WO2006019429A2 - Procedes d'identification et d'utilisation d'agents accroissant l'inhibition de l'infection au hcf ou la resistance a celle-ci - Google Patents

Procedes d'identification et d'utilisation d'agents accroissant l'inhibition de l'infection au hcf ou la resistance a celle-ci Download PDF

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WO2006019429A2
WO2006019429A2 PCT/US2005/012648 US2005012648W WO2006019429A2 WO 2006019429 A2 WO2006019429 A2 WO 2006019429A2 US 2005012648 W US2005012648 W US 2005012648W WO 2006019429 A2 WO2006019429 A2 WO 2006019429A2
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seq
sequence
cell
protein
sequences
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WO2006019429A3 (fr
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Hengli Tang
Flossie Wong-Staal
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Immusol Incorporated
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae

Definitions

  • the present invention is directed to methods of identifying agents that increase inhibition of or resistance to a viral infection, more particularly an HCV (hepatitis C virus) infection.
  • the invention is also directed to methods and compounds that increase such inhibition or resistance.
  • HCV infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to the development of hepatocellular carcinoma. It is estimated that 170 million people are chronically infected with HCV worldwide, with an additional 3-4 million people newly infected each year.
  • IFN interferon
  • the sustained response rate is suboptimal and highly variable. He and Katze, Viral Immunol, 15:95-119 (2002).
  • the present invention centers on the discovery of the involvement of a protein, pi 25 (SEQ ID NO:2; whose mRNA nucleotide sequence is SEQ ID NO:1), in HCV infection. Accordingly, the present invention provides methods of identifying agents useful for increasing the inhibition of or resistance to HCV infection in a cell by binding to SEQ ID NO:1
  • the present invention also provides agents that increase the inhibition of or resistance to HCV infection in a cell by binding to SEQ ID NOS :1 or 3, or portions thereof, or otherwise down-modulates SEQ ID NOS: 1 or 3, or portions thereof.
  • the agents of the invention include ribozymes.
  • the ribozymes of the invention have "substrate binding sequences" that hybridize to and cleave complementary sequences of the mRNA encoded by the genes and gene sequences disclosed herein, thereby reducing the expression of the genes which results in increased inhibition of or resistance to HCV infection.
  • the present invention further provides methods for increasing inhibition of or resistance to HCV infection in HCV-infected cells by introducing an agent into said cell which binds to SEQ ID NOS: 1 or 3, or portions thereof.
  • the agent can be, for example, a ribozyme or an siRNA.
  • the present invention further provides methods for identifying an agent that can increase inhibition of or resistance to HCV infection by binding SEQ ID NOS:1, 2 or 3, or portions thereof, or otherwise down-modulates SEQ ID NOS: 1, 2 or 3, or portions thereof.
  • the method includes introducing the agent into a cell infected with HCV and then measuring the level of HCV infection in the cell. A reduction in level indicates that the agent increases inhibition of or resistance to HCV infection.
  • Representative agents include antisense oligonucleotides, monoclonal and polyclonal antibodies, and small organic molecules.
  • the present invention centers on the discovery of the involvement of a protein, p 125
  • the present invention provides nucleic acid sequences and other agents that increase the inhibition of or resistance to HCV infection in a cell by binding SEQ ID NOS: 1, 2 or 3, or portions thereof, or otherwise down-modulates SEQ ID NOS: 1 or 3, or protions thereof, or down-regulates SEQ ID NO:2, or portions thereof.
  • pl25 protein has also been identified as a Sec23p interacting protein. Reduced pl25 expression inhibits HCV replication. See Example 3. The mechanism for this is thought to be inhibition of HCV internal ribosome entry site (IRES) directed translation. See Example 1. Conversely, increased pi 25 expression enhances HCV IRES mediated translation, resulting in increased replication of HCV upon infection. See Example 4.
  • Agents of the invention for increasing inhibition of or resistance to HCV infection include ribozymes.
  • the ribozymes of the invention have "substrate binding sequences" that hybridize to and cleave complementary sequences of the mRNA encoded by the genes and gene sequences disclosed herein thereby reducing expression of the genes. Included within the scope of the invention are expression vectors encoding the ribozymes, cells containing the vectors and cells expressing the ribozymes.
  • a ribozyme of the present invention can be introduced directly into a cell, i.e., without the use of a vector.
  • the present invention further provides methods for increasing inhibition of or resistance to HCV infection in HCV-infected cells by introducing an agent into said cell.
  • the agent can be, for example, a ribozyme, an siRNA, an antisense molecule or an antagonizing antibody.
  • the present invention further provides methods for identifying an agent that can increase inhibition of or resistance to HCV infection.
  • the method includes introducing the agent into a cell infected with HCV, where the agent binds to SEQ ID NOS: 1, 2 or 3, or portions thereof, or otherwise down-modulates SEQ ID NOS :1 or 3, or portions thereof, or down-regulates SEQ ID NO: 2, or portions thereof, and measuring the level of HCV infection in the cell. A reduction in level would indicate that the agent increases inhibition of or resistance to HCV infection.
  • Representative agents include, intrabodies, antisense molecules, ribozymes, siRNAs, small organic molecules or antibodies that antagonize pi 25 activity or expression.
  • a ribozyme of the invention has a "substrate binding sequence" that recognizes a target nucleic acid molecule involved in HCV replication.
  • the ribozymes of the invention are catalytic RNA molecules that bind to the target nucleic acid molecules and cleave them, thereby impairing their ability to function as cofactors of HCV replication.
  • the ribozymes of the invention are identified and selected by methods described herein. They may be "hairpin" ribozymes, "hammerhead” ribozymes or any other type of ribozyme known in the art.
  • Ribozymes of the invention bind to various target sequences.
  • target sequences include CATTAGTCCAGAACAG (SEQ ID NO:4), which corresponds to positions 3048 to 3063 of SEQ ID NO:1; TAGTTGTCAAAGTTAT (SEQ ID NO:5), which corresponds to positions 3364 to 3379 of SEQ ID NO:1; and AAGCGGTCTCTTAGCA (SEQ ID NO:6), which corresponds to positions 762 to 777 of SEQ ID NO:3.
  • the invention also includes a chimeric ribozyme based on the ribozyme's substrate binding sequence.
  • a chimeric hammerhead rib ⁇ 2yme i.e., a RNA/DNA hybrid
  • most or all of the binding arms and stem loop comprise DNA.
  • the catalytic domain, between the binding arms and stem loop comprises RNA.
  • Modification of the base composition at the stem loop or catalytic domain regions can increase the catalytic activity of the ribozyme, as assayed by in vitro cleavage See WO 00/32765.
