WO2013006722A1 - Hcv genotype 3 replicons - Google Patents
Hcv genotype 3 replicons Download PDFInfo
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- WO2013006722A1 WO2013006722A1 PCT/US2012/045593 US2012045593W WO2013006722A1 WO 2013006722 A1 WO2013006722 A1 WO 2013006722A1 US 2012045593 W US2012045593 W US 2012045593W WO 2013006722 A1 WO2013006722 A1 WO 2013006722A1
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- WIPO (PCT)
- Prior art keywords
- rna
- genotype
- cell
- hcv
- residue
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Classifications
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2770/24223—Virus like particles [VLP]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Definitions
- the disclosure is directed to hepatitis C replicons of genotype 3 and methods of preparing and using the replicons. STATE OF THE ART
- HCV hepatitis C virus
- the current standard of care is 24 to 48 week courses of pegylated interferon plus ribavirin. Due to the partial efficacy and poor tolerability of this regimen, the discovery and development of new antiviral agents has been intensely pursued. Recently, these efforts have culminated in the FDA approval of two NS3 protease inhibitors (boceprevir and telaprevir) for use in combination with pegylated interferon and ribavirin for the treatment of chronic genotype 1 HCV infection. Many other inhibitors are in advanced clinical development, however, the majority are being developed to treat genotype 1 infections.
- HCV is a positive-strand RNA virus that exhibits extraordinary genetic diversity.
- Six major genotypes i.e. genotype 1-6
- multiple subtypes e.g. genotype la, lb, lc etc.
- Genotypes 1, 2 and 3 have worldwide distributions. Genotypes la or lb are generally predominant in North America, South America, Europe and Asia. However, genotypes 2 and 3 are common and can constitute 20 to 50% of infections in many of these areas.
- Genotype 4a is the predominant in the Middle East and many African countries; up to 15% of the population of Egypt is infected with HCV and 93% of infections are genotype 4.
- Genotype 5 is prevalent in South Africa, while
- Genotype 6 is most common in Asia. Although most continents and countries have a "dominant" genotype, infected populations are almost universally made up of a mixture of multiple genotypes. Furthermore, the geographical distribution and diversity (epidemiology) of HCV infection is continuously evolving, due to large-scale
- genotype 4a has noticeably spread into central and northern Europe. This presents a clinical challenge, since it is well documented that individual genotypes respond differently to both direct antivirals and immunomodulatory therapies, including the current standard of care.
- HCV replicons are self-replicating RNA sequences derived from the HCV genome and have served as workhorses both for molecular virology studies and drug discovery. To date, replicons have been established from two genotypes and three subtypes (genotypes la, lb and 2a). These replicons have been crucial in multiple aspects of drug discovery and development including the identification of novel inhibitor classes, the optimization of clinical candidates and the characterization of clinical resistance. Recently, there has been increasing interest in developing next-generation drugs that are active against all major HCV genotypes. Ideally, the approval of "pan-genotypic" drugs and regimens will greatly simplify the treatment of HCV.
- a key step in the pursuit of pan-genotypic treatment regimens will be the development of in vitro tools that allow the study of all major genotypes and subtypes.
- Replicons derived from sequences of additional major genotypes ⁇ i.e. those other than genotype la, lb or 2a), however, have not been generated.
- genotype 3 HCV in the Middle East, North Africa and Europe, no genotype 3 replicons have been described.
- RNA sequence comprises a 5'NTR, an internal ribosome entry site (IRES), sequences encoding one or more of NS3, NS4A, NS4B, NS5A or NS5B, and a 3'NTR.
- the construct comprises an adaptive mutation in NS3, NS4A, NS4B, NS5A or NS5B.
- the mutation comprises an isoleucine at location 2204.
- the mutation comprises, in NS3, a serine at residue 607.
- the mutation comprises, in NS3, a leucine at residue 89.
- the mutation further comprises one or more of an arginine at residue 41, a threonine at residue 166, a threonine at residue 379, a glycine at residue 534, a glutamic acid at residue 583, and/or a cysteine at residue 1.
- the mutation further comprises one of more provided in Tables 7-9.
- DNA that transcribes to the RNA construct is also provided.
- viral particles that include the RNA construct are also provided.
- an NS3 protein of HCV genotype 3 that comprises a serine at residue 607 and/or a leucine at residue 89.
- an NS3 protein that further comprises an isoleucine at location 2204. Polynucleotides encoding these proteins and antibodies that specifically recognize the proteins are also provided.
- the present disclosure provides an isolated cell comprising a genotype 3 hepatitis C viral (HCV) RNA that replicates in the cell.
- HCV hepatitis C viral
- the cell comprises at least 10 copies, or alternatively at least about 100, 500, 1000, 2000, 5000, 10,000, 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 or 1 x 10 9 copies of the RNA.
- the RNA can be a subgenomic HCV sequence or a full-length HCV sequence and can include one or more of the adaptive mutations described above.
- Methods of improving the capability of a genotype 4 HCV viral RNA to replicate in a eukaryotic cell comprising substituting residue 607 of NS3 with a serine and/or substituting residue 89 of NS3 with a leucine.
- Further mutations can include one of more of an arginine at residue 41, a threonine at residue 166, a threonine at residue 379, a glycine at residue 534, a glutamic acid at residue 583, and/or a cysteine at residue 1 or those provided in Tables 7-9.
- a method of identifying an agent that inhibits the replication or activity of a genotype 3 HCV comprising contacting a cell of any of the above embodiments with a candidate agent, wherein a decrease of replication or a decrease of the activity of a protein encoded by the R A indicates that the agent inhibits the replication or activity of the HCV.
- the method comprises contacting the lysate of a cell of any of the above embodiments with a candidate agent, wherein a decrease of the activity of a protein encoded by the RNA indicates that the agent inhibits the activity of the HCV.
- FIG. 1 is a schematic diagram of genotype 3a replicon constructs.
- An S52 3a strain (Accession #: GU814264) replicon encodes a neomycin (A) or a Renilla luciferase (Rluc)-neomycin fusion reporter (B).
- A neomycin
- Rluc Renilla luciferase
- the synthesized replicon incorporated the following elements from 5' to 3': the S52 5'UTR; the neomycin phosphotransferase II gene (neo) or Rluc-Neo gene; the encephalomyocarditis virus (EMCY) IRES; the NS3 - NS5B polyprotein region of S52 including an NS5 A adaptive mutation (S2204I) and the 3'UTR of S52.
- Solid boxes indicate HCV core sequence. Dot shaded boxes indicate the HCV polyprotein sequence. "+" indicates the S2204I adaptive mutation.
- the 5' and 3' non-translated regions (NTR), and EMCV IRES are indicated. [0018] FIG.
- GT3a replicon NS3 protease activities in response to exposure to an NS3 protease inhibitor Compound C. 1 x 10 5 GT3a replicon cells were pelleted and lysed in 2.4 ml 1 x Promega luciferase lysis buffer supplemented with 150 mM NaCl at room temperature for 15 min. The cell lysate was then distributed to a 96-well plate with a volume of 80 ⁇ per well. Antiviral drug dilutions were prepared in DMSO at 10 times the final desired concentrations (5 nM or 50 nM).
