METHODS OF INHIBITING HCV REPLICATION

FIELD OF THE INVENTION

The present invention is directed to methods of inhibiting replication of hepatitis C virus (HCV). The invention is also directed to methods of identifying agents that inhibit HCV replication, and to compounds that inhibit such replication.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a positive stranded RNA virus that following entry into the cell replicates in the cytoplasm. The virus uses viral non-structural and cellular proteins for translation, processing, and RNA amplification. HCV replication is localized to ER/golgi membranes. Viruses such as HCV depend on cellular proteins to complement the viral proteins in translation and RNA amplification.

HCV infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to the development of hepatocellular carcinoma. It is the major etio logic agent of non-A, non-B hepatitis. It is estimated that 170 million people are chronically infected with HCV worldwide, with an additional 3-4 million people newly infected each year.

Several HCV genotypes are known. Genotypes Ia and Ib are the most prevalent in Western Europe and the US, each responsible for about 30-40% of the cases in the US population. The distribution between the different genotypes varies among different countries. In China, for example, genotype Ib is more prevalent than any other, while in Africa genotype 5a is the most prevalent.

The genetic variation is spread over the entire genome. This variation spread applies to all genotypes. However, there are regions with a lower level of variation, for example the 5'NTR region, the capsid protein region and the catalytic domains of the enzymes.

Currently, the standard therapy for HCV infection has been the use of interferon (IFN) alone or in combination with ribavirin. Di Bisceglie and Hoofnagle, Hepatology, 36:S 121-7 (2002). However, the sustained response rate is suboptimal and highly variable. He and Katze, Viral Immunol. 15:95-119 (2002).

Accordingly, alternative therapies as well as new target sites for testing drug candidates are greatly needed. The present invention satisfies this need and provides additional advantages as well.

SUMMARY OF THE INVENTION The present invention centers on the discovery that inhibiting the expression and/or enzymatic activity of phosphatidylinositol 4-kinase catalytic subunit alpha (PIK4CA), whose various nucleic acid and amino acid sequences are described below, inhibits HCV replication. Accordingly, the present invention provides methods for inhibiting HCV replication by inhibiting and/or down-modulating the expression and/or catalytic activity of PIK4CA.

The invention further provides methods of identifying an agent useful for inhibiting replication of HCV. The methods include introducing an agent into cells and measuring the expression level and/or level of enzymatic activity of PIK4CA both before and after introduction of the agent. An agent that causes a reduction in PIK4CA expression and/or enzymatic activity is useful for inhibiting HCV replication.

The present invention also provides agents useful for inhibiting replication of HCV. Included within the scope of the invention are agents that reduce the expression and/or enzymatic activity of PIK4CA, such as siRNAs, ribozymes, antisense oligonucleotides, monoclonal and polyclonal antibodies, and small organic molecules. The present invention further provides methods for conferring resistance to

HCV infection by inhibiting replication of HCV. The methods include introducing an agent into a cell that reduces the expression level and/or enzymatic activity of PIK4CA. The agent can be, for example, an siRNA, ribozyme, an antisense oligonucleotide, a monoclonal and polyclonal antibody or a small organic molecule. The present invention further provides methods for identifying agents that can confer resistance to HCV infection. The methods include introducing an agent into a cell infected with HCV and measuring the expression level and/or level of enzymatic activity of PIK4CA and/or the level of HCV RNA or HCV protein both before and after introduction of the agent. A reduction in level indicates that the agent is useful for conferring resistance to HCV infection. Representative agents include antisense oligonucleotides, monoclonal and polyclonal antibodies, and small organic molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of HCV NS5A based ELISA for the siRNAs on plate 9 of the Ambion Kinase library. Each target in the array is represented by 3 individual siRNAs. All siRNAs were reverse trans fected into Huh7 derived HCV Ib replicon cells #8- Ik. Five days post transfection a HCV NS5A based in situ ELISA was used to quantitate the level of HCV replication. The relative replication efficiency (RRE) is calculated relative to the untransfected controls. Positive control siRNAs target the NS3 and IRES regions of the replicon while a CD81 specific siRNA was included as a negative control. The location of the siRNAs in the Ambion kinase library are indicated at the x-axis. Figure 2 shows the validation of siRNAs at 20 and 200 nM concentrations.

