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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N2330/00—Production
  • C12N2330/10—Production naturally occurring

Definitions

• the present invention relates generally to a method and formulation for the treatment of Hepatitis C infection. More particularly, the present invention relates to regulating Bachl and/or HCV.
• Bachl and the HCV NS5A protein can be down -regulated by miR-196, while up-regulating HMOXl .
• Hepatitis C virus is a small (50 nm in size), enveloped, positive sense single strand RNA virus in the family Flaviviridae. Although Hepatitis A virus, Hepatitis B virus, and Hepatitis C virus have similar names because they all cause liver inflammation, each is a distinct and different virus both genetically and clinically. HCV causes the blood-borne (i.e. spread by blood-to-blood contact) infectious disease known as Hepatitis C. The infection is often asymptomatic, but once established, chronic infection can cause inflammation of the liver (chronic hepatitis). This condition can progress to scarring of the liver (fibrosis), and advanced scarring (cirrhosis). In some cases, those with cirrhosis will go on to develop liver failure or other complications of cirrhosis, including liver cancer.
• HCV Hepatitis C virus
• Acute hepatitis C refers to the first 6 months after infection with HCV. Around 60% to 70% of people infected develop no symptoms during the acute phase. In the minority of patients who experience acute phase symptoms, they are generally mild and nonspecific, and rarely lead to a specific diagnosis of hepatitis C. Symptoms of acute hepatitis C infection include decreased appetite, fatigue, abdominal pain, jaundice, itching, and flu-like symptoms. HCV is usually detectable in the blood within one to three weeks after infection, and antibodies to the virus are generally detectable within 3 to 12 weeks.
• LFTs liver function tests
• ALT alanine transaminase
• AST aspartate transaminase
• plasma HCV-RNA clearance plasma HCV-RNA clearance
• Chronic hepatitis C is defined as infection with HCV persisting for more than six months.
• the natural course of chronic hepatitis C varies considerably from one person to another. Virtually all people infected with HCV have evidence of inflammation on liver biopsy. However, the rate of progression of liver scarring (fibrosis) shows significant variability among individuals. Recent data suggests that among untreated patients, roughly one-third progress to liver cirrhosis in less than 20 years. Another third progress to cirrhosis within 30 years. Symptoms specifically suggestive of liver disease are typically absent until substantial scarring of the liver has occurred.
• hepatitis C is a systemic disease and patients may experience a wide spectrum of clinical manifestations ranging from an absence of symptoms to a more symptomatic illness prior to the development of advanced liver disease.
• Generalized signs and symptoms associated with chronic hepatitis C include fatigue, marked weight loss, flu-like symptoms, muscle pain, joint pain, intermittent low-grade fevers, itching, sleep disturbances, abdominal pain (especially in the right upper quadrant), appetite changes, nausea, diarrhea, dyspepsia, cognitive changes, depression, headaches, and mood swings.
• liver cirrhosis signs and symptoms may appear that are generally caused by either decreased liver function or increased pressure in the liver circulation, a condition known as portal hypertension.
• Possible signs and symptoms of liver cirrhosis include ascites (accumulation of fluid in the abdomen), bruising and bleeding tendency, bone pain, varices (enlarged veins, especially in the stomach and esophagus), fatty stools (steatorrhea), jaundice, and a syndrome of cognitive impairment known as hepatic encephalopathy.
• Hepatitis C chronic hepatitis C, more than other forms of hepatitis, is diagnosed because of extrahepatic manifestations associated with the presence of HCV such as thyroiditis (inflammation of the thyroid) with hyperthyreosis or hypothyreosis, porphyria cutanea tarda, cryoglobulinemia (a form of small-vessel vasculitis) and glomerulonephritis (inflammation of the kidney), specifically membranoproliferative glomerulonephritis (MPGN).
• Hepatitis C is also associated with sicca syndrome, thrombocytopenia, lichen planus, diabetes mellitus and with B-cell lymphoproliferative disorders.
• Hepatitis C virus infection is a world-wide health problem, for which a vaccine is currently not available.
• the current standard therapy for chronic hepatitis C (CHC) is a combination of pegylated interferon (IFN) and ribavirin, but only about 50% of patients respond to such treatment. Additionally, this treatment is expensive, prolonged, and accompanied by numerous unpleasant side-effects. An estimated 150-200 million people worldwide are infected with hepatitis C. As such, there remains a need to discover and develop anti-viral therapies and procedures targeting HCV and for the treatment of all forms of Hepatitis C.
• the present invention satisfies at least some of the aforementioned needs by providing a method of treating cells or a mammal suffering from HCV infection by reducing the expression of Bachl protein levels in human hepatoma cells expressing hepatitis C viral proteins.