- Portions (10 ⁇ ) of diluted Compound C was then added to each well of cell lysates, and the plates were incubated on a plate shaker for 10 minutes at room temperature. Then, 10 ⁇ of 1 ⁇ europium-labeled NS3 substrate was added to each well. Protease activity data were collected and the data were presented as the percentage of inhibition of DMSO control.
- FIG. 3 shows robust NS5A expression in genotype 3a replicon cell line.
- Stable GT3a, GTla, GTlb and GT2a replicon cells and 1C cells, 0.5 x 10 6 each were pelleted and completely lysed in 100 ⁇ SDS loading buffer. Twelve microliter lysates were subjected to SOS-PAGE and Western blot analysis. The blot was stained with primary anti-NS5A antibody (Apath; 1 :3000 dilution) and secondary anti-mouse antibody (IRDye 800CW Goat anti-Mouse IgG (H + L) from LI-COR, 1 : 10,000 dilution).
- the staining was then analyzed by Odyssey Imaging (LI-COR, Lincoln, Iowa).
- the blot was also co- stained with anti-BiP antibody (Abeam, 1 : 1000 dilution) and secondary anti-rabbit antibody (IRDye 800CW Goat anti-Rabbit IgG (H + L) from LI-COR, 1 : 10,000 dilution) as a loading control.
- 1C cells were included as a negative control. Strong expression of NS5A was detected in the genotype 3a replicon cell clone, confirming that these cells stably and robustly replicate this replicon.
- FIG. 4A-C show a schematic diagram of genotype 3 a replicon constructs in Example 3.
- Genotype 3a S52 strain replicons encode a neomycin phosphotransferase II gene (neo) (A), a Renilla luciferase (Rluc)-neo fusion reporter (B) or a Pi Renilla luciferase (PiRluc) fusion reporter (C).
- the synthesized replicon incorporated following elements from 5' to 3': the S52 5TJTR; the Neo, Rluc-neo or Pi-Rluc reporter gene; the EMCV IRES; the NS3 - NS5B polyprotein region of S52 including an NS5A adaptive mutation (S2210I) and the 3TJTR of S52. -. ⁇ indicates HCV S52 core sequence.
- FIG. 5A-B show NS5A expression in selected genotype 3a replicon cells.
- A The two stable genotype 3a replicon clones, as well as the genotype lb replicon cells, were pelleted and completely lysed in SDS loading buffer. Lysates were then subjected to SDS-PAGE and Western blot analysis.
- the blot was stained with primary anti-NS5A antibody (Apath; 1 :3000 dilution) and secondary anti-mouse antibody (IRDye 800CW Goat anti-Mouse IgG (H + L) from LI- COR, 1 : 10,000 dilution).
- the blot was also co-stained with anti-BiP antibody (Abeam, 1 : 1000 dilution) and secondary anti-rabbit antibody (IRDye 680CW Goat anti-Rabbit IgG (H + L) from LI-COR, 1 : 10,000 dilution) as a loading control.
- the staining was analyzed by Odyssey Imaging (LI-COR).
- B NS5A immunostaining of the selected replicon cells.
- the genotype 3a replicon clone #1 was stained with anti-NS5A antibody (red) and Hoechst 33342 (blue, indicating nuclei). 1C cells were stained as a negative control.
- FIG. 6 shows that selected genotype 3 a replicon clone acquires adaptive mutations.
- Total cellular RNA was extracted from the genotype 3a replicon cell clone #1 and then electroporated into Huh7-Lunet cells at the indicated amounts.
- Transfected cells were resuspended in complete DMEM medium, plated in a 100-mm diameter dish, and cultured with 0.5 mg/ml G418 (also known as Geneticin®, an aminoglycoside antibiotic). Three weeks later, colony plates were fixed with 4% formaldehyde and stained with 0.05% crystal violet. In vztro-transcribed GT3a replicon RNA was transfected in parallel as a control.
- FIG. 7 is a chart showing the establishment of stable and robust GT3a Rluc-Neo replicon cell lines.
- the P89L mutation in NS3 was introduced into the GT3a S52 Rluc- neo construct by site-directed mutagenesis.
- the GT3a-P89L-Rluc-neo mutant replicon RNA and the parental GT3a-Rlu-neo replicon RNAs (10 ⁇ g each) were transfected into 1C or Huh7-Lunet cells. The number of surviving colonies was counted for each selection.
- FIG. 8A-B indicate Luciferase expression in genotype 3a luciferase cell clones. (A).
- B Selected genotype 3a replicon cell clones derived from Huh7-Lunet cells. Genotype la and genotype lb stable replicon cells were included as references. Untransfected 1C or Huh7- Lunet cells were included as a negative control. 60,000 cells were used for for renilla luciferase activity assay.
- FIG. 9 shows that combined mutations P89L in NS3 and S232I in NS5A
- FIG. 10A-B present curves to show that the genotype 3 a cured cell lines are highly permissive to genotype 3a replication.
- Pi-Rluc-GT3a-P89L A
- Pi-Rluc-GTla B
- Pi-Rluc-GTlb B
- Luciferase activity was measured daily for 4 days post transfection.
- FIG. 11 A-B show that secondary mutations in NS3 or NS4A enhanced genotype 3a replicon replication.
- Secondary mutations Q41R, A166T, A379T, S534G, K583E in NS3, or SIC, in NS4A were introduced into the GT3a-P89L PiRluc construct by site-directed mutagenesis.
- Replicon RNAs (Pi-Rluc-GTlb as a control) were individually transfected into 1C (A) or 3a-C3 (B) cured cells, and 1 x 10 4 transfected cells were plated per well in a 96-well plate. Cells were analyzed for renilla luciferase activity at 4 hours and daily for 7 days post transfection,. Data presented in the figure are from a representative experiment of at least two independent experiments.
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.
- protein and "polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
- the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. , ester, ether, etc.
- a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
- a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
- polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
- Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
- the following are non- limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched
- polynucleotides plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
- a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
- the sequence of nucleotides can be interrupted by non-nucleotide components.
- a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules.
- any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
- a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is R A.
- A adenine
- C cytosine
- G guanine
- T thymine
- U uracil
- polynucleotide sequence is the alphabetical representation of a polynucleotide molecule.
- This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
- "Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
- an "unrelated" or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.
- the homologous peptide is one that shares the same functional characteristics as those described, including one or more of the adaptive mutations.
- a polynucleotide or polynucleotide region has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), 98%o or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
- This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
- default parameters are used for alignment.
- One alignment program is BLAST, using default parameters.
- a homolog of a nucleic acid refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof.
- a homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof.
- homo logs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.
- a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide or polypeptide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
- the term "express” refers to the production of a gene product.
- expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is
- polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
- encode refers to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
- the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
- Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
- a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells, or alternatively from a prokaryotic cells as described above. Non-limiting examples include simian, bovine, porcine, murine, rats, avian, reptilian and human.
- an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
- the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity
- the antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, e.g., murine, rat, sheep and canine.
- the terms "polyclonal antibody” or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.
- monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
- a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
- isolated refers to molecules or biological or cellular materials being substantially free from other materials or when referring to proteins or polynucleotides, infers the breaking of covalent bonds to remove the protein or polynucleotide from its native environment.
- isolated refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
- isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when
- an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
- isolated or recombinant means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
- an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
- isolated polynucleotide is separated from the 3 ' and 5 ' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
- a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
- isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
- Hepatitis C virus or "HCV” is a small (55-65 nm in size), enveloped, positive- sense single-stranded RNA virus of the family Flaviviridae. Hepatitis C virus is the cause of hepatitis C in humans.
- the hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell of protein, and further encased in a lipid (fatty) envelope of cellular origin.
- Two viral envelope glycoproteins, El and E2 are embedded in the lipid envelope.
- Hepatitis C virus has a positive sense single-stranded RNA genome.
- the genome consists of a single open reading frame that is 9600 nucleotide bases long. This single open reading frame is translated to produce a single protein product, which is then further processed to produce smaller active proteins.
- the 5 ' UTR has a ribosome binding site (IRES - Internal ribosome entry site) that starts the translation of a very long protein containing about 3,000 amino acids. This large pre-protein is later cut by cellular and viral proteases into the 10 smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles.
- IRS ribosome binding site
- Structural proteins made by the hepatitis C virus include Core protein, El and E2; nonstructural proteins include NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.
- nonstructural proteins include NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.
- the hepatitis C virus species is classified into six genotypes (1-6) with several subtypes within each genotype
- HCV genotypes are further broken down into quasispecies based on their genetic diversity. The preponderance and distribution of HCV genotypes varies globally. For example, in North America, genotype la predominates followed by lb, 2a, 2b, and 3a. In Europe, genotype lb is predominant followed by 2a, 2b, 2c, and 3a.
- Genotypes 4 and 5 are found almost exclusively in Africa. Genotype is clinically important in determining potential response to interferon-based therapy and the required duration of such therapy. Genotypes 1 and 4 are less responsive to interferon-based treatment than are the other genotypes (2, 3, 5 and 6). Duration of standard interferon- based therapy for genotypes 1 and 4 is 48 weeks, whereas treatment for genotypes 2 and 3 is completed in 24 weeks.
- Sequences from different HCV genotypes can vary as much as 33% over the whole viral genome and the sequence variability is distributed equally throughout the viral genome, apart from the highly conserved 5' UTR and core regions and the hypervariable envelope (E) region.
- HCV genotypes can be identified with various methods known in the art. PCR- based genotyping with genotype-specific primers was first introduced in 1992, in particular with primers targeting the core region. Commercial kits (e.g. , InnoLipa® by Innogenetics (Zwijwear, Belgium)) are also available. Direct sequencing, in the vein, can be used for more reliable and sensitive genotyping.
- Serologic genotyping uses genotype-specific antibodies and identifies genotypes indirectly.
- Two commercially available serologic genotyping assays have been introduced, including a RIBA SIA assay from Chiron Corp. and the Murex HCV serotyping enzyme immune assay from Nurex Diagnostics Ltd.
- genotype 3 HCV has been identified. For instance, GenBank accession # GU814264 provides the sequence of a subgenomic genotype 3a replicon based on the S52 infectious clone. Further discussion of the genotype 3 and their sequences are clinical impacts can be found at Zein Clin. Microbiol. Rev. 13(2):223-35 (2000).
- the term "replicon" refers to a DNA molecule or R A molecule, or a region of DNA or RNA, that replicates from a single origin of replication. For most prokaryotic chromosomes, the replicon is the entire chromosome.
- a replicon refers to a DNA or RNA construct that replicates in a cell in vitro.
- a replicon can replicate to produce at least about 10, or alternatively, at least about 100, 500, 1000, 2000, 5000, 10,000, 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 or 1 x 10 9 copies of the replicon in a cell in vitro.
- a replicon's replication efficiency can be measured by producing certain amount of viral RNA in total RNA that includes cellular RNA.
- a replicon can produce at least about 1000, 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 copies of the replicon per microgram of total RNA or cellular RNA.
- a "subgenomic" HCV sequence refers to a HCV sequence that does not include all sequences of a wild-type HCV.
- a subgenomic HCV or a subgenomic HCV replicon does not include the El, E2 or C regions.
- a subgenomic HCV or a subgenomic HCV replicon includes all or part of the 5' UTR, NS3, NS4A,
- a virus particle (or virion) consists of the genetic material made from either DNA or RNA of a virus and a protein coat that protects the genetic material.
- an envelope of lipids surrounds the protein coat when they are outside a cell.
- adaptive mutation of a HCV replicon of a certain genotype refers to a mutation, as compared to a wild-type HCV sequence of the genotype, that enables the wild-type replicon to replicate in a cell, in particular in a eukaryotic cell such as a mammalian cell and in vitro, or enhances a HCV replicon's ability to replicate. It is contemplated that an adaptive mutation can favorably influence assembly of the replicase complex with host cell-specific protein, or alternatively promote interactions of the protein that includes the adaptive mutation ⁇ e.g., NS3, NS4A, NS4B, NS5A etc) with cellular proteins involved in host cell antiviral defenses.
- reporter gene refers to a gene that can be attached to a regulatory sequence of another gene of interest in cell culture, animals or plants, to facilitate identification of this other gene. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population. Non-limiting examples of reporter gene include the luciferase gene and the green fluorescent protein gene.
- a "marker gene” or “selectable marker” refers to a gene that protects the organism from a selective agent that would normally kill it or prevent its growth.
- One non-limiting example is the neomycin phosphotransferase gene (Neo), which upon expression confers resistance to G418, an aminoglycoside antibiotic similar in structure to gentamicin Bl .
- the present disclosure relates, in general, to the unexpected discovery that clonal cell lines stably replicating genotype 3 replicons can be obtained by transcribing and electroporating subgenomic genotype 3 cDNAs into HCV permissive cell lines. From the clonal cells, adaptive mutations are then identified. One such adaptive mutation is N607S at NS3. Another adaptive mutation is P89L. The S2204I mutation is also applicable in this genotype.
- any one or more of these mutations can be further enhanced by one or more of Q41R (NS3), A166T (NS3), A379T (NS3 helicase domain), S534G (NS3 helicase domain), K583E (NS3 helicase domain), SIC (NS4A) or those provided in Tables 7-9.
- the present disclosure provides a genotype 3 hepatitis C viral (HCV) RNA that is capable of replication in a host cell.