  • Modification at the 2-position of the sugar of the base for example, substituting -OCH 3 at this position of an RNA base, can increase the stability of the ribozyme.
  • Other stabilizing substitutions include -OC 1-6 Alkyl, -F or other halogens, amino, azido, nitro and phenyl. See U.S. Pat. No. 5,298,612.
  • ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990; Pieken et ah, 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No.
  • substrate binding sequence of a ribozyme refers to that portion of the ribozyme which base pairs with a complementary sequence (referred to herein as a “ribozyme sequence tag” or "RST”) of a target nucleic acid.
  • RST ribozyme sequence tag
  • the general structure of the substrate binding sequence of a hammerhead includes three or four to nine bases at the 5' end of the ribozyme's binding arm and three or four to nine bases at the 3' end of the other binding arm.
  • the following approach may be used: 1) identify the ribozyme sequence tag (RST) of the RNA target of the specific hairpin ribozyme of interest; 2) specify the first six to nine nucleotides at the 5' end of the hammerhead as complementary to the first six to nine nucleotides at the 3' end of the RST; 3) specify the first 5 nucleotides at the 3' end of the hammerhead as complementary to nucleotides at the 5' end of the RST; and 4) specify nucleotides at positions 6 and 7 from the 3' end of the hammerhead as complementary to the RST, while base 8 from the 3' end is an A.
  • RST ribozyme sequence tag
  • a ribozyme of the present invention can comprise a) 3, 4, 5 or 6 contiguous bases as part of one binding arm of the ribozyme; and b) 3, 4, 5 or 6 contiguous bases of any of the remaining contiguous bases of that ribozyme substrate binding sequence as part of the other binding arm of the ribozyme.
  • a hammerhead ribozyme can be designed by incorporating sequences of one of the substrate binding sequences disclosed herein, for example bases 1 to 6 and 11 to 14 of the substrate binding sequence.
  • unmodified base means one of the bases adenine, guanine, cytosine, uracil or thymine attached to the 1 -carbon of the sugar (deoxyribose or ribo- furanose), with a phosphate bound to the 5-carbon of the sugar.
  • Bases are bound to each other via phosphodiester bonds between the 3-carbon of one base and the 5-carbon of the next base.
  • modified base means any base whose chemical structure is modified as follows.
  • Adenine can be modified to result in 6-dimethyl-amino-purine, 6- methyl-amino-purine, 2-amino-purine, 2,6-diamino-purine, 6-amino-8-bromo-purine or 6- amino-8-fluoro-purine.
  • Cytosine can be modified to result in 5-bromo-cytosine, 5-fluoro- cytosine, N,N-dimethyl-cytosine, N-methyl-cytosine, 2-thio-cytosine or 2-pyridone.
  • Guanine can be modified to result in 8-bromo-guanine, 8-fluoroguanine, 2-amino-purine, hypozanthine (inosine), 7-deaza-guanine or 6-thio-guanine.
  • Uracil can be modified to result in 3-methyl-uracil, 5,6-dihydro-uracil, 4-thio-uracil, thymine, 5-bromo-uracil, 5-iodo-uracil or 5-fluoro-uracil.
  • Thymine can be modified to result in 3-methyl-thymine, 5,6-dihydro- thymine, 4-thio-thymine, uracil, 5-bromo-uracil, 5-iodo-uracil or 5-fluoro-uracil.
  • Methods of making such modifications as well as other modifications, such as halogen, hydroxy, amine, alkyl, azido, nitro and phenyl substitutions are disclosed in U.S. Pat. No. 5,891,684; and U.S. Pat. No. 5,298,612.
  • the present invention encompasses sequences where one or more bases are modified:
  • the sugar moiety of a base can be modified as disclosed above regarding bases of a hammerhead ribozyme.
  • the present invention encompasses sequences where one or more bases are so modified.
  • nucleic acid or “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides, oligomers and polymers thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. For example, as disclosed herein, such analogues include those with substitutions, such as methoxy, at the 2-position of the sugar moiety. Unless otherwise indicated by the context, the term is used interchangeably with gene, cDNA and mRNA encoded by a gene.
  • a nucleotide sequence encoding refers to a nucleic acid which contains sequence information, for example, for a ribozyme, mRNA, structural RNA, and the like, or for the primary amino acid sequence of a specific protein or peptide.
  • sequence information for example, for a ribozyme, mRNA, structural RNA, and the like, or for the primary amino acid sequence of a specific protein or peptide.
  • the explicitly specified encoding nucleotide sequence also implicitly covers sequences that do not materially effect the specificity of the ribozyme for its target nucleic acid.
  • RNA correlate of a given DNA sequence means that sequence with "U” substituted for "T.”
  • the present invention encompasses the RNA correlate of SEQ ID NOS: 1 or 3.
  • sequence similarity in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are, when optimally aligned with appropriate nucleotide insertions or deletions, the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50% identity, 65%, 70%, 75%, 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence such as SEQ ID NO:2, or a nucleotide sequences such as SEQ ID NOS: 1 or 3, or portions thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and, visual inspection.
  • This definition also refers to the compliment of a test sequence.
  • the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • nucleic acid will hybridize under selective hybridization conditions, to a strand or its complement.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, more typically at least about 65%, preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa, Nuc. Acids Res. , 12 :203 -213 ( 1984), which is incorporated herein by reference.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, generally at least about 20 nucleotides, ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides.
  • Amino acid sequence homology, or sequence identity is determined by optimizing residue matches, if necessary, by introducing gaps as required. This changes when considering conservative substitutions as matches.
  • Conservative substitutions typically include substitutions within the following groups: [glycine, alanine]; [valine, isoleucine, leucine]; [aspartic acid, glutamic acid]; [asparagine, glutamine]; [serine, threonine]; [lysine, arginine]; and [phenylalanine, tyrosine].
  • Homologous amino acid sequences are intended to include natural allelic and interspecies variations in each respective receptor sequence.
  • Typical homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced), to 50-100% homology (if conservative substitutions are included).
  • Homology measures will be at least about 50%, generally at least 56%, more generally at least 62%, often at least 67%, more often at least 72%, typically at least 77%, more typically at least 82%, usually at least 86%, more usually at least 90%, preferably at least 93%, and more preferably at least 96%, and in particularly preferred embodiments, at least 98% or more.
  • homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin, " including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et ah, Cell, 50:667 (1987)).
  • the present invention naturally contemplates homologues of the pl25 protein, and polynucleotides encoding the same, as falling within the scope of the invention.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment ⁇ of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J MoL Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a. cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-395 (1984).
