Patent Grant
7786287
The references cited in the present application are not admitted to be prior art to the claimed invention.
It is estimated that about 3% of the world's population are infected with the Hepatitis C virus (HCV). (Wasley, et al., 2000. Semin. Liver Dis. 20, 1-16.) Exposure to HCV results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Iwarson, 1994. FEMS Microbiol. Rev. 14, 201-204.) In addition, epiderniological surveys indicate an important role of HCV in the pathogenesis of hepatocellular carcinoma. (Kew, 1994. FEMS Microbiol. Rev. 14, 211-220, Alter, 1995. Blood 85, 1681-1695.)
The HCV genome consists of a single strand RNA of about 9.5 kb in length, encoding a precursor polyprotein of about 3000 amino acids. (Choo, et al., 1989. Science 244, 362-364, Choo, et al., 1989. Science 244, 359-362, Takamizawa, et al., 1991. J. Virol. 65, 1105-1113.) The HCV polyprotein contains the viral proteins in the order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, E1, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima, et al., 1994. J. Virol. 68, 2731-2734, Hijikata, et al., 1993. P.N.A.S. USA 90, 10773-10777.)
The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein. A zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui, et al., 1993. J. Virol. 67, 1385-1395, Hijikata, et al., 1993. P.N.A.S. USA 90, 10773-10777.)
A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. (Barthenschlager, et al., 1993. J. Virol. 67, 3835-3844, Grakoui, et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10583-10587, Tomei, et al., 1993. J. Virol. 67, 4017-4026.) RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
NS4A provides a cofactor for NS3 protease activity. (Failla, et al., J. Virol. 1994. 68, 3753-3760, De Francesco, et al., U.S. Pat. No. 5,739,002.)
NS5A is a highly phosphorylated protein conferring interferon resistance. (De Francesco, et al., 2000. Semin Liver Dis., 20(1), 69-83, Pawlotsky, 1999. J. Viral Hepat. Suppl. 1, 4748.)
NS5B provides an RNA-dependent RNA polymerase. (De Francesco, et al., International Publication Number WO 96/37619, Behrens, et al., 1996. EMBO 15, 12-22, Lohmann, et al., 1998. Virology 249, 108-118.)
The present invention features assays employing a beta-lactamase reporter system, an HCV replicon enhanced cell, and/or a chimeric HCV replicon containing a 3′UTR based on the HCV-1a 3′ UTR. These features can be employed alone or together, and are preferably combined together to measure HCV replicon activity and the affect of compounds on such activity.
Thus, a first aspect of the present invention describes a method of measuring the ability of a compound to alter HCV replicon activity using a screening cell line comprising a first HCV replicon and a second HCV replicon. The first replicon comprises a selection sequence. The second replicon comprises a nucleotide sequence encoding for beta-lactamase. The method is performed using conditions supporting beta-lactamase activity. Such conditions provide the proper environment for beta-lactamase activity and include factors such as proper pH, temperature and buffer.
A selection sequence facilitates identification or isolation of cells containing the sequence. Examples of selection sequences include sequences encoding drug resistance and reporters compatible with cell sorting.
Another aspect of the present invention describes an HCV replicon enhanced cell comprising a first HCV replicon and a second HCV replicon. The first replicon comprises a selection sequence and is present in an amount compatible with replication of the second replicon. The second replicon is different from the first replicon and preferably encodes a reporter.
Another aspect of the present invention describes a method of producing an HCV replicon enhanced cell. The method comprises the steps of: (a) introducing into a cell a HCV replicon comprising a selection sequence; (b) obtaining a replicon enhanced cell; and (c) introducing into the replicon enhanced cell a replicon comprising a reporter.
Another aspect of the present invention describes an HCV replicon comprising a beta-lactamase reporter. In this aspect, the replicon does not contain a sequence coding for resistance to an agent that inhibits cell growth.
Another aspect of the present invention describes a chimeric HCV replicon containing an HCV-1a 3′ UTR. The chimeric replicon contains one or more HCV regions from different HCV strains. Reference to strains includes subtypes, which may be clinical isolates. Examples of chimeric replicons include replicons formed from one or more strains classified as HCV-1a, where the replicon contains a 3′ UTR from one strain and one or more HCV regions from a different strain(s); and the replicon being formed from two or more HCV strains classified in different groups, where the replicon contains a 3′ HCV-1a UTR.
Preferably, the chimeric replicon contains the HCV-1a 3′UTR provided by bases 8407-8641 of SEQ. ID. NO. 1. The remainder of such a chimeric replicon contains one or more HCV regions, preferably a non-structural region, from a HCV strain not containing the HCV-1a 3′ UTR provided by SEQ. ID. NO. 1.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples.
The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
The present invention features assays employing a beta-lactamase reporter system, an HCV replicon enhanced cell, and/or a chimeric HCV replicon containing a HCV-1a 3′ UTR. These different features are preferably employed together to evaluate the ability of a compound to inhibit HCV replication.
