WO2012161806A1

WO2012161806A1 - Modulation of stat3 expression

Info

Publication number: WO2012161806A1
Application number: PCT/US2012/026636
Authority: WO
Inventors: Brett P. Monia; Robert A. Macleod; Youngsoo Kim
Original assignee: Isis Pharmaceuticals Inc
Current assignee: Ionis Pharmaceuticals Inc
Priority date: 2011-02-25
Filing date: 2012-02-24
Publication date: 2012-11-29
Anticipated expiration: 2013-08-25

Abstract

Provided herein are methods, compounds, and compositions for reducing expression of a STAT3 mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for reducing inflammation in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of inflammatory disease such as an autoimmune disease, infectious disease or cancer or a symptom thereof.

Description

MODULATION OF STAT3 EXPRESSION

Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The

Sequence Listing is provided as a file entitled BIOL0140WOSEQ.txt, created on February 23, 2012 which is 226 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Field of the Invention

Provided herein are methods, compounds, and compositions for reducing expression of STAT3 mRNA and protein in an animal. Also, provided herein are methods, compounds, and compositions having a STAT3 inhibitor for reducing STAT3 related inflammatory diseases or conditions in an animal. Such methods, compounds, and compositions are useful, for example, to treat, prevent, delay or ameliorate any one or more of inflammatory diseases, cancers and infectious diseases, or a symptom thereof, in an animal.

Background The STAT (signal transducers and activators of transcription) family of proteins are DNA-binding proteins that play a dual role in signal transduction and activation of transcription. Presently, there are multiple distinct members of the STAT family (e.g, STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6). The activities of the STATs are modulated by various cytokines and mitogenic stimuli. Events mediated by cytokines through STAT activation include cell proliferation and differentiation and prevention of apoptosis.

STAT3 (also known as acute phase response factor (APRF)), in particular, has been found to be responsive to interleukin-6 (EL-6) as well as epidermal growth factor (EGF) (Darnell, Jr., J.E., et al, Science, 1994, 264, 1415-1421). In addition, STAT3 has been found to have an important role in signal transduction by interferons (Yang, C.-H., et al, Proc. Natl. Acad. Sci. USA, 1998, 95, 5568-5572). Evidence exists suggesting that STAT3 may be regulated by the MAPK pathway. ERK2 induces serine phosphorylation and also associates with STAT3 (Jain, N., et al, Oncogene, 1998, 17, 3157-3167).

STAT3 is expressed in most cell types (Zhong, Z., et al, Proc. Natl Acad. Sci. USA, 1994, 91, 4806-4810). It induces the expression of genes involved in response to tissue injury and inflammation.

STAT3 has also been shown to prevent apoptosis through the expression of bcl-2 (Fukada, T., et al, Immunity, 1996, 5, 449-460).

STAT3 may play a role in several forms of cancer, including colon cancer, myeloma, breast carcinomas, prostate cancer, brain tumors (e.g., glioblastomas and medulloblastomas), head and neck carcinomas, melanoma, leukemias and lymphomas, chronic myelogenous leukemia and multiple myeloma (Niu et al, Cancer Res., 1999, 59, 5059-5063; Sartor, C.I., et al, Cancer Res., 1997 57, 978-98^' Garcia, R., et al., Cell Growth and Differentiation, 1997, 8, 1267-1276; Cattaneo, E., et al., Anticancer Res., 1998, 18, 2381-2387; Corvinus et al, Neoplasia, 2005, 7(6): 545-555).

STAT3 may play a role in autoimmune diseases including rheumatoid arthritis (Sengupta, T.K., et al, J. Exp. Med., 1995, 181, 1015-1025; Wang, F., et al, J. Exp. Med., 1995, 182, 1825-1831).

STAT3 may play a role in infectious diseases (Lin and Bost, Biochemical and Biophysical Research Communications, 2004, 321, 828-834; Matsuzaki et al., J Immunol, 2006, 177, 527-537) and endotoxin-induced inflammation (Hosoi et al, Brain Res. 2004, 1023(1), 48-53; Kano et al, JEM, 2003, 198(10), 1517-1525.

