Cells and cell lines which replicate HCV of non-hepatic human and non human origin are disclosed. Also provided are methods of using such cells and cell lines to identify anti-HCV agents for the treatment of HCV infection.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides HCV replicating cells and cell lines derived from human non-hepatic cells or non-human cells. According to one embodiment of the invention, the cells are human epithelial cells of non-liver origin, such as, HeLa cells. According to another embodiment of the invention, the cells capable of replicating HCV are hepatoma and hepatocyte cells of mouse origin, such as, Hepa1-6 cells, or AML12 cells respectively.

The present invention also provides a non-human host animal comprising cells infected with HCV. In one embodiment of the invention, the host animal is a mouse. In another embodiment of the invention, the cells infected with HCV are mouse hepatoma cells.

Also provided by the present invention are methods for producing human non-hepatic cells or non-human cells that are capable of replicating HCV, and cell lines comprising the same. Such methods include transfection with total HCV RNA or an HCV replicon which comprises one or more adaptive mutations which facilitate replication in a cell of interest.

The present invention further provides methods for screening an agent that modulates HCV replication by incubating the agent with the aforementioned cells or administering the agent to the aforementioned host animal comprising cells replicating HCV and assessing said agent for modulation of HCV replication. Such agents may inhibit or enhance production of HCV. These agents may be cytopathic or non-cytopathic to HCV infected cells. Agents which activate aspects of the JAK/STAT pathway may also be screened using the cells and cell lines of the invention.

Also provided by the present invention are HCV derived polynucleotides comprising adaptive mutations. The present inventor has discovered that these mutations are associated with expanded tropism of HCV.

Additionally, the present invention provides polypeptides encoded by the mutated HCV polynucleotides described above.

DETAILED DESCRIPTION OF THE INVENTION

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations (OLT). Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether non-primate cells are permissive for RNA replication. However, because viral replication leads to the accumulation of mutations, it is conceivable that variants can emerge with novel properties such as the potential to replicate in different cell types of various species. Furthermore, accumulation of a large number of quasispecies may also contribute to resistance to IFN-.alpha. treatment. Therefore, it is important to determine the properties of HCV variants, and the effect such variation has on the efficacy of IFN-.alpha. therapy.

Provided herein is evidence that subgenomic HCV RNAs can replicate in mouse hepatoma and non-hepatic human epithelial cells. Moreover, efficient replication requires adaptation of the virus to cell-type specific environmental conditions. These results show that HCV RNA replication can lead to the accumulation of mutants with altered tissue and host tropism thereby facilitating the development of small animal models for HCV infection.

In accordance with the present invention, there are provided nucleic acids and stably-transfected human non-hepatic, and murine hepatic cell lines that replicate HCV. Also provided are methods of use for such cells for identifying therapeutic anti-viral agents for the treatment of HCV infection. Additionally, the availability of a murine line which replicates HCV enables the production of a greatly needed mouse model of HCV infection. Furthermore, the invention provides polynucleotides and their corresponding polypeptides which have adaptive mutations which results in expanded tropism of HCV.

The detailed description set forth below describes preferred methods for making and using the nucleic acids and cell lines of the present invention, and for practicing the methods of the invention. Any molecular cloning or recombinant DNA techniques not specifically described are carried out by standard methods, as generally set forth, for example, in Sambrook et al., "DNA Cloning, A Laboratory Manual," Cold Spring Harbor Laboratory, 1989 and Ausubel et al. Current Protocols in Molecular Biology, J. Wiley & Sons, 1995.

Methods for Obtaining HCV RNA and Producing Non-Hepatic Human Cell Lines and Non-Human Hepatic Cell Lines that Replicate HCV

The HCV replicating non-hepatic human cell-based and non-human hepatic cell-based systems are prepared according to the general methods set forth below for isolation of nucleic acids, transformation of cultured cells, and maintenance of cell lines.

A. Nucleic Acids

The HCV replicons of the present invention comprise adaptive mutations which alter the ability of HCV to replicate in different cell types. Surprisingly, the present inventors have identified mutations which are associated with expanded viral tropism.

The HCV nucleic acid molecules of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate chemical starting materials, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence information enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a 3 kilobase double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be ligated such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 3 kilobase double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

HCV nucleic acid sequences may be isolated from appropriate biological sources using methods known in the art. For example, total RNA can be extracted with TRIzol reagent from Gibco BRL, although other reagents are also available for this purpose.

In some cases, it may be desirable to synthesize HCV subgenomic RNA wherein a selectable marker gene is substituted for a HCV structural gene.