Compounds inhibiting HCV replication have research and therapeutic applications. Research applications include the study of HCV and the production of replicon enhanced cells. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat or inhibit onset of HCV in a patient.
I. Beta-Lactamase Reporter Systems
Assays employing a beta-lactamase reporter system can be used to measure the activity of a nucleic acid or polypeptide coupled to a beta-lactamase and the effect of a compound on such activity. Nucleic acid coupled to a beta-lactamase contains a target nucleotide region and a region encoding a beta-lactamase. Beta-lactamase is the byproduct of target nucleic acid expression and measuring Bla activity provides a mean to measure target nucleic acid activity.
The target nucleotide region is a region of interest and can provide regulatory activities or encode for a polypeptide. Reference to polypeptide and polypeptide region does not provide a size limitation and includes smaller length polypeptides, enzymatic proteins, structural proteins, and polyproteins.
Activities that can be measured using a target nucleotide region and a region encoding a beta-lactamase include one or more of the following: target nucleotide region production, target nucleotide region processing, target nucleotide region transport, activity of encoded for polypeptides involved in replication, and production of encoded for polypeptides. Nucleotide region transport includes extracellular transport that may be viral mediated.
A polypeptide coupled to beta-lactamase is a chimeric polypeptide containing a region of interest and a beta-lactamase. Polypeptide regions of interest include those providing enzymatic and structural proteins.
Polypeptide activities that can be measured using a beta-lactamase reporter system include one or more of the following: polypeptide production, polypeptide processing and polypeptide transport. Polypeptide transport includes extracellular transport that may be viral mediated.
A preferred target nucleic acid is an HCV replicon. HCV replicons containing a region encoding a beta-lactamase can be used to measure the effect of a compound on both HCV nucleic acid and HCV protein activity. HCV proteins are involved in processing the HCV polyprotein and in HCV replication. Inhibiting HCV proteins involved in HCV processing or more directly involved in HCV replication reduces the production of HCV replicons encoding beta-lactamase.
II. Beta-Lactamase Activity
Beta-lactamases are enzymes catalyzing the cleavage of the beta-lactam ring present in cephalosporins. Different naturally occurring beta-lactamases and functionally derivatives of naturally occurring beta-lactamases are well known in the art. References such as Ambler, Phil. Trans R. Soc. Lond Ser. B. 289:321-331, 1980, provide examples of naturally occurring β-lactamases.
Beta-lactamases that are functional derivatives of a naturally occurring beta-lactamase can be produced by altering a naturally occurring sequence to produce an enzyme retaining beta-lactamase activity. Examples of common alterations include substitutions, deletions, and additions of amino acids or amino acid regions.
One method of designing altered proteins is to take into account amino acid R-groups. An amino acid R group affects different properties of the amino acid such as physical size, charge, and hydrophobicity. Amino acids can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine); basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid).
Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing a change in polypeptide functioning.
Changes outside of different amino acid groups can also be made. Preferably, such changes are made taking into account the position of the amino acid to be substituted in the polypeptide. For example, arginine can substitute more freely for nonpolar amino acids in the interior of a polypeptide than glutamate because of its long aliphatic side chain. (See, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C.)
Derivatives can be produced to enhance intracellular activity. An example of such a derivative is TEM-1 beta-lactamase. (Kadonaga et al., J. Biol. Chem. 259:2149-2154, 1984.) TEM-1 beta-lactamase is a derivative of E. coli beta-lactamase where the signal sequence is deleted. The deletion of the signal sequence increases cytoplasmic accumulation.
Beta-lactamase encoding sequences can be incorporated into nucleic acids taking into account the genetic code. The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990). Amino acids are encoded by codons as follows:
Intracellular beta-lactamase activity is preferably measured using a fluorogenic substrate that is cleaved by beta-lactamase. Preferred substrates are membrane permeant fluorogenic substrates that become membrane impermeant inside a cell, and that are cleaved by beta-lactamase to produce a detectable signal. Examples of such substrates are provided in Zlokarnik, et al., 1998. Science 279:84-88, and Tsien et al., U.S. Pat. No. 5,741,657.
In an embodiment of the present invention, a cell-permeant fluorescent β-lactamase substrate such as CCF2-AM or CCF4-AM (Aurora Biosciences, Inc., San Diego, Calif.) is loaded into a cell. These substrates contain an ester group facilitating transport across the cell membrane. Inside the cell, the ester group is cleaved rendering the substrate membrane impermeant. The intact substrates when stimulated with light of ˜405 nm, emit green fluorescence (˜530 nm) due to resonant energy transfer from a coumarin to fluorescein dye molecule. Cleavage of the substrate by beta-lactamase disrupts the resonance energy transfer and, the fluorescence emission changes to a blue color (˜460 nm) of only the coumarin. The fluorescence emissions of the substrate present in the cells can be detected by, for example, fluorescence microscopy or by a fluorometer in conjunction with appropriate emission and excitation filters.