There are currently several approaches for inhibiting STAT3 expression. US Patent Nos. 5,719,042 and 5,844,082 to Akira, S. and Kishimoto, T. disclose the use of inhibitors of APRF, including antibodies, antisense nucleic acids and ribozymes for the treatment of EL-6 associated diseases, such as inflammatory diseases, leukemia, and cancer. Schreiber, R.D., et al, in US Patent Nos. 5,731,155; 5,582,999; and

5,463,023, disclose methods of inhibiting transcriptional activation using short peptides that bind p91.

Darnell, J.E., et al, in US Patent No. 5,716,622, disclose peptides containing the DNA binding domain of STATs, chimeric proteins containing the DNA binding domain, and antibodies to STATs for inhibiting STAT transcriptional activation. US Patent Nos. 6,159,694, 6,727,064, 7,098,192 and 7,307,069 to Karras disclose oligonucleotides targeting STAT3 and methods for reducing STAT3 expression. The use of an antisense oligonucleotide targeted to the translation start region of human STAT3 has been disclosed (Grandis, J. R., et al, J. Clin. Invest, 1998, 102, 1385-1392). In this report, a phosphorothioate

oligodeoxynucleotide complementary to the translation start region of STAT3 inhibited TGF-β stimulated cell growth mediated by the epidermal growth factor receptor (EGFR).

However, there remains an unmet need for therapeutic compositions and methods targeting expression of STAT3, and preventing, ameliorating or treating inflammatory diseases associated therewith.

Summary of the Invention

Provided herein are methods of preventing, ameliorating or treating an inflammatory disease in an animal comprising administering to the animal a STAT3 inhibitor. In certain embodiments, the STAT3 inhibitor is an antisense compound targeting STAT3. In certain embodiments, the antisense compound is a modified oligonucleotide. In certain embodiments the STAT3 inhibitor reduces STAT3 expression in the animal thereby preventing, ameliorating or treating inflammatory disease in the animal. In certain embodiments, the compound is a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3. Certain embodiments provide a method of reducing CRP levels in an animal comprising administering to the animal a compound targeted to STAT3, wherein the level of CRP is reduced in the animal.

Certain embodiments provide a method of reducing a cytokine level in an animal comprising administering to the animal a compound targeted to STAT3, wherein the cytokine level is reduced in the animal.

Certain embodiments provide a method for treating an animal with inflammatory disease comprising: a) identifying said animal with inflammatory disease, and b) administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 16 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NO: 1 -18 as measured over the entirety of said modified oligonucleotide.

Certain embodiments provide use of a STAT3 inhibitor to prevent, ameliorate or treat an

inflammatory disease in an animal.

Certain embodiments provide use of a STAT3 inhibitor to reduce cytokine levels in an animal Certain embodiments provide use of a STAT3 inhibitor to reduce CRP levels in an animal. Certain embodiments provide use of a STAT3 inhibitor to reduce cytokine levels in an animal.

Detailed Description of the Invention

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

"2'-0-methoxyethyl" (also 2'-MOE, 2'-0-(2-methoxyethyl and 2'-0(CH₂)₂-OCH₃) refers to an O- methoxy-ethyl modification of the 2' position of a furosyl ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.

"2'-0-methoxyethyl nucleotide" means a nucleotide comprising a 2'-0-methoxyethyl modified sugar moiety.

"3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3 '-most nucleotide of a particular antisense compound.

"5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5 '-most nucleotide of a particular antisense compound.

"5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. A 5- methylcytosine is a modified nucleobase.

"About" means within ±10% of a value. For example, if it is stated, "the compounds affected at least about 70% inhibition of STAT3", it is implied that the STAT31evels are inhibited within a range of 63% and 77%.

"Active pharmaceutical agent" means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to STAT3 is an active pharmaceutical agent.

"Active target region" or "target region" means a region to which one or more active antisense compounds is targeted. "Active antisense compounds" means antisense compounds that reduce target nucleic acid levels or protein levels.

"Administered concomitantly" refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

"Administering" means providing an agent to an animal, and includes, but is not limited to, administering by a medical professional and self-administering.

"Agent" means an active substance that can provide a therapeutic benefit when administered to an animal. "First Agent" means a therapeutic compound of the invention. For example, a first agent can be an antisense oligonucleotide targeting STAT3. "Second agent" means a second therapeutic compound of the invention (e.g. a second antisense oligonucleotide targeting STAT3) and/or a non-STAT3 therapeutic compound.