- the replication is in vitro. In one aspect, the replication is productive replication. In another aspect, the cell is a eukaryotic cell such as a mammalian cell or a human cell. In yet another aspect, the cell is a hepatoma cell. In some aspects, the RNA can replicate to produce at least 10 copies of the RNA in a cell. In another aspect, the number of copies is at least about 100, 500, 1000, 2000, 5000, 10,000, 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 or 1 x 10 9 .
- the HCV RNA can be a subgenomic HCV sequence. It is specifically contemplated that a full-length HCV replicon containing any or more of such adaptive mutations is also capable to replicate. Still further, an entire HCV virus of the corresponding genotype containing the adaptive mutation(s) would be infectious and capable to replicate.
- RNA can include one or more of 5'NTR, an internal ribosome entry site (IRES), sequences encoding NS3, NS4A, NS4B, NS5A and NS5B, and a 3 'NTR.
- the RNA includes, from 5' to 3' on the positive-sense nucleic acid, a functional HCV 5 ' non-translated region (5 'NTR) comprising an extreme 5 '-terminal conserved sequence; an HCV polyprotein coding region; and a functional HCV 3' non-translated region (3'NTR) comprising an extreme 3 '-terminal conserved sequence.
- a functional HCV 5 ' non-translated region 5 'NTR
- an HCV polyprotein coding region comprising an extreme 3 '-terminal conserved sequence.
- the HCV RNA can include an adaptive mutation that enables the RNA to replicate in the cell.
- Such adaptive mutations can include an isoleucine at location 2204 at NS5A, and/or a serine at residue 607 for NS3.
- Another adaptive mutation is P89L.
- the effect of any one or more of these mutations can be further enhanced by one or more of Q41R (NS3), A166T (NS3), A379T (NS3 helicase domain), S534G (NS3 helicase domain), K583E (NS3 helicase domain), SIC (NS4A) or those provided in Tables 7-9.
- the HCV RNA can be a RNA sequence that has at least about 75%, or about 80%, 85%, 90%, 95%, 98%, 99%, or about 99.5% sequence identity to any of the disclosed sequences, so long as it retains the corresponding adaptive mutation(s) and/or activities.
- a genotype 3 HCV RNA construct comprising a 5 'NTR, an internal ribosome entry site (IRES), sequences encoding NS3, NS4A, NS4B, NS5A and NS5B, and a 3'NTR, wherein the construct is capable to replicate in a eukaryotic cell.
- the construct comprises an adaptive mutation in NS3, NS4A, NS4B, NS5A or NS5B.
- the mutation in one aspect, comprises an isoleucine at location 2204 in NS5A, and in another aspect, comprises, in NS3, a serine at residue 607 and/or a leucine at residue 89.
- the genotype 3 is genotype 3a.
- the HCV R A can further comprise a marker gene for selection.
- a non-limiting example of such marker gene is a neomycin phosphotransferase gene. Other examples are well known in the art.
- the HCV RNA can further comprise a reporter gene.
- a non-limiting example of such marker gene is a luciferase gene. Other examples are well known in the art.
- RNA construct of any of the above embodiment can further comprise sequences encoding one or more of C, El or E2.
- the RNA construct is a full-length HCV replicon.
- the disclosure also provides a single or double-stranded DNA that can be transcribed to a RNA construct of any of the above embodiment, a viral particle comprising a RNA construct of any of the above embodiment, or an isolated cell comprising a RNA construct of any of the above embodiment.
- the genotype 3 is genotype 3a.
- the polynucleotide can be RNA or DNA.
- provided is an RNA or DNA construct comprising the polynucleotide.
- a cell comprising the polynucleotide.
- provided is an antibody that specifically recognizes a protein of any of the above embodiments.
- Another embodiment of the present disclosure provides an isolated cell comprising a hepatitis C viral (HCV) RNA that is genotype 3, wherein the HCV RNA replicates in the cell.
- HCV hepatitis C viral
- the cell comprises at least 10 copies of the RNA. In another aspect, the cell comprises at least 100, 500, 1000, 2000, 5000, 10,000, 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 or 1 x 10 9 copies of the RNA.
- the HCV RNA can be subgenomic HCV sequence or a full-length HCV sequence. In either case, RNA can include one or more of 5'NTR, an internal ribosome entry site (IRES), sequences encoding NS3, NS4A, NS4B, NS5A and NS5B, and a 3'NTR.
- the HCV RNA can include an adaptive mutation that enables the RNA to replicate in the cell. Such adaptive mutations can include an isoleucine at location 2204 at NS5A.
- HCV RNA can be a RNA sequence that has at least about 75%, or about 80%, 85%, 90%, 95%, 98%, 99%, or about 99.5% sequence identity to any of the disclosed sequences, so long as it retains the corresponding adaptive mutation(s).
- the cell is a eukaryotic cell such as a mammalian cell and in particular a human cell.
- the cell is hepatoma cell, such as but not limited to a Huh7 cell (e.g., Huh7-Lunet, 51C and 1C).
- the cell is placed at an in vitro or ex vivo condition.
- HCV genotype 3 replicons are identified, as shown in Example 1 , introduction of the relevant adaptive mutation into a corresponding genotype HCV RNA can result in the RNA's capability to replicate, in particular in a mammalian cell in vitro. Accordingly, the present disclosure provides a method of improving the capability of a genotype 3 HCV viral RNA to replicate in a eukaryotic cell, comprising substituting residue 607 of NS3 with a serine or residue 89 of NS3 with a leucine. In one aspect, an S2204I mutation or any secondary mutation provided herein can further be introduced into the RNA.
- the present disclosure also provides, in one embodiment, a method of identifying an agent that inhibits the replication or activity of a genotype 3 HCV, comprising contacting a cell of any embodiment of the present disclosure with a candidate agent, wherein a decrease of replication or a decrease of activity of a protein encoded by the RNA indicates that the agent inhibits the replication or activity of the HCV.
- the protein is a protease, such as any or more of NS3, NS4A, NS4B, NS5A or NS5B.
- Replication of the RNA in one aspect, can be measured by a reporter gene on the RNA, such as the luciferase gene.
- a method of identifying an agent that the activity of a genotype 3 HCV comprising contacting the lysate of a cell of any embodiment of the present disclosure with a candidate agent, wherein a decrease of the activity of a protein encoded by the RNA indicates that the agent inhibits the activity of the HCV.
- the protein is a protease, such as any or more of NS3, NS4A, NS4B, NS5A or NS5B.
- the method further comprises measuring the replication of the RNA or the activity of the protein encoded by the RNA.
- a HCV inhibitor can be a small molecule drug that is an organic compound, a peptide or a protein such as antibodies, or nucleic acid-based such as siRNA.
- a peptide or a protein such as antibodies
- nucleic acid-based such as siRNA.
- Boceprevir is a protease inhibitor that binds to the HCV NS3 active site on hepatitis C genotype 1.
- Telaprevir inhibits the hepatitis C virus NS3.4A serine protease.
- More conventional HCV treatment includes a combination of pegylated interferon-alpha-2a or pegylated interferon-alpha-2b (brand names Pegasys or PEG- Intron) and the antiviral drug ribavirin.