The indicated siRNAs were reverse transfected into #8-lk replicon cells at 20 or 200 nM final concentrations. Five days post transfection the cells were assayed for HCV replication by in situ ELISA (Figure 2A) and for toxicity by Alamarblue assay (Figure 2B). The plate and location of the siRNAs is located on the x-axis. Control siRNA targeting HCV NS3 was included in the assay as were interferon controls.

Figure 3 shows the confirmation of inhibition with HCV genotypes Ia and Ib. Inhibition of HCV replicons is shown in Figure 3A while toxicity is evaluated in Figure 3B. The location in the Ambion kinase library is listed on the x-axis.

Figure 4 shows the dose response between PIK4CA siRNA and HCV NS5A levels. HCV Ia or Ib replicon cell lines were transfected with a range of PIK4CA siRNA concentrations and assayed for HCV NS5A levels by NS5A ELISA. Each of the three siRNAs showed a dose dependent inhibition of HCV NS5A levels.

Figure 5 shows the results of a colony formation inhibition assay of HCV replication using Huh 7 cells stably transduced with lentiviruses expressing PIK4CA specific shRNAs.

DETAILED DESCRIPTION OF INVENTION

While it was known that PIK4CA binds to HCV NS5A (Ahn et al., Ahn et al, J. Biochem. and MoL Biol., 37:741-48 (2004)), no biological relevance for the observed interaction was found prior to the present invention. Indeed, many proteins have been found

to bind to individual HCV proteins. However, the biological function of most of these proteins has not been elucidated.

The present invention centers on the discovery that reducing the expression and/or enzymatic activity of PIK4CA inhibits HCV replication. Accordingly, the present invention provides methods of inhibiting HCV replication by inhibiting the expression and/or catalytic activity of PIK4CA. The invention further provides methods for identifying agents useful for inhibiting replication of HCV. The methods include introducing agents into cells and measuring the expression level and/or level of enzymatic activity of PIK4CA both before and after introduction of the agents. An agent that causes a reduction in PIK4CA expression and/or enzymatic activity is useful for inhibiting HCV replication.

The present invention also provides agents useful for inhibiting replication of HCV. Included within the scope of the invention are agents that reduce the expression of and/or the enzymatic activity of PIK4CA. Such agents include siRNAs, ribozymes, antisense oligonucleotides, monoclonal and polyclonal antibodies and small organic molecules. The present invention further provides methods for conferring or increasing resistance to HCV infection by inhibiting replication of HCV in cells. The methods include introducing an agent into the call that reduces the expression level and/or enzymatic activity of PIK4CA. The agent can be, for example, an siRNA, ribozyme, an antisense oligonucleotide, a monoclonal and polyclonal antibody or a small organic molecule. The present invention further provides methods for identifying an agent that confers or increases resistance to HCV infection. The methods include introducing agents into cells infected with HCV and measuring the expression level and/or level of enzymatic activity of PIK4CA both before and after introduction of each agent. A reduction in the expression level and/or level of enzymatic activity of PIK4CA indicates that the agent is useful for conferring or increasing resistance to HCV infection. Representative agents include antisense oligonucleotides, monoclonal and polyclonal antibodies, and small organic molecules.

Agents of the invention useful for inhibiting HCV replication include siRNAs. Examples of such siRNAs include the following three pairs: A) GGUUUAAGAACACAGAAGCtt (SEQ ID NO :26) and GCUUCUGUGUUCUUAAACCtg (SEQ ID NO:27);

B) GGCAUGUCUAAGAAAACCAtt (SEQ ID NO:28) and UGGUUUUCUUAGAC AUGCCtg (SEQ ID NO:29); and

C) GGCUGGAUC AAC AC AUACCtt (SEQ ID NO: 30) and GGUAUGUGUUGAUCCAGCCtt (SEQ ID NO:31). See Examples. Included within the scope of the invention are expression vectors encoding such agents and cells containing the vectors encoding such agents.

