WO2004035076A1 - Treatment of tnf-alpha-mediated disorders with casein kinase i epsilon inhibitors - Google Patents

Abstract

Methods for modulating tumor necrosis factor-alpha (TNF-α)-mediated pathways, more particularly, TNF-α-induced NF-λB activation and TNF-α-induced apoptosis, in a cell or in a subject are provided. The methods of the present invention comprise contacting a cell, or administering to a patient, a composition comprising a CKIε inhibitor in an amount sufficient to modulate a TNF-α-mediated pathway. These methods are useful for treatment of TNF-α-mediated disorders, particularly those associated with TNF-α induced NF-λB activation or TNF-α-induced apoptosis, including inflammatory diseases.

Description

TREATMENT OF TNF-ALPHA-MEDIATED DISORDERS WITH CASEIN KINASE I EPSILON INHIBITORS

FIELD OF THE INVENTION

The present invention relates to methods for modulating tumor necrosis factor- alpha (TNF-α)-mediated pathways using inhibitors of casein kinase I epsilon (CKIε). These methods find use in the treatment of TNF-α-mediated disorders, particularly inflammatory diseases and disorders involving apoptosis.

BACKGROUND OF THE INVENTION

Casein kinase I (CKI) is a ubiquitous protein kinase that was first described as one of the two protein kinases responsible for the Serine/Thr eonine protein kinase activity on acidic rather than basic polypeptides in total cell extracts (Matsumara

(1972) Biochem. Biophys. Acta 289:237-241). Since then, CKI homologs have been identified in eukaryotes ranging from yeast to humans. Several isoforms are known, and most organisms contain more than one isoform. In vertebrates, seven CKI isoforms have been reported (α, β, γl, γ2, γ3, δ, and ε). They range in size from 34 to 49 kDa (Fish et al. (1995) J Biol. Chem. 270:14875-14883; Graves et al. (1993) J Biol. Chem. 268:6394-6401; Rowles et l. (1991) Proc. Natl. Acad. Sci. USA 88:9548-9552; Zhai et al. (1992) Biochem. Biophys. Res. Comm. 189:944-949). CKIδ and ε isoforms share 98% identity in the kinase domain and are 53% identical in a C- terminal domain that is not present in other CKI isoforms (Skanaka et al. (1999) Proc. Natl. Acad. Sci. USA 96:12548-12552). This C-terminal domain appears to negatively regulate kinase activity (Fish et al. (1995) J. Biol. Chem. 270:14875- 14883).

Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine, a class of compounds that recruit and stimulate cellular components of the immune system and which play a role in host defense. TNF-α is produced primarily by macrophages and monocytes in response to infection, injury, or inflammation. Like other proinflammatory cytokines, TNF-α acts to increase its own production, as well as the synthesis of other inflammatory mediators, such as platelet-activating factor, eicosanoids, and oxidative radicals. This cytokine mediates proinflammatory actions, including stimulation of the release of platelet activating factor from neutrophils, macrophages, and vascular endothelial cells (Camussi et al. (1987) J. Exp. Med. 166:1390), increasing the adherence of neutrophils and lymphocytes (Pober et al. (1987) J Immunol. 138:3319), and induction of procoagulant activity on vascular endothelial cells (Pober et al. (1986) J Immunol. 136:1680). It also plays a role in the induction of apoptosis, i.e., programmed cell death. TNF-α has been implicated in acute disease states such as gram-negative sepsis and endotoxic shock (Michie et al. (1989) Brit. J. Surg. 76:670-671; Simpson et al. (1989) Crit. Care Clin. 5:27-47), traumatic injury, cardiac and cerebral ischemia, asthma, and burns (Cairns et al. (2000) Academic Emergency Medicine 7:930-941). TNF has also been associated with infections (Cerami et al. (1988) Immunol. Today 9:28), neoplastic pathologies (Oliff et al. (1987) Cell 50:555), autoimmune pathologies and graft-versus-host pathologies (Piguet et al. (1987) J Exp. Med. 166:1280), and cachexia, the extensive wasting away associated with cancer and other diseases.

TNF-α interacts with cell membrane receptors, including TNF type 1 receptor, (TNFR1), TNF type 2 receptor (TNFR2), and Fas (Cairns et al. (2000) Academic Emergency Medicine 7:930-941; Meldrum (1998)_^4m. J Physiol. 274 (Regulatory Integrative Comp. Physiol. 43):R577-R595. Binding of TNF-α to TNFR1 or Fas leads to a pathway favoring apoptosis, whereas TNF-α binding to TNFR2 leads to the activation of the transcription factor nuclear factor kappa-B (NF-κB). Although TNF- α binding to TNFR1 can lead to apoptosis, it can also cause the activation of NF-κB, which leads to cell survival. Whether the cell progresses to apoptosis or survives after TNFR1 stimulation is thought to depend upon cell type and the balance of various factors recruited to the receptor complex (Yeh et al. (1999) Immunological Reviews 169:283-302).

Nuclear factor kappa-B activation, besides protecting a cell from apoptosis, is also involved in TNF-α induction of genes involved in inflammatory processes. This transmission factor is highly activated at sites of inflammation in many human disorders, including rheumatoid arthritis, atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuritis, asthma, inflammatory bowel disease, helicobacter pylori-associated gastritis, and systemic inflammatory response syndrome (Tak and Feinstein (2001) J. Clin Invest 107:7-11). Its activation increases expression of proinflammatory cytokines, chemokines such as TNF-α, IL-1B, IL-6, and 1L-8, adhesion molecules, such as E-selection, VCAM-1, and ICAM-1, matrix metalloproteinases (MMPs), cyclooxygenase 2 (Cox-2), and inducible nitric oxide (INOS). Further, some of the effects of corticosteroids, used in the treatment of inflammatory bowel disease, asthma, psoriasis, and rheumatoid arthritis, are thought to be mediated through the inhibition of NF-κB activation (Tak and Firestein (2001) supra). Once a cell enters the apoptotic pathway, the process continues through the cleavage of intracellular proteins, mediated by proteases called caspases (Cairns et al. (2000) Academic Emergency Medicine 7:930-941). Caspases remain inactive until a signal activates one, thereby starting a cascade of proteolytic activation. A cell undergoing apoptosis develops cytoplasmic blebbing, chromatin condensation, nuclear fragmentation, and, finally, cellular fragmentation into apoptotic bodies that are phagocytized by macrophages. Inflammatory responses normally induced by the release of cellular contents are therefore avoided.

Thus, controlling TNF-α activity has become a focus of clinical studies. However, there remains an unmet need for compositions and methods for modulating the activity of TNF-α and processes associated therewith.

SUMMARY OF THE INVENTION

Methods for modulating tumor necrosis factor-alpha (TNF-α)-mediated pathways, more particularly, TNF-α-induced-expression of genes involved in inflammation, particularly those genes induced by NF-κB activation, and TNF-α- induced apoptosis, in a cell or in a subject are provided. The methods of the present invention comprise contacting a cell, or administering to a patient, a composition comprising a CKIε inhibitor in an amount sufficient to modulate a TNF-α-mediated pathway. These methods are useful for treatment of TNF-α-mediated disorders, particularly those associated with TNF-α induced NF-κB activation or TNF-α-induced apoptosis, including inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of casein kinase I (CKIε) antisense oligonucleotides on tumor necrosis factor-alpha (TNF-α)-induced apoptosis. Cancer cells were transfected with CKIε antisense oligonucleotides (antiCK) or a control oligonucleotide (RC). DNA fragmentation, indicative of apoptosis, was measured using the Roche™ Cell Death Detection ELISA^PLUS kit, Cat. No. 1 920 685. The manufacturer's assay instructions were followed.

Figure 2 shows the effect of CKIε antisense oligonucleotides on staurosporine- induced apoptosis in cancer cells transfected with CKIε antisense oligonucleotides (antiCK) or a control oligonucleotide (RC). Apoptosis was measured as described above.

Figure 3 shows the effect of CKIε antisense oligonucleotides on ultraviolet radiation-induced apoptosis in cancer cells transfected with CKIε antisense oligonucleotides (antiCK) or a control oligonucleotide (RC). Apoptosis was measured as described above.

Figure 4 shows the effect of CKIε antisense oligonucleotides on TNF-α- induced apoptosis in cancer cells transfected with control oligonucleotide (first panel; RC) or CKIε antisense oligonucleotides (second panel; AS). Apoptosis (A) and necrosis (N) were measured using the Roche™ Annexin-V-FLOUS Staining Kit, Cat. No. 1 858 777.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for modulating tumor necrosis factor-alpha (TNF-α)-mediated pathways. By "modulating" or "modulation" of a TNF-α-mediated pathway is intended the inhibition of or interference with a biochemical pathway that is regulated by TNF-α. Methods of the invention particularly target the pathways associated with TNF-α-induced apoptosis and TNF-α- induced expression of gene products involved in inflammation, where expression results from TNF-α-induced activation of nuclear factor-kappa B (NF-κB). The methods comprise contacting a cell, or administering to a subject in need thereof, a composition comprising an inhibitor of casein kinase I epsilon (CKIε) in an amount sufficient to modulate a TNF-α-mediated pathway. By "inhibitor of CKIε" or "CKIε inhibitor" is intended any agent that effectively inhibits CKIε activity, including, but not limited to, antisense agents, ribozymes, antibodies, and kinase inhibitors. By "inhibit," or "inhibiting," or "inhibition" of CKIε activity is intended a reduction in CKIε kinase activity, interference with binding of CKIε to other molecules and/or proteins, and/or interference with the expression of the CKIε gene product in the presence of the CKIε inhibitor. The methods find use in the treatment of TNF-α- mediated disorders.