IV. Use Of Beta-Lactamase Inhibitor
Beta-lactamase inhibitors such as clavulanic acid can be used to enhance a beta-lactamase reporter system by being present throughout the assay. For example, clavulanic acid being present throughout an assay involving an HCV replicon beta-lactamase reporter sensitizes the assay towards HCV replication inhibitors.
Clavulanic acid can be used to enhance the signal-to-background ratio from the beta-lactamase reporter system wherein, a larger signal to noise ratio facilitates detection of HCV replication inhibitors by better reflecting changes in the intracellular concentration of beta-lactamase. Because clavulanic acid is an irreversible inhibitor, it is a more potent inhibitor of beta-lactamase at low enzyme concentrations than at high enzyme concentrations and therefore its inhibitory activity has a more pronounced effect in the background signal than in the foreground signal of the assay.
In different embodiments concerning the level of signal-to-background reduction, the beta-lactamase inhibitor is provided in an amount sufficient to enhance the signal-to-background ratio by 2 to 5 fold; the amount of inhibitor is about 10 nM to 1.0 μM; and/or the inhibitor is an irreversible inhibitor, preferably clavulanic acid.
V. HCV Replicons
An HCV replicon is an RNA molecule able to autonomously replicate in a cultured cell and produce detectable levels of one or more HCV proteins. The HCV replicon expresses the HCV derived components of the replication machinery and contains cis-elements required for replication in a cultured cell.
The production and use of HCV replicons, and the effect of different mutations on replicon activity are described in different references. (See, for example, Lohmann, et al., 1999. Science 285, 110-113, Bartenschlager, European Patent Application 1 043 399, published Oct. 11, 2000, Blight, et al., 2000. Science 290:1972-1974, Lohmann, et al., 2001. Journal of Virology 75:1437-1449, and Pietschmann, et al., 2001. Journal of Virology 75:1252-1264.)
The basic subunit of an HCV replicon encodes for a HCV NS3-NS4A-NS4B-NS5A-NS5B polyprotein (also referred to as NS3-NS5B) along with a suitable 5′-UTR-partial core (PC) region and 3′ UTR. NS3-NS5B may contain different regions from different HCV strains. The 5′-UTR-PC region is made up of a 5′-UTR region and about 36 nucleotides of the beginning of the core. Additional regions may be present including those coding for HCV proteins or elements such as the complete core, E1, E2, p7 or NS2; and those coding for other types of proteins or elements such as a encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), a reporter protein, or a selection protein.
The HCV 5′-UTR-PC region provides an IRES for protein translation and elements needed for replication. The HCV 5′-UTR-PC region includes naturally occurring HCV 5′UTR extending about 36 nucleotides into a HCV core encoding region, and functional derivatives thereof. The IRES and PC can be present in different locations such as site downstream from a sequence encoding a selection protein, a reporter, protein, or an HCV polyprotein.
Functional derivatives of the 5′-UTR-PC region able to initiate translation and assist replication can be designed taking into account structural requirements for HCV translation initiation. (See, for example, Honda, et al., 1996. Virology 222, 31-42). The effect of different modifications to a 5′ UTR-PC region can be determined using techniques measuring replicon activity.
In addition to the HCV 5′ UTR-PC region, other types of IRES elements can also be present in the replicon. The other types of IRES elements can be present in different locations including immediately upstream the region encoding an HCV polyprotein. Examples of non-HCV IRES elements that can be used are the EMCV IRES, poliovirus IRES, and bovine viral diarrhea virus IRES.
The HCV 3′ UTR assists HCV replication. HCV 3′ UTR includes naturally occurring HCV 3′ UTR and functional derivatives thereof. Naturally occurring 3′ UTR's include a poly U tract and an additional region of about 100 nucleotides. (Tanaka, et al., 1996. J. Virol. 70, 3307-3312, Kolykhalov, et al., 1996. J. Virol. 70, 3363-3371.) At least in vivo, the 3′ UTR appears to be essential for replication. (Kolykhalov, et al., 2000. J. Virol. 4, 2046-2051.) Examples of naturally occurring 3′ UTR derivatives are described by Bartenschlager International Publication Number EP 1 043 399.
The NS3-NS5B polyprotein encoding region provides for a polyprotein that can be processed in a cell into different proteins. Suitable NS3-NS5B polyprotein sequences that may be part of a replicon include those present in different HCV strains and functional equivalents thereof resulting in the processing of NS3-NS5B to produce functional replication machinery. Proper processing can be measured by assaying, for example, HCV protein production.
An HCV replicon may contain non-HCV sequences in addition to HCV sequences. The additional sequences should not prevent replication and expression, and preferably serve a useful function. Sequences that can be used to serve a useful function include a selection sequence, a reporter sequence, transcription elements and translation elements.