"Amelioration" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators can be determined by subjective or objective measures, which are known to those skilled in the art.

"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

"Anti-inflammatory drug" refers to compounds used to decrease inflammation locally or

systemically. Anti-inflammatory drugs include steroids, NSAIDS (nonsteroidal anti-inflammatory drugs), therapeutic antibodies against TNFa (e.g., infliximab, etanercept, adalimumab, etc.) and against IL-6 (e.g., tocilizumab), chemotherapeutic drugs and anti-infection drugs. NSAIDS include aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like. Anti-infection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs

"Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

"Antisense compound" means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. As used herein, the term "antisense compound" encompasses pharmaceutically acceptable derivatives of the compounds described herein

"Antisense inhibition" means the reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to the target nucleic acid levels or target protein levels in the absence of the antisense compound.

"Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. As used herein, the term "antisense oligonucleotide" encompasses pharmaceutically acceptable derivatives of the

oligonucleotides described herein

"Bicyclic sugar" means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

"Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

"cEt" or "constrained ethyl" refers to a bicyclic nucleoside having a furanosyl sugar that comprises a methyl(methyleneoxy) (4'-CH(CH₃)-0-2') bridge between the 4' and the 2' carbon atoms. "C-reactive protein" or "CRP" is protein that is raised when there is inflammation present in an animal.

"Cap structure" or "terminal cap moiety" means chemical modifications, which have been incorporated at either terminus of an antisense compound.

"Cardiovascular disorder" refers to a group of conditions related to the heart, blood vessels, or the circulation. Examples of cardiovascular diseases include, but are not limited to, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease (stroke), coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, and hypercholesterolemia.

"Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'- O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0- methoxyethyl modifications.

"Chemotherapeutic agents" agents are drugs used to prevent, ameliorate or treat cancer or a symptom of cancer. Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5- fiuorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al, eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5- FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide) or a combination thereof.

"Chimeric antisense compound" means an antisense compound that has at least two chemically distinct regions, each region can include a plurality of subunits.

"Co-administration" means administration of two or more agents to an individual. The two or more agents can be in a single pharmaceutical composition, or can be in separate pharmaceutical compositions. Each of the two or more agents can be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration. "Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, complementarity between the first and second nucleic acid may be between two DNA strands, between two RNA strands, or between a DNA and an R A strand. In certain embodiments, some of the nucleobases on one strand are matched to a complementary hydrogen bonding base on the other strand. In certain embodiments, all of the nucleobases on one strand are matched to a complementary hydrogen bonding base on the other strand. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid. In certain such embodiments, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.

"Contiguous nucleobases" means nucleobases immediately adjacent to each other.

"Comprise," "comprises" and "comprising" are to be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

"Cross-reactive" means an oligomeric compound targeting one nucleic acid sequence can hybridize to a different nucleic acid sequence. For example, in some instances an antisense oligonucleotide targeting human STAT3 can cross-react with a murine STAT3. Whether an oligomeric compound cross-reacts with a nucleic acid sequence other than its designated target depends on the degree of complementarity the compound has with the non-target nucleic acid sequence. The higher the complementarity between the oligomeric compound and the non-target nucleic acid, the more likely the oligomeric compound will cross- react with the nucleic acid.

"Cure" means a method that restores health or a prescribed treatment for an illness.

"Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

"Diluent" means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.

"Disease modifying drug" refers to any agent that modifies the symptoms and/or progression associated with an inflammatory disease, disorder or condition, including autoimmune diseases (e.g. arthritis, colitis or diabetes), trauma or surgery-related disorders, sepsis, allergic inflammation and asthma. Disease modifying drugs modify one or more of the symptoms and/or disease progression associated with these diseases, disorders or conditions.

"Dosage unit" means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense

oligonucleotide. "Dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month. Doses can be expressed, for example, as mg/kg.

"Duration" means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.

"Effective amount" or "therapeutically effective amount" means the amount of active

pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

"Efficacy" means the ability to produce a desired effect.

"Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.

"Fully complementary" or "100% complementary" means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid. In certain such embodiments, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.

"Gapmer" means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region can be referred to as a "gap segment" and the external regions can be referred to as "wing segments."