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. MoI Biol. 215:403-410 (1990), respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • each method of the invention described herein encompasses: a) all compounds having about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NOS: 1, 2 or 3; b) all compounds having about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with positions 1 to 3768 or positions 1 to 3075 of SEQ ID NO: 1 ; c) all compounds with 50, 60, 70, 80, 90 or more amino acids and having about 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:2; and d) all compounds with 100, 150, 200, 250, 300 or more nucleotides and having about 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NOS :1 or 3.
  • the invention also encompasses the encoding DNA of SEQ ID NO:1, as well as
  • Moderately stringent conditions means hybridization conditions that permit a nucleic acid molecule to bind to a second nucleic acid molecule that has substantial identity to the sequence of the first. Moderately stringent conditions are those equivalent to hybridization of filer-bound nucleic acid in 50% formamide, 5 X Denhart's solution, 5 X SSPE, 0.2% SDS at 42 0 C, followed by washing in 0.2 X SSPE, 0.2% SDS at 5O 0 C.
  • Highly stringent conditions are those equivalent to hybridization of filer-bound nucleic acid in 50% formamide, 5 X Denhart's solution, 5 X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2 X SSPE, 0.2% SDS at 65°C.
  • Other suitable moderately stringent and highly stringent conditions are known in the art and described, for example, in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992), and Ansubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore MD (1998).
  • a nucleic acid molecule that hybridizes to a second one under moderately stringent conditions will have greater than about 60% identity, preferably greater than about 70% identity and, more preferably, greater than about 80% identity over the length of the two sequences being compared.
  • a nucleic acid molecule that hybridizes to a second one under highly stringent conditions will have greater than about 90% identity, preferably greater than about 92% identity and, more preferably, greater than about 95%, 96%, 97%, 98% or 99% identity over the length of the two sequences being compared.
  • an "isolated” when used in conjunction with a nucleic acid or protein denotes that the nucleic acid or protein has been isolated with respect to the many other cellular components with which it is normally associated in the natural state.
  • an "isolated" gene of interest may be one that has been separated from open reading frames which flank the gene and encode a gene product other than that of the specific gene of interest. Such genes may be obtained by a number of methods including, for example, laboratory synthesis, restriction enzyme digestion or PCR.
  • an "isolated" protein may be substantially purified from a natural source or may be synthesized in the laboratory.
  • a “substantially purified” nucleic acid or protein gives rise to essentially one band in an electrophoretic gel, and is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • the term "intrabody” refers to a class of neutralizing molecules with applications in gene therapy (vonMehren M,Weiner L M. (1996) Current Opinion in Oncology. 8:493-498, Marasco Wash. (1997) Gene Therapy. 4:11-15, Rondon I J, Marasco Wash. (1997) Annual Review of Microbiology. 51:257-283). Intrabodies are engineered antibodies that can be expressed within a cell and target an intracellular molecule of molecular domain.
  • intrabodies directed against pl25 will bind to the nascent pi 25 protein and direct it to the ubiquitin pathway for catalytic degradation, rather than to the cellular membrane.
  • intrabodies provide yet another approach to down regulating pi 25 expression and activity.
  • the intrabody method is analogous to the inactivation of proteins by deletion or mutation, but is directed at the level of gene product rather than at the gene itself. Using the intrabody strategy even molecules involved in essential cellular pathways can be targeted, modified or blocked.
  • Antibody genes for intracellular expression can be derived either from murine or human monoclonal antibodies or from phage display libraries. For intracellular expression small recombinant antibody fragments, containing the antigen recognizing and binding regions, can be used. Intrabodies can be directed to different intracellular compartments by targeting sequences attached to the antibody fragments. The construction and use of intrabodies is discussed, for example, in U.S. Pat. No. 6,004,940.
  • the term "expression vector” includes a recombinant expression cassette that has a nucleotide sequence that can be transcribed into RNA in a cell.
  • the cell can further translate transcribed mRNA into protein.
  • An expression vector can be a plasmid, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes the encoding nucleotide sequence to be transcribed (e.g. a ribozyme), operably linked to a promoter, or other regulatory sequence by a functional linkage in cis.
  • an expression vector comprising a nucleotide sequence encoding ribozymes of the invention can be used to transduce cells suitable as hosts for the vector.
  • procaryotic cells including bacterial cells such as E. coli and eukaryotic cells including mammalian cells may be used for this purpose.
  • promoter includes nucleic acid sequences near the start site of transcription (such as a polymerase binding site) and, optionally, distal enhancer or repressor elements (which may be located several thousand base pairs from the start site of transcription) that direct transcription of the nucleotide sequence in a cell.
  • the term includes both a “constitutive” promoter such as a pol III promoter, which is active under most environmental conditions and stages of development or cell differentiation, and an “inducible” promoter, which initiates transcription in response to an extracellular stimulus, such as a particular temperature shift or exposure to a specific chemical.
  • Promoters and other regulatory elements may be incorporated into an expression vector encoding ribozymes of the present invention as described in WO 00/05415 to Barber et al.
  • LTRs retroviral long terminal repeats
  • AAV adeno associated viral inverted terminal repeats
  • promoters may be incorporated into an expression vector encoding ribozymes of the present invention as described in WO 00/05415 to Barber et al.
  • the term "expresses” denotes that a given nucleic acid comprising an open reading frame is transcribed to produce an RNA molecule. It also denotes that a given nucleic acid is transcribed and translated to produce a polypeptide.
  • a ribozyme typically is not translated into a protein since it functions as an active (catalytic) nucleic acid.
  • gene product refers either to the RNA produced by transcription of a given nucleic acid or to the polypeptide produced by translation of a given nucleic acid.
  • the term "transduce” denotes the introduction of an exogenous nucleic acid molecule (e.g., by means of an expression vector) inside the membrane of a cell.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transduced cell is generally one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication, or one which includes stably maintained extrachromosomal plasmids. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • transfection means the genetic modification of a cell by uptake of an exogenous nucleic acid molecule (e.g., by means of an expression vector).
  • ribozyme gene vector library denotes a collection of ribozyme-encoding genes, typically within expression cassettes, in a collection of viral or other vectors. The vectors may be naked or contained within a capsid. Propagation of the ribozyme gene vector library can be performed as described in WO 00/05415 to Barber et al. The ribozyme-encoding genes of a ribozyme gene vector library, after transduction and transcription in appropriate cells, produce a collection of ribozymes.
  • siRNA small interfering RNAs
  • RNAi RNA interference
  • Zamore Phillip et al, Cell 101:25-33(2000); Elbashir, Sayda M. et al, Nature 411:494-497 (2001).