Tumor necrosis factor-alpha is associated with a number of pathologies, including, but not limited to, chronic inflammatory diseases, autoimmune diseases, neurodegenerative disorders, and bacterial, viral, or parasitic infections. TNF-α plays a role in cachexia in patients with advanced chronic heart failure (CHF) (Ceconi et al. (1998) Prog. Cardiovascular Dis. 41 :25-30) and reportedly mediates left ventricular dysfunction. This proinflammatory cytokine is also a contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury, sepsis, viral myocarditis, and cardiac allograft rejection. (Meldrum-Ferrari (1999) Pharmacol. Res. 40:97-105). Recent data also suggest that TNF-α-mediated apoptosis is a significant contributor to myocyte cell death in heart failure syndromes (Ceconi et al. (1988) Prog. Cardiovascular Dis. 4:25-30) and is associated with arryhthmogenic right ventricular dysplasia and sudden cardiac death (Runge et al. (2000) Am. J. Med. Sci. 320:310-319; Kolodgie et al. (2000) Am. J. Pathol. 157:1259-1268).

TNF-α-induced hepatocyte apoptosis is also associated with liver damage in, for example, lipopolysaccharide-induced Kupffer-cell activation, ischemia- reperfusion injury, and septic shock (Kanzler et al. (2000) Cancer Biology 10:173- 184). TNF-α is thought to mediate hepato cellular apoptosis in viral hepatitis. Further, patients with alcoholic hepatitis display increased serum levels of TNF-α. In the rat, hepatic necrosis and inflammation caused by chronic alcohol exposure can be reduced by antibodies to TNF-α. TNF-α also plays a significant role in concavalin A liver injury (a model for inflammatory liver injury); patients with fulminant hepatitis also show raised levels of TNF-α serum levels (Kanzler et al. Cancer Biol. 10:173- 184; Bradham et al. (1998) Am. J. Physiol. 275 (Gastrointest. Liver. Physiol. 38):G387-G392).

TNF-α has also been associated with autoimmune disorders of the nervous system (Weishaupt et al. (2000) J. Neuropath. Exp. Neurology 59:368-376). In an animal model of human demyelinating neuropathies, effects of antigen treatment are thought to involve TNF-α and TNF-α-induced apoptosis.

TNF-α has also been associated with various malignancies, including, but not limited to those affecting the colon, breast, lung, prostate, kidney, pancreas, liver, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory-system carcinomas, gastrointestinal-system carcinomas, genitourinary-system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine-system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. TNF-α can mediate cachexia in cancer. Cachexia in cancer patients is the wasting away of an individual, characterized by progressive weight loss, anorexia, and persistant erosion of body mass in response to a malignant growth.

Thus, by "TNF-α-mediated disorders" is intended rheumatoid arthritis, atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuritis, asthma, inflammatory bowel disease, helicobacter pylori- associated gastritis, systemic inflammatory response syndrome, psoriasis, rheumatoid arthritis, cachexia in patients with advanced chronic heart failure (CHF), left ventricular dysfunction, myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury, sepsis, viral myocarditis, cardiac allograft rejection, heart failure syndromes, arryhthmogenic right ventricular dysplasia, sudden cardiac death, lipopolysaccharide-induced Kupffer-cell activation, septic shock, viral hepatitis, alcoholic hepatitis, fulminant hepatitis, autoimmune disorders of the nervous system, human demyelinating neuropathies, malignancies and carcinomas, such as those noted above, and cachesia associated with cancer.

The methods of the present invention are applicable to any subject suffering from or predisposed to a TNF-α-mediated disorder. Thus subject as used herein refers to a mammal, e.g., a human, or to an experimental animal or disease model.

Exemplary subjects included but are not limited to cats, dogs, horses, cows, goats, sheep, pigs or other domestic animals, and more preferably humans.

In accordance with the methods of the present invention, modulation of a TNF-α-mediated pathway in a cell or subject in need thereof is achieved by contacting the cell, or administering to a subject in need thereof, a composition comprising an inhibitor of CKIε in an amount sufficient to modulate the TNF-α-mediated pathway of interest. Thus, in one embodiment, a CKIε inhibitor is used to modulate TNF-α- induced apoptosis in a cell or a subject in need thereof. In another embodiment of the invention, a CKIε inhibitor is used to modulate TNF-α-induced activation of NF-κB in a cell or a subject in need thereof. In this manner, inhibition or interference with activation of NF-κB results in inhibition of the expression of a number of NF-κB- activated gene products involved in the inflammatory response, including, but not limited to, cytokines, chemokines, such as TNF-α, IL-1B, IL-6, and 1L-8, adhesion molecules, such as E-selection, NCAM-1, and ICAM-1, matrix metalloproteinases (MMPs), cyclooxygenase 2 (Cox-2), and inducible nitric oxide (iΝOS).

For purposes of the present invention, CKIε inhibitors include any agent that inhibits CKIε activity, specifically human CKIε or variant thereof, either by inhibiting or interfering with its expression or its kinase protein activity. Human CKIε is a kinase protein of 416 amino acids (see SEQ ID ΝO:2) with a molecular mass of about 47.3 kDa (Fish et al. (1995) J. Biol. Chem. 270(25): 14875-14883). It has a core kinase domain of 285 amino acids (residues 9 to 293 of SEQ ID NO:2) and a carboxyl-terminal tail of 123 amino acids. It shares closest similarity with the CKI delta (CKIδ) isoform, having 97.5% amino acid identity with human CKIδ over the kinase domain, and 53% identity over the carboxyl-terminal region (residues 294-416 of SEQ ID NO:2). The nucleotide sequence encoding human CKIε is known (see GenBank Accession No. L37043; also set forth in SEQ ID NO:l). This protein kinase is expressed in multiple human cell lines (Fish et al, supra).

It is recognized that the methods of the invention can be directed to inhibition of CKIε polypeptides that are variants or homologues of human CKIε. By "variant CKIε polypeptides" is intended polypeptides having CKIε activity and which have sequences that are substantially similar to that disclosed in SEQ ID NO:2. By "substantially similar" is intended the sequences share at least 70%, 75%, 80%, preferably 85%, 90%, more preferably at least 95%, 98%, up to 99% or more sequence identity to SEQ ID NO:2. Variant CKIε polypeptides are encoded by nucleotide sequences that share at least 70%), 75%, 80%, preferably 85%, 90%, more preferably at least 95%, 98%, up to 99% or more sequence identity to SEQ ID NO:l.

For instance, those of skill in the art appreciate that DN A sequence polymorphisms that lead to changes in the amino acid sequences of CKIε proteins may exist within a population (e.g., the human population). Such genetic polymorphism in a CKIε gene may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes that occur alternatively at a given genetic locus. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a CKIε protein, preferably a mammalian CKIε protein. By "allelic variant" is intended a nucleotide sequence that occurs at a CKIε locus or a polypeptide encoded by that nucleotide sequence. Such natural allelic variations can typically result in 1-5%) variance in the nucleotide sequence of the gene. Moreover, nucleic acid molecules encoding CKIε proteins from other species (CKIε homologues), which have a nucleotide sequence differing from that of the CKIε sequences disclosed herein, are known in the art. See, for example, Sakanaka et al. (1999) Proc. Natl. Acad. Sci. USA 96(22):12548-12552; and Vielhaber et al. (2000) Mol. Cell Biol. 20:4888-4899. For example, the murine CKIε homologue shares 98.8% identity with human CKIε at the nucleotide sequence level (see SEQ ID NO:3 for the murine CKIε nucleotide sequence, SEQ ID NO:4 for the murine CKIε amino acid sequence). Thus, the methods of the invention contemplate the use of inhibitors to homologues of human CKIε, such as the murine CKIε homologue set forth in SEQ ID NO:4.

In addition to naturally occurring allelic variants of the CKIε sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence encoding known CKIε proteins, such as human CKIε shown in SEQ ID NO: 2, encoded by SEQ ID NO:l, or murine CKIε shown in SEQ ID NO:4₃ encoded by SEQ ID NO:3, thereby leading to changes in the amino acid sequence of the encoded CKIε polypeptide, without altering the biological activity of the CKIε polypeptide. Thus, an isolated nucleic acid molecule encoding a CKIε polypeptide having a sequence that differs from that of SEQ ID NO:2 or SEQ ID NO:4 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, i.e., SEQ ID NO:l or SEQ ID NO:3, respectively, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); U.S. Patent No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the CKIε polypeptide of interest may be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly Ala, Val <=>Ile <=>Leu, Asp Glu, Lys = Arg, Asn <=>Gln, and Phe Trp Tyr. Thus, the methods of the invention can be targeted to human CKIε or variant thereof having CKIε activity. Such variant CKIε polypeptides include those having at least 70%, 75%, 80%, preferably 85%, 90%, more preferably at least about 95%, 98%), up to 99%) or more sequence identify to the full-length amino acid sequence set forth in SEQ ID NO:2. By "sequence identity" is intended the same amino acid residues are found within the variant and the reference CKIε molecule when a specified, contiguous segment of the amino acid sequence of the variant is aligned and compared to the amino acid sequence of the reference CKIε molecule, which serves as the basis for comparison. A CKIε polypeptide that is a variant of human CKIε may differ from this reference molecule by as few as 1-15 amino acids, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. The percentage sequence identity between two amino acid sequences is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the segment undergoing comparison to the reference molecule, and multiplying the result by 100 to yield the percentage of sequence identity.

For purposes of optimal alignment of the two sequences, the contiguous segment of the amino acid sequence of the variant polypeptide may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference polypeptide molecule, i.e., human CKIε. The contiguous segment used for comparison to the reference amino acid sequence will comprise at least twenty (20) contiguous amino acid residues, and may be 30, 40, 50, 100, or more residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art for both amino acid sequences and for the nucleotide sequences encoding amino acid sequences.

