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• • A—HUMAN NECESSITIES
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  • C12N2330/00—Production
  • C12N2330/10—Production naturally occurring

Definitions

• 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.
• 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.
• 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.
• 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 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.
• 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)
• 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.
• 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).
• 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 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.

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