"Gap-widened" means a chimeric antisense compound having a gap segment of 12 or more contiguous 2'-deoxyribonucleosides positioned between and immediately adjacent to 5' and 3' wing segments having from one to six nucleosides.

"Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target. "Identifying" or "selecting an animal with an inflammatory disease" means identifying or selecting a subject having been identified as having an inflammatory disease or disorder or identifying or selecting a subject having any symptom of an inflammatory disease or disorder.

"Induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g. denote quantitative differences between two states. For example, "an amount effective to inhibit the activity or expression of

STAT3" means that the level of activity or expression of STAT3 in a treated sample will differ from the level of STAT3 activity or expression in untreated cells. Such terms are applied to, for example, levels of expression, and levels of activity.

"Inhibiting the expression or activity" refers to a reduction, blockade of the expression or activity of the target and does not necessarily indicate a total elimination of expression or activity.

"Immediately adjacent" means there are no intervening elements between the immediately adjacent elements. For example, between regions, segments, nucleotides and/or nucleosides.

"Individual" or "subject" or "animal" means a human or non-human animal selected for treatment or therapy.

"Inflammation" refers to a complex biological response of a body to a stimulus (e.g., a pathogen, cellular damage or an irritant). Clinical signs of inflammation include increased redness (rubor), temperature {color), swelling (tumor), pain (dolor) and/or loss of function (functio laesa) in a tissue. Inflammation can be local (e.g., vascular inflammation) or systemic. Inflammation, when prolonged, can lead to an inflammatory disease or disorder. Factors elicited during an inflammatory reaction include CRP, pro-inflammatory cytokines (e.g., TNF-a, IL-1 (e.g., IL-1 β), IL-4, EL-5, IL-6, INF-γ, MCP-1), cellular migration (e.g., monocytes, macrophages, lymphocytes, plasma cells) and serum proteins (e.g., serum amyloid A (SAA) and serum amyloid P (SAP)). Pro-inflammatory cytokine and CRP levels have been linked to various types of inflammatory diseases or conditions. For example, changes in cytokine and/or CRP levels has been linked to infection (Hotoura et al., Scand J Immunol. 2011, 73(3), 250-5; Chundadze et al, Clin Biochem. 2010, 43(13- 14), 1060-3 ; Sage et al, Int J STD AIDS. 2010, 21 (4), 288-92), cardiovascular disease (Gotto, Am J Cardiol, 2007, 99(5), 718-725; Eisenhardt et al, Trends Cardiovasc Med. 2009, 19(7), 232-7), trauma (Sogut et al, J Int Med Res. 2010, 38(5), 1708-20.), burns (van de Goot et al, J Burn Care Res. 2009, 30(2), 274-80), surgery (Honsawek et al, Int Orthop. 2011, 35(1), 31-5), autoimmune diseases (Nalesnik et al, Med Glas Ljek komore Zenicko-doboj kantona. 2011, 8(1), 163-168), atopic conditions (Pellizzaro and Heuertz, Clin Lab Sci. 2010, 23(4), 223-Ί), cancer (Chundadze et al, Clin Biochem. 2010, 43(13-14), 1060-3; Wang and Sun, Chang GungMed J. 2009, 32(5), 471-82), diabetes (Goldberg, J Clin Endocrinol Metab. 2009, 94(9), 3171 -82), anemia (Chawla and Krishnan, Hemodial Int. 2009, 13(2), 222-34), depression (Howren et al, Psychosom Med. 2009, 71(2), 171-86) and the like.

"Inflammatory response" refers to any disease, disorder or condition related to inflammation in an animal. Examples of inflammatory responses include an immune response by the body of the animal to clear the injury or stimulus responsible for initiating the inflammatory response. Alternatively, an inflammatory response can be initiated in the body even when no known injury or stimulus is found such as in autoimmune diseases. Inflammation can be mediated by a Thl or a Th2 response. Thl and Th2 responses include production of selective cytokines and cellular migration or recruitment to the inflammatory site. Cell types that can migrate to an inflammatory site include, but are not limited to, eosinophils and macrophages. Thl cytokines include, but are not limited to IL-1, EL-6, TNFoc, INFy and keratinocyte chemoattractanct (KC). Th2 cytokines include, but are not limited to, IL-4 and IL-5. A decrease in cytokine(s) level or cellular migration can be an indication of decreased inflammation. Accordingly, cytokine level or cellular migration can be a marker for certain types of inflammation such as Thl or Th2 mediated inflammation.