"PIK4CA" as used herein includes the amino acid sequences SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, 16 or 18; and the nucleic acid sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, 22, 23, 24 or 25. Exemplary amino acids sequences are variants 1 or 2 (SEQ ID NOS :2 or 4). Exemplary nucleic acids sequences are variants 1 or 2 (SEQ ID NOS:l or 3).

As used herein, the term "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. As used herein, the phrase "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. In reference to an siRNA or ribozyme, unless otherwise indicated, the explicitly specified encoding nucleotide sequence also implicitly covers sequences that do not materially effect the specificity of the ribozyme or siRNA for its target nucleic acid. In reference to a protein or peptide, unless otherwise indicated, the explicitly specified encoding nucleotide sequence also implicitly encompasses variations in the base sequence encoding the same amino acid sequence (e.g., degenerate codon substitutions). The invention also contemplates proteins or peptides with conservative amino acid substitutions. The identity of amino acids that may be conservatively substituted is well known to those of skill in the art. Degenerate codons of the native sequence or sequences may be chosen to conform with codon preference in a specific host cell.

The terms "sequence similarity", "sequence identity", or "percent identity," 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 complement of a test sequence. Preferably, 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. These relationships hold, notwithstanding evolutionary origin (Reeck et ah, Cell, 50:667 (1987)). When the sequence identity of a pair of polynucleotides or polypeptides is greater or equal to 65%, the sequences are said to be "substantially identical."

Alternatively, substantial identity will exist when a nucleic acid will hybridize under selective hybridization conditions, to a strand or its complement. Typically, 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, as described, 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.

In relation to proteins, the term "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 al, Cell, 50:667 (1987)). The present invention naturally contemplates homologues of the PIK4CA protein, and polynucleotides encoding the same, as falling within the scope of the invention. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, 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. The 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. MoL Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics

Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. MoI. 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. Using 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).

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which 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 (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for

initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. 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). For amino acid sequences, 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. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

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. For example, 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. An indication that two 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. Thus, 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.

It should be understood that, when referring to PIK4CA, each method of the invention described herein encompasses: a) all amino acid sequences having about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with PIK4CA; and b) all nucleic acid sequences having about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with PIK4CA.

The term "moderately stringent conditions," as used herein, 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 50 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 ⁰ C. Other suitable moderately stringent and highly stringent conditions are known in the art and described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992), and Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore MD (1998). In general, 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.

As used herein, the term "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. For example, 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. Likewise, 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, Werner L M. (1996) Current Opinion in Oncology. 8:493-498, Marasco Wash. (1997) Gene Therapy. 4: 11-15, Rondon l 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. Using this technique, intracellular signals and enzyme activities can be inhibited, or their transport to cellular compartments prevented. Marasco, W. A., et ai, Proc. Natl. Acad. Sci. USA 90:7889-7893 (1993). In particular, intrabodies directed against PIK4CA will bind to the nascent PIK4CA protein and direct it to the ubiquitin pathway for catalytic degradation, rather than to the cellular membrane. Thus intrabodies provide yet another approach to down regulating PIK4CA 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.

As used herein, 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. Typically, the recombinant expression cassette portion of an expression vector includes the encoding nucleotide sequence to be transcribed (e.g. a siRNA), operably linked to a promoter, or other regulatory sequence by a functional linkage in cis. In accordance with the present invention, an expression vector comprising a

nucleotide sequence encoding siRNAs of the invention can be used to transduce cells suitable as hosts for the vector. Both procaryotic cells including bacterial cells such as E. coli and eukaryotic cells including mammalian cells may be used for this purpose.

As used herein, the term "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 (e.g., an origin of replication), and/or chromosome integration elements such as retroviral long terminal repeats ("LTRs"), or adeno associated viral (AAV) inverted terminal repeats ("ITRs"), may be incorporated into an expression vector encoding ribozymes of the present invention as described in WO 00/05415 to Barber et al.

As used herein, 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. Although the term may be used to refer to the transcription of a ribozyme, a ribozyme typically is not translated into a protein since it functions as an active (catalytic) nucleic acid. As used herein, the term "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.