  • SiRNAs are assembled into a multi-component complex known as the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the siRNAs guide RISC to homologous mRNAs, thus targeting them for destruction.
  • RISC RNA-induced silencing complex
  • siRNA is a double-stranded RNA that is preferably between 16 and 25, more preferably 17 and 23 and most preferably between 18 and 21 base pairs long, each strand of which has a 3' overhang of 2 or more nucleotides.
  • siRNA comprises a sequence capable of specifically inhibiting genetic expression of a gene or closely related family of genes by a process termed RNA interference.
  • siRNA molecules are small (typically 16-25bp) double-stranded RNAs that elicit a process known as RNA interference (RNAi), a form of sequence-specific gene inactivation.
  • RNAi action proposes an ATP-dependent cleavage of mRNA molecules activated by a short double-stranded RNA.
  • the nucleotide sequence of the cleaved mRNA molecules are reported to contain a sequence fragment homologous to that of the double-stranded RNA.
  • Zamore, Phillip et al (2000) Cell, 101:25-33.
  • RNA interference has been shown to exist in mammalian cell lines, oocytes, early embryos and some cell types (see e.g., Elbashir, Sayda M. et al (2001) Nature 411:494-497).
  • siRNAs for use in the present invention can be produced from a pi 25 encoding nucleic acid sequence. For example, short complementary DNA strands are first prepared that represent portions of both the "sense” and “antisense” strands of the pl25 coding region. This is typically accomplished using solid phase nucleic acid synthesis techniques, as known in the art.
  • the short duplex DNA thus formed is ligated into a suitable vector that is then used to transfect a suitable cell line.
  • Other methods for producing siRNA molecules are known in the art. (See, e.g., Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498).
  • Libraries of siRNAs specific for pl25 can be constructed by, for example, mechanically shearing pi 25 cDNA, ligating the resulting fragments into suitable vector constructs and transfecting a suitable host cell with the vectors.
  • RNAi and siRNA expression see Hammond, Scott M. et ah, Nature Genetics Reviews, 2:110-119; Fire, Andrew (1999) TIG, 15(9):358-363; Bass, Brenda L. (2000) Cell, 101:235-238.
  • antisense oligonucleotides to mRNA is another mechanism of decreasing protein synthesis, and, consequently, represents a powerful and targeted approach to diminishing pi 25 expression.
  • the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Pat. Nos. 5,739,119 and 5,759,829, each specifically incorporated herein by reference in its entirety).
  • antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDGl), ICAM-I, E-selectin, STK-I, striatal GABA.sub.A receptor and human EGF (Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et at., 1998; U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288, each specifically incorporated herein by reference in its entirety).
  • Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U.S. Pat. Nos.
  • the invention provides therefore oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to a polynucleotide sequence described herein, or a complement thereof.
  • the antisense oligonucleotides comprise DNA or derivatives thereof.
  • the oligonucleotides comprise RNA or derivatives thereof.
  • the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone.
  • the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof.
  • preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of SEQ ID NOS :1 or 3.
  • antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
  • Highly preferred target regions of the mRNA are those which are at or near the AUG translation initiation codon, and those sequences which are substantially complementary to 5' regions of the mRNA.
  • Non-native promoter refers to any promoter element operably linked to a coding sequence by recombinant methods. Non-native promoters include mutagenized native reporters, when mutagenesis alters the rate or control of transcriptional events.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • the invention also encompasses vectors in which a pl25 nucleic acid is cloned into a vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, of the nucleic acid sequences described herein, including both coding and non- coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • Antisense nucleic acids may be obtained from libraries encoding pi 25 or synthesized synthetically. Transfection of suitable host cells with pi 25 is performed in a manner analogous to that described for siRNAs above.
  • Recombinant expression cassette refers to a DNA sequence capable of directing expression of a nucleic acid in cells.
  • a "DNA expression cassette” comprises a promoter, operably linked to a nucleic acid of interest, which is further operably linked to a termination region.
  • polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art.
  • polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro- protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro- protein sequence.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N- formylmethionine.
  • the modifications can be a function of how the protein is made.
  • the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • Candidate protein-based compounds for binding or down-modulating pi 25 include, for example, 1) peptides such as soluble peptides, including fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of L- and/or D-conf ⁇ guration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et ah, Cell 12:161-11% (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies, including intrabodies, as well as Fab, F(ab').sub
  • Soluble full-length receptors, or fragments of the same, that compete for ligand binding are also considered candidate reagents.
  • Other candidate compounds include mutant receptors or appropriate fragments containing mutations that affect receptor function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.
  • the receptor polynucleotides are also useful for constructing host cells expressing a part, or all, of the receptor polynucleotides and polypeptides.
  • the receptor polynucleotides are also useful for constructing transgenic animals expressing all, or a part, of the receptor polynucleotides and polypeptides. These animals are useful as model systems for HCV infection and can be used to test compounds for their effect, through the receptor gene or gene product, on the development or progression of the disease.
  • the term "down-modulation” means a decrease in the rate or level of mRNA production.
  • the term “down-regulation” means a decrease in the rate or level of protein expression.
  • ribozymes of the present invention were shown to down- regulate the level of the target protein pl25 (SEQ ID NO:2) by up to almost 80% (see Example 3).
  • the present invention also provides a method of increasing the inhibition of or resistance to HCV infection in a cell infected with HCV.
  • This method comprises introducing an agent of the invention into a cell infected with HCV.
  • Such an agent can be, for example, a ribozyme of the present invention, an antisense molecule, an antagonizing antibody or an siRNA.
  • This method can comprise transducing the infected cell with an expression vector encoding the agent.
  • the agent can be introduced into a cell directly, i.e., without using a vector. This method can also be used to prevent HCV infection in the cell.
  • the method of the invention can be accomplished by the agent binding to a target, for instance SEQ ID NOS: 1, 2 or 3, or portions thereof.
  • the method of the invention can also be accomplished by the agent down-modulating SEQ ID NOS :1 or 3, or portions thereof, or down-regulating SEQ ID NO:2, or portions thereof.
  • the present invention also provides methods of identifying agents that prevent HCV infection in a cell or increase inhibition of or resistance to HCV infection in a cell infected with HCV.
  • Such methods include introducing an agent that binds to SEQ ID NOS : 1 , 2 or 3 , or any portion thereof, into a cell. If the cell is infected with HCV, after the agent is introduced, the cell can be measured for the level of HCV infection or replication, where a decrease in the level indicates that the agent increases inhibition of or resistance to HCV infection. If the cell is not infected with HCV, the agent can be introduced into the cell and then introduced to HCV..