- Pegylated interferon-alpha-2a plus ribavirin may increase sustained virological response among patients with chronic hepatitis C as compared to pegylated interferon-alpha-2b plus ribavirin according to a systematic review of randomized controlled trials.
- HCV inhibitors can be tested with the disclosed methods for their efficacy in inhibiting HCV genotype 3.
- the cells are then incubated at a suitable temperature for a period time to allow the replicons to replicate in the cells.
- the replicons can include a reporter gene such as luciferase and in such a case, at the end of the incubation period, the cells are assayed for luciferase activity as markers for replicon levels. Luciferase expression can be quantified using a commercial luciferase assay.
- efficacy of the HCV inhibitor can be measured by the expression or activity of the proteins encoded by the replicons.
- Luciferase or NS3 protease activity level is then converted into percentages relative to the levels in the controls which can be untreated or treated with an agent having known activity in inhibiting the HCV.
- a decrease in HCV replication or decrease in NS3 activity, as compared to an untreated control indicates that the candidate agent is capable of inhibiting the corresponding genotype of the HCV.
- a larger decrease in HCV replication or larger decrease in NS3 activity, as compared to a control agent indicates that the candidate is more efficacious than the control agent.
- Huh7-Lunet was obtained from ReBLikon GmbH (Mainz, Germany)
- This clonal line showed the highest permissivity to GTla and lb replicons out of screened 50 clones and was 5-10 folds more permissive than Huh7-Lunet and 51C cells overall.
- All cell lines were propagated in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX-I (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 1 unit/ml penicillin (Invitrogen), 1 ⁇ g/ml streptomycin (Invitrogen), and 0.1 mM non-essential amino acids (Invitrogen); this media formulation is referred to as complete DMEM.
- Replicon cell lines were selected and maintained in complete DMEM containing 0.5 mg/ml G418 (Geneticin; Invitrogen). Construction ofPlasmids Encoding Genotype 3a HCV Subgenomic Replicons
- a plasmid (pGT3aS52SG) encoding a subgenomic genotype 3a replicon based on the S52 infectious clone was prepared by DNA synthesis (GeneScript, Piscataway, NJ) and cloning. The synthesized replicon
- Plasmid p/zRlucNeoSG2a was derived from the plasmid pLucNeo2a (Cheng et al., Antimicrob Agents Chemother 55:2197-205 (2011)).
- the hRenilla Luciferase- Neomycin fusion gene (hRluc-Neo) was PCR amplified from pF9 CMV hRluc-neo Flexi(R) (Promega, Madison, WI) by PCR using Accuprime Super Mix I (Invitrogen) and a primer set of Afel hRLuc Fwd and NotI Neo Rev. These two primers had the following sequence and introduced restriction sites for subsequent cloning: Afel hRLuc: 5'
- hRluc-Neo amplification product was subcloned into pCR2.1-TOPO (Invitrogen).
- the resulting plasmid was digested with Afel and NotI, and the excised fragment (hRluc-Neo) was ligated with T4 DNA ligase (Promega) into pLucNeo2a digested with the same enzymes.
- the resulting vector, phRlucNeoSG2a was sequenced to ensure correct orientation and sequence of the hRluc-Neo fusion gene.
- Plasmids encoding genotype 3a subgenomic HCV replicons were linearized with Xbal and purified using a PCR purification kit (Qiagen). RNA was synthesized and purified with T7 MEGAScript (Ambion, Austin, TX) and RNeasy kits, respectively, according to the manufacturer's instructions. RNA concentrations were measured using optical density at 260 nm and confirmed by 0.8% agarose gel electrophoresis (Invitrogen).
- Replicon RNA was added to 400 ⁇ of cell suspension in a Gene Pulser (BioRad,
- Replicon Colony Formation Assays [0101] To determine the efficiency of G418 -resistant colony formation, cells were electroporated with indicated amounts of replicon RNA or cellular RNA extract, and plated at multiple densities ranging from 2 x 10 5 to 2 x 10 6 cells/ 100mm dish. Forty-eight hours after plating, medium was replaced with complete DMEM supplemented with 0.5 mg/ml G418 which was refreshed twice per week. Three weeks later, colony plates were used for cell expansion or G418 -resistant foci were fixed with 4% formaldehyde and stained with 0.05% crystal violet in H 2 0.
- HCV RNA isolation, RT-PCR, and sequencing were performed by TACGen (Hayward, CA).
- HCV replicon cellular RNA was extracted and purified using an RNeasy kit (Qiagen) according to the manufacturer's protocol.
- RT-PCR was performed using the Superscript III first-strand synthesis system (Invitrogen). PCR products were sequenced by TACGen (Hayward, CA).
- Replicon cells were plated in 96-well plates at a density of 1 x 10 4 cells per well. After cultured for 24 hours, cells were then stained for NS5A protein as described previously (Cheng et ah, Antimicrob Agents Chemother 55:2197-205 (2011)). Briefly, cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were then washed three times with PBS, blocked with 3% bovine serum albumin, 0.5% TritonX-100, and 10%> FBS and then stained with anti-NS5A antibody. Staining was performed using a 1 : 10,000 dilution of mouse monoclonal antibody 9E10 (Apath, Brooklyn, NY).
- a secondary anti-mouse antibody conjugated to Alexa Fluor 555 was used to detect anti-NS5A antibody labeled cells (Invitrogen). Nuclei were stained with 1 ⁇ g/ml Hoechst 33342 (Invitrogen). Cells were washed with PBS and imaged with a Zeiss fluorescence microscope (Zeiss, Thornwood, NY).
- Replicon cell IS S3 protease assay for replicon RNA replication
- Genotype 3 a clonal replicons cells were seeded in 96-well plates at a
- 2,000 cells/well were seeded in 384-well plates in 90 ⁇ of DMEM culture medium, excluding G418. Compounds were added to cells at a 1 :225 dilution, achieving a final concentration of 0.44% in a total volume of 90.4 ⁇ . Three-fold serial drug dilutions with 10 concentrations were used, and starting concentrations were 4.4 ⁇ or 0.44 ⁇ for all the tested compounds, except Compound A whose starting concentrations was 44.4 nM. Cell plates were incubated at 37°C for 3 days, after which culture medium was removed and cells were assayed for luciferase activity as markers for replicon levels.
- Luciferase expression was quantified using a commercial luciferase assay (Promega). Luciferase or NS3 protease activity levels were converted into percentages relative to the levels in the untreated controls (defined as 100%), and data were fitted to the logistic dose response equation y _ a/[l_(x/b)c] using XLFit4 software (IDBS, Emeryville, CA) (y is the amount of normalized luciferase signal, x is the drug concentration, a represents the curve's amplitude, b is the x value at its transition center [EC 50 ], and c is a parameter which defines its transition width).
- Huh7-Lunet cell colonies containing genotype 3 replicons were identified.