• a reduction in the expression of Bachl protein levels can be achieved by transfecting the cells with miRNA-196 mimic so that the miRNA-196 binds with the 3'-UTR of Bachl mRNA to reduce the expression of Bachl.
• the miRNA merely needs to include the matching "seed region" to effectively bind with the 3'-UTR of Bachl mRNA.
• the miRNA is up-regulated or over- expressed to increase the reduction in Bachi expression levels.
• the level of miRNA-196 is up-regulated by a synthesized miRNA mimic, which enters the miRNA pathway and acts as a mature miRNA-196.
• a mammal suffering from HCV infection can also be treated by up-regulating
• HMOXl gene expression in cells expressing HCV non-structural proteins in various embodiments, the up-regulation of HMOXl gene expression is accompanied by the down- regulation of Bachl gene expression in the cells.
• the miRNA-196 indirectly up- regulates HMOXl by binding with Bachl, which negatively regulates HMOXl. The regulation of each can be achieved by transfecting the cells with miRNA-196 mimic.
• cells, such as hepatocytes, infected with HCV can be treated by reducing the expression of HCV in the infected cells by transfecting the cells with miRNA-196 mimic.
• the invention provides a pharmaceutical formulation adapted for administering miRNA-196 mimic to a mammal so that cells infected with HCV can be transfected with miRNA-mimic. The administration of these formulations translationally represses the expression of Bachl, up-regulates HMOXl, and regulates HCV replication in hepatocytes.
• the present invention demonstrates the functional miRNA-196 binding sites in the 3 '-UTR of Bachl , which lead to down regulation of Bachl gene expression, up regulation of HMOXl gene expression, and down regulation of HCV gene expression.
• the use of miRNA- 196 provides new additional therapies for treating cells infected with HCV and mammals having Hepatitis C (e.g. chronic HCV infection) and, perhaps, for other diseases characterized by increased oxidative stress.
• Figure IA shows a schematic of a first seed region match between miR-196 and the first putative Bachl 3 '-UTR site targeted
• Figure IB shows a schematic of second seed region match between miR-196 and the putative Bachl 3'-UTR site targeted
• FIG. 1A illustrates the down-regulated Bachl protein levels associated with transfection with miRNA-196 mimic
• Figure 2B illustrates up-regulated Bachl protein levels associated with transfection with miRNA-196 inhibitor
• FIG. 2C shows that Bachl mRNA levels were not altered by miRNA-196 mimic transfection
• Figure 3 A illustrates the up-regulation of HMOXl mRNA levels associated with miRNA-196 mimic
• Figure 3B shows that transfection with miRNA-196 mimic did not alter Cullin 3 mRNA levels
• Figure 4A illustrates the down-regulation of HCV NS5A protein levels associated with transfection with miRNA-196 mimic
• Figure 4B illustrates the up-regulation of HCV NS5A protein levels associated with transfection with miRNA-196 inhibitor
• Figure 4C shows the down-regulation of HCV NS5A and core mRNA levels by transfection with miRNA-196 mimic in the Con 1 (subtype Ib) full length replicon cells;
• Figure 5 A shows the two Bachl 3'-UTR seed match sites for miRNA-196;
• Figure 5B illustrates a schematic representation of pGL3-Bachl, the firefly luciferase if-luc) reporter construct utilized in co-transfectin cells with pGL3-Bachl, pRL- TK (renilla) and with miR-196 mimic or inhibitor by Lipofectamine 2000.
• Figure 5C shows that miRNA-196 mimic inhibited the f-luc activities of pGL3- Bachl reporter
• Figure 5D shows that miRNA-196 inhibitor slightly increased the f-luc activity of pGL3 -Bachl reporter
• Figure 6A illustrates the replacement of four nucleotides in the two seed match sites of Bachl 3'-UTR;
• Figure 6B illustrates that miRNA-196 mimic decreased the /-/we activity of pGL3-
• Figure 6C illustrates that miR-155 mimic decreased the f-luc activities of both pGL3-Bachl-WT and pGL-Bachl-Mut reporter in cells co-transfected with mutant pGL3- Bachl or pGL3-Bachl, with pRL-TK (renilla), and with miR-155 mimic;
• Figure 7A illustrates the four nucleotide mutations that were introduced to the seed match sites of miRNA-196;
• Figure 7B shows the Luciferase activity of cells co-transfected with pGL3 -Bachl, pRL-TK and with increasing concentrations of mimic negative control, miR-196 mimic or mutant miR-196;
• Figure 8 A illustrates the restoration of the seed match between mutant miRNA-196 and mutant Bachl 3'-UTR
• Figure 8B shows the measured luciferase activities of cells co-transfected with mutant reporter (pGL3-Bachl-Mut), pRL-TK, and with mutant miRNA-196 or wild type miRNA- 196 mimic for 48 h;
• Figure 9A shows down-regulation of HCV J6/JFHI RNA levels by miRNA-196 mimic in Huh-7.5 cells transfected with J6/JFH1 RNA;
• Figure 9B shows in Huh-7.5 cells infected with J6/JFH1 hepatitis C virus secreted into the culture supernatant; and Figure 9C shows down-regulation of HCV J6/JFH1 protein levels by miRNA-196 mimic in Huh-7.5 cells infected with J6/JFH1 hepatitis C virus secreted into the culture supernatant.