As used herein, 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. In eukaryotic cells, 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.

The term "transfection," as used herein, means the genetic modification of a cell by uptake of an exogenous nucleic acid molecule (e.g., by means of an expression vector).

As used herein, "small interfering RNAs" (siRNA) are short double-stranded RNA fragments that elicit a process known as "RNA interference" (RNAi), a form of sequence-specific gene silencing. 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). The siRNAs guide RISC to homologous mRNAs, thus targeting them for destruction. Hammond et al., Nature Genetics Reviews 2: 110-119(2000). RNAi has been observed in a variety of organisms including plants, insects and mammals.

An "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. Functionally, the characteristic distinguishing an siRNA over other forms of dsRNA is that the 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. Examples of such dsRNA siRNA are SEQ ID NOS:26/27, 28/29 and 30/31, as described above. 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. A proposed mechanism for 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). The development of efficient methods for screening effective siRNAs offers a means for identifying the functional characteristics of genes targeted by such siRNAs, through a process of subtractive phenotypic analysis. Thus by creating and expressing in a cell infected or

challenged with HCV, siRNAs specific for sequences found in PIK4CA mRNA, its expression can be down regulated. siRNAs for use in the present invention can be produced from a PIK4CA 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 PIK4CA 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 PIK4CA can be constructed by, for example, mechanically shearing PIK4CA cDNA, ligating the resulting fragments into suitable vector constructs and transfecting a suitable host cell with the vectors. For a review of RNAi and siRNA expression, see Hammond, Scott M. et al, Nature Genetics Reviews, 2: 110-119; Fire, Andrew (1999) TIG, 15(9):358-363; Bass, Brenda L. (2000) Cell, 101:235-238.

The targeting of antisense oligonucleotides to mRNA is another mechanism of decreasing protein synthesis, and, consequently, represents a powerful and targeted approach to diminishing PIK4CA expression. For example, the synthesis of polygalacturonase and the muscarine type 2 acetylcholine receptor are each 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). Further, examples of 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 al, 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. 5,747,470; 5,591,317 and 5,783,683, each specifically incorporated herein by reference in its entirety).

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. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, 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 a PIK4CA nucleic acid sequence, as described above. Selection of 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. These secondary structure analyses and target site selection considerations were performed using v.4 of the OLIGO primer analysis software (Rychlik, 1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997). "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. With regard to the present invention, the term "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. Thus, 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 PIK4CA nucleic acid molecule is cloned into a vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, 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 PIK4CA or synthesized synthetically. Transfection of suitable host cells with PIK4CA 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.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

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.

Accordingly, the 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.

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.

Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. {Ann. N. Y. Acad. Sci. 663:48-62 (1992)).

As is also well known, polypeptides are not always entirely linear. For instance, 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. For recombinant polypeptides, for example, 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 PIK4CA or portions thereof, 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-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, 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.2, Fab expression library fragments, and epitope- binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries). As used herein, the term "down-modulation" or "down-regulation" means a decrease in the rate or level of mRNA production, or a decrease in the rate or level of protein expression, or a decrease in the level of enzymatic activity.

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, an siRNA, a ribozyme, an antisense molecule, an antagonizing antibody or small organic molecule. This method can comprise transducing the infected cell with an expression vector

encoding an appropriate agent. Alternatively, the agent can be introduced into a cell directly, i.e., without using a vector.

The method of the invention can be accomplished by the agent binding to PIK4CA, or portions thereof. The method of the invention can also be accomplished by the agent down-modulating PIK4CA, or portions thereof, or down-regulating PIK4CA, or portions thereof.

The present invention also provides methods of identifying agents that confer or increase resistance to HCV infection in cells infected with HCV by inhibiting HCV replication. Such methods include introducing an agent that binds to PIK4CA, 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, where a decrease in the level indicates that the agent increases resistance to HCV infection. If the cell is not infected with HCV, the agent can be introduced into the cell with HCV introduced thereafter. The cell can then be measured for the level of viral protein, 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 level of HCV replication can be measured, for example, by an ELISA assay or PCR. See Examples 1 and 2.