- Huh7-Lunet, 51C and 1C cells were transfected with the GT3a replicon R A as described in the Materials and Methods. The numbers of surviving colonies were counted for each selection. The data represent the total number of colonies selected from at least 6 independent transfections in each cell line.
- Huh7-Lunet was obtained from ReBLikon GmbH (Mainz, Germany). The derivation of 51C cells was previously described (Robinson et ah, Antimicrob Agents Chemother 54:3099-106 (2010).
- 1C cells were derived by curing a GS-5885-resistant genotype la replicon clone derived from 51C cells. Transfection of Huh7-Lunet yielded two colonies that replicated the GT3a replicon and could subsequently be expanded into cell lines. Transfection of the other two cell lines did not yield any viable colonies (Table
- Total cellular R A from 50000 GT3a replicon cells (expanded from a colony established in Huh7-Lunet cells described in Table 1) was extracted using a virus RNA QIAamp kit (Qiagen) as recommended by the manufacturer. Half of the total cellular RNA was then electroporated into Huh7-Lunet cells and the other half into IC cells. Transfected cells were resuspended in complete DMEM medium and plated at a density of 1 x 10 6 and 3 x 10 6 cells in 150 mm-diameter dishes. Forty-eight hours after plating, medium was replaced with complete DMEM supplemented with 0.25 mg/ml G418 which was refreshed twice per week.
- GT3a replicon clone was expanded and subjected to genotypic analysis.
- Total cellular RNA was extracted and purified using a RNeasy kit (Qiagen).
- RT-PCR was performed using the Superscript III first-strand synthesis system
- HCV inhibitors that target NS5 A, NS5B active site, NS3 protease, NS5B non-active sites, NS4A and host factors, were evaluated for their antiviral activities against stable genotype lb and genotype 3a Rluc-Neo replicon cells. Like in stable genotype lb replicon cells, EC 5 o values against genotype 3a replicon were generated successfully for all the inhibitors in a high throughput 384-well format by measuring NS3 protease activity.
- subgenomic replicon cDNAs based on the genotype 3a S52 strain were synthesized, cloned, transcribed and electroporated into HCV permissive cell lines. Clonal cell lines stably replicating genotype 3a replicons were selected with G418.
- Adaptive mutations were identified by RT-PCR amplification and DNA sequencing and engineered into the parental replicons by site-directed mutagenesis. [0114] Numerous electroporations into multiple different permissive cell lines allowed the identification of a few colonies that replicated either genotype 3 replicons. Expansion and sequencing of these replicons clones revealed adaptive mutations in viral proteins. One mutation identified so far was located in NS3 (N607S). [0115] The establishment of robust genotype 3a replicon systems provides powerful tools to facilitate drug discovery and development efforts. Use of these novel replicons in conjunction with those derived from other genotypes will aid in the development of pan- genotypic HCV regimens.
- Example 1 shows that agents known to be HCV inhibitors for other genotypes, such as genotype 1, can be tested with the genotype 3 replicons for their efficacy in inhibiting genotype 3 HCV. It is also contemplated that agents not yet known to be inhibitory of HCV can be screened with these genotype 3 replicons as well.
- the candidate HCV inhibitor can be a small molecule drug, a peptide or a protein such as antibodies, or nucleic acid-based such as siRNA.
- the candidate HCV inhibitor is incubated with cells that contain a genotype 3 replicon, at a suitable temperature for a period time to allow the replicons to replicate in the cells.
- the replicons can include a reporter gene such as luciferase and in such a case, at the end of the incubation period, the cells are assayed for luciferase activity as markers for replicon levels. Luciferase expression can be quantified using a commercial luciferase assay.
- efficacy of the HCV inhibitor can be measured by the expression or activity of the proteins encoded by the replicons.
- proteins encoded by the replicons One example of such proteins is the NS3 protease, and detection of the protein expression or activity can be carried out with methods known in the art, e.g., Cheng et al, Antimicrob Agents Chemother 55:2197-205 (2011).
- Luciferase or NS3 protease activity level is then converted into percentages relative to the levels in the controls which can be untreated or treated with an agent having known activity in inhibiting the HCV.
- a decrease in HCV replication or decrease in NS3 activity, as compared to an untreated control indicates that the candidate agent is capable of inhibiting the corresponding genotype of the HCV.
- a larger decrease in HCV replication or larger decrease in NS3 activity, as compared to a control agent indicates that the candidate is more efficacious than the control agent.
- genotype 3a replicons that efficiently replicate in vitro. This study demonstrates that robust replication was achieved based on an adaptive mutation P89L in the NS3 protease domain, which could be further augmented by mutations in NS3, NS4A, and NS5A.
- P89L into luciferase encoding constructs
- this example generated stably replicating genotype 3 a replicon cell lines, and by combing with selected host cells cured of genotype 3 replicons, efficient replication of genotype 3 a HCV RNA was established in a transient-trans fected cell culture.
- This system is fully capable of supporting potency profiling of antiviral compounds and selecting and phenotyping clinical resistance mutants emerging in HCV genotype 3 patients. These replicons should also serve as a valuable system for molecular virology studies of genotype 3 HCV, including a better understanding of its association with a high incidence of liver steatosis.
- Huh7-Lunet cells were obtained from ReBLikon GmbH (Mainz, Germany) (Friebe et al., J Virol 2005;79:380-92). 51C cells were derived by curing a HubJ-Lunet-based genotype la replicon clone and were described in Robinson et al.,
- 1C cells were derived by curing a GS- 5885 -resistant genotype la replicon clone derived from 51C cells, and showed much higher permissiveness to genotype la replicon replication. All cell lines were propagated in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX-I (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 1 unit/ml penicillin (Invitrogen), 1 ⁇ g/ml streptomycin (Invitrogen), and 0.1 mM nonessential amino acids (Invitrogen) (complete DMEM). Replicon cell lines were selected and maintained in complete DMEM containing 0.25 to 0.5 mg/ml G418 (Geneticin;
- the cell lines GT3a-Cl, GT3a-C2, and GT3a- C3 are 1C clones stably replicating the genotype 3a pGT3aS52SG-Neo replicon.
- To cure these cell lines of HCV RNA they were cultured in the presence of IFN (1000 IU/ml), the NS5B nucleoside inhibitor 2'-CMeA (2 ⁇ ), the NS5A inhibitor BMS-790052 (500nM), and the non-nucleoside NS5B inhibitor HCV-796 (1 ⁇ ). Cells were passaged in medium containing the four drugs twice a week for a total of eight passages.
- Cured cells were fully sensitive to G418 (500 mg/ml, also known as Geneticin®, an aminoglycoside antibiotic) and lacked detectable NS5A staining, confirming the absence of the replicon.
- Cured cell lines were expanded and cryopreserved at early passage levels. Cured cells were designated 3a-Cl, 3a-C2, and 3a-C3.
