
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`15
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`density lipoproteins (VLDL).
`
`The present invention further includes a method of screening for anti-HCV agents by
`assessing the effect of a candidate agent on apoE and/or apoC-1 gene expression, assembly,
`and/or secretion.
`
`The present invention also includes a method of screening for anti-HCV agents by
`assessing the effect of a candidate agent.on the blockage of the interaction and/or incorporation
`
`ofHCV nonstructural proteins (e.g., NS5A) and/or their fusion forms with reporter proteins (e.g.,
`Luciferase, alkaline phosphotase, GFP, etc.) into HCV virions.
`
`20
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`25
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`Turning now to FIGURE 1, an exemplary method 100 of the present invention for
`treating and/or preventing HCV infection in a patient includes the following: providing a
`
`composition capable of suppressing apolipoprotein E (apoE) and/or apolipoprotein C-1 (apoC-1)
`levels in the patient 102; and administering to the patient an effective amount of the composition
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`104. An effective amount of tlie composition to be used in accordance with the present invention
`is intended to mean a nontoxic but sufficient amount of the composition, such that the desired
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`prophylactic or therapeutic effect is produced. The exact amount of the composition that is an
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`effective amount will vary from patient to patient, depending, for example, on the age, and
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`general condition of the patient, the severity of the infection being treated, the particular carrier
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`or adjuvant being used and its mode of administration, and the like. Accordingly, the effective
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`amount of any particular composition will vary based on the particular circumstances, and an
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`appropriate effective amount may be determined in each case of application by one of ordinary
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`skill in the art using only routine experimentation.
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`With continued reference to FIGURE 1, the administered composition may include one
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`or more agents capable of suppressiQ.g apoE and/or apoC-I. For example, apoE and/or apoC-I
`may be pre-transcriptionally suppressed 106, by providing a composition capable of decreasing
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`apoE and/or apoC-I gene expression, such that the relative amount of apoE and/or apoC-I mRNA
`that is transcribed is decreased 108. By so reducing apoE and/or apoC-I gene expression, the
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`amount of apoE and/or apoC-I protein that may be produced is decreased, thereby limiting the
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`amount of apoE and/or apoC-I protein that is available to participate in the HCV assembly and
`production process 110, resulting in the prevention and/or treatment of an HCV infection in the
`patient 112. Examples of agents that may pre-transcriptionally suppress apoE and/or apoC-I
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`include peroxisome proliferator-activated receptors (PPARs), antagonists and interferon.
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`ApoE and/or apoC-1 may also be suppressed post-transcriptionally 114, for example, by
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`providing a composition capable of degrading the apoE and/or apoC-I m.RNA or otherwise
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`blocking translation of apoE and/or apoC-I mRNA, thereby reducing the amount of apoE and/or
`apoC-I mRNA that is available for translation 116. By so reducing the amount of mRNA
`available for translation, the amount of apoE and/or apoC-I protein that may be produced is
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`decreased, thereby limiting the amount of apoE and/or apoC-1 protein that is available to
`participate in the HCV assembly and production process llO, resulting in the prevention and/or
`treatment of an HCV infection in the patient 112. Examples of agents that may post(cid:173)
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`transcriptionally suppress apoE and/or apoC-I include compounds capable of RNA interference,
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`such as small interfering RNAs (siRNA). RNA interference is a post-transcriptional gene
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`silencing tool, i.e., specifically degrading or destroying the mRNA encoding the product of the
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`gene of interest. Short RNA duplexes that have been shown to successfully interfere with
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`expression of specific genes of interest in cells are referred to as small interfering RNAs
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`(siRNAs). An siRNA is a double stranded RNA oligonuleotide that is typically less than about
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`30 nucleotide bases in length. One strand of an siRNA is complementary to a portion of an
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`mRNA of a gene of interes4 binds thereto, and degrades mRNAs and/or prevents translation,
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`thereby suppressing expression of the gene.