For purposes of the present application, percent identity of an amino acid sequence is determined using the Smith- Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. The Smith- Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2:482-489, herein incorporated by reference. Alternatively, percent identity of a nucleotide sequence is determined using the Smith- Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic. Corrections for sequence identity associated with conservative residue substitutions or gaps can be made (see Smith- Waterman homology search algorithm).

The variant CKIε polypeptides whose activity is targeted by the methods of the present invention are further characterized by having CKIε kinase activity. Methods for assaying a polypeptide for such activity are known in the art. Activities of particular interest for the present invention are those associated with CKIε induction of TNF-α-mediated pathways, more particularly TNF-α-induced apoptosis and TNF-α-induced NF-κB activation. Thus, in the absence of an inhibitor targeting activity of the variant CKIε polypeptide, TNF-α-induced apoptosis and TNF-α- induced NF-κB activation can occur following CKIε induction of the respective TNF- α-mediated pathway. Methods for assaying the ability of a variant CKIε polypeptide to induce these TNF-α-mediated pathways are known in the art, including the assays described in the Examples disclosed herein. See also the kinase bioassays disclosed in Fish et al. (1995) J. Bio. Chem. 270-(25):US75-U883, herein incorporated by reference in its entirety.

Thus, it is understood that use of the term CKIε in the following discussion is intended to include human CKIε and variant CKIε polypeptides described above. Where inhibitors of CKIε are sequence-specific inhibitors, their sequences are designed based on the nucleotide sequence encoding the specific CKIε polypeptide of interest.

Preferred CKIε inhibitors for use in the methods of the invention include those targeting CKIε gene expression, particularly antisense agents and ribozymes, and those targeting CKIε kinase activity, particularly anti-CKIε antibodies and kinase inhibitors. As used herein the term "antisense agent" refers to a sequence-specific regulator of gene expression and target protein function. Suitable antisense agents for use with the methods of the invention include, but are not limited to, isolated polynucleotide s, synthetic antisense oligonucleotides, antisense polynucleotides produced in vivo from an expression vector, and antisense peptide nucleic acids (PNAs). The effectiveness of an antisense agent depends upon numerous factors, including the type of cell that comprises the target mRNA or protein, the local concentration of the agent at the endogenous target mRNA or protein, the rate of synthesis and degradation of the target mRNA and its encoded protein, the accessibility of the target sequence, the specificity of the antisense agent, and the nature of the mechanism of action (e.g., inhibiting mRNA translation, affecting RNA splicing, or inducing RNase H-mediated degradation of the target mRNA). In addition, the type of agent will also influence its characteristics and mechanism of cellular uptake. In one embodiment, the polynucleotide agent comprises a short synthetic oligonucleotide or an oligonucleotide mimic (e.g., PNA molecule) that is a sequence-specific regulator of nucleic acid utilization. For purposes of the present invention, "antisense molecule" and "antisense agent" are used interchangeably to refer to a molecule comprising a nucleotide sequence designed, according to the rules of Watson-Crick base pairing, to be complementary to an endogenous nucleic acid (e.g., DNA or RNA) target that can hydrogen bond to the target nucleic acid under physiological conditions and thereby inhibit cellular utilization of the target nucleic acid. It is to be understood that the administration of an antisense agent ultimately regulates (e.g., modulates) the amount of target protein. This is accomplished by providing antisense agents that "specifically hybridize" with the endogenous target polynucleotide molecule. Generally, the target nucleic acid is an endogenous mRNA molecule encoding CKIε, for example human CKIε (SEQ ID NO:2, encoded by SEQ ID NO: 1) or variant thereof when the subject is a human or the cell is of human origin.

As used herein, the term "antisense molecule" encompasses linear oligomers of natural or modified monomers or linkages, including deoxyribonucleo sides, ribonucleosides, polyamide nucleic acids, and the like, capable of specifically hybridizing to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions (e.g., nucleoside-to-nucleoside). Ideally, the antisense molecule should not hybridize to any other nucleic acid sequence in the cell except the target sequence and should not bind nonspecifically to other cellular constituents such as proteins. "Specifically hybridizable" and "complementary" are terms that are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the endogenous target nucleic acid and the antisense agent. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to participate in specific hybridization.

In one embodiment, a suitable antisense nucleic acid molecule for use in the method of the invention can be complementary to a contiguous region of ribonucleotide sequence that comprises a portion of the coding region of a target mRNA, in this case RNA encoding CKIε, such as human CKIε or variant thereof. The term "coding region" is understood to refer to the portion of a mRNA sequence that consists of the codons that are translated into the amino acid sequence of a polypeptide. In an alternative embodiment, the antisense nucleic acid molecule is antisense (i.e., complementary) to a noncoding sequence of the target mRNA. It is to be understood that as used in the context of this invention, the term "mRNA" includes not only the coding region but also the flanking noncoding sequences of contiguous ribonucleotides located upstream and downstream of the coding region. These regions are known to a person of skill in the art to include the 5 '-untranslated region, the 3 '-untranslated region, the 5' cap region, intron regions, exon regions, and intron/exon or splice junction ribonucleotides. Thus, for example, oligonucleotides designed for use in accordance with the methods of the present invention can target wholly or partially these flanking ribonucleotide sequences as well as the sequence of the coding ribonucleotides. In another embodiment, the antisense nucleic molecule is targeted to the translation initiation site or the "start codon region" or sequences in the 5'- or 3'- untranslated region of the mRNA molecule. The terms "start codon region," AUG region," and "translation initiation codon region" are used synonymously herein and refer to a portion of a mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. This region is a preferred RNA-binding site for the design of antisense agents. There is substantial guidance in the literature for the design and identification of antisense agents, and it is well known by one of skill in the art that a preferred antisense agent will have the following characteristics: a unique complementary sequence that is specific for an accessible target RNA-binding site; efficient cellular uptake; in vivo biological stability; and an antisense mechanism of action that successfully reduces the mRNA and/or target protein level (see, for example, Sezakiel et al. (2000) Frontiers in Bioscience 5:dl94). Because there are no a priori rules to predict the most desirable antisense sequence, one of skill in the art will recognize the need to empirically design effective antisense agents. Accordingly, it would not be unreasonable to design at least ten or more different antisense sequences that are complementary to sequences contained in the target nucleotide sequence. One of skill in the art could readily employ the principles of Watson-Crick base pairing to design oligonucleotides that maximize hybridization while avoiding sequences with regions of polyguanosine or G-C arms that could potentially form strong hairpins. See, for example, An Antisense Oligonucleotide Primer, by Richard I. Hogrete, available at www.trilink.com.

In fact, empirical testing or "walking the gene" is the routine method by which antisense oligonucleotides have been selected. In this technique, large numbers of sequences complementary to various sites in the target mRNA are tested to determine which are best able to inhibit the target. Other methods are also available, including RNase H mapping, in which a random library of oligonucleotides is allowed to hybridize to the mRNA target. The DNA-RNA hybrid is then digested with RNase H, and sequences of the resulting fragments are analyzed to determine which sites are accessible. Other techniques available include combinatorial oligonucleotide arrays, in which all antisense oligonucleotide sequences up to a predetermined length are tested for hybridization to the target mRNA. The limitation of this technique is that secondary structure analysis cannot be performed. Computational methods are available for designing antisense oligonucleotides, as well as other methods and techniques. See, for example, Smith et al. (2000) Euro. J. Pharm. Sci. 11:191-198. In short, methods for designing antisense oligonucleotides are available and routine for those of skill in the art. The preparation of a suitable antisense agent for use in the method of the invention is a multistep process that begins with the selection of a target RNA-binding site (or sites) within the nucleic acid sequence encoding CKIε or variant thereof for the oligonucleotide interaction to occur such that the desired effect, inhibition of CKIε gene expression (e.g., inhibition of mRNA processing or of translation) will occur. Once the RNA-binding site has been identified, a complementary oligonucleotide (or an oligonucleotide mimic) is designed to specifically hybridize to the endogenous nucleic acid sequence under physiological conditions. In order to be an effective therapeutic agent, binding of the antisense agent to its target sequence must interfere with the transcription or translation of the targeted DNA or mRNA in a manner that is sufficient to inhibit the intracellular level of the target protein, i.e., CKIε or variant thereof. In general, target sequences encoding initiation sequences, termination sequences, and splice regions are considered to have the potential to produce the most effective inhibition. Depending upon the type of the antisense agent, the final step required for the preparation of a suitable antisense oligonucleotide for exogenous administration may also involve the introduction of a modification into the backbone of the oligonucleotide to produce a polynucleotide analogue. In general, chemically modified antisense agents (e.g., phosphorothioate or morpholino polynucleotide analogues) demonstrate increased stability to nuclear degradation compared to unmodified sequences. As a result, chemically modified oligonucleotides are more effective both in vitro and in vivo. Although targeting to mRNA is preferred and exemplified in the description below, it will be appreciated by those skilled in the art that other forms of nucleic acid, such as pre-mRNA or genomic DNA, may also be targeted. As used herein the term "oligonucleotide" includes single-stranded oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally occurring (e.g., modified) backbones that function similarly. Thus, as used herein the term "oligonucleotide" encompasses natural oligόmers and the chemical analogue and chimeric molecules described below. Use of a modified or substituted oligonucleotide may be preferable to the use of a native oligomer because the modification could confer a desirable property such as, for example, enhanced binding to the target polynucleotide or resistance to nuclease degradation. See, for example, Agrawal et al. (1997) Proc. Natl. Acad. Sci. USA 94(6):2620, and Proc. Natl. Acad. Sci. USA 94(6):2620. If present, modifications to the nucleotide can be introduced either before or after assembly of the polymer. For example, an antisense agent (antisense oligonucleotide) suitable for use in the method of the invention can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides or monomers. Suitable oligonucleotides for use in the methods of the invention should be of sufficient length to specifically hybridize to the CKIε target nucleotide sequence and to modulate the information transfer from a gene to a protein (e.g., inhibition of translation, or splicing). The binding of an oligodeoxynucleotide to the target nucleic acid may inhibit the interaction of the nucleic acid with other nucleic acids or proteins required for cellular utilization of the mRNA transcript. Appropriate oligonucleotides preferably comprise from about 8 to about 50 monomers (e.g., nucleobases). It is known in the art that a nucleoside is a base-sugar combination in which a heterocyclic base (e.g., a purine or a pyrimidine) normally comprises the base component of the combination. Particularly preferred are antisense oligonucleotides comprising from about 10 to about 30 nucleobases (i.e., from about 10 to about 30 linked nucleosides). Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. In the context of antisense molecules that comprise non-naturally occurring monomeric units, it is to be understood that suitable antisense agents comprise 8 to 50 monomers. Accordingly, suitable antisense oligonucleotides may be of any appropriate length, e.g., from about 10 to 50 nucleotides in length (e.g., 10, 12, 14, 15, 17, 18, 20, 25, 30, 35, 40, 45 or 50 nucleobases or monomers) and may contain phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages. However, it should be noted that a higher in vivo intracellular concentration of the antisense agent is more likely to be achieved with the use of a relatively small (e.g., less than 12 nucleobases) oligonucleotide because of a higher efficiency of uptake by cells in vivo. It is well known that an antisense oligonucleotide comprising 13-15 complementary nucleotides is statistically predicted to bind to a single sequence. Preferably, antisense oligonucleotides should be at least 15 nucleotides long, to achieve adequate specificity. In a preferred embodiment, 25-nucleotide antisense molecules are utilized, for example, those set forth in SEQ ID NOS:5, 6, and 7.