In addition to the assays described in the examples below, the level of HCV infection (or resistance to or inhibition of HCV infection) can be determined using the cell- based replicon system as described by Bartenschlager, R., Nat Rev Drug Discov, 1 :911-16 (2002).

Moreover, 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 al, 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 al., Chembiochem, 3:766-771 (2002); and Richman et al., Curr. Prot. Immun., pp. 1-21 (Wiley & Sons 1993). Dose-dependent activity can also be determined by measuring the decrease in HCV protein expression using ELISA. See Example 5; and Luedtke et al. and Richman et al., supra.

In accordance with another embodiment of the present invention, reducing the level of PIK4CA protein in the cell can be accomplished by contacting the cell with a peptide that is an antagonist of PIK4CA.

Combinatorial peptide libraries or nucleic acid based aptamer libraries can be screened to identify antagonists of PIK4CA, 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 Merrifϊeld, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield, et al, J. Am. Chem. Soc. 85: 2149-2156 (1963), and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, III. (1984). 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,N'-dicycylohexylcarbodiimide) are known to those of skill in the art. The PIK4CA proteins useful in this invention, or portions or domains thereof, 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 be labeled, as described below, or may be used in the screening assays of the present invention to ascertain their ability to modulate PIK4CA expression or activity.

PIK4CA polypeptides or domains can be useful in competition binding assays in methods designed to discover compounds that interact with PIK4CA polypeptides. Thus, a compound is exposed to a PIK4CA polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. A soluble PIK4CA polypeptide is also added to the mixture. If the test compound interacts with the soluble PIK4CA polypeptide, it decreases the amount of complex formed or activity from the PIK4CA target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific

regions of PIK4CA. Thus, the soluble polypeptide that competes with the target PIK4CA region is designed to contain peptide sequences corresponding to the region of interest.

The compounds tested as modulators of PIK4CA 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 PIK4CA. Typically, test compounds will be small chemical molecules and peptides. Essentially 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. For example, 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.

To perform cell-free drug screening assays, it is desirable to immobilize either the PIK4CA protein, or fragment, or its target molecule to facilitate separation of complexes

from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Using high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, 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. 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, microfluidic 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. In general, 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.

A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate 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).

Similarly, 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. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody that recognizes the first antibody. In addition to antibody-antigen interactions, PIK4CA-ligand interactions are also appropriate as tag and tag-binder pairs. Similarly, toxins and venoms, viral epitopes, 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), drugs, lectins, sugars, nucleic

acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

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.

Common 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. Such flexible linkers are known to persons of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or hetero functional 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. For example, 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., Merrifϊeld, J. Am. Chem. Soc. 85:2149- 2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al, J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al, Science, 251:767-777 (1991); Sheldon et al, Clinical

Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). 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. ScL, 87:6378-6382, 1990; Devlin et al, 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 al., 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) and Rutter et al. (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al, Proc. Natl. Acad. Sci. USA 90: 10700-4, 1993; Ohlmeyer et al, Proc. Natl. Acad. Sci. USA 90:10922-10926, 1993; Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 9428028) and the like can be used to screen for PIK4CA ligands according to the present invention.

The screening can be performed with recombinant cells that express PIK4CA, or alternatively, using purified protein, e.g., produced recombinantly, as described above. For example, the ability of labeled, soluble or solubilized PIK4CA that includes the ligand- binding portion of the molecule, to bind ligand can be used.

Yet another assay for compounds that modulate or regulate PIK4CA activity involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of PIK4CA 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 PIK4CA 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 (or a nucleotide sequence encoding 10 amino acids) 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. 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. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.

Once the structure has been generated, 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 PIK4CA protein to identify ligands that bind to PIK4CA. 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 PIK4CA 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 PIK4CA. Modulators can also be genetically altered versions of PIK4CA. Such modulators are useful in the treatment and diagnosis of HCV infection.