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`ApoE and/or apoC-1 may additionally be suppressed post-translationally 118, for
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`example, by providing a composition capable of degrading the apoE and/or apoC-I protein or
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`scavenging the apoE and/or apoC-I protein, thereby limiting the amount of apoE and/or apoC-I
`protein that is available to participate in the HCV assembly and production process 110. By so
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`limiting the amount of protein available for participation in the HCV assembly and production
`process, an HCV infection in the patient may be prevented and/or treated 112. Examples of
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`agents that may post-translationally suppress apoE and/or apoC-I include acyl coenzyme
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`A:cholesterol acyltransferase (ACAT) inhibitors (e.g., glybenclamide and naringenin),
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`Cholesteryl Ester Transfer Protein (CETP) inhibitors (e.g., torcetrapib) and microsomal
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`triglyceride transfer protein (MTP) inhibitors (e.g., CP346086 and BMS-200150). It is also
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`possible that post-translational modification of apoE will affect its incorporation into HCV
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`virions, for instance phosphorylation, and therein results in inhibition ofHCV production.
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`Turning now to FIGURE 2, an ·exemplary screening method 200 of the present invention
`for identifying anti-HCV agents includes the following: providing cells in culture 202;
`uninfecting the cells, infecting the cells with HCV, or HCV-producing cells 204; treating the
`cells with a candidate anti-HCV agent 206; measuring apoE expression in the cells and/or cell
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`culture supernatant or HCV nonstructural proteins (e.g., NSSA) and reporter gene (e.g.,
`luciferase) that is fused with NSSA in the culture supernatant 208; and identifying the candidate
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`agent as an actual anti-HCV agent when apoE gene expression in the cells and/or supernatant or
`HCV nonstructural proteins in the culture supernatant is below a predetermined level 210.
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`The cells that may be provided 202 include any cells that may be grown in culture,
`produce apolipoproteins, and/or are capable of being infected with HCV 204. The cells may be
`treated with a candidate anti-HCV agent 206 before and/or after infection with HCV, or without
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`HCV infection, as desired. For example, if an assessment of the abilities of a candidate agent to
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`prevent HCV infection is being made, it may be desirable to treat the cells with the candidate
`agent before infection with HCV. For another example, if an assessment of the abilities ofa
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`candidate agent to treat HCV infection is being made, it may be desirable to treat the cells with
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`the candidate agent after infection with HCV. Additionally, cells without HCV infection can be
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`treated with the candidate agent to assess its effect on apoE expressio~ assembly, and/or
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`production (secretion).
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`The apoE and/or apoC-1 gene expression level in the cells and/or in the culture
`supernatant or HCV nonstructural proteins and/or their fusion forms with reporter proteins (e.g.,
`· NSSA, NS5A-luciferase, NS5A-GFP, NS5A-alkaline phosphotase, and etc.) in the culture
`supernatant may be measured 208 by any method known to those skilled in the art. For. example,
`the apoE and/or apoC-I gene expression level could be measured by quantifying the
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`concentration of apoE and/or apoC-1 mRNA. For another example, the apoE and/or apoC-I gene
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`expression level could be measured by quantifying the concentration of apoE and/or apoC-I
`protein in the cells and/or in the cell culture supernatant. For another example, HCV
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`nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NSSB) in the cell culture
`supernatant of the HCV-infected cells could be measured by quantifying the levels of NS (e.g.,
`NS5A) proteins and/or the levels of reporter activity (e.g., luciferase, alkaline phosphotase, GFP,
`and etc.) of the NS-reporter fusion proteins (e.g., NSSA-luciferase, NS5A-a1kaline phosphotase,
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`NS5A-GFP, and etc.) in the culture supernatant.
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`A candidate agent may be identified as an actual anti-HCV agent when the apoE and/or
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`apoC-1 gene expression level and/or HCV nonstructural protein (e.g., NS5A) and the reporter
`gene that is fused with HCV nonstructural protein (e.g., NS5A-luciferase) is below a
`predetermined level 210. The predetermined level may be identified, for example, by
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`quantifying the apoE and/or apoC-1 mRNA and/or protein in the cells and/or supernatant using
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`methods known to those skilled in the art and comparing the quantified apoE and/or apoC-1
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`mRNA and/or protein level to a standard curve plotting level of apoE and/or apoC-1 mRNA
`and/or protein. For another example, the predetermined level may be identified using a control
`sample that does not contain the candidate anti-HCV agent. In this regard, when a control
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`sample that does not contain the candidate