Numerous mechanisms have been proposed to explain how an antisense oligonucleotide regulates the activity of its target mRNA, including the inhibition of the processing of the primary RNA transcript (e.g., capping, methylation, splicing, 3'- polyadenylation), inhibition of mRNA transport out of the nucleus, and inhibition of translation (e.g., cellular utilization) by hybridization arrest. Alternatively, an oligodeoxynucleotide can activate the destruction of the target mRNA by an RNase H-dependent mechanism. Although the mechanism of action of antisense oligonucleotides may differ from cell type to cell type and may vary depending on the nature of the endogenous nucleotide sequence that is targeted for binding, there is strong evidence that the predominant mechanism of action in vitro is mediated by the enzymatic cleavage of the target RNA by RNase H (Dash et al. (1987) Proc. Natl. Acad. Sci. USA 84:7896-7900; Walder and Walder (1988) Proc. Natl. Acad. Sci. USA 85:5011-5015). RNase H is a ubiquitous enzyme that specifically degrades the RNA strand of an RNA-DNA heteroduplex (i.e., hybrid). It is well known that RNase H enzymes do not require long hybrid regions as substrates; thus it is not possible to increase the specificity of an antisense agent by increasing the length of the oligonucleotide. It has been estimated that as few as ten base pairs are likely to be sufficient in human cells (Branch (1998) Trends Biochem. Sci. 23(2):45-50). It is well known that cells contain a variety of endo- and exonucleases and that oligonucleotides in their natural form are subject to rapid enzymatic digestion in vivo. In one embodiment, the antisense agent is an antisense oligonucleotide that is modified to improve the biophysical, biochemical, pharmacokinetic, or safety profile of a native phosphodiester oligonucleotide. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated relatively more resistant to nuclease degradation. Phosphodiester nucleotides were initially studied in cell free systems and in vitro cell cultures, however as a class of molecules they are not very stable against nuclease s and therefore have limited potential as in vivo agents. In an alternative embodiment, an oligonucleotide is modified to enhance its inherent nuclease resistance. Improved nuclease stability confers favorable changes in the in vivo stability and biodistribution of the polynucleotide analogue. Accordingly, chemical analogues that are suitable for use according to the methods of the present invention include, but are not limited to, analogues in which, for example, the phosphodiester bonds have been modified (e.g., to a methylphosphonate, a phosphotriester, a phosphorothioate, a phosphorodithioate, or a phosphoramidate) so as to render the oligonucleotide more stable in vivo. For example, oligodeoxyribonucleotide phosphorothioates (e.g., where one of the phosphate oxygen atoms not involved in the phosphate bridge is replaced by a sulphur atom) or oligodeoxyribonucleotide methylphosphonates (e.g., in which a nonbridging oxygen atom at the phosphorous is replaced with a methyl group) embody common chemical analogues that impart improved stability with respect to nuclease degradation. See Cohen, ed. (1989) Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression (CRC Press, Inc., Boca Raton, FL). The half-life of a phosphodiester oligomer introduced into the peripheral circulation of a mouse is about 1 minute, while the half-life of a phosphorothioate oligomer is about 48 hours (Agrawal et α/ .(1991) Proc. Natl. Acad. Sci. USA 88:7595).

However, it should be noted that there are some problems with the in vivo use of phosphorothioate oligonucleotides. For example, the backbone is chiral, resulting in a racemic mixture of 2ⁿ oligonucleotide species (where n = number of phosphorothioate internucleotide linkages) instead of a single compound. Furthermore, the binding affinity of a phosphorothioate oligomer is lower than the affinity of its corresponding phosphodiester oligonucleotide (Agrawal et α..(1998) Antisense and Nucleic Acid Drug Dev. 8:135; LaPlanche et α/.(1986) Nucleic Acids Res. 14:9081-9093). In addition, because they are negatively charged, phosphorothioate oligonucleotides have been known to bind nonspecifically to cellular proteins, lipids, and carbohydrates, which can consequently mediate non- antisense effects that can result in toxicity or which can be mistakenly attributed to an antisense effect. Phosphorothioates also have a reputation for being toxic although that may be a sequence specific phenomenon or due to contamination in early oligonucleotide preparations (Srinivasan and Iverson (1995) J. Lab. Anal. 9:129-137). In addition, the administration of phosphorothioate oligonucleotides comprising particular sequences and structural motifs has been reported to have undesirable immunostimulatory effects.

Preferred modified oligonucleotide backbones (e.g., polynucleotide analogues) that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, and S component parts. For example, morpholino oligomers are a class of chemically modified oligonucleotides in which the ribose moiety is replaced with a morpholino group (U. S. Patent No. 5,185,444, the teachings of which are incorporated herein by reference). The morpholino modification renders an oligomer resistant to enzymatic degradation and morpholino antisense nucleotides have been successfully utilized to inhibit the production of target proteins in vivo. See Qin et al. (2000) Antisense and Nucleic Acid Drug Dev. 10:11. Nuclease resistance is routinely measured by incubating oligonucleotides with isolated nuclease solutions or cellular extracts and determining (e.g., by gel electrophoresis) the extent of intact oligonucleotide remaining over time. Oligonucleotides that have been modified to enhance their nuclease resistance survive intact for a longer time relative to the native oligonucleotides.

Appropriate antisense oligonucleotides for use with the method of the invention also include "chimeric oligonucleotides." As used herein the term

"chimeric oligonucleotide" connotes a mixed -backbone polynucleotide analo ue that comprises a mixture of different sugar and/or backbone chemistries. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance or increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. The most common chimeric oligonucleotides are also referred to as "second generation" oligonucleotides. This nomenclature derives from the fact that phosphorothioates are usually considered to be the first generation antisense agents.

Chimeric or mixed-backbone oligonucleotides vary considerably in their specific construction, but generally all of them have the same basic design characteristics a phosphodiester or phosphorothioate central region surrounded by nuclease resistant arms. More specifically, a chimeric or mixed-backbone oligonucleotide suitable for use in the method of the invention may comprise phosphorothioate segments at the 5' and 3' ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligomer. See Agrawal et al. (1997) Proc. Natl. Acad. Sci. USA 94(6): 2620. The art teaches that a good starting point is to use an oligonucleotide eighteen nucleotides in length that has six 2'-OMe nucleotides at both the 5' and 3' ends, leaving a core of six 2'-deoxyribose nucleosides with phosphorothioate internucleotide linkages (Monia et al. (1996) Nat. Med. 2 668-675). The arms may or may not contain phosphorothiate linkages. Removal of phosphorothiate linkages is favorable from the point of view that it may reduce toxicity, however it will also reduce nuclease resistance. The underlying principles driving the design of a suitable chimeric oligonucleotide suitable for use in the methods of the invention are two fold: increased stability and retention of RNase H activity. Many of the chimeric oligonucleotides reported in the literature have improved properties compared to the properties of phosporothioate oligomers with respect to affinity for RNA, RNase H activation, and pharmacokinetic profiles.

Alternatively, other molecular designs that depend on extreme hybridization enhancement using highly modified oligonucleotides such as 2'-MOEs (Monia (1997) Ciba Found. Symp. 209:107-123), N3' —> P5' phosphoramidates (Gryaznov and Chen (1994) J. Amer. Chem. Soc. 116:3143-3144; Mignet and Gryaznov (1998) Nucleic Acids Res. 26:431-438), PNA's (Hanvey et al. (1992) Science 258: 1481-1485), chirally pure methylphosphonates (Reynolds et al. (1996) Nucleic Acids Res. 24:4584-4591), and MMIs (Morvan et «/ . (1996) J. Amer. Chem. Soc. 118:255; Swayze (1997) Nucleosides Nucleotides 16:971-972) represent alternative embodiments that may be particularly useful for the inhibition of CKI protein expression by hybrid arrest. In one embodiment, a chimeric oligonucleotide suitable for use in the method of the invention comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNase H. A common design is to have nuclease resistant arms (such as 2'-O-methyl (OMe) nucleosides) surrounding a phospodiester- or phosphorothioate-modified central core region (Agrawal and Goodchild (1987) J. Tetrahedron Letters 28:3539- 3542; Giles and Tidds (1992) Nucleic Acids Res. 20:753-770). See the examples disclosed herein and the chimeric oligonucleotide methodology disclosed in International Publication No. WO 01/16306 A2. Also see the methodology disclosed in U.S. Patent No. 5,714,170, and U.S. Application Serial Nos. 60/151,246, filed August 27, 1999, and 09/648,254, filed August 25, 2000, both entitled "Chimeric Antisense Oligonucleotides and Cell Transfecting Formulations Thereof," herein incorporated by reference in their entirety. The antisense molecules of the present invention include bioequivalent compounds, including but not limited to pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al. (1977) J Pharma. Sci. 66:1-19).