"Inflammatory disorder" or "inflammatory disease" refers to a condition characterized by

inflammation in a cell, tissue or body. Inflammatory diseases and disorders include, but are not limited to, atopic conditions (e.g., hypersensitivities such as allergies or asthma), autoimmune disease (e.g., rheumatoid arthritis, lupus, multiple sclerosis), cancer, diabetes, inflammatory bowel disease (IBD) or infectious disease.

"Inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity of a RNA or protein and does not necessarily indicate a total elimination of expression or activity.

"Internucleoside linkage" refers to the chemical bond between nucleosides.

"Intravenous administration" means administration into a vein.

"Linked nucleosides" means adjacent nucleosides which are bonded together.

"Mismatch" or "non-complementary nucleobase" refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

"Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

"Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

"Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.

"Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.

"Modified oligonucleotide" means an oligonucleotide comprising at least one modified

internucleoside linkage, modified sugar or modified nucleobase. A modified oligonucleotide can also have a nucleoside mimetic or nucleotide mimetic.

"Modified sugar" refers to a substitution or change from a natural sugar. "Modulation" means a perturbation of function, for example, one associated with either an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression.

"Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occuring or modified.

"Motif means the pattern of chemically distinct regions in an antisense compound.

"Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

"Natural sugar moiety" means a sugar found in DNA (2'-H) or RNA (2' -OH).

"NSAID" refers to a Non-Steroidal Anti-Inflammatory Drug. NSAIDs reduce inflammatory reactions in a subject but in general do not necessarily ameliorate or prevent a disease from occurring or progressing.

"Nucleic acid" refers to molecules composed of monomelic nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid can also comprise a combination of these elements in a single molecule.

"Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid. "Nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the oligonucleotide and the target nucleic acid are considered to be complementary at that nucleobase pair.

"Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.

"Nucleoside" means a nucleobase linked to a sugar.

"Nucleoside mimetic" includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound; for example, nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics such as non furanose sugar units.

"Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

"Nucleotide mimetic" includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=0)-0- or other non-phosphodi ester linkage). "Oligomeric compound" or "oligomer" refers to a polymeric structure comprising two or more substructures and capable of hybridizing to a region of a nucleic acid molecule. In certain embodiments, oligomeric compounds are oligonucleosides. In certain embodiments, oligomeric compounds are

oligonucleotides. In certain embodiments, oligomeric compounds are antisense compounds. In certain embodiments, oligomeric compounds are antisense oligonucleotides. In certain embodiments, oligomeric compounds are chimeric oligonucleotides.

"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

"Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular

administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. Administration can be continuous, or chronic, or short or intermittent.

"Peptide" means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.

"Pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to STAT3 is pharmaceutical agent.

"Pharmaceutical composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a sterile aqueous solution.

"Pharmaceutically acceptable carrier" or "Pharmaceutically acceptable diluent" means a medium or diluent that does not interfere with the structure or function of the oligonucleotide. Certain, of such carries enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. Certain of such carriers enable pharmaceutical compositions to be formulated for injection, infusion or topical administration. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.

The term "pharmaceutically acceptable derivative" encompasses pharmaceutically acceptable salts of the compounds described herein. For example, pharmaceutically acceptable derivatives can include, but is not limited to, solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelled variants and the like.

"Pharmaceutically acceptable salts" or "salts" mean physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" can include a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. Pharmaceutically acceptable salts of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH,

Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. Accordingly, in one embodiment the compounds described herein are in the form of a sodium salt.

"Phosphorothioate linkage" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified intemucleoside linkage.

"Portion" means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

"Prevent" refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.

"Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., a drug) within the body or cells thereof by the action of endogenous enzymes or non-endogenous enzymes or other chemicals or conditions.

"Region" or "target region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

"Ribonucleotide" means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides can be modified with any of a variety of substituents.

"Second agent" or "second therapeutic agent" means an agent that can be used in combination with a "first agent". A second therapeutic agent can be any agent that inhibits or prevents inflammation, infection or cancer. A second therapeutic agent can include, but is not limited to, an siRNA or antisense oligonucleotide including antisense oligonucleotides targeting STAT3. For example, a second agent can be a NS JD, disease-modifying anti-rheumatic drug (DMARD), chemotherapeutic agent, antifection agent and the like.