The availability of HCV replicon encoding nucleic acids enables the production of strains of laboratory mice carrying part or all of the HCV sequence or mutated sequences thereof. Such mice provide an in vivo model for studying HCV infection, and analyzing possible treatment modalities for the same.

Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. the incorporation of genetically manipulated embryonic stem cells into an early embryo.

A transgenic mouse carrying an HCV replicon comprising the adaptive mutations is generated by genomic integration of exogenous genomic sequence encoding HCV. These transgenic animals are useful for drug screening studies as animal models for human diseases.

The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term "transgenic animal" is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.

A type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.

B. Cell Lines

The cell lines of the invention include any cell which supports production of HCV components. These cells include human, non hepatic cells and/or non-human hepatic cells, such as murine hepatic cells. Cell lines useful for practice of the invention include, but are not limited to HELA, a non-hepatic epithelial cell line (ATCC CRL number CCL-2.2), Hepa1-6, a murine hepatoma cell line (ATCC CRL number-1830), and AML-12, a murine hepatocyte cell line (ATCC CRL number-2254).

To achieve stable gene transfer, HCV subgenomic RNA is introduced into host cells. This may be accomplished according to numerous methods known in the art, including, but not limited to: (1) calcium phosphate transfection; (2) transfection with DEAE-dextran; (3) electroporation; and (4) liposome-mediated transfection. For general protocols, see, e.g., chapter 9 in Current Protocols in Molecular Biology, Ausubel et al. (editors), John Wiley & Sons, Inc. 1987-1995. For stable transfer of nucleic acids into mammalian cells, the liposome-mediated transfection method may be used in the present invention because of the large amount of nucleic acid that can be introduced into the cells, thereby increasing the possibility of integration of the nucleic acid into the host genome.

Cells are grown according to standard methods known in the art, such as those set forth in Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, 4th ed. Edition, available from the ATCC.

Stable transfectants are selected by the ability of an individual cell colony to grow in the presence of a selection agent, e.g., an antibiotic, by virtue of a resistance-encoding gene on HCV RNA or by isolating cells using FACS and antibodies directed any HCV protein.

Detection and quantitation of expression of HCV gene products in stably-transfected cell lines of the invention can be accomplished using a variety of known assays. For instance, cells transformed with the RNA subgenomes of HCV can be selected with an antibiotic such as G418 (neomycin). Alternatively, cells may be selected based on the presence and accumulation of HCV RNA or HCV gene products. As another example, the starting HCV encoding nucleic acids may be modified to also comprise a hygromycin or puromycin or any other resistance or reporter gene, such that cells transfected with the nucleic acids can be selected by their ability to grow on hygromycin- or puromycin-containing medium. Alternatively, selectable markers including luciferase, beta lactamase etc., may be utilized which allow for the selection of cells by FACS and related procedures. In an alternative embodiment, a separate plasmid may be constructed that comprises an antibiotic resistance gene, and can be used to co-transfect cells along with the subgenomic RNA molecules. Further, as described in detail in the following Examples, cells stably transfected with the subgenomic HCV RNA are grown in the appropriate medium for a selected period of time, the medium is then collected and analyzed for the presence of HCV RNA by dot blot hybridization or by conventional Northern hybridization, using a radioactively labeled probe having HCV DNA or RNA complementary sequences. Alternatively, viral gene products may be detected in the cells of the invention using conventional methods, including, without limitation, immunoassay and Western blotting.

Using the assays described above, stably-transfected cell lines can be selected which possess optimum characteristics for use in cell-based assays for screening potential anti-viral compounds.

Another aspect of the invention includes a non-human host animal which comprises the HCV expressing cells of the invention. These animals may be produced by administration of a HCV replicating cell, an HCV encoding nucleic acid having one or more adaptive mutations which permit replication in mice. The cells or viral nucleic acid could be directly injected intravenously (e.g. via tail vein injection), intramuscularly, subcutaneously, or via-intrahepatic injection. Alternatively, transgenic mice could be produced using the HCV replicons of the inventions, as described above.

III. Uses of Cell Lines for Cell-Based Assays of Potential Anti-HCV Agents

The human non-hepatic and murine hepatic cell lines of the invention which replicate HCV may be used in research, diagnostic, and therapeutic applications, including cell-based assays to evaluate the effectiveness of potential anti-HCV compounds, utilizing methodologies known in the art. Typical assays are summarized herein below. These cell-based assays may be performed in standard cell culture media utilizing commonly-available equipment, reagents and culture containers.