  • the cell can then be measured for the level of HCV infection or replication, where a decrease in the level, as compared to a cell introduced to HCV but not agent, indicates that the agent can promote prevention of HCV infection.
  • the methods of the invention can also include assessing the binding capability of the agent with SEQ ID NOS:1, 2 or 3, or any portion thereof.
  • the level of HCV infection can also include assessing the binding capability of the agent with SEQ ID NOS:1, 2 or 3, or any portion thereof.
  • resistance to or inhibition of HCV can be determined, for example, by measuring the concentration required to reduce the cytopathic effect of the virus, as described by Santosh et ah, Bioorg. Med. & Chem. Lett, . , 10:2505-08 (2000).
  • Such resistance or inhibition can also be determined using a plaque formation assay, and measuring the dose-dependent decrease in plaques, as described by Luedtke et ah, Chembiochem, 3:766-771 (2002); and Richman et ah, Curr. Prot. Immum, pp. 1-21 (Wiley & Sons 1993). Dose-dependent activity can also be determined by measuring the decrease in HCV protein expression using ELISA. See Luedtke et al. and Richman et al, supra.
  • high-throughput screening assays can be performed to identity, for example, potential inhibition of HCV integration into the host cell chromosome. See Vandergraaf et al., Antimicrobial Agents and Chemotherapy, 45:2510-16 (2001).
  • the step of reducing the level of the target protein in the cell can be accomplished by contacting the cell with an antisense compound or molecule.
  • Combinatorial peptide libraries can be screened to identify antagonists of pl25, which can increase the inhibition of or resistance to HCV infection.
  • Combinatorial peptide libraries can be constructed from genomic or cDNA libraries, or by using non-cellular synthetic methods. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis,
  • Proteins may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N 5 N 1 - dicycylohexylcarbodiimide) are known to those of skill.
  • the proteins useful in this invention may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer- Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Pat. No. 4,511,503.
  • Peptide and protein reagents can optionally labeled, as described below, or may be used in the screening assays of the present invention to ascertain their ability to modulate p 125 expression or activity.
  • p 125 polypeptides or domains can be useful in competition binding assays in methods designed to discover compounds that interact with the receptor.
  • a compound is exposed to a receptor polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide.
  • Soluble receptor polypeptide is also added to the mixture. If the test compound interacts with the soluble receptor polypeptide, it decreases the amount of complex formed or activity from the receptor target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the receptor.
  • the soluble polypeptide that competes with the target receptor region is designed to contain peptide sequences corresponding to the region of interest.
  • the compounds tested as modulators of pi 25 can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Screening combinatorial libraries of small organic molecules offers an approach to identifying useful therapeutic compounds or precursors targeted to pi 25.
  • test compounds will be small chemical molecules and peptides.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika- Biochemica Analytika (Buchs Switzerland) and the like. In one embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds).
  • Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
  • the compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • the invention provides soluble assays using molecules such as a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; pl25; or a cell or tissue expressing pl25, either naturally occurring or recombinant.
  • the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, pl25, or cell or tissue expressing pl25 is attached to a solid phase substrate.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible using the integrated systems of the invention. More recently, micro fluidic approaches to reagent manipulation have been developed, e.g., by Caliper Technologies (Palo Alto, CA).
  • the molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag.
  • the tag can be any of a variety of components.
  • a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the taste transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody that recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book /(1993).
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.,
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to persons of skill in the art.
  • poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science 249:386-390, 1990; Cwirla, et al, Proc. Natl. Acad. Sci., 87:6378-6382, 1990; Devlin et ah, Science, 49:404-406, 1990), very large libraries can be constructed (10 6 -10 8 chemical entities).
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et ah, Molecular Immunology 23:709-715, 1986; Geysen et al. J. Immunologic Method 102:259-274, 1987; and the method of Fodor et al. (Science 251:767-773, 1991) are examples.
  • Furka et al. 14th International Congress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int. J. Peptide Protein Res. 37:487-493, 1991
  • Houghton U.S. Pat. No. 4,631,211, issued December 1986
  • Rutter et al. U.S. Pat. No. 5,010,175, issued Apr. 23, 1991
  • synthetic libraries (Needels et al, Proc. Natl. Acad. Sci. USA 90:10700-4, 1993; Ohlmeyer et ah, Proc. Natl. Acad. Sci. USA 90:10922-10926, 1993; Lam et ah, International Patent Publication No. WO 92/00252; Kocis et ah, International Patent Publication No. WO 9428028) and the like can be used to screen for P 125 ligands according to the present invention.
  • the screening can be performed with recombinant cells that express pi 25, or alternatively, using purified protein, e.g., produced recombinantly, as described above.
  • purified protein e.g., produced recombinantly, as described above.
  • the ability of labeled, soluble or solubilized pi 25 that includes the ligand-binding portion of the molecule, to bind ligand can be used.
  • Radioligand binding assays allow further characterization of hits from high throughput screens as well as analogs of neurotensin agonists and antagonists.
  • membranes from cells stably expressing each neurotensin receptor subtype one point binding assays are first performed to determine how well a particular concentration, such as 25 .mu.M, of each hit or analog displaces specific [ 3 H] NT binding from the receptor. If the hit or analog displaces >50% of the [ 3 H] NT bound, a competition binding assay is performed.
  • Competition binding assays evaluate the ability of increasing concentrations of competitor (the hit or any test compound analog) to displace [ H] NT binding at each neurotensin receptor subtype.
  • the resulting Ki value indicates the relative potency of each hit or test compound for a particular receptor subtype.
  • Yet another assay for compounds that modulate or regulate pi 25 activity involves computer assisted drug design, in which a computer system is used to generate a three- dimensional structure of pi 25 based on the structural information encoded by the amino acid sequence.
  • the input amino acid sequence interacts directly and actively with a pre- established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein.
  • the models of the protein structure are then examined to identify regions of the structure that have the ability to bind, e.g., ligands. These regions are then used to identify ligands that bind to the protein.
  • the three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a pi 25 polypeptide into the computer system. Contiguous portions of SEQ ID NO: 1 , and conservatively modified versions thereof, can be used for this purpose.
  • the amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein.
  • At least 10 residues of the amino acid sequence are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM.
  • electronic storage media e.g., magnetic diskettes, tapes, cartridges, and chips
  • optical media e.g., CD ROM
  • the three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
  • the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential ligand binding regions are identified by the computer system.
  • Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of the pi 25 protein to identify ligands that bind to pi 25. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
  • the activity of pi 25 polypeptides or domains can be assessed using a variety of in vitro and in vivo assays that determine functional, physical and chemical effects, e.g., measuring ligand binding (e.g., by radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like. Furthermore, such assays can be used to test for inhibitors and activators of pi 25. Modulators can also be genetically altered versions of p 125. Such modulators are useful in the treatment and diagnosis of HCV infection.