• HCV Hepatitis C virus
• HMOXl heme oxygenase 1
• bZip basic leucine zipper
• Bachl is a gene that encodes a transcription factor that belongs to the cap'n'collar type of basic region leucine zipper factor family (CNC-bZip).
• the encoded protein contains broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domains, which is atypical of CNC-bZip family members. These BTB/POZ domains facilitate protein-protein interactions and formation of homo- and/or hetero-oligomers.
• this encoded protein forms a heterodimer with MafK, it functions as a repressor of Maf recognition element (MARE) and transcription is repressed.
• MARE Maf recognition element
• Bachl is a mammalian transcriptional repressor of HMOXl that negatively regulates HMOXl gene expression.
• Bachl forms antagonizing heterodimers with the Maf-related oncogene family. These heterodimers bind to Maf recognition elements (MAREs) and suppress expression of genes (e.g., HMOXl and NQOl) that respond to Maf-containing heterodimers and other positive transcriptional factors.
• miRNAs are small non-coding RNAs (-22 nt) that are generally considered to be important regulators of gene expression. Prior to the present invention, whether and how microRNAs regulate Bachl or HCV was largely unknown.
• the present invention recognizes, for the first time, that miRNA-196 directly acts on the 3'-UTR of Bachl mRNAs and translationally represses the expression of this protein, and up-regulates HMOXl . Additionally, miR-196 also inhibits HCV NS5A protein expression. Thus, miRNA-196 plays an important, perhaps even critical, role in the regulation of HCV replication and HMOXl/Bachl expression in hepatocytes. Accordingly, cells infected with HCV or mammals suffering from Hepatitis can be treated by the administration of miRNA-196 mimic so that the infected cells are transfected with miRNA-196 mimic.
• over-expression of miRNA-196 and tranfection thereof into infected cells can beneficially provide an approach for preventing or ameliorating hepatitis C infection.
• the level of miRNA-196 is up-regulated by a synthesized miRNA mimic, which enters the miRNA pathway and acts as a mature miRNA-196.
• miRNAs are small non-coding RNAs ( ⁇ 22 nt) that are generally considered to be important regulators of gene expression. More specifically, miRNAs are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression primarily through translational repression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem- loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (niRNA) molecules, and their main function is to downregulate gene expression. The function of miRNAs appears to be in gene regulation.
• niRNA messenger RNA
• a miRNA is complementary to a part of one or more messenger RNAs (mRNAs).
• Animal miRNAs are usually complementary to a site in the 3' UTR.
• the annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA.
• the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi).
• RNAi RNA interference
• the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded.
• the exact mechanism by which target genes are down-regulated remains unclear.
• MicroRNA mimics are double-stranded RNA oligonucleotides chemically modified with ON- TARGET® to increase their stability and to improve their activities.
• the microRNA mimics mimic endogenous precursor miRNAs to enter the miRNA pathway and act as mature miRNA species.
• miRNA-196 was first recognized to have extensive and evolutionarily conserved complementarity to homeobox (HOX) clusters, groups of related transcription factor genes crucial for numerous of developmental programs in animals, and to regulate HOX gene expression.
• HOX homeobox
• miRNA-196 targets HCV genome as well as u ⁇ -regulation of the HMOXl gene by targeting the 3'-UTR of Bachl niRNA, Accordingly, cells infected with HCV can be treated by tranfecting the infected cells with miRNA-196 mimic. By transfecting the HCV infected cells, Bachl expression levels can be reduced while increasing HMOXl levels. Beneficially, the down-regulation of Bachl, which negatively regulates HMOXl , aids in increasing the expression of HMOXl . As noted above, HMOXl provides anti-oxidative effects to mitigate the oxidative stress induced by HCV. Additionally, miRNA-196 up-regulation can be induced with IFN ⁇ treatment. This induction of miRNA-196 can be seen in the human hepatoma cell line Huh-7 and in primary murine hepatocytes.