A selection sequence in an HCV replicon facilitates the identification and/or isolation of a cell containing the replicon. Selection sequences providing for resistance to an agent that inhibits cell growth can be used in conjunction with some selective pressure that inhibits growth of cells not containing the selection sequence. Examples of selection sequences include sequences encoding for antibiotic resistance, and ribozymes; and reporters compatible with cell sorting such as green fluorescence protein and beta-lactamase.
Antibiotic resistance can be used in conjunction with an antibiotic to select for cells containing replicons. Examples of selection sequences providing for antibiotic resistance are sequences encoding resistance to neomycin, hygromycin, puromycin, or zeocin.
A ribozyme serving as a selection sequence can be used in conjunction with an inhibitory nucleic acid molecule that prevents cellular growth. The ribozyme recognizes and cleaves the inhibitory nucleic acid.
A reporter sequence can be used to detect replicon replication or protein expression. Preferred reporter proteins are enzymatic proteins whose presence can be detected by measuring product produced by the protein. Examples of reporter proteins include luciferase, beta-lactamase, secretory alkaline phosphatase, beta-glucuronidase, green fluorescent protein and its derivatives. In addition, a reporter nucleic acid sequence can be used to provide a reference sequence that can be targeted by a complementary nucleic acid. Hybridization of the complementary nucleic acid to its target can be determined using standard techniques.
Replicons containing reporter sequences may or may not also contain a selection sequence. Selection sequences providing resistance to an agent that inhibits cell growth can be used in conjunction with selective pressure to select for cells maintaining the replicon. The Examples provides below illustrate that replicons containing a beta-lactamase reporter sequences are sufficiently maintained in the absence of selective pressure. In an embodiment of the present invention the replicon contains a reporter sequence (preferably, beta-lactamase), and does not contain a sequence coding for resistance to an agent that inhibits cell growth.
Additional sequences can be part of the same cistron as the HCV polyprotein or can be a separate cistron. If part of the same cistron, additional sequences coding for a protein should result in a product that is either active as a chimeric protein or is cleaved inside a cell so it is separated from HCV protein.
Selection and reporter sequences encoding a protein when present as a separate cistron should be associated with elements needed for translation. Such elements include an IRES 5′ to the selection or reporter sequence.
A preferred construct is a bicistronic replicon, where one cistron encodes for a selection or reporter sequence and the second cistron encodes for HCV proteins. More preferably, the first cistron contain a HCV 5′-UTR-PC region joined to the selection or reporter sequence; and the second cistron contains the EMCV internal ribosome entry site, NS2-NS5B or NS3-NS5B, and a 3′ UTR. Examples of bicistronic constructs are illustrated by SEQ. ID. NOs. 1 and 2.
Preferred cells for use with a HCV replicon are Huh-7 cells and Huh-7 derived cells. “Huh-7 derived cells” are cells produced starting with Huh-7 cells and introducing one or more phenotypic and/or genotypic modifications.
VI. HCV Replicon Sequences
HCV sequences for use in HCV replicons include HCV sequences present in different HCV strains and functional derivatives thereof. Functional derivatives can process the HCV polyprotein and provide for HCV replication.
Replicon activity from naturally occurring sequences providing for little or no detectable replicon activity in a cell can be enhanced by producing functional derivatives containing adaptive mutations. Adaptive mutations and techniques for selecting for adaptive mutations are well known in the art. (See, for example, Lohmann, et al., 1999. Science 285, 110-113, Bartenschlager, European Patent Application 1 043 399, published Oct. 11, 2000, Blight, et al., 2000. Science 290:1972-1974, Lohmann, et al., 2001. Journal of Virology 75:1437-1449, and Pietschmann, et al., 2001. Journal of Virology 75:1252-1264, 2001.)
Numerous examples of naturally occurring HCV isolates are well known in the art. HCV isolates can be classified into the following six major genotypes comprising one or more subtypes: HCV-1/(1a, 1b, 1c), HCV-2/(2a, 2b, 2c), HCV-3/(3a, 3b, 10a), HCV-4/(4a), HCV-5/(5a) and HCV-6/(6a, 6b, 7b, 8b, 9a, 11a). (Simmonds, 2001. J. Gen. Virol., 82, 693-712.) Examples of HCV sequences have been deposited in GenBank and described in various publications. (See, for example, Chamberlain, et al., 1997. J. Gen. Virol., 78, 1341-1347).
VII. Chimeric HCV Replicons and Resistance Phenotyping
Chimeric HCV replicons contain HCV regions from different HCV strains and can be used, for example, to measure the effect of a compound on HCV activity and to provide a template for HCV resistance phenotyping. The starting template for HCV resistance phenotyping is a functional replicon. One or more different regions of the functional replicon can be replaced with an HCV region from a different strain, including HCV obtained from a patient infected with the virus.
The template used for HCV resistance phenotying can contain regions from different HCV strains. Preferably, the template contains a HCV-1a 3′ UTR. Bases 8407-8641 of SEQ. ID. NO. 1 provide the nucleotide sequence of the HCV-1a 3′ UTR.