"Segments" are defined as smaller, sub-portions of regions within a nucleic acid. For example, a "target segment" means the sequence of nucleotides of a target nucleic acid to which one or more antisense compounds is targeted. "5' target site" refers to the 5 '-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment.

"Shortened" or "truncated" versions of antisense oligonucleotides or target nucleic acids taught herein have one, two or more nucleosides deleted.

"Side effects" means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum can indicate liver toxicity or liver function abnormality. For example, increased bilirubin can indicate liver toxicity or liver function abnormality.

"Single-stranded oligonucleotide" means an oligonucleotide which is not hybridized to a

complementary strand.

"siRNA" is defined as a double-stranded compound having a first and second strand and comprises a central complementary portion between said first and second strands and terminal portions that are optionally complementary between said first and second strands or with a target mRNA. In one non-limiting example, the first strand of the siRNA is antisense to the target nucleic acid, while the second strand is complementary to the first strand. Once the antisense strand is designed to target a particular nucleic acid target, the sense strand of the siRNA can then be designed and synthesized as the complement of the antisense strand and either strand can contain modifications or additions to either terminus.

"Sites," as used herein, are defined as unique nucleobase positions within a target nucleic acid. "Slows progression" means a decrease in the development of a disease, condition or symptom. "Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.

"STAT3" (also known as acute-phase response factor or APRF) means any nucleic acid or protein of STAT3. For example, in certain embodiments, STAT3 includes a STAT3 nucleic acid sequence or a STAT3 peptide sequence.

"STAT3 expression" means the level of mRNA transcribed from the gene encoding STAT3 or the level of protein translated from the mRNA. STAT3 expression can be determined by art known methods such as a Northern or Western blot.

"STAT3 nucleic acid" means any nucleic acid encoding STAT3. For example, in certain embodiments, a STAT3 nucleic acid includes a DNA sequence encoding STAT3, an RNA sequence transcribed from DNA encoding STAT3 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding STAT3. "STAT3 mRNA" means an mRNA encoding a STAT3 protein.

"STAT3 inhibitor" means anything that specifically inhibits or reduces STAT3 expression including, but not limited to, antibodies, antisense compounds, polypeptides, small molecule inhibitors and the like.

Examples of STAT3 inhibitors can be found in U.S. Patent Nos. 6,159,694, 6,727,064, 7,098,192, 7,307,069, 6,514,725 and US Serial Nos. 20060127502, 20100210661.

"Subcutaneous administration" means administration just below the skin.

"Targeting" or "targeted" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect. "Target nucleic acid," "target RNA," and "target RNA transcript" all refer to a nucleic acid capable of being targeted by antisense compounds.

"Target segment" means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5 '-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment.

"Thl related disease, disorder or condition" means an inflammatory disease, disorder or condition mediated by a Thl immune response. Examples of Thl diseases include, but is not limited to, allergic diseases (e.g., allergic rhinitis), autimmune diseases (e.g, multiple sclerosis, arthritis, scleroderma, psoriasis, celiac disease), cardiovascular diseases, colitis, diabetes (e.g., type 1 insulin-dependent diabetes mellitus), hypersensitivities (e.g., Type 4 hypersensitivity), infectious diseases (e.g., viral infection, mycobacterial infection) and posterior uveitis.

"Th2 related disease, disorder or condition" means an inflammatory disease, disorder or condition mediated by a Th2 immune response. Examples of Th2 diseases include, but is not limited to, allergic diseases (e.g, chronic rhinosinusitis), airway hyperresponsiveness, asthma, atopic dermatitis, colitis, endometriosis, infectious diseases (e.g., helminth infection), thyroid disease (e.g., Graves' disease), hypersensitivities (e.g, Types 1 , 2 or 3 hypersensitivity) and pancreatitis.