• miRNA-122 was identified as the most abundant miRNA expressed in hepatocytes and shown to have major effects on several enzymes of cholesterol metabolism. miRNA-122 was also shown to be required for HCV expression. Generally, the effects of miRNA-122 depend upon the context and location of its cognate seed sequence binding sites with the sites in the 5' region mostly associated with up-regulation of expression, whereas those in the 3' untranslated region mostly associated with repression of expression.
• Embodiments of the present invention comprise utilizing miRNA-196 mimic as a down-regulator of HCV NS5A protein expression. This protein is essential for the full and normal expression of HCV.
• embodiments of the present invention comprise methods of treating infected cells or mammals suffering from Hepatitis C with a therapeutically effective amount of miRNA-196 mimic.
• a therapeutically effective amount can be provided by known administrative routes.
• therapeutic agents comprising miRNA-196 mimic can be applied by topical, enteral or parenteral administration routes according to various embodiments of the present invention.
• HMOXl helps to protect numerous cells and tissues against the potentially damaging effects of excess oxidative stress.
• HMOXl is a key cytoprotective enzyme that has anti-oxidant and anti-inflammatory activities that may negate or mitigate the oxidative stress induced by HCV, More specifically, HMOXl is a key cytoprotective enzyme, catalyzing heme degradation and generating ferrous iron, carbon monoxide and biliverdin, which have anti-oxidant and anti-inflammatory activities in vivo.
• HMOXl beneficial properties are based upon the ability of HMOXl to decrease "free" or loosely-bound heme, which can act as a potent prooxidant, and to increase production of carbon monoxide, biliverdin, and bilirubin, which have potent antioxidant and antiinflammatory and antifibrogenic effects.
• HMOXl Humuscular Activated protein responsive elements
• MARE Maf protein responsive elements
• MPRE metalloporphyrin-responsive elements
• BBl is a member of the cap n' collar family zinc, leucine zipper proteins. It plays a key role in tonic repression of expression of the HMOXl gene. It does so by forming heterodimers with small Maf proteins and blocking transcriptional activation of the gene.
• Bachl contains several consensus binding sites (all containing CP motifs) which, when they bind heme, lead to a change in conformation of the protein with marked reduction in affinity for Maf proteins and subsequent depression and increase in activity of HMOXl gene expression.
• One of the major stimulatory Maf proteins is Nrf2. Up regulation of Nrf2 is associated with increased expression of the HMOXl gene.
• HMOXl activity might be increased in HCV infection.
• clinical studies have identified a decrease in expression of HMOXl in the setting of chronic hepatitis C.
• patients with genetic or other factors that lead to lower levels of HMOXl gene expression may be at increased risk for development of chronic hepatitis C infection after acute HCV exposure and/or with greater risks of development of more rapidly progressive liver disease due to HCV infection.
• one embodiment of the present invention comprises a method for treating a mammal suffering from chronic hepatitis C infection by transfecting the cells with miRNA-196 mimic so that the miRNA-196 binds with the 3'-UTR of Bachl mRNA to reduce the expression of Bachl and up-regulate HMOXl .
• the miRNA-196 is over expressed to provide more miRNA-196 for binding to Bachl and up-regulating HMOXl.
• a mammal suffering from HCV infection e.g. chronic hepatitis C
• the infected cells are transfected with miRNA-196 mimic, which down-regulates the expression of Bachl.
• Bachl protein expression levels in infected cells expressing HCV non-structural protein can be reduced by transfecting the cells with miRNA-196 mimic so that the miRNA-196 binds with the 3'-UTR of Bachl mRNA to reduce the expression of Bachl.
• the miRNA-196 which binds with the 3'-UTR of Bachl mRNA is over-expressed.
• miRNA-196 is up-regulated by also administering interferon beta
• miRNA-196 is also up-regulated by the administration of miRNA- 196 mimic alone or in combination with interferon beta.
• a daily therapeutic amount of the miRNA- 196 mimic for the treatment of HCV infection can generally range from 0.5 to 5 ⁇ mol/kg of body weight, or from 1.0 to 4 ⁇ mol/kg of body weight, or from 2 to 3 ⁇ mol/kg of body weight.
• the therapeutic amount of miRNA-196 mimic can range from 0.1 to 1 ⁇ mol/kg of body weight, or from 0.25 to 1 ⁇ mol/kg of body weight, or form 0.25 to 0.75 ⁇ mol/kg of body weight.
• the expression of Bachl protein levels can be reduced by about 10% to about 75%, or by about 25% to about 65%.
• the expression of Bachl protein levels 24 hours after transfection comprises a reduction of about 25% to about 75%, or about 50% to about 60%.
• the expression of Bachl protein levels 48 hours after transfection provides a reduction of about 40% to about 80%, or about 60% to about 70%.