  • the p 125 of the assay will be selected from a polypeptide having a sequence of SEQ ID NO:2, a portion of 10, 20, 30 , 40, 50 or more contiguous amino acids thereof, a domain as described above or conservatively modified variant thereof.
  • the pi 25 of the assay will be derived from a eukaryote and include an amino acid subsequence were the homology will be at least 60%, preferably at least 75%, more preferably at least 90% and most preferably between 95% and 100% that of SEQ ID NO:2.
  • the polypeptide of the assays will comprise a domain of pl25. Either pl25 or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
  • Modulators or regulators of pi 25 activity are tested using pi 25 polypeptides as described above, either recombinant or naturally occurring.
  • the protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring.
  • Samples or assays that are treated with a potential pl25 inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation.
  • Control samples (untreated with activators or inhibitors) are assigned a relative p 125 activity value of 100.
  • Inhibition of pi 25 is achieved when the pi 25 activity value relative to the control is about 90%, preferably 50%, more preferably 25-0%.
  • Activation of pi 25 is achieved when the pi 25 activity value relative to the control is 110%, preferably 150%, 200-500%, or 1000-2000%.
  • Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing pi 25.
  • polarization i.e., electrical potential
  • One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the "cell- attached” mode, the "inside-out” mode, and the "whole cell” mode (see, e.g., Ackerman et al, New Engl. J. Med. 336:1575-1595 (1997)).
  • Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al, PFlugers. ArcHCV. 391:85 (1981).
  • radiolabeled ion flux assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al, J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al, J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al, J. Membrane Biology 137:59-70 (1994)).
  • the compounds to be tested are present in the range from 1 pM to 100 niM.
  • reporter gene The practice of using a reporter gene to analyze nucleotide sequences that regulate transcription of a gene-of-interest is well documented.
  • the demonstrated utility of a reporter gene is in its ability to define domains of transcriptional regulatory elements of a gene-of- interest. Reporter genes express proteins that serve as detectable labels indicating when the control elements regulating reporter gene expression are up or down-regulated in response to outside stimuli.
  • the first is a scorable reporter gene, whose expression can be quantified, giving a proportional indication of the level of expression supported by the genetic construct comprising the reporter gene.
  • the second example is a selectable reporter gene. When expressed, the selectable reporter gene allows the host cell harboring the reporter gene to survive under restrictive conditions that would otherwise kill (or retard the growth of) the host cell. Scorable reporter genes are typically used when the relative activity of a genetic construct is sought, whereas selectable reporters are used when confirmation of the presence of the reporter expression construct within the cell is desired.
  • Firefly luciferase expression systems have become widely used for quantitative analysis of transcriptional modulation in living cells (see, e.g., Wood, K.V. (1998) Promega Notes 65: 14).
  • recombinant cells comprising this reporter construct enable libraries of small molecules to be rapidly screened for those affecting specific aspects of cellular physiology, such as receptor function or intracellular signal transduction.
  • the luciferase assay could be used to screen any of the potential reagents listed above. For example, by placing the luciferase gene under the control of the pl25 promoter, reagents that bind to the pi 25 protein can trigger a feedback loop modulating expression of the luciferase gene. Similarly, by creating a fusion protein comprising the luciferase and p 125 coding sequences, siRNAs, antisense sequences and ribozymes targeted against the pl25 gene can be screened, as any reagent acting on the pl25 transcript will necessarily disrupt expression of the luciferase enzyme encoded in the same transcript.
  • Modulators will manifest themselves by altering the amount of light emitted by the luciferase-catalyzed hydrolysis of ATP, with up-modulators increasing the amount of light emitted (they induce increased luciferase production) and down-modulators decreasing the amount of light emitted (by inhibiting luciferase production) in proportion to the degree of expressional modulation (at least within the linear range limits of the assay).
  • Luciferase assay kits and other reporter gene constructs suitable for use in the present invention are well known in the art and commercially available, e.g., Invitrogen and Promega.
  • a number of selectable marker systems can be used in the present invention, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et ah, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad.
  • adenine phosphoribosyltransferase genes can be employed in tk “ , hgprt " or aprf cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et ah, 1980, Natl. Acad. ScI USA 77:3567; O'Hare, et ah, 1981, Proc. Natl. Acad.
  • selectable markers are included in expression cassettes comprising the target gene to or construct to be incorporated into the host cell.
  • the selectable marker may be under the control of the same promoter as the target construct, e.g., as part of a fusion protein or polycistronic transcript; or may be under the control of an independent promoter.
  • the purpose of the selectable marker is to confer selectable growth characteristics on cells that are able to express it.
  • the selectable marker By including the selectable marker in the same nucleic acid comprising the target gene or construct, the selectable marker will be included in any cell transformed with the target. Therefore, by selecting for the growth characteristics conferred by the selectable marker, cells transfected with the target can be selected.
  • detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al, Science 241:1077-1080 (1988); and Nakazawa et al, PNAS 91:360- 364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al, Nucleic Acids Res. 23:675-682 (1995)).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.
  • nucleic acid e.g., genomic, mRNA or both
  • Real-time PCR assays take advantage of those cycles of a normal PCR reaction where the DNA being amplified is increasing at a logrythmic rate and hence proportional to the amount of DNA present.
  • kits are commercially available for performing real ⁇ time PCR.
  • One such kit is the TaqMan assay.
  • the TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.
  • TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time. In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations.
  • Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acid molecules in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore.
  • a conformational reorganization that restores the fluorescence of an internally quenched fluorophore.
  • Northern blot methods allow RNA isolated from cells of interest to be separated using gel electrophoresis techniques. After separation, nucleic acids are transferred to membranes and hybridized with radio-labeled nucleotide probes. For analysis of expression maps, poly A (adenylyl) probed are used, which hybridize to mRNA species present on the blot.
  • the present invention includes both traditional and expression map Northern blotting. Expression of the pi 25 gene can be tracked using probes specific for this gene. Expression mapping can be used to monitor alterations in gene expression in response to pl25-specific binding agents.
  • RNA isolation Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
  • Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.
  • High density oligonucleotide arrays are particularly useful for monitoring expression control at the transcriptional, RNA processing and degradation level.
  • the fabrication and application of high density arrays in gene expression monitoring have been disclosed previously in, for example, WO 97/10365, WO 92/10588, U.S. Pat. No. 6,040,138 incorporated herein for all purposes by reference.