Administration of pharmaceutically acceptable salts of the polynucleotides described herein is included within the scope of the invention. Such salts may be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like. For a helpful discussion of pharmaceutical salts, see Berge et al. (1977) J. Pharm. Sci. 66:1-19), the disclosure of which is hereby incorporated by reference.

For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, and calcium; (b) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, and the like; (c) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and the like; and (d) salts formed from elemental anions such as chlorine and bromine.

There is substantial guidance in the literature for selecting particular sequences for complementary oligonucleotides given a knowledge of the sequence of the target polynucleotide and the accessibility of the binding site. See, for example, Ulmann et al. (1990) Chem. Rev. 90:543-584; Crooke (1992) Ann. Rev. Pharmacol. Toxicol. 32:329-376; and Zamecnik and Stephenson (1974) Proc. Natl. Acad. Sci. USA 75:280-284. Preferably, the synthetic oligonucleotide sequence is designed so that the G-C content is at least 60%. Oligonucleotides suitable for use in the methods of the invention may be conveniently and routinely produced and purified using chemical synthesis, enzymatic ligation reactions and purification procedures that are well known in the art. Equipment for such synthesis is sold by several vendors including Applied Biosystems.

In general, antisense agents can comprise from about 10 to about 50 nucleotides (or monomers), from about 15 to about 40 nucleotides, preferably from about 20 to about 30 nucleotides, more preferably from about 18 to about 25 nucleotides, even more preferably about 25 nucleotides. For example, a suitable CKIε antisense oligonucleotide can include, but is not limited to, a modified chimeric oligonucleotide or PNA that is antisense to a target sequence set forth in Table I, i.e., SEQ ID NOS:5-24, preferably antisense to SEQ ID NOS:5, 6, and 7. The target sequences set forth in SEQ ID NOS:5, 6, and 7 correspond to nucleotides 807-831, 703-727, and 766-790 of SEQ ID NO:l, respectively. The relative position of the remaining target sequences is shown in Table I. Examples of suitable CKIε antisense oligonucleotides include 5'-cgtaggtaagagtagtcgggcttgt-3' (SEQ ID NO:25), which targets the CKIε sequence set forth in SEQ ID NO:5; 5'-cgttgacatcttcttctcgctgatc-3' (SEQ ID NO:26), which targets the CKIε sequence set forth in SEQ ID NO:6; and 5'-gcggcagaagttgaggtatgttgag-3' (SEQ ID NO:27), which targets the CKIε sequence set forth in SEQ ID NO:7.

Table I - Examples of CKIε -derived Sequences Targeted by CKIε Antisense Oligonucleotides

In addition to the use of exogenous antisense agents, the methods of the invention contemplate endogenous production of antisense agents to modulate TNF- α-mediated pathways in a cell or subject of interest. Thus in an alternative embodiment, a viral vector-mediated or nonviral vector-mediated delivery method can be used for the delivery of a nucleotide sequence encoding a sequence capable of directing the endogenous production of an antisense agent, for example an oligonucleotide, designed to inhibit production of CKIε. See Luo and Saltzman (2000) Nature Biotechnology 18:33.

The use of an expression vector or eukaryotic expression plasmid to generate antisense agents intracellularly (e.g., endogenously) offers several potential advantages over the exogenous administration of an antisense agent. For example, an antisense RNA that is produced in vivo can be more effectively delivered (e.g., achieve higher copy number) to specific cells and or tissues relative to the efficiency of an exogenous delivery protocol, particularly in light of the fact that enzymatic degradation of native oligonucleotides is prominent in vivo. Thus, the duration or residence time of the antisense agent will likely be longer when it is delivered in the context of a delivery method that facilitates endogenous production, particularly if the vector-mediated transfer of the sequence results in the sequence becoming incorporated in the genome of the recipient, but also if in vivo production occurs as a result of episomal expression. In addition, the opportunity to select a particular expression control element, such as promoter sequences, affords the opportunity to accomplish tissue-specific, site-specific (e.g., nuclear or cytoplasmic), or inducible (e.g., by the administration of a transcription activator) production of the antisense agent.

Eukaryotic expression plasmids or viral vectors represent suitable vehicles for use with the antisense methods of the invention. Suitable plasmids for use with this embodiment of the invention include nonintegrative plasmids known in the art, as well as plasmids that are designed to integrate a polynucleotide sequence into the genome of a recipient cell. The choice of an appropriate vector will be dictated by the identity of the tissue or cell which is targeted for delivery.

Consistent with these observations, a viral vector can be utilized for the localized delivery of a replication-deficient adenovirus comprising a DNA sequence encoding an antisense agent. In one embodiment, a viral vector comprising a nucleotide (e.g., DNA) sequence encoding an antisense oligonucleotide agent is delivered to the cell targeted for modulation of a TNF-α-mediated pathway. In a second embodiment, a viral vector comprising an antisense olignucleotide is delivered to the targeted cell. Viral vectors and methods for delivering plasmids and viral vectors are well known in the art. See, for example, the viral vectors and methods described below.

Antisense agents useful in the invention can include peptide nucleic acids. As used herein the terms "peptide nucleic acids" or "PNAs" refer to polynucleotide mimics in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone to which the four native nucleobases are linked. More specifically, the phosphodiester backbone of DNA or RNA is replaced by a homomorphous backbone consisting of (N-2 aminoethyl) glycine units bearing nucleobases attached via methylenecarbonyl linker. (Nielsen et al. (1991) Science 254: 1497; Larsen et al. (1999) Biochem. Et Biophysica Acta 1489:159-166). The nucleobases are maintained to mediate sequence -specific hybridization with the targeted endogenous nucleic acid molecule. Chemically, PNA agents have a homomorphous, charge neutral, achiral polyamide backbone that is relatively flexible (Larsen et al. (1999) Biochem. Et Biophysica Acta 1489: 159-166). The uncharged nature of the PNA oligomer enhances the stability of the hybrid PNA/DNA (mRNA) duplex. Accordingly, PNA agents embody a DNA mimic that is only remotely chemically related to DNA. Although PNA agents are in fact more closely related to proteins (peptides) than to nucleic acids, they provide alternative sequence-specific regulators of nucleic acid function.

As discussed above, the antisense effect of conventional oligonucleotides and their chemical analogs rely on the activation of RNase H. However it is well known that morpholino-mRNA complexes and PNA-mRNA complexes are not substrates for RNase H activity. Thus, the proposed mechanism of action of morpholino oligomers and PNA molecules is believed to be translation arrest due to steric interference with assembly or progression of the translation machinery. PNA oligomers have been successfully used to inhibit target protein expression at both the transcriptional and translocational level. More specifically, PNA oligomers that are complementary to nucleotide sequences present at the translation start site of 5 '-untranslated regions of targeted mRNA sequences have been shown to efficiently inhibit translation both in vitro and in vivo (Pooga et al. (1998) Nature Biotechnology 16:857). Appropriate target regions for PNAs reside both within and outside of the AUG region. The identification of suitable PNA targets is achieved using an empirical determination of an optimal target based on the results obtained from mRNA walks (e.g., testing a series of oligonucleotides designed to be complementary to different regions of the targeted mRNA sequence). See Nielsen (1999) Current Opinion in Structural Biol. 9:353-357; Monia et al. (1996) Nat. Med. 2: 668-675. It should be noted that the observation that in vitro PNA/mRNA hybrids are not a substrate for RNase H does not exclude the possibility that PNA binding in vivo could mediate degradation of the targeted mRNA by an alternative catalytic mechanism of action. However, it is likely that the efficiency of the antisense activity of a PNA antisense agent may rely on a mechanism that is related to the stability of the resulting PNA/mRNA hybrid. PNA molecules are characterized by extremely desirable nucleic acid hybridization properties (e.g., high affinity and specificity) enabling them to form extremely stable duplex hybrids with complementary DNA, RNA, or PNA oligomer sequences. In fact, the sequence discrimination (i.e., specificity) of PNA/DNA binding has been systematically determined to be as high or even higher than that of DNA (Larsen et al. (1999) Biochem. Et Biophysica Acta 1489:159-166). In addition, the peptide (or amide) bonds in PNAs are sufficiently distinct from the alpha-amino acid peptide bonds present in protein to confer protease- and peptidase-resistance to PNA. Thus, PNA oligomers are highly stable in biological environments.

These inherent characteristics (e.g., high affinity, specificity, and biological stability) make PNA molecules attractive alternative agents for use as an antisense agent for the sequence-specific (i.e., based on specific hybridization) regulation of a target mRNA and its encoded protein, specifically CKIε or variant thereof. However, unlike other nucleic acid analogs, PNA molecules are not spontaneously taken up by all cell types. This limitation can be obviated by the use of a cell -penetrating transit peptide (e.g., transportan or antennapedia (pAntp). See, for example, Pooga et al. (1998) Nature Biotechnology 16: 877. It has recently been demonstrated that PNA- peptide conjugates are efficiently taken up by certain eukaryotic cells in vitro (Aldrian-Herrada et al. (1998) Nucleic Acids Res. 26(21):4910).