• one embodiment of the present invention comprises a method of treating cells infected with HCV or a mammal suffering from HCV infection by up-regulating HMOXl gene expression in cells expressing HCV non-structural proteins.
• HMOXl gene expression is up-regulated by transfecting the infected cells with a therapeutically effective amount of miRNA-196 mimic.
• the up-regulating of HMOXl is accompanied with the down- regulating of Bachl gene expression in the cells.
• the method includes up-regulating or over-expressing miRNA-196 to treat hepatocytes.
• the expression of HMOXl is increased by about 2 to about 3 fold over the level of HMOXl expression in cells that have not been transfected with miRNA- 196 mimic.
• HCV non- structural protein 5a (HCV NS5A) expression in human hepatocytes expressing HCV non- structural proteins is also repressed.
• embodiments of the present invention comprise treating HCV infected cells with miRNA mimic.
• the infected cells can be tranfected with miRNA-196 mimic to down-regulate the expression of HCV NS5A.
• the miRNA-196 is over- expressed.
• Certain embodiments of the present invention comprises treating cells infected with HCV or a mammal suffering from HCV infection by reducing the expression of HCV NS5A in cells expressing HCV non-structural proteins by transfecting the cells with miRNA-196 mimic. That is, in certain embodiments a mammal suffering from HCV infection is treated by reducing the expression of HCV RNA and/or protein expression in HCV repiicon cells and HCV infected cells by transfecting the cells with miRNA-mimic. In one such embodiment, the expression of HCV NS5A protein levels is reduced by 10 to 60 percent, or by 20 to 50 percent. In another embodiment, the expression of HCV NS5A protein levels 24 hours after transfection comprise a reduction of about 45 to about 55 percent, while the expression of HCV NS5A protein levels 48 hours after transfection comprise a reduction of about 35 to about 45 percent.
• transfection describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer.
• Transfection of animal cells typically involves opening transientpores or 'holes' in the cell plasma membrane, to allow the uptake of material.
• Genetic material such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected.
• transfection can be carried out by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.
• One possible method of transfection can utilize calcium phosphate.
• a HEPES- buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the material to be transfected.
• HeBS HEPES- buffered saline solution
• a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the material to be transfected on its surface.
• the suspension of the precipitate is then added to the cells to be transfected. Although the process is not completely understood, the cells take up some of the precipitate, and with it, the material to be transfected.
• lipid-cation based transfection is more typically used, because the cells are more sensitive.
• Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine.
• the negatively charged genetic material binds to the polycation and the complex is taken up by the cell via endocytosis.
• a direct approach to transfection is the gene gun, where the genetic material is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus.
• the genetic material can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and, the cells are said to be transduced.
• Other methods of transfection include nucleofection, electroporation, heat shock, magnetofection and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect.
• U.S. Patent No. 5,942,634 incorporated herein by reference, describes the use of cationic amphiphiles to facilitate transport of biologically active (therapeutic) molecules into cells.
• U.S. Patent No. 5,942,634 also teaches how to make therapeutic compositions incorporating a therapeutic molecule by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules.
• the therapeutic molecules that can be delivered into cells include DNA, RNA, and polypeptides.
• Such compositions can be used to provide gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells.
• the present invention provides a pharmaceutical formulation for the treatment of cells or mammals having hepatitis C, preferably chronic hepatitis C.
• Formulations according to the present invention should comprise a therapeutically effective amount of miRNA-196 mimic so the Bachl and/or HCV NS5A are down- regulated while HMOXl expression is increased.
• Embodiments according to the present invention are adapted to enable the transfection of miRNA-196 mimic into hepatocytes expressing HCV.
• the formulations of the present invention can include or utilize, but are not limited to, any of the aforementioned means for transfecting genetic material into a target cell.
• formulations of the present invention can be provided in a form for enteral administration,
• a formulation according to embodiments of the present invention can be provided in the form of a tablet, capsule or liquid preparation for oral administration.
• the formulation is provided as a liquid preparation for intravenous injection,
• the formulation can be provided in a form for injection or infusion.
• various formulations according to the present invention can be administered intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), subcutaneous (under the skin), intradermal, (into the skin itself), intrathecal (into the spinal canal), or intraperitoneal, (infusion or injection into the peritoneum)
• the treatment of infected cells individually, or mammals carrying such infected cells, with the formulations according to embodiments of the present invention can provide a significant reduction in the expression of Bachl and HCV NS5A protein levels and an increase in HMOXl levels upon the administration of a therapeutically effective amount of miRNA-196 mimic.
• therapeutically effective amount it is meant an amount of miRNA-196 mimic to an infected cell or mammal having Hepatitis effective to treat and/or prevent one or more targeted disorders.