"Thl" or "Th2" responses include production of selective cytokines and cellular migration or recruitment to an inflammatory site. Cell types that can migrate to an inflammatory site include, but are not limited to, eosinophils and macrophages. Accordingly, cytokine level or cellular migration can be a marker for certain types of inflammation such as Thl or Th2 mediated inflammation. Thl markers include, but are not limited to cytokines IL-1 , IL-6, TNFa, INFy and keratinocyte chemoattractanct (KC). Th2 markers include, but are not limited to, eosinophil infiltration, mucus production and cytokines IL-4 and DL-5. A decrease in cytokine(s) level or cellular migration can be an indication of decreased inflammation.

"Therapeutically effective amount" means an amount of an agent, such as an antisense compound, that provides a therapeutic benefit to an individual. "Effective amount" in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of active ingredient or pharmaceutical agent such as an antisense compound to a subject in need of such modulation, such as inhibition, treatment or prophylaxis, either in a single dose or as part of a series of doses, that is effective for modulating that activity, such as inhibition of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount will vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of subjects to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

"Treat" refers to administering a pharmaceutical composition to an animal to effect an alteration or improvement of a disease, disorder, or condition. "Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and intemucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside). Certain Embodiments

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3. The STAT3 target can have a sequence selected from any one of SEQ ID NOs: 1 -18.

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide consisting of 10 to 30 nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NOs: 1-18.

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NOs: 1 -18.

In certain embodiments, the compounds or compositions of the invention can consist of 10 to 30 linked nucleosides and have a nucleobase sequence comprising at least 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 49, 50, 57 and 58.

In certain embodiments, the compounds or compositions of the invention comprise a salt of the modified oligonucleotide.

In certain embodiments, the compounds or compositions of the invention further comprise a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to any one of SEQ ID NO: 1-18 as measured over the entirety of the modified oligonucleotide.

In certain embodiments, the compound of the invention consists of a single-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.

In certain embodiments, at least one intemucleoside linkage of the modified oligonucleotide is a modified intemucleoside linkage. In certain embodiments, each intemucleoside linkage is a phosphorothioate intemucleoside linkage. In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, the modified oligonucleotide comprises at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces a furanose ring. In certain embodiments each of the tetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety. In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar comprises a 2'-0-methoxyethyl or a 4'- (CH₂)_n-0-2' bridge, wherein n is 1 or 2. In certain embodiments, at least one modified sugar comprises a constrained ethyl (cEt).

In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises: a) a gap segment consisting of linked deoxynucleosides; b) a 5' wing segment consisting of linked nucleosides; and c) a 3' wing segment consisting of linked nucleosides. The gap segment is positioned between the 5' wing segment and the 3' wing segment and each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5' wing segment consisting of five linked

nucleosides, the 3 ' wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5' wing segment consisting of three linked nucleosides, the 3' wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a cEt sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NO: 1-18, wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5' wing segment consisting of five linked nucleosides; and c) a 3' wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, each intemucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NO: 1-18, wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5' wing segment consisting of three linked nucleosides; and c) a 3 ' wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a cEt sugar, each intemucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

Certain embodiments provide methods, compounds, and compositions for inhibiting STAT3 expression.

Certain embodiments provide a method of reducing STAT3 expression in an animal comprising administering to the animal a compound of the invention described herein. In certain embodiments, the compound comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3.

Certain embodiments provide a method of preventing, ameliorating or treating an inflammation in an animal comprising administering to the animal a compound of the invention described herein. In certain embodiments, the compound comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3. In certain embodiments, reducing inflammation ameliorates an inflammatory disease or disorder. Examples of inflammatory diseases or disorders include, but are not limited to, atopic conditions (e.g., hypersensitivities such as allergies or asthma), autoimmune disease (e.g., rheumatoid arthritis, lupus, multiple sclerosis), cardiovascular disease (e.g., aneurysm, angina, arrhythmia, atherosclerosis,

cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia, hyperlipidemia,

hypercholesterolemia), cancer (e.g., colon cancer, multiple myeloma), diabetes, inflammatory bowel disease (IBD) or infectious disease.