  • high density oligonucleotide arrays are synthesized using methods such as the Very Large Scale Immobilized Polymer Synthesis (VLSIPS) disclosed in U.S. Pat. No. 5,445,934.
  • VLSIPS Very Large Scale Immobilized Polymer Synthesis
  • Each oligonucleotide occupies a known location on a substrate.
  • a nucleic acid target sample is hybridized with a high density array of oligonucleotides and then the amount of target nucleic acids hybridized to each probe in the array is quantified.
  • One preferred quantifying method is to use confocal microscope and fluorescent labels.
  • the GeneChip.RTM. system (Affymetrix, Santa Clara, Calif.) is particularly suitable for quantifying the hybridization; however, it will be apparent to those of skill in the art that any similar systems or other effectively equivalent detection methods can also be used.
  • High density arrays are suitable for quantifying a small variations in expression levels of a gene in the presence of a large population of heterogeneous nucleic acids.
  • Such high density arrays can be fabricated either by de novo synthesis on a substrate or by spotting or transporting nucleic acid sequences onto specific locations of substrate.
  • Nucleic acids are purified and/or isolated from biological materials, such as a bacterial plasmid containing a cloned segment of sequence of interest. Suitable nucleic acids are also produced by amplification of templates.
  • polymerase chain reaction, and/or in vitro transcription are suitable nucleic acid amplification methods. Synthesized oligonucleotide arrays are particularly preferred for this invention. Oligonucleotide arrays have numerous advantages, as opposed to other methods, such as efficiency of production, reduced intra- and inter array variability, increased information content and high signal-to-noise ratio.
  • an “antisense compound or molecule” refers to such compound or molecule that includes a polynucleotide that is complementary to a target sequence of choice and capable of specifically hybridizing with the target molecules.
  • the term antisense includes a "ribozyme,” which is a catalytic RNA molecule that cleaves a target RNA through ribonuclease activity.
  • Antisense nucleic acids hybridize to a target polynucleotide and interfere with the transcription, processing, translation or other activity of the target polynucleotide.
  • An antisense nucleic acid can inhibit DNA replication or DNA transcription by, for example, interfering with the attachment of DNA or RNA polymerase to the promoter by binding to a transcriptional initiation site or a template.
  • RNA transcript can interfere with processing of niRNA, poly (A) addition to mRNA or translation of mRNA by, for example, binding to regions of the RNA transcript such as the ribosome binding site. It can promote inhibitory mechanisms of the cells, such as promoting RNA degradation via RNase action.
  • the inhibitory polynucleotide can bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Methods of inhibition using antisense polynucleotides therefore encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms (see, e.g., Helene & Toulme, Biochim. Biophys. Acta., 1049:99-125 (1990)).
  • the antisense compounds that may be used in connection with this embodiment of the present invention preferably comprise between about 8 to about 30 nucleobases (i.e., from about 8 to about 30 linked nucleosides), more preferably from about 12 to about 25 nucleobases, and may be linear or circular in configuration. They may include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Methods of preparing antisense compounds are well known in the art (see, for example, U.S. Patent No
  • the present invention also provides a method of increasing inhibition of or resistance to HCV infection in a cell, the method comprising introducing into the cell an effective amount of an expression vector comprising a sequence of nucleotides that encodes a ribozyme binding to a target sequence of SEQ ID NOS: 1 or 3, such as those disclosed herein.
  • the expression vector is preferably administered in combination with a suitable carrier. After the vector has been administered, the ribozyme is expressed in the cell.
  • This method can be applied to a subject with HCV infection.
  • Administration of the vector into the subject can be by any suitable route including oral, sublingual intravenous, subcutaneous, transcutaneous, intramuscular, intracutaneous, and the like.
  • Any of a variety of non-toxic, pharmaceutically acceptable carriers can be used for formulation including, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, dextrans, and the like.
  • the formulated material may take any of various forms such as injectable solutions, sterile aqueous or non- aqueous solutions, suspensions or emulsions, tablets, capsules, and the like.
  • the phrase "effective amount” refers to a dose of the deliverable sufficient to provide circulating concentrations high enough to impart a beneficial effect on the recipient, which is an increase of inhibition of or resistance to HCV infection.
  • the specific therapeutically effective dose level for any particular subject and deliverable depends upon a variety of factors including the severity of the infection, the activity of the specific compound administered, the route of administration, the rate of clearance of the specific compound, the duration of treatment, the drugs used in combination or coincident with the specific compound, the age, body weight, sex, diet and general health of the patient, and like factors well known in the medical arts and sciences. Dosage levels typically range from about 0.001 up to 100 mg/kg/day; with levels in the range of about 0.05 up to 10 mg/kg/day.
  • the present invention also provides an antibody with binding specificity for a protein that is involved in HCV infection, such as SEQ ID NO:2, or any molecule with 80%, 85%, 90% or 95% or more sequence identity with SEQ ID NO:2, or any fragment of 10 or more contiguous amino acids of SEQ ID NO:2.
  • the antibody can have a binding specificity for a protein or peptide (i.e., amino acid sequence) encoded by SEQ ID NO:1, or any molecule with 80%, 85%, 90% or 95% or more sequence identity with SEQ ID NO:1.
  • the term "antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • Igy antibody generated against a sequence in the N-terminus region of SEQ ID NO:2, as described below.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2, a dimer of Fab which itself is a light chain joined to VH-C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)).
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • the term antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990)). Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art.
  • transgenic mice or other organisms such as other mammals, may be used to express humanized antibodies.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens ⁇ see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
  • a number of pi 25 comprising immunogens may be used to produce antibodies specifically reactive with pi 25.
  • recombinant pi 25 or an antigenic fragment thereof can be isolated, as is known in the art.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
  • Recombinant protein is one embodiment of an immunogen for the production of monoclonal or polyclonal antibodies.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Naturally occurring protein may also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies.
  • Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
  • Methods of production of polyclonal antibodies are known to those of skill in the art.
  • An inbred strain of mice e.g., BALB/C mice
  • rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to pi 25.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow & Lane, supra).
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, Science 246:1275-1281 (1989).
  • Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • an immunoassay for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non- pi 25 proteins or even other related proteins from other organisms, using a competitive binding immunoassay.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a K d of at least about 0.1 mM, more usually at least about 1 _M, optionally at least about 0.1 _M or better, and optionally 0.01 _M or better.
  • P 125 can be detected by a variety of immunoassay methods.
  • immunoassay methods see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991).
  • the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed. 3 1980); and Harlow & Lane, supra.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include
  • the antibodies are also useful for inhibiting receptor function, for example, blocking ligand binding. These uses can also be applied in a therapeutic context in which treatment involves inhibiting receptor function.