• a daily therapeutic amount of the miRNA-196 mimic for the treatment of HCV infection can generally range from 0,5 to 5 ⁇ mol/kg of body weight, or from 1.0 to 4 ⁇ mol/kg of body weight, or from 2 to 3 ⁇ mol/kg of body weight.
• the therapeutic amount of miRNA-196 mimic can range from 0.1 to 1 ⁇ mol/kg of body weight, or from 0.25 to 1 ⁇ mol/kg of body weight, or form 0.25 to 0.75 ⁇ mol/kg of body weight.
• BCA protein assay reagents were obtained from Pierce Biotechnology (Rockford, IL). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from HyClone (Logan, UT).
• the Dual-GIo® Luciferase Assay System was from Promega (Madison, WI), TRIzol was purchased from Invitrogen (Carlsbad, CA) and geneticin (G- 418) was from Gibco (Grand Island, NY). Primers were synthesized by Integrated DNA Technologies (Coralville, IA). 4-15% gradient SDS-PAGE gels and ImmunBlot PVDF membranes were purchased from Bio-Rad (Hercules, CA).
• Mouse anti-HCV NS5A and mouse anti-HCV NS3 monoclonal antibodies were purchased from Virogen (Watertown, MA). Goat anti -human Bachl and GAPDH polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). ECL-Plus was from Amersham (Piscataway, NJ).
• 9-13 cells were provided by University of Heidelberg, Heidelberg, Germany.
• the 9-13 cell line a human hepatoma Huh-7 cell population, harbors a replicating HCV non- structural region and stably expresses HCV non-structural protein NS3 to NS5B.
• the cells were maintained in DMEM supplemented with 10% (v/v) FBS, 100 u/mL penicillin, 100 ⁇ g/mL streptomycin, and 500 ⁇ g/mL G-418.
• the Con 1 (subtype Ib) full length replicon Huh-7.5 cells was from the Rockefeller University (New York, NY).
• the Conl cell line is a Huh-7.5 cell population containing the full-length HCV genotype Ib replicon with the highly adaptive serine to isoleucine substitution at amino acid 2204 of the polypeptide.
• the Conl cells were maintained in DMFM supplemented with 10% (v/v) FBS and 0.ImM nonessential amino acids, 100 units/mL penicillin, lOO ⁇ g/mL streptomycin, and selection antibiotic 750 ⁇ g/mL G4I8.
• the cells were maintained in a humidified atmosphere of 95% room air and 5% CO 2 at 37 0 C.
• the miRIDIAN microRNA mimics for has-miRNA-196, has-miRNA-16, customized mutant has-miRNA-196, and miRNA mimic negative control (MMNC) were obtained from Dharmacon (Lafayette, CO).
• MicroRNA mimics are double-stranded RNA oligonucleotides chemically modified with ON-TARGET® to increase their stability and to improve their activities.
• the microRNA mimics mimic endogenous precursor miRNAs to enter the miRNA pathway and act as mature miRNA species. Additionally, the following miRNA inhibitors, has-miR-196 and miRNA inhibitor negative control (MINC), were also from Dharmacon.
• miRIDIAN microRNA hairpin inhibitors are the newest generation synthetic oligonucleotide inhibitors designed to suppress the native miRNAs activity.
• pRL-TK vector was from Promega.
• the pRL-TK reporter vector contains a cDNA (Rluc) encoding Renilla luciferase as an internal control reporter.
• pGL3-Bachl luciferase reporter construct containing an 1837 bp fragment of Bachl 3'-UTR, was provided by University of Florida, Gainesville, FL. Mutant pGL3-Bachl was generated by GENEWIZ, Inc. (South Plainfield, NJ). Constructs were confirmed by restriction enzyme digestion and sequencing.
• HCV infectious clone pJ6/JFHl the full-length chimeric genome with the core-NS2 regions from the infectious J6 (genotype 2a) and NS3-NS5B regions from the infectious JFHl (genotype 2a), was generously provided by C. M. Rice (the Rockefeller University, New York, NY).
• the production of J6/ JFHl -based cell culture-generated HCV (HCVcc) has been reported previously.
• the pJ6/JFHl plasmid was linearized with Xbal, and purified by ethanol precipitation, digestion with proteinase K, and phenol-chloroform extraction.
• the linearized plasmid was used as a template for in vitro transcription using the MEGAscript T7 kit (Ambion, Austin, TX).
• Huh-7.5 cells were plated in 24-well plates one day prior to transfection and transfected at 70-80% confluence. Cells were transfected at an RNA/lipofectamine ratio of 1 :2 by using 2 ⁇ g/well of HCV RNA and 4 uL/well Lipofectamine 2000 for 48 h.