The synthesis of polynucleotide mimics contemplated for use in the methods of the present invention can be performed either with Boc-, Fmoc-, or -protected monomers according to conventional solid-phase peptide technologies and are purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using techniques that are well known to one of skill in the art. In addition, because PNA oligomers are synthesized by conventional peptide chemistry protocols, a peptide can be conjugated to a particular PNA oligomer thereby producing a PNA- peptide conjugate. For example, a peptide embodying a carrier moiety can be conjugated to a PNA oligomer to facilitate cellular uptake or membrane transport of the oligomer. Alternatively, PNA monomers and/or oligomers designed for regulation of a target RNA can be prepared by a commercial supplier.

Ribozymes may also be used in the methods of the present invention. A ribozyme is a bioengineered catalytic RNA molecule that possesses sequence-specific RNA cleavage activity. In theory, a ribozyme can be designed to specifically base pair with and cleave any given RNA target, thereby mediating specific gene inactivation, in this case the gene encoding human CKIε or variant thereof. Ribozymes are desirable sequence-specific regulators of gene expression because they inhibit translation by specifically cleaving and, thus, destroying the targeted mRNA sequence. The specificity of ribozyme binding and cleaving of target RNA has been demonstrated, and highly efficient intracellular cleavage (approaching 100%) has been reported for many cellular RNAs. The two types of ribozymes that have been most extensively studied are hammerhead and hairpin ribozymes. Each type of ribozyme has its own minimal target sequence requirement; hammerhead ribozymes require NUH (where N denotes any base, and H denotes A, C or U), and hairpin ribozymes require a GUC at the site of cleavage (James and Gibson (1998) Blood 91:371-382). To date, ribozymes have been used as therapeutic agents to target viral RNA in infectious diseases, oncogene expression in malignant tumors, and specific mutations in certain genetic disorders. Both hammerhead and hairpin ribozymes are currently being tested in human clinical trials. Either an expression vector comprising a transcriptional unit designed to direct the endogenous production of a ribozyme or an exogenous synthetic ribozyme can be administered to a cell or subject for the purpose of catalytically cleaving CKIε mRNA transcripts. The delivery of ribozyme genes provides for stable intracellular expression, and the promoter choice (e.g., inducible, repressible, or tissue-specific) offers the opportunity to confer temporal, cell-specific expression. In addition, the incorporation of a selectable marker into the vector also allows for the possibility of inducing high-level expression. Further, because typical hammerhead and hairpin ribozymes are usually less than 60 nucleotides in length, multiple ribozymes can be expressed from the same transcriptional unit. Synthetic ribozymes can be chemically stabilized with various base substitutions or 5' and 3' modifications. See, for example, Burlina et al. (1997) Bioorg. Med. Chem. 5:1999-2010; Earnshaw and Gait (1997) Antisense Nucleic Acid Drug Dev. 7:403-411; and Eckstein. (1997) Ciba Found. Symp. 209:207-212. One of skill in the art will recognize that the successful use of a ribozyme is dependent upon numerous factors such as target site selection and accessibility, efficiency of ribozyme gene delivery and expression, intracellular colocalization of the ribozyme and the target sequence, and in vivo stability of the ribozyme. The use of random ribozyme libraries in cells and cell extracts is the most direct approach to target identification. An alternative screening method for the identification of a suitable target sequence involves the binding of a library of DNA oligonucleotides designed to be complementary to potential ribozyme cleavage sites to native protein- associated RNA isolated from cellular extracts, followed by incubation with RNase H. Because RNase H cleaves the RNA component of a DNA/RNA hybrid, an investigator can utilize this technique to identify nucleotide sequences of the target RNA that are most accessible to hybridization by quantifying the cleaved fragments and assuming that the most accessible target sites will generate the strongest cleavage products. See, for example, Scherr and Rossi (1998) Nucleic Acids Res. 26:5079; Birikh et al. (1997) RNA 3:429. Standard techniques such as Northern gel, RNase protection, primer extension, and RT-PCR can be performed to analyze in vivo ribozyme function. One of skill in the art would acknowledge that RNA analysis alone is not conclusive because of the possibility that cleavage could occur during RNA extraction, and the assays described in the Examples below would also be necessary to confirm that the ribozymes confer the desired modulation of a TNF-α- mediated pathway. Kinase inhibitors can also be used in the methods of the invention. CKI kinase inhibitors include inositol hexasulphate, isoquinoline sulfonamide compounds, such as N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (also known as CKI7), and ribofuranosyl-benzimidazoles (Torres-Quantana et al. (2000) J. Dent. Res. 79:1794-1801; Chijawa et al. (1989) J. Biol. Chem. 264:4924-4927; Meggio et al. (1990) Eur. J. Biochem. 187:89-94). Selective inhibitors of CKI have been isolated by screening small molecule libraries. See, for example, Mashoon et al. (2000) J. Biol. Chem. 275:20052-20060, where the kinase inhibitor IC261 is disclosed. Methods for screening inhibitors are described in Mashoon et al. (2000), supra, herein incorporated by reference in its entirety. The present invention contemplates the use of any suitable selective CKI kinase inhibitor that is identified from the screening of such small molecule libraries.

Suitable inhibitors for use in the methods of the invention include, but are not limited to, Ν-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI 7) and 3- [(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (also known as IC261). See also Xu et al. (1996) Proc. Natl. Acad. Sci. 93 :6308-6313 ; Mashhoon et al. (2000) J. Biol. Chem. 275:20052-50060.

Thus, modulation of a TNF-α-mediated pathway in a cell can be achieved by contacting a cell with a kinase inhibitor whereby the kinase inhibitor inhibits the activity of CKIε in the cell. Kinase inhibitors can also be administered to a subject in need of modulation of a TNF-α-mediated pathway, whereby treatment of a TNF-α- mediated disorder is achieved.

Antibodies can also be used to inhibit CKIε activity, thereby modulating a TNF-α-mediated pathway. Antibodies can be prepared against any region of the CKIε polypeptide but preferably are prepared against the kinase domain. Antibodies can also be used to prevent binding of CKIε to its substrate or prevent the binding of CKIε to an up-stream molecule interacting with an activating CKIε. Antibodies selectively binding to CKIε polypeptide or variant thereof can be developed against the entire protein, or against regions thereof, such as the kinase region. Thus, antibodies to CKIε polypeptides include those raised against the native proteins or against variants thereof. An antibody is considered to selectively bind even if it also binds to other polypeptides not substantially homologous with the CKIε. Antibodies can be polyclonal or more preferably monoclonal. An intact antibody, or fragment thereof, can be used.

Accordingly, another aspect of the invention pertains to the use of anti-CKIε polyclonal and monoclonal antibodies that bind to CKIε or variant thereof, thereby inhibiting CKIε activity. Polyclonal anti-CKIε antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a CKIε. The anti- CKIε antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CKIε. At an appropriate time after immunization, e.g., when the anti-CKIε antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, NY), pp. 77-96), or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al. eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension in Biological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-CKIε antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CKIε to thereby isolate immunoglobulin library members that bind the

CKIε. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (\993) EMBOJ. 12:725-734. Additionally, recombinant anti- CKIε antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and nonhuman portions, which can be made using standard recombinant DNA techniques, can be used in the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication Nos. WO 86/101533 and WO 87/02671; European Patent Application Nos. 184,187, 171,496, 125,023, and 173,494; U.S. Patent Nos. 4,816,567 and 5,225,539; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al.

(\985) Nature 314:446-449; Shaw et al. (1988) J Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93; and U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. This technology is described by Jespers et al. (1994) Bio/Technology 12:899-903).

Thus, modulation of a TNF-α-mediated pathway can also be achieved through contacting a cell with anti-CKIε antibodies, where the antibodies disrupt the activity of CKIε. These antibodies can also be administered the antibodies to a subject, whereby modulation of a TNF-α-mediated pathway is useful in treatment of a TNF-α- mediated disorder.

The CKIε inhibitors described herein are useful for treating a subject having a pathology or condition associated with abnormal levels of TNF-α activity, exemplified by TNF-α induced apoptosis and inflammation responses activated by NF-κB activation. Abnormal levels may be in excess of, or less than levels present in a normal healthy subject, where such excess or diminished levels occur in a systemic, localized, or particular tissue type or location in the body. Exemplary tissue types include, but are not limited to, blood, lymph, CNS, liver, kidney, spleen, heart muscle or blood vessels, brain or spinal cord white matter or grey matter, cartilage, ligaments, tendons, lung, pancreas, ovary, testes, and prostrate. Increased or decreated TNF-α concentrations relative to normal levels can also be localized to specific regions or cells in the body, such as joints, nerve, blood vessel junctions, bones, specific tendons or ligaments, or sites of infection, such as bacterial or viral infections. Diseases that are treated with the CKIε inhibitors include, but are not limited to, rheumatoid arthritis, atherosclererosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneurits, asthma, inflammatory bowel disease, heliobacter pylori- associated gastritis, systemic inflammatory response syndrome, psoriasis, rheumatoid arthritis, cachexia in patients with advanced chronic heart failure (CHF), left ventricular dysfunction, myocardial dysfunction and cariomyocyte death in ischemia- reperfusion injury, sepsis, viral myocaritis, cardiac allograft rejection, heart failure syndromes, arryhthmogenic right ventricular dysplasia, sudden cardiac death, kipopolysaccharide-induced Jupffer-cell activation, septic shock, viral hepatitis, alcoholic hepatitis, fulminant hepatitis, autoimmune disorders of the nervous system, human demyelinating neuropathies, malignancies and carcinomas, such as those noted elsewhere herein, and cachesia associated with cancer.