- a plasmid (pGT3aS52NeoSG) encoding a subgenomic genotype 3a replicon based on the S52 infectious clone was prepared by DNA synthesis (GeneScript, Piscataway, NJ) and cloning. The synthesized replicon incorporated following elements from 5' to 3' (FIG.
- a second plasmid (pGT3aS52RlucNeoSG) encoding a subgenomic replicon that incorporated the humanized Renilla luciferase (hRluc) reporter gene was generated as follows: The pGT3aS52NeoSG plasmid (described above) was cut using Ascl and Mlul restriction enzymes (to remove the neo gene) and gel purified using a commercial kit (Qiagen, Valencia, CA).
- a gene fragment encoding the hRluc gene fused with the neo gene along with the EMCV region from p/zRlucNeoSG2a plasmid were PCR amplified using Accuprime super mix I (Invitrogen) with the following primers: 2aRlucNeoAscIFor: 5'-AAC ACC ATC GGC GCG CCC ATG GCT TCC AAG GTG TAC GAC -3' (SEQ ID NO: 8, AscI site is introduced by the primer and is underlined), 2aEMCVIRESMluIRev: 5' -TCGGGG CCA TAC GCG TAT CGT GTT TTT CAA AGG -3' (SEQ ID NO: 9, Mlul site underlined).
- the subsequent PCR fragment was cut with AscI and Mlul and gel purified using a commercial kit (Qiagen).
- the vector and insert pieces were ligated using the LigaFast Rapid DNA Ligation System per the
- pGT3aS52RlucNeoSG was sequenced to confirm the correct orientation and sequence of the hRluc-Neo.
- the p/zRlucNeoSG2a plasmid was constructed by replacing the Luc-Neo fragment in the plasmid pLucNeoSG2a with the hRluc-Neo gene amplified from the plasmid hRluc-Neo Flexi(R) (Promega) as previously described (Robinson et ah,
- a third plasmid (Pi-GT3aS52RlucSG), encoding a bicistronic replicon with the hRluc reporter gene downstream of the poliovirus IRES (PI) and the genotype 3 a (S52 strain) HCV nonstructural genes (NS3-NS5B) downstream of the EMCV IRES was used for transient transfection studies.
- the plasmid was generated as follows: The
- pGT3aS52RlucNeoSG plasmid (described above) was cut using AscI and Mlul restriction enzymes (to remove the Rluc-Neo gene) and gel purified using a commercial kit
- a gene fragment encoding the PI, hRluc gene and EMCV region was PCR amplified from a genotype lb plasmid ( using Accuprime super mix I (Invitrogen) with the following primers: 3aPiRlucAscIFor: 5'- AAC ACC ATC GGC GCG CCA AAC CAA GTT CAA TAG-3' (SEQ ID NO: 10, AscI site is introduced by the primer and is underlined), IbEMCVIRESMluIRev: 5'-TCG GGG CCA TAC GCG TAT CGT GTT TTT CAA AGG -3' (SEQ ID NO: 11, Mlul site underlined).
- the subsequent PCR fragment was cut with AscI and Mlul and gel purified using a commercial kit (Qiagen).
- the vector and insert pieces were ligated using LigaFast Rapid DNA Ligation System per the manufacturer's protocol (Promega).
- the resulting vector, Pi-GT3aS52RlucSG was sequenced to confirm the correct orientation and sequence of the PI-hRluc region of the gene.
- mutant replicons Construction of mutant replicons. Adaptive mutations were introduced into the pGT3aS52RlucNeoSG or Pi-Rluc-GT3aS52 replicons by site-directed mutagenesis using a QuikChange Lightening kit (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing by TACGen (Hayward, CA).
- RNA transcription Plasmids encoding genotype 3a subgenomic HCV replicons were linearized with Xbal and purified using a PCR purification kit (Qiagen). RNA was synthesized and purified with T7 MEGAScript (Ambion, Austin, TX) and RNeasy kits (Qiagen), respectively, according to the manufacturer's instructions. RNA concentrations were measured using optical density at 260 nm and confirmed by 0.8% agarose gel electrophoresis (Invitrogen).
- RNA transfection and isolation of stable replicon cell lines Ten micrograms of in v tro-transcribed RNA were transfected into Huh7-Lunet or 1C cells by
- Cells were electroporated at 270 V and 960 ⁇ , incubated at room temperature for 10 minutes, resuspended in 30 ml complete DMEM and then plated into two 100-mm- diameter dishes. Forty-eight hours after plating, medium was replaced with complete DMEM supplemented with 0.25 mg/ml G418, which was refreshed twice per week. After three weeks, cell clones were isolated, expanded with 0.5 mg/ml G418, and cryopreserved at early passages.
- Replicon colony formation assays To determine the efficiency of G418- resistant colony formation, cells were electroporated with the indicated amounts of replicon RNA or extracted cellular RNA, and plated at multiple densities ranging from 2 10 5 to 2 10 6 cells/100 -mm dish. Forty-eight hours after plating, media were replaced with complete DMEM supplemented with 0.5 mg/ml G418, which was refreshed twice per week. Three weeks later, colony plates were used for cell expansion or G418- resistant foci were fixed with 4% formaldehyde and stained with 0.05% crystal violet.
- HCV RNA isolation, RT-PCR, and population sequencing were performed by TACGen. Briefly, HCV replicon cellular RNA were extracted and purified using an RNeasy kit (Qiagen) according to the manufacturer's protocol. RT-PCR was performed using the Superscript III first-strand synthesis system (Invitrogen), and PCR products were subsequently sequenced by TACGen.
- NS5A protein by indirect immunofluorescence. Replicon cells were plated in 96-well plates at a density of 1 x 10 4 cells per well. After incubation for 24 hours, cells were then stained for NS5A protein as described previously (Cheng et ah, Antimicrob Agents Chemother 2011;55:2197-205). Briefly, cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were then washed three times with PBS, blocked with 3% bovine serum albumin, 0.5% Triton X- 100, and 10% FBS and stained with anti- NS5A antibody.
- Staining was performed using a 1 : 10,000 dilution of mouse monoclonal antibody 9E10 (Apath, Brooklyn, NY). After washing in PBS three times, a secondary anti-mouse antibody conjugated to Alexa Fluor 555 was used to detect anti-NS5A antibody labeled cells (Invitrogen). Nuclei were stained with 1 ⁇ g/ml Hoechst 33342 (Invitrogen). Cells were washed with PBS and imaged with a Zeiss fluorescence microscope (Zeiss, Thornwood, NY).
- NS5A protein by Western blot.
- the blot was also co-stained with anti-BiP antibody (Abeam, 1 : 1000 dilution) and secondary anti-rabbit antibody (IRDye 800CW Goat anti-Rabbit IgG (H + L) from LI-COR, 1 : 10,000 dilution) as a loading control. Staining was analyzed by Odyssey Imaging (LI-COR).
- Replicon antiviral assays Replicon RNA were electroporated into 1C cells as described above. After transfection, cells were quickly transferred into 100 mL of pre- warmed culture medium, and 90 ⁇ was seeded in 384-well plates at a density of 2,000 cells/well.