The PIK4CA of the assay will be selected from a polypeptide having a sequence of SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, 16 or 18, a portion of 10, 20, 30 , 40, 50 or more contiguous amino acids thereof, a domain as described above or conservatively modified variant thereof. Alternatively, the PIK4CA 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 NOS:2, 4, 6, 8, 10, 12, 16 or 18. Preferably, the polypeptide of the assays will comprise a domain of PIK4CA. Either PIK4CA 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 PIK4CA activity are tested using PIK4CA 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 PIK4CA 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 PIK4CA activity value of 100. Inhibition of PIK4CA is achieved when the PIK4CA activity value relative to the control is about 90%, preferably 50%, more preferably 25-0%. Activation of PIK4CA is achieved when the PIK4CA 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 PIK4CA. 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). Other known 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)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.

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.

By way of example, two types of reporter gene assay are discussed below. 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). In particular, 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. deWet et al. (1987) MoI. Cell. Biol. 7:725; Wood, K.V. (1991) In: Bioluminescence and Chemiluminescence: Current Status, eds. P. Stanley and L. Kricka, John Wiley and Sons, Chichester, 11.

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 PIK4CA promoter, reagents that bind to the PIK4CA protein can trigger a feedback loop modulating expression of the luciferase gene. Similarly, by creating a fusion protein comprising the luciferase and PIK4CA coding sequences, siRNAs, antisense sequences and ribozymes targeted against the PIK4CA gene can be screened, as any reagent acting on the PIK4CA 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. See, e.g., Steady- GIoTM Luciferase Assay Reagent Technical Manual Luciferase Assay Reagent Technical Manual #TM051, Promega Corporation.

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 ai, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,

1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al, 1980, Cell 22:817) genes can be employed in tk ^" , hgprt ^" or aprt ^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, 1980, Natl. Acad. ScL USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. MoI. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes. Typically, selectable markers are included in expression cassettes comprising the target gene 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.

As suggested above, the purpose of the selectable marker is to confer selectable growth characteristics on cells that are able to express it. 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.

In certain embodiments, 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)). 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.

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. Several 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. (See, e.g., Heid, C.A., Stevens, J., Livak, KJ. and Williams, P.M. Real time quantitative PCR. Genome Res. 6:986-994 (1996); Gibson, U.E.M., Heid, CA. and Williams, P.M. A novel method for real time quantitative RT-PCR. Genome Res. 6:995-1001 (1996)).

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 PIK4CA gene can be tracked using probes specific for this gene. Expression mapping can be used to monitor alterations in gene expression in response to PIK4CA- specific binding agents. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et ah,

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. Through the use of high density oligonucleotide arrays, expression profiles for individual cells can be rapidly obtained and compared. High density 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. In some embodiments using high density arrays, 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. 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. As a nonlimiting illustration, 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. It can interfere with processing of mRNA, 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, phosphorodiamidate morpholino oligomers (PMO), 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'. Also included are derivatives of the above having moieties linked to them for facilitating delivery. Methods of preparing antisense compounds are well known in the art (see, for example, U.S. Patent No. 6,210,892).

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 or siRNA binding to a target sequence of PIK4CA, as disclosed herein. The expression vector is preferably administered in combination with a suitable carrier. After the vector has been administered, the ribozyme or siRNA 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.

As used herein, 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 or replication, such as PIK4CA (SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, 16 or 18), or any molecule with 80%, 85%, 90% or 95% or more sequence identity with PIK4CA, or any fragment of 10 or more contiguous amino acids of PIK4CA. The antibody can have a binding specificity for a protein or peptide (i.e., amino acid sequence) encoded by PIK4CA (SEQ ID NOS: l, 3, 5, 7, 9, 11, 13, 15 or 17), or any molecule with 80%, 85%, 90% or 95% or more sequence identity with SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16 or 18.

As used herein, 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. Also encompassed is the Igy antibody generated against a sequence in the N-terminus region of a PIK4CA protein, 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 (V _L ) 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. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab) ⁵ 2, a dimer of Fab which itself is a light chain joined to V _H -C _H I by a disulfide bond. The F(ab) ⁵ 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)). While various 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.

Thus, the term antibody, as used herein, 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 ah, Nature 348:552-554 (1990)).

Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art. See, e.g. , Coligan ( 1991 ) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA; Huse et al. (1989) Science 246:1275-1281; and Ward et al. (1989) Nature 341 :544-546, and references cited therein. Techniques for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce antibodies to polypeptides of this invention.

Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, 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)).

Methods of producing polyclonal and monoclonal antibodies that react specifically with PIK4CA are known to those of skill in the art (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al. , Nature 341:544-546 (1989)).

A number of PIK4CA comprising immunogens may be used to produce antibodies specifically reactive with PIK4CA. For example, recombinant PIK4CA 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. Alternatively, 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) or 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 PIK4CA. When appropriately high titers of antibody to the immunogen are obtained, 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 UusQ 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. Typically, polyclonal antisera with a titer of 10 ⁴ or greater are selected and tested for their cross reactivity against non- PIK4CA 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 niM, more usually at least about 1 μM, optionally at least about 0.1 μM or better, and optionally 0.01 μM or better.

Using PIK4CA specific antibodies, PIK4CA can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds. , 7th ed. 1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include

luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵ 1, ⁱ³ⁱ j _^ ³⁵ g _or ³ J_J Additional labels are discussed at the end of this section, below.

The antibodies are also useful for inhibiting PIK4CA function, for example, blocking ligand binding. These uses can also be applied in a therapeutic context in which treatment involves inhibiting PIK4CA 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.

As used herein, the phrase "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. Thus, under designated immunoassay conditions, the specified antibody binds to a particular protein and does not bind significantly to other proteins present in the sample. The following examples are intended to illustrate but not limit the present invention.

Example 1

This example shows the results of screening an HCV cell line with a human kinase siRNA library.

A. Materials

The Ambion human kinase library consisting of an array of 3 siRNAs targeting each of 726 identified human kinases was reverse transfected into an HCV replicon cell line. Transfections conditions for this experiment were optimized using the Ambion GAPDH based siRNA transfection optimization kit.

B. Method i. siRNA transfection. HCV replicon cell line (#8-lK), which is a Huh7 cell line containing a HCV genotype Ib based replicon, was used for the siRNA library screening. The replicon cells were cultured in the presence of 1 mg/ml Neomycin until the day of transfection. Cell density was carefully observed. Transfection conditions per well were as follows: 2 ul of 2500 nM siRNA was added to each well (final cone = 50 nM). 0.5 ul of

Oligofectamine in 37.5 ul OptiMem was then added to allow a lipid/siRNA complex to form. Following a 20 minute incubation at room temperature of this mixture, 3000 replicon cells in 60 ul of growth media were added. The cell/lipid/siRNA mixture was allowed to incubate overnight at 37°C with 5% CO ₂ . The next day the transfection reagent and media was removed and replaced with fresh growth media (DMEM, 10% serum). ii. ELISA. HCV replication was measured by an indirect HCV NS5A based in situ ELISA 5 days post transfection. Changes in the level of HCV replicon amplification due to the siRNA mediated target knockdown resulted in altered levels of HCV proteins being expressed from the replicon. The colorimetric readout of the ELISA was measured in a plate reader and the relative replication efficiency (RRE) was calculated as the ELISA optical density (OD) reading observed for a specific siRNA divided by the OD value for a control sample (Figure 1).

C. Result. PIK4CA is required for HCV replication. Of 726 siRNA sets, only the three siRNAs specific for target 9-F8 induced a significant knockdown in HCV replication with all 3 siRNAs. These siRNAs target PIK4CA, phosphatidylinositol 4-kinase catalytic subunit alpha (PIK4CA).

Example 2

This example confirms the results of Example 1, that PIK4CA is required for HCV replication.

To confirm the initial hit from the primary screen, the PIK4CA specific siRNAs and control siRNAs were tested in a second assay to validate the specificity. In this assay, the siRNAs were tested at concentrations of 20 and 200 nM. The PIK4CA specific siRNAs were able to reduce HCV replication at both 20 and 200 nM concentrations indicating the validity of this target (Figure 2A).

Example 3

This example shows that reduced levels of PIK4CA are not toxic to the cells.