HCV resistance phenotyping can be performed by examining the sensitivity of different HCV targets isolated from a clinical sample to experimental or approved HCV drugs using a chimeric replicon. HCV targets that can be analyzed include HCV encoded enzymes such as NS2/3 protease, NS3 protease, NS3 helicase, and NS5B, as well as polynucleotide regions important for HCV replication. In different embodiments concerning HCV chimeric replicons, the replicon comprises:
a) a HCV 3′UTR, preferably HCV-1a UTR,
b) HCV non structural proteins from any one genotype or any combination of genotypes as long as the combination supports HCV replication, different HCV genotypes include HCV-1, HCV-2, HCV-3, HCV-4, HCV-5, and HCV-6; preferably, the non-structural regions contain one region from a clinical isolate;
c) restriction sites that are silent with respect to coding that facilitate introduction of HCV sequences obtained from a clinical isolate; and
d) a reporter gene, preferably beta-lactamase.
Resistance phenotyping can be performed by isolating an HCV target region from a clinical isolate, and transferring the region to a template replicon. An example of a procedure for constructing a chimeric HCV replicon for use in resistance phenotyping is as follows:
a) Isolation and purification: isolation and purification of HCV genomic RNAs from infected patient serum can be achieved using well known techniques. (Examples of such techniques are described in Chamberlain, et. al., 1997. J. Gen. Virol., 78, 1341-1347, Grahovac, et. al., 2001. J. Clin. Virol. 20, 85-89, Castelain, et al., 2002. JID 185, 573-583, Farci, et. al., 2002. PNAS 99, 3081-3086.)
c) Amplification of desired target sequences. Polymerase chain reaction amplification can performed to amplify the target sequence using primers 5′ and 3′ of the target sequence. The primers preferably contain restriction sites that facilitate cloning. Examples of useful restriction sites include BstZ17 I and Cla I and Bcl I; and
d) Subcloning of target sequence: Restriction enzymes are used to subclone the target sequence into a HCV replicon.
Two examples of HCV template sequences useful for resistance phenotyping are provided by SEQ. ID. NO. 1 and SEQ. ID. NO. 2. SEQ. ID. NO. 1 contains an NS2-NS5B region from HCV-1b consensus (con-1) while SEQ. ID. NO. 2 contains a NS3-NS5B region from HCV-1b consensus (con-1). SEQ. ID. NO. 2 also contains useful restriction sites for subcloning NS5B. SEQ. ID. NOs. 1 and 2 both contain a beta-lactamase reporter and a HCV-1a 3′ UTR.
In different embodiments of the present invention, one or more HCV regions from SEQ. ID. NOs. 1 or 2 are replaced with an HCV region from a clinical isolate. Preferably, the region from the clinical isolate is either NS2/3 protease, NS3 protease, NS3 helicase, NS5B or a combination thereof.
VIII. Replicon Enhanced Cells
HCV replicon enhanced cells provide a preferred system for measuring HCV replicon activity. HCV replicon enhanced cells can be isolated based on their ability to support replication of a first replicon and the resulting cell population supports more efficient replication of a second replicon compared to parental cells.
The replicon enhanced cell supports chronic or persistent replication of the second HCV replicon. Enhanced cells may or may not contain the first HCV replicon used obtain the replicon enhanced cell.
The first HCV replicon is defined as the replicon used to select the enhanced cell population and the second replicon is defined as the replicon used to measure HCV replication. Preferably, replicon enhanced cells contain a first HCV replicon having a drug resistance gene and a second HCV replicon having a reporter. The first replicon is present in an amount (copy number) compatible with efficient replication of the second HCV replicon. The combination of the two replicons in a replicon enhanced cell is particularly useful for high throughput screening.
Different HCV replicons can be constructed for use as the first replicon. A drug resistance gene can be used to isolate cells that support replication of the first replicon. Alternatively, the first replicon can encode a reporter gene that is compatible with cell sorting which allows isolation of cells that support replication of the first replicon.
The first replicon, if present in a cell containing the second replicon, should be present in an amount compatible with efficient replication of the second HCV replicon. The enhanced phenotype of a replicon enchanced cell can be masked or inhibited if the copy number of the first replicon is too high.
If needed, the copy number of the first replicon can be reduced by treating cells with inhibitors of HCV replication or by using cell culture conditions that are not compatible with replicon replication. The latter includes maintaining the cells at high cell densities for prolonged periods of time. The second replicon can be used to monitor HCV replication in enhanced cells.
Inhibitors of HCV replicon replication include IFN-α and HCV inhibitor compounds targeting a HCV protein. Examples of HCV inhibitory compounds are described in Llinas-Brunet, et al., 2000. Bioorg Med Chem. Lett. 10(20), 2267-2270.