- VX-950 telaprevir
- boceprevir 2-C-methyl adenosine (2-CMeA) were purchased from Acme Bioscience (Belmont, CA).
- Cyclosporine A was purchased from Sigma-Aldrich (St. Louis, MO).
- the Wyeth HCV NS5B site IV inhibitor HCV-796 was synthesized by Curragh Chemistries
- Gilead compounds GS-5885, GS-9190, GS-9451, GS-9669, and GS- 7977, Pfizer NS5B thumb site II inhibitor filibuvir, Merck NS5B thumb site I inhibitor MK-3281 and protease inhibitor MK-5172, and the Bristol-Myers Squibb NS5A inhibitor (BMS-790052) were synthesized by Gilead Sciences.
- a subgenomic genotype 3a replicon was constructed as previously described by Lohmann et ah, Science 1999;285: 110-3 and based on the consensus sequence (GenBank accession #GU814263) of the genotype 3a S52 strain
- the S52 strain was selected due to its robust infection in chimpanzees.
- an NS5A mutation S2210I (equivalent to S2204I in genotype la) was incorporated.
- Huh7-Lunet, 51C and 1C cells were transfected with genotype 3 a replicon RNA. The number of surviving colonies was counted for each selection transfected with 10 ⁇ g RNA.
- genotype 3a replicon cell line using anti-NS5A antibody confirmed the expected expression pattern for NS5A, particularly in the perinuclear region.
- untransfected parental 1C cells did not show detectable NS5A protein (FIG. 5B).
- Genotypic analyses of genotype 3a replicon clones To identify adaptive mutations, genotype 3a replicon clones were subjected to genotypic analyses. Total cellular RNA was extracted, and HCV replicon RNA was subsequently amplified by RT- PCR. PCR products that cover the entire NS3-NS5B region were sequenced by population sequencing. Amino acid changes in each clone are summarized in Table 2. All clones had an amino acid substitution of leucine with proline at residual position 89 (P89L) within the viral NS3 protease domain. Clone #3 had an additional mutation,
- luciferase activity of the Rluc-Neo-GT3a-P89L replicon continuously decreased to a level slightly above the background ( ⁇ 100 relative luminescence units (RLU)), suggesting the replicon did not replicate efficiently in the transient assay, likely due to insertion of the large-size Rluc-Neo gene.
- Pi-Rluc-GT3a- P89L conferred a meaningfully higher level of replication.
- HCV RNA replication reached a level that was two orders of magnitude higher than that achieved with Rluc-Neo-GT3a-P89L.
- the three replicon cell lines (Table 6) were cured using a high-dose cocktail of IFN and three direct antivirals for 4 weeks.
- the resulting cured cell lines designated 3a-Cl, 3a- C2, and 3a-C3, lacked detectable HCV replication.
- To assess the ability of the cured cell lines to support genotype 3 a replication they were transfected with the Pi-Rluc-GT3a- P89L replicon, and luciferase activity was measured daily for 4 days.
- two cured replicon clones, 3a-C2 and 3a-C3 but not 3a-Cl exhibited approximately 10-fold higher permissiveness to genotype 3a replication than 1C cells (FIG. 10A).
- genotype la and lb replicons were transfected into 1C and the 3a-C3 cell lines.
- genotype la and lb replicons were transfected into 1C and the 3a-C3 cell lines.
- genotype la and lb replicon replication between 1C cells and the cured cell line (FIG. 10B).
- P89L/A166T or P89L/K583E double mutation replicon was transfected into genotype 3a- permissive 3a-C3 cells, the replicon replication efficiency was further stimulated about 5- fold to a level that is comparable to the highly adapted genotype la or lb replicons (FIG. 11B).
- a combination of selected host cells e.g. 3a-C3 cells
- secondary adaptive mutation e.g. A166T in NS3 established an efficient system to support genotype 3 HCV replication in cell culture that is comparable to those for genotype 1.
- Table 9 Mutations analyzed in Rluc-neo-GT3a-P89L replicon cells lines
- genotype 3a Pi-Rluc replicon NS3 protease, NS5A, NS5B active site, NS5B non-active site, and host factor inhibitors.
- a standard genotype lb Pi-Rluc replicon was tested in parallel.
- the transient replicon replication assays were performed in 1C cells to minimize the potential influence of host cell difference on compound antiviral activities.
- EC50 values against both genotype lb and genotype 3 a replicons were successfully generated for all inhibitors using a high- throughput 384-well assay measuring Rluc activity (Table 10).
- Inhibitors targeting the NS5B active site (2- CMeA or GS-7977) were 3- to 6-fold more active against genotype 3a compared with lb.
- NS5B thumb site II inhibitors filibuvir and GS-9669 and the palm site 1/2 inhibitor GS-9190 were approximately 17-, 60-, and 17-fold, respectively, less active
- NS5A inhibitor BMS-790052 was 11-fold less active against genotype 3a, it was still quite potent with EC 50 values of 0.31- 0.45 nM against genotype 3a.
- Another NS5A inhibitor, GS-5885 available from Gilead
- genotype 3a compared with genotype lb (EC 50 values for genotype 3a: 40-92 nM).
- NS3 protease inhibitors boceprevir and telaprevir were slightly less potent (1.5- to 3.3- fold) against genotype 3a versus genotype lb.
- the protease inhibitors BILN- 2061, GS-9451, and MK-5127 were 28- to 478-fold less potent against genotype 3a versus genotype lb.
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WO2019113462A1 (en) | 2017-12-07 | 2019-06-13 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
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Cited By (6)
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EP2752485A4 (en) * | 2011-08-31 | 2015-06-24 | Japan As Represented By The Director General Of Nat Inst Of Infectious Diseases | NUCLEIC ACID CONSTRUCT INCLUDING NUCLEIC ACID DERIVED FROM GENOTYPE 3a HCV GENOME |
US9453056B2 (en) | 2011-08-31 | 2016-09-27 | Japan As Represented By Director-General Of National Institute Of Infectious Diseases | Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 3a |
US11628181B2 (en) | 2014-12-26 | 2023-04-18 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
WO2019113462A1 (en) | 2017-12-07 | 2019-06-13 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
US11331331B2 (en) | 2017-12-07 | 2022-05-17 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
US11903959B2 (en) | 2017-12-07 | 2024-02-20 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
Also Published As
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NZ619298A (en) | 2016-02-26 |
CN103687869A (en) | 2014-03-26 |
HK1197075A1 (en) | 2015-01-02 |
US20130052633A1 (en) | 2013-02-28 |
MX2014000124A (en) | 2014-02-17 |
US8889848B2 (en) | 2014-11-18 |
EP2729489A1 (en) | 2014-05-14 |
AU2012278960A1 (en) | 2013-05-02 |
CA2840828A1 (en) | 2013-01-10 |
AU2012278960B2 (en) | 2015-08-27 |
JP2014522648A (en) | 2014-09-08 |
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