Persons skilled in the art will appreciate that these assays represent exemplary embodiments, and may be varied to provide similar/equivalent equipment or reaction conditions. For example, a variety of genes encoding antibiotic resistance are available, and can be utilized in accordance with the present invention in the generation of the cell lines of the invention. In a preferred embodiment, RNA isolated from parental human hepatic or untransformed cells is also utilized as a control in the assays described herein below to determine the effects of potential anti-viral compounds on HCV expressed in the cells. The control RNA is obtained in a manner similar to the HCV RNA. This cell line is treated in the assays described herein below as a negative control, to assure that any effects observed are due to the action of the compound being tested on HCV, and not non-specific effects due to the introduction of RNA into the cells.

A. General Cell-Based Assay for Inhibitors of HCV Replication

96-well microtiter plates are seeded with an appropriate amount of cells which replicate HCV in a standard cell culture medium containing G418 (e.g., 400 .mu.g/ml), as well as standard concentrations of penicillin, streptomycin and kanamycin or gentamicin to prevent bacterial and mycoplasma contamination. The cells are incubated at 37.degree. C. in a humidified 5% CO.sub.2 incubator. On day 0 wells are washed three times with warm phosphate-buffered saline (PBS). The culture medium is then replaced with fresh medium containing 0.3% dimethylsulfoxide (DMSO), 10% fetal calf serum (FCS), penicillin, streptomycin, kanamycin/gentamicin, containing one of the following ingredients: (1) various concentrations of a known HCV inhibitor, such as interferon alpha, as a positive control; and (2) various concentrations of one or more of the compounds to be tested. The plates are incubated at 37.degree. C. in humidified, 5% CO.sub.2 incubator for 24, 48, and 72 hours. The plates are washed twice with PBS and then with a solution of methanol and acetone (1:1) to fix the cells. The cells are then incubated with an antibody specific for a viral protein (i.e. NS5A) according to the standard methods, such as enzyme linked immunosorbent assay (ELISA). Briefly, following incubation with the primary antibody, the plates are washed to remove unbound antibody and then incubated with a second, enzyme-conjugated antibody that can bind to the primary antibody. The plates are washed again, followed by an incubation with a colorless substrate that upon hydrolysis (cleavage) by the enzyme yields a colored product, the concentration of which can be determined with a spectrophotometer (microtiter plate reader). The concentration of the product corresponds to the levels of viral replication in cells and can be used to determine the activity of a given drug to inhibit HCV replication.

B. Cytotoxicity Assays

A cytotoxicity assay may be conducted to evaluate potential anti-HCV agents, utilizing a protocol similar to that described above. Instead of measuring HCV replication levels, however, cytoxicity of the various test agents is assessed by standard procedures to determine cell viability, proliferation and levels of cellular metabolism including but not restricted to cell membrane permeability, lysosomal mass-pH, cell density or mitochondrial activity. For example, the CytoTox-ONE.TM. Assay from Promega is a rapid, fluorescent measure of the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium is measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into resorufin. Since the CytoTox-ONE.TM. Reagent mix does not damage healthy cells, released LDH can be measured directly in assay wells containing a mixed population of viable and damaged cells.

IV. Identification of Cell Lines Permissive for HCV Infection

As shown herein, it is possible to produce HCV carrying adaptive mutations that confer broad tissue and species tropism. Using such virus stocks it will be possible to screen cell lines of human and non-human origin for virus infection. Briefly, probes which correspond to unique portions of the sequence, may be used in detection methods. This method will lead to the identification of novel cell lines that are permissive for a complete cycle of HCV replication.

V. Screening for the HCV Receptor(s)

The ability to replicate HCV in different cell lines facilitates the isolation of the HCV receptor. Virus stocks similar to the ones described in section IV can be used to isolate the HCV receptor(s). For this purpose virus stocks carrying replicons with a selectable marker, such as neomycin or hygromycin will be used. Cells that are non-permissive for infection, will be transfected with DNA isolated from cells that are known to express the receptor (i.e. human hepatocytes, cells identified with the procedure described in section III) and subsequently infected with recombinant HCV carrying the selectable marker. Cells that express the receptor can then be selected through the addition of an antibiotic (i.e. G418 or hygromycin) to the culture medium. Once cells are identified, the transfected DNA can be isolated, cloned, and sequenced. The sequence information can then be used to identify the gene(s) encoded by transfected DNA.
 

Claim 1 of 23 Claims

1. A hepatitis C virus (HCV) replicating cell line, wherein said cell line is a mouse cell line comprising an HCV genome.
 

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