The CKIε inhibitors described herein are delivered to a cell derived from a subject or administered to a subject in need thereof in an amount sufficient to modulate a TNF-α-mediated pathway, particularly TNF-α-induced apoptosis or TNF- α-induced activation of NF-κB. Efficacy of a particular CKIε inhibitor in modulating the TNF-α-mediated pathway of interest, and determination of amounts sufficient to achieve this objective, are readily assessed using bioassays known in the art. Such bioassays for monitoring the efficacy of a CKIε inhibitor in modulating a TNF-α- mediated pathway include in vitro and in vivo assays. Suitable in vitro assays can include a TNF cytotoxicity assay, such as a radioimmunoassay that determines a decrease in cell death by contact with TNF, such as human TNF-α in isolated or recombinant form, wherein the concurrent presence of a CKIε inhibitor reduces the degree or rate of cell death. Cell death can be determined using inhibitor dose (ID) 50 values, which represent the concentration of a CKIε inhibitor that decrease the cell death rate by 50%. See also the assays described in the Examples below. Other suitable in vitro and in vivo assays are known in the art. See, for example, the assays described in U.S. Patent No. 5,919,452, herein incorporated by reference. Thus, modulation of TNF-α-induced apoptosis in a cell can be monitored using the bioassays described in the examples below. Likewise, modulation of TNF-α-induced activation of NF-κB in a cell can be monitored using one or more assays. For instance, the degradation of transcription factor IkB induced by TNF-α can be monitored using antibodies specific to IkB, such as New England Biolabs IkB-a antibody (Catalog No. Ab#9242), using techniques known to those of skill in the art. For example, degradation could be monitored by using gel-electrophoresis, followed by detection of the smaller degradation product with the antibodies specific to IkB. Alternatively, a luciferase reporter gene assay, such as PathDetect® Cis-Reporting Systems by Stratagene (Catalog No. 219077), can be used to monitor activation of NF-κB induced by TNF-α. In this system, the luciferase gene is driven by a promotor having a basic promotor element (TATA box) plus a cis-enhancer element with an NF-κB binding site. Increased expression of luciferase indicates the presence of active NF-κB. Finally, quantitative PCR could also be used to detect mRNA of genes known to be induced by NF-κB, such as Cox-2 (see page 3, above).

Generally, delivery of antisense agents for both ex vivo and in vitro cell applications can be accomplished by any suitable delivery method known in the art. Exemplary methods include, but are not limited to, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

When the methods of the invention are directed to treatment or prevention of a TNF-α-mediated disorder in a subject, a therapeutical ly effective amount of the CKIε inhibitor will be administered. By "therapeutically effective amount" is intended an amount of a CKIε inhibitor sufficient to exhibit a detectable preventive, ameliorative, curative or other therapeutic effect with respect to treatment or prevention of a TNF- α-mediated disorder. The effect may include, for example, treatment, amelioration, or prevention of any physical or biochemical condition, for example, including but not limited to rheumatoid arthritis, atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneurits, asthma, inflammatory bowel disease, helicobacter pylori-associated gastritis, systemic inflammatory response syndrome, psoriasis, rheumatoid arthritis, cachexia in patients with advanced chronic heart failure (CHF), left ventricular dysfunction, myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury, sepsis, viral myocarditis, cardiac allo graft rejection, heart failure syndromes, arryhthmogenic right ventricular dysplasia, sudden cardiac death, lipopolysaccharide-induced Kupffer-cell activation, septic shock, viral hepatitis, alcoholic hepatitis, fulminant hepatitis, autoimmune disorders of the nervous system, and human demyelinating neuropathies. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. For purposes of the present invention, an effective dose of an antisense agent is about 0.01 mg/ kg to about 50 mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered. Where the agent is a ribozyme, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered. Where the CKIε inhibitor is an anti-CKIε antibody, treatment of a subject with a therapeutically effective amount of a antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. Where the CKIε inhibitor is a kinase inhibitor, the effective dose will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the CKIε inhibitor to have. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. .

The CKIε inhibitors are formulated as pharmaceutical compositions for administering to a subject in need of modulation of a TNF-α-mediated pathway, whereby treatment or prevention of a TNF-α-mediated disorder is effected. In addition to a therapeutically effective dose or amount of the particular CKIε inhibitor, a pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991). Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

When administration is for the purpose of treatment, administration may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the substance is provided in advance of any symptom. The prophylactic administration of the substance serves to prevent or attenuate any subsequent symptom. When provided therapeutically, the substance is provided at (or shortly after) the onset of a symptom. The therapeutic administration of the substance serves to attenuate any actual symptom. The pharmaceutical compositions comprising the therapeutically effective amount of CKIε inhibitor can be administered directly to a subject by any means that enables the active therapeutic agent to reach the agent's site of action in the body of the subject. For example, where anti-CKIε antibodies are utilized, administration is carried out such that the antibodies can reach and bind with the CKIε in target cells expressing this kinase protein. Administration can be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into an organ. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a site of inflammation is located, and the therapeutic composition comprising a therapeutically effective amount of a CKIε inhibitor is injected several times in several different locations within the inflamed area. Alternatively, the CKIε inhibitor composition is directly administered to the surface of the inflamed area, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the aforementioned delivery methods.

Receptor-mediated targeted delivery of therapeutic compositions described herein to specific tissues can also used in the methods of the present invention.

Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al. (1993) Trends in Biotechnol. 11:202-205; Chiou et α/. (1994) Gene Therapeutics: Methods and Applications of Direct Gene Transfer (ed. Wolff); Wu and Wu (1988) J. Biol. Chem. 263:621-24; Wu et al. (1994) J. Biol. Chem. 269:542-46; Zenke et al. (1990) Proc. Natl. Acad. Sci. USA 87:3655-59; Wu et al. (1991) J. Biol. Chem. 266:338-42. Preferably, receptor-mediated targeted delivery of therapeutic compositions containing antibodies of the invention is used to deliver the antibodies to specific tissue.

The therapeutic polynucleo tides of the present invention may be utilized in gene delivery vehicles. The gene delivery vehicle may be of viral or non- viral origin (see generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 1:185-193; and Kaplitt (1994) Nature Genetics 6:148-153). Gene therapy vehicles for delivery of constructs including an antisense sequence or coding sequence for a ribozyme targeting the CKIε gene or variant thereof can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches.

Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

Methods of the present invention can employ recombinant retroviruses that are constructed to carry or express a selected CKIε sequence-specific inhibitor of interest, such as an antisense sequence or ribozyme targeting CKIε expression. Retrovirus vectors that can be employed include those described in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 93/10218; Vile and Hart (1993) Cancer Res. 53:3860-3864; Vile and Hart (1993) Cancer Res. 53:962-967; Ram et al. (1993) Cancer Res. 53:83-88;

Takamiya et al. (1992) J. Neurosci. Res. 33:493-503; Baba et al. (1993) J. Neurosurg. 79:729-735; U.S. Patent No. 4,777,127; GB Patent No. 2,200,651; and EP 0 345 242. Preferred recombinant retroviruses include those described in WO 91/02805.

Packaging cell lines suitable for use with the above -described retroviral vector constructs may be readily prepared (see PCT publications WO 95/30763 and WO

92/05266), and used to create producer cell lines (also termed vector cell lines) for the production of recombinant vector particles. Within particularly preferred embodiments of the invention, packaging cell lines are made from human (such as HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that can survive inactivation in human serum.

Alphavirus-based vectors that can function as gene delivery vehicles can be used in the methods of the present invention. Such vectors can be constructed from a wide variety of alphaviruses, including, for example, Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532). Representative examples of such vector systems include those described in U.S. Patent Nos. 5,091,309; 5,217,879; and 5,185,440; and PCT Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO 95/07994.

Gene delivery vehicles for use in the methods of the present invention can also employ parvo virus such as adeno-associated virus (AAV) vectors. Representative examples include the AAV vectors disclosed by Srivastava in WO 93/09239, Samulski et al. (1989) J. Virol. 63:3822-3828; Mendelson et al. (1988) Virol. 166:154-165; and Flotte et al, (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617. Representative examples of adenoviral vectors include those described by Berkner (1988) Biotechniques 6:616-627; Rosenfeld et al. (1991) Science 252:431- 434; WO 93/19191; Kolls et al. (1994) Proc. Natl. Acad. Sci USA 91:215-219; Kass- Eisler et al. (1993) Proc. Natl. Acad. Sci USA 90:11498-11502; Guzman et al. (1993) Circulation 88:2838-2848; Guzman et al. (1993) Cir. Res. 73:1202-1207; Zabner et al. (1993) Cell 75:207-216; Li et al. (1993) Hum. Gene Ther. 4:403-409; Cailaud et al. (1993) Eur. J. Neurosci. 5:1287-1291; Vincent et al. (1993) Nat. Genet. 5:130- 134; Jaffe et al. (1992) Nat. Genet. 1 :372-378; and Levrero et al. (1991) Gene 101 : 195-202. Exemplary adenoviral gene therapy vectors employable in this invention also include those described in WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655. Administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed.

Other gene delivery vehicles and methods may be employed, including polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel (1992) Hum. Gene Ther. 3:147-154; ligand-linked DNA, for example see Wu (1989) Biol. Chem. 264:16985-16987; eukaryotic cell delivery vehicles cells, for example see U.S. Serial No. 08/240,030, filed May 9, 1994, and U.S. Serial No. 08/404,796; deposition of photo polymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; ionizing radiation as described in U.S. Patent No. 5,206,152 and in WO 92/11033; nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol. 14:2411-2418, and in Woffendin (1994) Proc. Natl. Acad. Sci. USA 91 :1581-1585.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Nos. WO 95/13796, WO 94/23697, and WO 91/14445; and EP No. 0 524 968.

Further non- viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al. (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photo polymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; and use of ionizing radiation for activating transferred gene, as described in U.S. Patent No. 5,206,152 and PCT No. WO 92/11033.

Therapeutic compositions containing antisense agents can be administered in a range of about 100 mg to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense agents, or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a site of inflammation, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Application Serial No. 08/869,309, which is expressly incorporated herein. The invention is further illustrated by the following examples, which should not be construed as limiting.