  • An antibody can be used, for example, to block ligand binding.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact receptor associated with a cell.
  • binding specificity in relationship to an antibody that binds to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified antibody binds to a particular protein and does not bind significantly to other proteins present in the sample.
  • Example 1 shows that a ribozyme of the invention (RzHCV4) specifically inhibits HCV IRES mediated translation.
  • RzHCV4 a ribozyme of the invention specifically inhibits HCV IRES mediated translation.
  • a selection system was established to select ribozymes targeting cellular factors required for HCV IRES-mediated translation. Kruger et al, MoI Cell Biol, 21:8357-64 (2001); Lu and Wimmer, Proc Natl Acad Sci USA, 93:1412-7 (1996).
  • HeLa5'tk an HCV- IRES reporter cell line was used in this selection assay.
  • HeLa5'tk contains a bicistronic reporter gene which encodes hygromycin B phosphotransferase and HSV-tk. Expressed from this reporter gene, the translation of hygromycin B phosphotransferase is cap- dependent while the HCV IRES mediates the translation of HSV-tk protein.
  • ribozymes targeting cellular factors required for the HCV IRES activity were selected by ganciclovir (GCV).
  • GCV ganciclovir
  • RzHC V4 is one of the ribozymes selected by this assay, and targeted positions 3364 to 3379 of SEQ ID NO:1.
  • this ribozyme was cloned into pLPR and reintroduced into HeLa 5 'tk cells by transfection.
  • the regenerated RzHCV4 expressing HeLa5'tk cells was then challenged by Pv/HCV.
  • Pv/HCV is a poliovirus with its IRES replaced by the HCV IRES while the rest of the viral genome remains unchanged.
  • HeLa 5'tk-RzHCV4 cells were challenged by wild-type poliovirus and assayed for plaque formation.
  • HeLa 5'tk-RzHCV4 cells developed the same number of plaques to HeLa 5'tk- BRl and HeLa 5'tk cells.
  • the ribozyme expressing HeLa Hl cells were also infected with human rhinovirus (HRV) and showed no inhibition effect.
  • HRV human rhinovirus
  • Example 2 This example shows that ribozymes of the invention target pi 25 (SEQ ID NO:1).
  • RzHCV4 targets a human EST-BG392542 TSEO ID NO:3)
  • the RNA binding sequence of RzHCV4 was used to identify the target RNA for RzHCV4.
  • a hairpin ribozyme substrate RNA binding sequence contains 6 ⁇ 8 nt in the helix 1, 4 nt in the RNA cleavage site (NGTC) and 4 nt in the helix 2.
  • NGTC RNA cleavage site
  • BG392542 SEQ ID NO:3 was identified as one of the potential targets for RzHC V4, with 4 perfect matches at helix 1 and 7 perfect matches at helix 2.
  • BG392542 (SEQ ID NO:3) is the target for RzHCV4
  • three additional ribozymes were designed.
  • CV4-VR1 and CV4-VR3 target additional sites (SEQ ID NOS: 4 and 5) of BG392542 (SEQ ID NO:3), while CV4-VR2 targets to the same site as RzHCV4 with a 16/16 match to the RNA binding site (SEQ ID NO:6).
  • These validation ribozymes were cloned into pLPR and the stable cell lines expressing these validation ribozymes were generated by transfecting HeLa5'tk cells with these pLPRs followed by puromycin selection.
  • BG392542 (SEQ ID NO:3) is clustered to Hs.300208 in NCBFs UniGene database.
  • Hs.300208 contains mRNA and EST sequences for pl25. Based on two sequences in
  • pl25 is the cellular target for RzHCV4 and the validation ribozymes.
  • Example 3 shows that the ribozymes of the invention (RzHC V4 and the validation ribozymes) also down-regulate the expression of pi 25 protein (SEQ ID NO:2).
  • a direct consequence of down regulating mRNA is the reduced expression of the protein.
  • pl25 protein SEQ ID NO:2
  • a chicken polyclonal IgY was generated against a peptide sequence located at the N- terminus of pl25.
  • Cell lysates from HeLa5'tk cells expressing the ribozymes were analyzed by Western Blot using these anti-pl25 IgY.
  • RzHCV4 and the validation ribozyme expressing HeLa5'tk cells showed noticeably reduced pl25 expression when compared to the BRl expressing HeLa5'tk cell and the parental HeLa5'tk.
  • CV4-2 and CV4-3 HeLa5'tk cells showed almost 80% down regulation of p 125 protein expression. These results further confirmed pi 25 mRNA as the target of RzHCV4 and the other ribozymes of the invention. It also showed the inhibition effect observed from these cells after Pv/HCV infection was due to the reduced pi 25 expression.
  • Example 4 This example shows that over-expressed pi 25 promotes Pv/HCV replication in HeLa cells.
  • a pi 25 expression plasmid vector was constructed and was stably transfected into HeLa and HeLa5'tk cells. These cells were then challenged by Pv/HCV and analyzed by plaque assay. Both HeLa and HeLa5'tk cells with over- expressed pi 25 developed bigger plaques compared to the respective parental cells, indicating that pi 25 is not only required by HCV IRES, it is also a determinative factor for the Pv/HCV replication efficiency.

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Abstract

L'invention concerne des procédés d'identification d'agents accroissant l'inhibition d'une infection virale ou la résistance à celle-ci, plus précisément à une infection au HCV (virus de l'hépatite C). L'invention concerne également des procédés et des composants accroissant une telle inhibition ou résistance.
PCT/US2005/012648 2004-04-22 2005-04-15 Procedes d'identification et d'utilisation d'agents accroissant l'inhibition de l'infection au hcf ou la resistance a celle-ci WO2006019429A2 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BHOPALE ET AL.: 'Emerging drugs for chronic hepatitis C' HEPATOLOGY RESEARCH vol. 32, no. 3, July 2005, pages 146 - 153, XP004999344 *
KAPADIA ET AL.: 'Interference of hepatitis C virus RNA replication by short interfering RNAs' PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA vol. 100, no. 4, February 2003, pages 2014 - 2018, XP002251050 *
TANG ET AL.: 'Novel technologies for studying virus-host interaction and discovering new drug targets for HCV and HIV' CURRENT OPINION IN PHARMACOLOGY vol. 12, no. 5, October 2002, pages 541 - 547, XP003002407 *
TANI ET AL.: 'p125 is a novel mammalian Sec23p-interacting protein with structural similarity to phospholipid-modifying proteins' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 274, no. 29, July 1999, pages 20505 - 20512, XP002211138 *

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