The first replicon can also be made less active to reduce its copy number. A less active first replicon can be produced in different ways such as by employing regulatory elements or HCV coding sequences that provide for less activity. Regulatory elements that can be altered include ribosome binding sequences, 5′ UTR, and 3 ′UTR sequences. The activity of a replicon can readily be tested by, for example, measuring HCV nucleic acid or protein production.
Different HCV replicons can be constructed for use as the second replicon. The second replicon in addition to containing an HCV sequence preferably contains a reporter sequence. More preferably, the second replicon contains a reporter sequence such as beta-lactamase, beta galactosidase, green fluorescence protein or luciferase.
IX. Detection Methods
Methods for detecting replicon activity include those measuring the production or activity of replicon RNA and encoded protein. Measuring includes qualitative and quantitative analysis. Preferably, replicon activity is measured using a reporter protein. A preferred reporter system is the beta-lactamase reporter system.
Beta-lactamase activity can be measured, for example, by direct visualization of cells using a fluorescence microscope. Quantitation of HCV replication can be accomplished using a CCD camera that acquires digital images and suitable software that quantitates the number of blue and green cells present in such images. This method quantitates the number of cells in a population that harbors HCV replicons expressing beta-lactamase and this measurement is typically expressed as percentage blue cells (% Blue cells).
Another method for measuring beta-lactamase activity is using a fluorescence plate reader that quantitates the amount and green (˜530 nm) or blue (˜460 nm) fluorescence emitted by cells stimulated with light of ˜405 nm. This method can be used for high throughput screening.
Quantitation of beta-lactamase activity can also be accomplished by FACS. This method quantitates the number of blue and green cells in a given cell population as well as the amount of blue and green fluorescence. Instruments capable of cell sorting can be used to isolate cells harboring HCV replicons expressing beta-lactamase.
Techniques suitable for measuring RNA production include those detecting the presence or activity of RNA. The presence of RNA can be detected using, for example, complementary hybridization probes or quantitative PCR. Techniques for measuring hybridization between complementary nucleic acid and quantitative PCR are well known in the art. (See for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and U.S. Pat. No. 5,731,148.)
RNA enzymatic activity can be provided to the replicon by using a ribozyme sequence. Ribozyme activity can be measured using techniques detecting the ability of the ribozyme to cleave a target sequence.
Techniques for measuring protein production include those detecting the presence or activity of a produced protein. The presence of a particular protein can be determined by, for example, immunological techniques. Protein activity can be measured based on the activity of an HCV protein or a reporter protein sequence.
Techniques for measuring HCV protein activity vary depending upon the protein that is measured. Techniques for measuring the activity of different non-structural proteins such as NS2/3, NS3, and NS5B, are well known in the art. (See, for example, references provided in the Background of the Invention.)
Assays measuring replicon activity also include those detecting virion production from a replicon that produces a virion; and those detecting a cytopathic effect from a replicon producing proteins exerting such an effect. Cytopathic effects can be detected by assays suitable to measure cell viability.
Assays measuring replicon activity can be used to evaluate the ability of a compound to modulate HCV activities. Such assays can be carried out by providing one or more test compounds to a cell expressing an HCV replicon and measuring the effect of the compound on replicon activity. If a preparation containing more than one compound is used and found to modulate replicon activity, individual compounds or smaller groups of compounds can be tested to identify replicon active compounds.
HCV inhibitory compounds can be used to produce replicon enhanced cells and compounds having the appropriate pharmacological properties may be therapeutic compounds. The ability of a compound to serve as a therapeutic compound can be confirmed using animal models such as a chimpanzee to measure efficacy and toxicity.
X. Examples
Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
This example illustrates the production of replicon enhanced cells. It is not necessary to cure cells from the first replicon to obtain a cell population that is enhanced for replication of a second replicon. A preferred method to achieve this is to use a replicon (first replicon) with replication properties and replicon copy number that are compatible with enhanced replication of a second replicon.
Replicons used in the Example section have the general gene organization: HCV 5′UTR-drug resistance and/or reporter-EMCV IRES-HCV NS proteins-HCV 3′UTR. The replicon nomenclature used in the examples is as follows: the drug resistance and/or reporter gene under control of the HCV RES is indicated first, the start of the HCV polyprotein is indicated next and the presence of adaptive mutations is indicated last. For example, a replicon harboring the S2204I adaptive mutation, expressing the neo gene and containing the NS3-NS5B HCV NS region is referred to as neo-NS3/si. Replicons expressing Bla instead of neor and containing the NS2-NS5B HCV NS region are referred to as Bla-NS2/si.
The activity of a beta-lactamase replicon (bla-NS3/si) and a replication deficient replicon (bla-NS3/si/gaa), was compared in parental Huh-7 cells, neo-NS3 cells (Huh-7 cells harboring neo-NS2 replicons), neo-NS2 (Huh-7 enhanced cells harboring neo-NS2 replicons) partially cured neo-NS2 cells harboring a reduced number of neo-NS2 replicons and cured neo-NS2 cells that do not contain replicons. Neo-NS2 cells were partially cured using interferon alpha and are referred to as JGEM cells. Neo-NS2 cells were completely cured using a potent inhibitor of HCV replication and are referred to in the examples below as JG884 cells.