• To infect na ⁇ ve Huh-7.5 cells cell culture supernatants from the cells transfected with HCV RNA for 48 h were collected and filtered through a 0.20 ⁇ m filter, and added to cultures of na ⁇ ve Huh-7.5 cells.
• RNA from tested cells was extracted and cDNA was synthesized as described previously.
• Primers used were as follows: Bach- 1 -specific sense primer 5'- GGACACTCCTTG CCAAATGCAG-3' (22 bp), anti-sense primer 5'- TGACCTGGTTCTGGGCTCTCAC-3' (22 bp); HMOXl -specific sense primer 5'- CGGGCCAGCAACAAAGTG-3' (18 bp), anti-sense primer 5'-
• AGTGT AAGGACCCATCGGAGAA-3' (22 bp); Cul3-specific sense primer 5'- GTGCTCACGACAGGATA-3' (17 bp), anti-sense primer 5'- GTTTGGCTAAGTAGAACCTTC-3' 21 bp); GAPDH-specific sense primer 5'- TTGTTGCCATCAATGACCC-3' (19 bp), anti-sense primer 5'- CTTCCCGTTCTCAGCCTTG-3 ' ( 19 bp).
• Real time quantitative RT-PCR was performed using a CFX96TM Real-Time PCR Detection System (Bio-Rad) and iQTM SYBR Green Supermix Real-Time PCR kit (Bio-Rad).
• Bioinformatic approaches were utilized to identify potential miRNA targets.
• An online search of the TargetScan 4.0 data base demonstrated that at least two putative miRNA- 196 seed match sites were harbored in the 3'-UTR of Bachl mRNA.
• one of the predicted binding site (2280-2286 nt) was highly conserved in human, mouse, rat, chicken and dog, whereas the other putative sites (2161-2166 nt) was poorly conserved across species.
• No predicted miRNa-196 binding sites were found in the Nrf2 and HMOXl gene, and no putative miRNA- 196 binding sites were found in the coding region of Bachl gene (data not shown).
• miRNA-196 down-regulates the transcriptional repressor Bachl and up-regulates HMOXl in 9-13 cells expressing HCV non-structural proteins
• 9-13 cells were transfected with miRNA-196 specific mimic or inhibitor, Bachl protein and mRNA levels were assessed by Western blots and qRT-PCR, respectively.
• 9-13 cells transfected with miRNA-196 mimic showed a significant reduction in the expression of Bachl protein levels by about 55% after 24 h transfection and about 64% after 48 h transfection compared with miRNA mimic negative control (MMNC).
• MMNC miRNA mimic negative control
• No effects on Bachl protein levels were detectable in cells transfected with miRNA mimic negative control compared with mock transfection (Fig. 2A).
• Down-regulation of miRNA- 196 by miRNA- 196 specific inhibitor significantly increased Bachl protein levels in contrast to miRNA inhibitor negative control (Fig. 2B).
• HMOXl and Cullin 3 (CuI 3, non-specific gene control) mRNA were quantified by qRT-PCR.
• miRNA- 196 mimic significantly up-regulated HMOXl mRNA levels by 2.4 fold compared with the same amount of miRNA mimic negative control as shown in Fig. 3A.
• CuI 3 mRNA levels were not up-regulated as shown in Fig. 3B.
• miRNA-196 represses HCV non-structural NS5A protein expression in 9-13 cells expressing HCV non-structural proteins
• miRNA-196 alters HCV nonstructural NS5A protein levels in human hepatoma cells expressing HCV non-structural proteins.
• 9-13 cells were transfected with either miRNA-196 mimic or inhibitor.
• Western blots were performed to analyze NS5A and GAPDH protein levels.
• miRNA-196 mimic resulted in a significant reduction in the expression of NS5A protein levels by about 52% after 24 h transfection, and about 37% after 48 h, compared with the same amount of miRNA mimic negative control as shown in Fig. 4A.
• miRNA-196 specific inhibitor significantly increased NS5 A protein levels by 2.2 fold as shown in Fig. 4B.
• Fig. 4C 5 miRNA- 196 resulted in a significant reduction in viral RNA levels, in comparison with miRNA mimic negative control (MMNC).
• miR-196 inhibits HCV expression in the HCV JFHl-based cell culture system
• Huh-7.5 cells were transfected with 2 ⁇ g/well of HCV J6/JFH 1 RNA by
• Lipofectamine 2000 After 48 h, cells were transfected with miRNA- 196 mimic, or miRNA mimic negative control (MMNC),
• miRNA- 196 mimic or miRNA mimic negative control (MMNC)
• MMNC miRNA mimic negative control
• HCV infection na ⁇ ve Huh-7.5 were cultured with 1 mL of cell culture supernatants harvested from J6/JFH1 -transfected cells, as previously described. After 48 hours of exposure to the supernatants, cells were transfected with miRNA- 196 mimic, or miRNA mimic negative control (MMNC) for 48 h. Cells were harvested and total RNA and proteins were extracted. HCV RNA was quantified by qRT-PCR, and HCV NS3 and GAPDH protein levels were measured by Western blots.