EXPERIMENTAL

Example 1: Effect of CKIε Antisense Oligonucleotides on

Induction of Apoptosis of Cancer Cells Protocol

Colon cancer cell lines were transfected with either a CKIε antisense chimeric oligonucleotide or a control oligonucleotide (the antisense oligonucleotide in reverse orientation). Cells were transfected by complexing the oligonucleotides with lipitoids to facilitate their uptake by the cell. See, e.g., WO 01/16306. The antisense chimeric oligonucleotides were constructed with a 5' 0-methyl thymidine block and consisted of a core region of 9 phosphorothioate-linked deoxribonucleo tides flanked on either end by 8 phosphodiester-linked 2'-0-methyl ribonucleotides. The chimeric oligonucleotide was antisense to the target sequence set forth in SEQ ID NO:18. The cells were then subjected to one of the following apoptosis-inducing events: exposure to TNF-α and cycloheximide (10 μg/ml), staurosporine, or ultraviolet radiation. DNA fragmentation, indicative of apoptosis, was then measured using the Roche™ Cell Death Detection ELISA^PLUS kit, Cat. No. 1 920 685. The manufacturer's assay instructions were followed. Results Figures 1, 2, and 3 show the results of each of these apoptosis-inducing events.

As can be seen, DNA fragmentation increased 2-fold in cells receiving the control oligonucleotide after exposure to 25 ng/ml or 250 ng/ml of TNF-α and cyclosporine (10 μg/ml). By contrast, DNA fragmentation was lower in cells receiving the antisense oligonucleotide. The amount of DNA fragmentation in cells receiving the antisense oligonucleotide exposed to 25 ng/ml of TNF-α was indistinguishable from that observed in cells that were not exposed to TNF-α; only at 250 ng/ml of TNF-α was an increase in DNA fragmentation observed (Figure 1). In the staurosporine and ultraviolet induced apoptosis experiments, DNA fragmentation increased with increasing staurosporine and ultraviolet radiation levels by roughly the same amount regardless of whether the cell had been transfected with the antisense oligonucleotide or the control oligonucleotide (Figures 2 and 3).

These results demonstrate that CKIε antisense oligonucleotides selectively protect a cell from TNF-α-induced apoptosis.

In a second experiment, colon cancer cell lines were transfected with either a CKIε antisense oligonucleotide or a control oligonucleotide (the antisense oligonucleotide in reverse orientation) as noted above. The cells were then exposed to 0, 2, or 20 ng/ml of TNF-α causing the induction of apoptosis and eventual cellular necrosis. Apoptosis and necrosis were measured using the Roche™ Annexin- V- FLOUS Staining Kit, Cat. No. 1 858 777. The manufacturer's assay instructions were followed. Cells transfected with the control oligonucleotide showed roughly double the amount of eventual necrosis as compared with cells transfected with the CKIε oligonucleotide. These results confirm the CKIε antisense oligonucleotides confer a protective effect from TNF-α-induced apoptosis.

Example 2: Effect of CKIε Inhibitors on NF-κB Activation Cells are contacted with a CKIε-inhibiting agent selected from the following embodiments: CKIε antisense molecule, CKIε kinase inhibitor, ribozyme targeting mRNA encoding CKIε, or anti-CKIε antibody. The effect on TNF-α-induced apoptosis is monitored by DNA fragmentation or Annexin V staining using the Roche™ Cell Death Detection ELISA^PUJS kit, Cat. No. 1 920 685 or the Roche™ Annexin- V-FLOUS Staining Kit, Cat. No. 1 858 777, as described above. Activation of NF-κB pathway is monitored indirectly by detecting the degradation of IkB or by using quantitative PCR to detect mRNA of genes known to be induced by NF-κB, and directly by using a luciferase reporter gene assay.

Example 3 : Effect of CKIε Inhibitors on a TNF-α-Mediated Disorder A subject with a TNF-α-mediated disorder is administered a CKIε-inhibiting agent selected from the following embodiments: CKIε antisense molecule, CKIε kinase inhibitor, ribozyme targeting mRNA encoding CKIε, or anti-CKIε antibody. The effect on the TNFα-related disease is monitored clinically. Subjects receiving CKIε inhibitor exhibit amelioration of the symptoms associated with the TNF-α- mediated disorder relative to control subjects.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention as disclosed herein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for modulating a tumor necrosis factor-alpha (TNF-α)- mediated pathway in a cell, said method comprising contacting said cell with a composition comprising an amount of an inhibitor of casein kinase I epsilon (CKIε) sufficient to modulate said TNF-α-mediated pathway in said cell, wherein said CKIε is a polypeptide selected from the group consisting of: a) the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4; and b) a polypeptide having CKIε activity and which shares at least about 80% sequence identity with the polypeptide set forth in SEQ ID NO:2 or SEQ ID

NO:4.

2. The method of claim 1, wherein said inhibitor is selected from the group consisting of a kinase inhibitor, an antisense agent that binds to a target sequence within a nucleic acid molecule encoding said CKIε, and a ribozyme polynucleotide that cleaves a mRNA encoding said CKIε.

3. The method of claim 1, wherein said TNF-α-mediated pathway is selected from the group consisting of TNF-α-induced apoptosis and TNF-α-induced nuclear factor kappa-B (NF-κB) activation.

4. The method of claim 3, wherein said inhibitor is selected from the group consisting of a kinase inhibitor, an antisense agent that binds to a target sequence within a nucleic acid molecule encoding said CKIε, and a ribozyme polynucleotide that cleaves a mRNA encoding said CKIε.

5. The method of claim 4, wherein said kinase inhibitor is selected from the group consisting of CKI7 and IC261.

6. The method of claim 4, wherein said antisense agent is selected from the group consisting of an oligonucleotide, a chemically modified oligonucleotide, and a peptide nucleic acid molecule.

7. The method of claim 6, wherein said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, and wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO:3.

8. The method of claim 7, wherein said antisense agent is the oligonucleotide set forth in SEQ ED NO:25, SEQ ID NO:26, or SEQ ID NO:27.

9. The method of claim 4, wherein said inhibitor is said ribozyme polynucleotide and said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein SEQ ID NO:2 is encoded by the nucleotide sequence set forth in SEQ ID NO.T and SEQ ID NO:4 is encoded by the nucleotide sequence set forth in SEQ ID NO-.3.

10. A method for modulating a TNF-α-mediated pathway in a subject, said method comprising administering to said subject a composition comprising an inhibitor of CKIε in an amount sufficient to modulate said TNF-α-mediated pathway in said subject, wherein said CKIε is a polypeptide selected from the group consisting of: a) the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4; and b) a polypeptide having CKIε activity and which shares at least about 80% sequence identity with the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4.

11. The method of claim 10 wherein said inhibitor is selected from the group consisting of a kinase inhibitor, an antisense agent that binds to a target sequence within a nucleic acid molecule encoding said CKIε, and a ribozyme polynucleotide that cleaves a mRNA encoding said CKIε.

12. The method of claim 10, wherein said TNF-α-mediated pathway is selected from the group consisting of TNF-α-induced apoptosis and TNF-α-induced nuclear factor kappa-B (NF-κB) activation.

13. The method of claim 12, wherein said inhibitor is selected from the group consisting of a kinase inhibitor, an antisense agent that binds to a target sequence within a nucleic acid molecule encoding said CKIε, and a ribozyme polynucleotide that cleaves a mRNA encoding said CKIε.

14. The method of claim 13, wherein said kinase inhibitor is selected from the group consisting of CKI7 and IC261.

15. The method of claim 13, wherein said antisense agent is selected from the group consisting of an oligonucleotide, a chemically modified oligonucleotide, and a peptide nucleic acid molecule.

16. The method of claim 15, wherein said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, and wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO:3.

17. The method of claim 16, wherein said antisense agent is the oligonucleotide set forth in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

18. The method of claim 13, wherein said inhibitor is said ribozyme polynucleotide and said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein SEQ ID NO:2 is encoded by the nucleotide sequence set forth in SEQ ID NO:l and SEQ ID NO:4 is encoded by the nucleotide sequence set forth in SEQ ID NO.3.

19. A method for treating a TNF-α-mediated disorder in a subject, said method comprising administering to said subject a composition comprising a therapeutically effective amount of an inhibitor of CKIε, wherein said CKIε is a polypeptide selected from the group consisting of: a) the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4; and b) a polypeptide having CKIε activity and which shares at least about 80% sequence identity with the polypeptide set forth in SEQ ID NO:2 or SEQ ID

NO:4.

20. The method of claim 19, wherein said TNF-α-mediated disorder is selected from the group consisting of chronic heart failure, ischemia, arrhythmogenic right ventricular dysplasia, viral myocarditis, sudden cardiac death, viral hepatitis, alcoholic hepatitis, sepsis, septic shock, psoriasis, cardiovascular disease, hepatic toxicity, multiple sclerosis, rheumatoid arthritis, Chron's disease, malignancy, and cachexia associated with cancer.

21. The method of claim 19, wherein said inhibitor is selected from the group consisting of a kinase inhibitor, an antisense agent that binds to a target sequence within a nucleic acid molecule encoding said CKIε, and a ribozyme polynucleotide that cleaves a mRNA encoding said CKIε.

22. The method of claim 21, wherein said kinase inhibitor is selected from the group consisting of CKI7 and IC261.

23. The method of claim 21, wherein said antisense agent is selected from the group consisting of an oligonucleotide, a chemically modified oligonucleotide, and a peptide nucleic acid molecule.

24. The method of claim 23, wherein said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, and wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO:3.

25. The method of claim 24, wherein said antisense agent is the oligonucleotide set forth in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

26. The method of claim 21, wherein said inhibitor is said ribozyme polynucleotide and said CKIε is the polypeptide set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein SEQ ID NO:2 is encoded by the nucleotide sequence set forth in SEQ ID NO:l and SEQ ID NO:4 is encoded by the nucleotide sequence set forth in SEQ ID NO.3.