Quantitation of the beta-lactamase reporter was used to measure HCV sub-genomic replication and is expressed as the percentage of beta-lactamase positive cells (% blue cells). The percentage of blue cells (% BC) at 24 hours after transfection is close to 100% in both cell lines indicating similar transfection efficiencies (Table 1). The % BC at days 3 & 6 post transfection is a measure of persistent replication.
The experiment described in Table 1 shows that the bla-NS3/si/gaa replicon did not replicate in any of the cell lines analyzed but replication of the bla-NS3/si replicon was readily detected in all cell lines both at day 3 and day 6. The activity of the bla-NS3/si replicon was significantly lower in Huh-7 and neo-NS3 cells than in the neo-NS2 cell lines.
Importantly, the replication efficiency of the bla-NS3/si replicon was comparable in neo-NS2, JGEM, and JG884 cells demonstrating that cells harboring neo-NS2 replicons do not have to be cured to support enhanced replication of a second replicon. In contrast, the activity of the bla-NS3/si replicon in the neo-NS3 cell line was similar to that observed in Huh-7 cells indicating that neo-NS3 cells were not enhanced (Table 1).
Quantitation of replicon RNA in the different cell lines indicate that neo-NS3 cells produce significantly more RNA per cell than neo-NS2 cells suggesting that the replicon copy number in neo-NS3 cells might be too high for efficient replication of a second replicon (
The enhanced phenotype of JGEM and JG884 cells is similar to neo-NS2 cells and is manifested as both increased permissiveness (i.e., a greater percentage of transfected cells support replication) and increased persistence (ie., replicon maintenance). The enhanced phenotype is not dependent on the sequence of the second replicon. The observation that JGEM cells support enhanced replication of replicons harboring adaptive mutants S2321 (SI), A227T (AT) and, R465G (RG) (
Examples of techniques that can be employed to create a stable cell line harboring a second replicon expressing the beta-lactamase reporter and assaying for activity is provided in this example. Replicon enhanced cells containing a first replicon can be produced as described in Example 1.
Enhanced Huh-7 cells are transfected with HCV replicon RNA encoding beta-lactamase. The resulting cell population is expanded using standard cell culture conditions (DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1×non-essential amino acids, Pen/Strep) until the desired number of replicon harboring cells is obtained.
An alternative approach involves cell sorting. Cell sorting can be performed, for example, by separating cell harboring replicons three to six days after transfection from cells that do not harbor the replicon using flow cytometry assisted cell sorting. The population of cells stably maintaining the replicon expressing beta-lactamase is expanded using standard cell culture techniques until sufficient cells (>2×109) to carry out the screen is obtained. Replicon harboring cells are then aliquoted, frozen and stored in liquid nitrogen.
Prior to use, frozen cells are thawed fresh in the morning of the assay or the day before and diluted in pre-warmed media to 40,000 cells/ml. Cell suspension (50 μl) is plated into 384-well micro-titer plates. The cells are allowed to recover for at least 6 hours at 37° C. in the plates before beginning an assay. 1 μl of 25 μM clavulanic acid in PBS and 0.25 μl of 500 μM test compounds in DMSO are added to the cells. The assay cell plates are then incubated for 24 hours at 37° C. To the assay plate, 10 μl of 6×CCF4/AM substrate in loading buffer is added with a Multidrop (Labsystems), and the plate incubated for 90 minutes at ambient temperature. The plate is then read from the bottom on a Teacn Spectrafluor Plus (340 nm excitation, 460 nm and 530 nm emissions).
In the example described below, clavulanic acid is present throughout the assay at a concentration that slightly reduces beta-lactamase activity instead of using it at high concentration that would complete inhibit beta-lactamase activity. Because clavulanic acid is an irreversible inhibitor, it is a more potent inhibitor of beta-lactamase at low enzyme concentrations than at high enzyme concentrations and therefore its inhibitory activity has a more pronounced effect in the background signal than in the foreground signal which in turn explains the enhancement of the signal-to-background ratio from the beta-lactamase reporter.
Table 2 provides results obtained using different concentrations of clavulanic acid and an HCV NS5B inhibitor.
The experiment in
JGEM cells were lipotransfected with bla-NS2/si (BK 3′-UTR) replicon RNA using DMRIE-C (transfection reagent, Invitrogen Life Technologies) and expanded under standard growth conditions. Cells were stained with CCF4/AM for 90 minutes, dissociated from the flask with 0.25% trypsin/1 mM EDTA, and then suspended in sort buffer (PBS with 0.1% bovine serum albumin and pen/strep).