An Alamarblue™ assay was performed to evaluate whether the siRNA transfection- induced reduced levels of PIK4CA are toxic to the cells. Neither the 20 nM or 200 nM concentration of PIK4CA induced noticeable levels of cell toxicity (Figure 2B). This indicates that reduced levels of PIK4CA are not toxic to the cells over the 5 day time frame of the experiment.

Example 4

This example shows that it is specifically PIK4CA, and not various isoforms, that is required for HCV replication.

PIK4CA catalyzes the reaction in which phosphorylation of 1-phosphatidylinositol to 1-phosphatidylinositol 4-phosphate occurs. This is an intermediate to the synthesis of 1- phosphatidylinositol 4,5-phosphate. This product is subsequently cleaved by Phospholipase C to 1 ,2-diacylglycerol and inositol 1 ,4-phosphate. The phosphatidyl-inositol 4-kinase enzymatic activity is catalyzed by several isoforms and splice variants. Isoforms, proteins catalyzing the same reaction as PIK4CA and PI4K230 but encoded by different genes include PIKII, PI4K-2B (PMKIIb), and PIK4CB. The screen included siRNAs targeting these isoforms (PI4KII, PI4k-2B, PIK4CB).

However, no effect on HCV replication was observed due to the transfection of siRNAs targeting these isoforms. This indicates that it is specifically PIK4CA that is required for HCV replication.

In addition, kinases up and downstream in the phosphatidyl inositol phosphorylation cascade (PI4K5, or PIK3) had no effect on replication. This finding further confirms the specificity of this enzyme, or its enzymatic activity, for HCV viral replication.

Example 5

This example shows that PIK4CA is required for both genotype Ib and 1 a replication.

It is possible that the clonal cell line #8- Ik, containing the HCV Ib replicon, is specifically sensitive to reduced levels of PIK4CA due to genetic changes accrued during the selection process. Alternatively, PIK4CA could be specifically important for genotype Ib replication and not be required for replicons derived from other genotypes. To address this issue, the effect of siRNA mediated knockdown of PIK4CA on a HCV genotype Ia replicon cell line was tested.

This cell line was independently established from Huh7 cells electroporated with a HCV genotype Ia derived replicon. The Genotype Ia replicon cells were reverse transfected with the PIK4CA specific siRNAs and assayed for HCV replication and toxicity (Figure 3). The Genotype Ia replicon expressed NS5A protein was reduced 2.5 to 5-fold in the presence of each of the 3 PIK4CA siRNAs (Figure 3A), while no toxicity was observed for the same siRNAs (Figure 3B). This clearly shows that PIK4CA is essential for both genotype Ib and Ia replication and that the observed effects are not due defects in the replicon cells that were selected during the expansion of the cells.

Example 6

This example shows that inhibition of HCV replication is dependent on the level of PIK4CA mRNA.

To confirm that inhibition of HCV replication is dependent on the level of PIK4CA mRNA, we performed a titration of the three PIK4CA siRNAs. The siRNAs were reverse transfected into HCV Ia and Ib replicon cell lines and assayed for HCV NS5A levels by NS5A ELISA. The highest concentration of siRNA used was 30 nm and a 2-fold dilution series was used. A good dose response was observed for each of the siRNAs (Figure 4).

Example 7

This example shows that stable knockdown of PIK4CA by siRNAs inhibits HCV replication as measured by a colony formation assay.

Huh 7 cell lines were transduced with lentiviruses expressing the following shRNA sequences for stable knockdown of PIK4CA: 105 ICA, 2535CA and 1428CA. shRNA 954 (targeting type PIK4-II) was used as a control. The cells were electroporated with 5 ug of in vitro RNA transcript of an HCV Ib replicon and subjected to G418 selection for two weeks. Neo resistant cells were stained with 2% crystal violet. Figure 5 shows that knockdown of PIK4CA by all three siRNAs resulted in a significant reduction of colony formation compared to control, with two of the three causing almost complete inhibition.

As a further control, all Huh 7 cell lines were also electroporated with the GND replication mutant of Hut Ib replicon, and these yielded no neo resistant colonies after two weeks of drug selection.

All references made herein, including articles, patent applications and any other publications, are incorporated by reference in their entirety.