• a perfect match for miRNA- 196 was found in the coding region of the HCV NS5A gene in HCV JFHl genome.
• a down-regulatory effect of miRNA- 196 on HCV expression in the HCV J6/JFH1 cell culture system was observed.
• 50 nM of miRNA- 196 led to a significant decrease of HCV J6/JFH1 RNA by nearly 70% in J6/JFH1 transfected Huh-7.5 cells (as shown in Fig 9A), -50% in J6/JFH1 infected Huh-7.5 cells (as shown in Fig. 9B), and -60% reduction of HCV NS3 protein in J6/JFH1 infected Huh-7.5 cells (as shown Fig. 9C).
• miRNA-196 directly interacts with the 3'-UTR of Bachl mRNA in 9-13 cells expressing HCV non-structural proteins
• miRNA-196 targets the 3 '-UTR of Bachl mRNA, which contains two predicted seed match sites for miR-196 as shown in Fig. 5A.
• a reporter construct called pGL3-Bachl with Bachl 3'-UTR downstream of the firefly luciferase (f-luc) open reading frame (Fig. 5B) was used.
• 9-13 cells were co -transfected with pGL3-Bachl (f-luc), pRL-TK (renilla, to normalize for transfection efficiencies), and with miRNA-negative controls, miRNA-196 mimic or inhibitor or miRNA-196 (a "negative" miR with no predicted binding sites in the 3'-UTR of Bachl mRNA).
• miRNA-196 mimic transfection significantly decreased reporter activity by about 53%, whereas miRNA mimic negative control and miRNA-196 mimic had no effect on reporter luciferase activity. Additionally, no significant change of reporter activity was observed in cells transfected with miRNA-196 inhibitor.
• miRNA- 196 mimic inhibited the f-luc activities of pGL3-Bachl reporter, while miRNA-196 inhibitor slightly increased the f-luc activity of ⁇ GL3-Bachl reporter.
• 9-13 cells were co-transfected with Luc reporter construct containing a four nucleotide mutant Bachl 3'-UTR 3 which was called pGL3-Bachl-Mut (Fig. 6A), and with pRL-TK, and with miRNA-196 mimic or miRNA-155 mimic (a "negative" miR, with no changes of predicted miR-155 binding sites in pGL3 -Bachl -WT and pGL3-Bachl-Mut).
• the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega, firefly luciferase activities were normalized to renilla luciferase activities and total protein. As shown in Fig.
• miRNA-196 mimic decreased the f-luc activity of pGL3-Bachl-WT but not pGL-Bachl-Mut reporter.
• ⁇ u ' RNa-196 mimic transfection significantly decreased luciferase activity in a dose- dependent fashion, whereas miRNA- 196 did not change reporter activity in cells transfected with the reporter construct containing mutant binding sites for miRNA-196.
• miRNA-155 significantly decreased reporter activity in both pGL3-Bachl-WT and pGL3- Bachl -Mut as shown in Fig. 6C.
• miRNA- 196 resulted in a significant reduction in luciferase activity in a dose-dependent fashion, which was constant with our previous observations, whereas miRNA negative controls and mutant miR- 196 did not affect luciferase activity, further indicating the direct interaction between miR-196 and the 3'-UTR of Bachl mRNA.
• Mutant miRNA-196 (miR-196-Mut), containing base complementary with mutant pGL3-Bachl (pGL3 -Bachl -Mut), should restore its effect on mutant reporter (Bachl -3'- UTR-Mut) activity since they again match perfectly in their seed regions as shown in Fig. 8 A.
• Mutant miRNA-196 mimic significantly inhibited luciferase activity in cells transfected with mutant pGL3-Bachl, which was mutated to "fit" mutant miRNA-196.
• no significant effects of miRNA-196 wild type on mutant reporter (pGL3-Bachl-Mut) luciferase activity were observed.
• miRNA-196 mimic significantly down-regulated Bachl and HCV non- structural NS5A protein levels, and up-regulated HMOXl gene expression.
• miRNA-196 can play an important, perhaps even critical, role in the regulation of HCV replication and HMOXl/Bachl expression in hepatocytes. Accordingly, up-regulation of miR- 196 can provide an additional new therapeutic approach for therapy of chronic hepatitis C and, perhaps, other hepatic disorders.

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