Cells were then sorted using Becton-Dickenson FACS Vantage SE cell sorter equipped with Coherent Innova 70C-4 (488 nm) and Coherent 302C Krypton (407 nm) lasers. Optical filters used on the cell sorter were 530/30 and 450/30 bandpass filters separated by a 490 dichroic mirror. Cells were sorted at a rate of 10,000 cells/s.
Cells staining positive for beta-lactamase expression were collected in complete medium containing 20% fetal calf serum. Medium was replaced the following day with complete medium containing 10% FCS. Cells were assayed for % blue cells by digital image processing at the indicated intervals. Cells maintained replicon for at least two weeks as indicated by the high percentage of cells that stain positive for the beta-lactamase reporter (Table 3).
HCV BK is a genotype 1b clone that is infectious in chimpanzees. BK replicons with and without the NS5A S2204I adaptive mutation were generated and tested for their ability to replicate using the beta-lactamase reporter. The replicon without the S2204I adaptive mutation failed to replicate in JGEM cells, while the replicon with the mutation replicated with modest efficiency.
A series of BK and con1 chimeras were generated to identify the region(s) that accounts for the different replicative activities of the two replicons. JGEM cells were lipotransfected with chimeric replicon RNAs and then cultured for 4 days under standard growth conditions. As shown in Table 4, replacement of the BK NS3 sequence with that from the con1 replicon resulted in a dramatic improvement in replication. In other BK/con1 chimeras, most of the differences in replication efficiency tracked with the NS3 sequence used while other parts of the genome had more modest or no effects on the observed differences in replication efficiency.
This example illustrates resistance phenotyping with NS5B. BstZ17 I and Cla I restrictions sites were introduced at the 5′-and 3′-ends of the NS5B coding region, respectively. The nucleotide substitutions made to introduce these restriction sites do not affect amino acid coding. The BstZ17 I and Cla I sites are unique in the resulting construct and facilitate rapid subcloning of NS5B sequences from clinical isolates for phenotypic analysis. As shown in Table 5, the nucleotide changes required to introduce the restriction sites do not affect replication.
Viral RNA was purified from 140 μl of infected patient serum (103-106 copies/ml) using the Qiagen viral RNA purification kit according to the manufacturer's directions followed by phenol:chloroform extraction and ethanol precipitation. cDNAs were generated by reverse transcription of 1/20 to ⅕ of the sample using a poly(A) oligonucleotide primer and Superscript II reverse transcriptase (Gibco-BRL) according to the manufacturer's guidelines.
NS5B coding regions were amplified by nested PCR using Expand High Fidelity polymerase (Roche). The initial amplification reaction was carried out using a primer that anneals immediately upstream of the NS5B coding region and the poly(A) primer used for reverse transcription. The second amplification used primers (sequences) that anneal at the 5′- and 3′-ends of the NS5B coding region internal to the primers used in the initial PCR reaction.
Amplification products were cloned directly into pGEM T-vector (Promega). The NS5B coding regions were subsequently amplified using a sense primer that included Bcl I restriction site and an antisense primer designed to introduce a Cla I site at the 3′-end. Amplification products were subcloned into the corresponding sites of SEQ. ID. NO. 2 to generate chimeric viruses containing the NS5B sequences derived from the clinical samples.
Replication of the chimeric replicons was characterized by using the beta-lactamase reporter assay as described above. As shown in Table 5, functional replicons with NS5B sequences from another genotype 1b isolate and from a genotype 1a isolate were functional in the context of the resistance phenotyping replicon.
NS2 replicon constructs that expressed a beta-lactamase reporter and that contained either the con1 or HCV 1a 3′-UTR were generated using techniques well known in the art. Replication of the resulting replicons was analyzed by digital image processing or using a fluorescent plate reader. As shown in Table 6, the replicon containing the HCV 1a 3′-UTR replicates more efficiently than the corresponding replicon containing the con1 3′-UTR.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.
The present application claims priority to provisional application U.S. Ser. No. 60/372,847, filed Apr. 16, 2002, hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/12509 | 4/11/2003 | WO | 00 | 10/8/2004 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO03/089672 | 10/30/2003 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5739002 | De Francesco et al. | Apr 1998 | A |
| 5741657 | Tsien et al. | Apr 1998 | A |
| 5783669 | Hawkins et al. | Jul 1998 | A |
| 5885779 | Sadowksi et al. | Mar 1999 | A |
| 6063562 | Melnick et al. | May 2000 | A |
| 6297003 | Rice et al. | Oct 2001 | B1 |
| 6630343 | Bartenschlager | Oct 2003 | B1 |
| 20020142350 | Kukolj et al. | Oct 2002 | A1 |
| 20040018529 | Li et al. | Jan 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 199 15 178 | May 2000 | DE |
| 1 043 399 | Oct 2000 | EP |
| WO 9637619 | Nov 1996 | WO |
| WO 0189364 | Nov 2001 | WO |
| WO 02059321 | Aug 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20050181356 A1 | Aug 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60372847 | Apr 2002 | US |