Certain embodiments provide a method of reducing C-reactive protein (CRP) levels in an animal comprising administering to the animal a compound of the invention described herein. In certain

embodiments, the compound comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3. In certain embodiments, reduction of CRP levels in an animal prevents, ameliorates or treats an inflammatory disease. In certain embodiments, reduction of CRP levels in an animal prevents, ameliorates or treats multiple myeloma. In certain embodiments, the CRP level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method of reducing one or more cytokine levels in an animal comprising administering to the animal a compound of the invention described herein. In certain

embodiments, the compound comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3. In certain embodiments, the cytokine is IL-1 (e.g., IL-lbeta), IL-6, IL-lbeta, IL-4, IL-5, IFN-gamma, TNF-alpha or MCP-1. In certain embodiments, reduction of the cytokine level(s) in an animal prevents, ameliorates or treats an inflammatory disease. In certain embodiments, the cytokine level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method for treating an animal with a STAT3 related disease or condition comprising: a) identifying said animal with the STAT3 related disease or condition, and b) administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 16 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NO: 1 -18 as measured over the entirety of said modified oligonucleotide. In certain embodiments, the therapeutically effective amount of the compound administered to the animal treats or reduces the STAT3 related disease or condition, or a symptom thereof, in the animal. In certain embodiments, the STAT3 related disease or condition is an inflammatory disease.

In certain embodiments, STAT3 has the sequence as set forth in any of the GenBank Accession Numbers listed in Table 1 (incorporated herein as SEQ ED NOs: 1-18). In certain embodiments, STAT3 has the human sequence as set forth in SEQ ID NOs: 1-10. In certain embodiments, STAT3 has the murine sequence as set forth in SEQ ID NOs: 1 1-18).

Table 1: Gene Target Names and Sequences

In certain embodiments, the animal is a human.

In certain embodiments, the compounds or compositions of the invention are designated as a first agent. In certain embodiments, the methods of the invention comprise administering a first and second agent. In certain embodiments, the first agent and the second agent are co-administered. In certain embodiments the first agent and the second agent are co-administered sequentially or concomitantly.

In certain embodiments, the second agent is also a compound or composition of the invention. In certain embodiments, the second agent is different from a compound or composition of the invention.

Examples of second agents include, but are not limited to, an anti-inflammatory agent, chemotherapeutic agent or anti-infection agent.

In certain embodiments, the second agent is an anti-inflammatory agent (i.e., an inflammation lowering therapy). In certain embodiments the inflammation lowering therapy can include, but is not limited to, a therapeutic lifestyle change, a steroid, a NSAID or a DMARD. The steroid can be a corticosteroid. The NSAJOD can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like. The DMARD can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate and the like.

In certain embodiments, the second agent is a chemotherapeutic agent (i.e., a cancer treating agent). Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone,

hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,

pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5- fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and diethylstilbestrol (DES).

In certain embodiments, the second agent is an anti-infection agent. Examples of anti-infection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs.

In certain embodiments, administration comprises parenteral administration.

Certain embodiments provide the use of a STAT3 inhibitor for preventing, ameliorating or treating an inflammatory disease, or symptom thereof, in an animal. In certain embodiments, the STAT3 inhibitor comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3 as shown in any of SEQ ID NO: 1-18. Certain embodiments provide the use of a STAT3 inhibitor for reducing CRP or cytokine levels in an animal. In certain embodiments, the STAT3 inhibitorcomprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3 as shown in any of SEQ ID NO: 1-18.

Certain embodiments provide the use of a STAT3 inhibitor in the manufacture of a medicament for treating, ameliorating, delaying or preventing an inflammatory disease in an animal.

Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for reducing CRP or cytokine levels in an animal.

Certain embodiments provide a kit for treating, preventing, or ameliorating an inflammatory disease, or a symptom thereof, as described herein wherein the kit comprises: a) a compound as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate the inflammatory disease.

Antisense Compounds

In certain embodiments, the STAT3 specific compounds provided herein are inhibitory compounds. The STAT3 specific compounds provided herein include, but are not limited to, oligomeric compounds such as oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound can be "antisense" to a target nucleic acid, meaning that it is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the

5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to STAT3 nucleic acid is 10 to 30 nucleotides in length. In other words, antisense compounds are from 10 to 30 linked nucleobases. In other embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8 to 80, 10-80. 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, 16 or 20 linked nucleobases. In certain such embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked nucleobases in length, or a range defined by any two of the above values. In some embodiments, the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides. In certain embodiments, a shortened or truncated antisense compound targeted to a STAT3 nucleic acid has a single subunit deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated antisense compound targeted to a STAT3 nucleic acid can have two or more subunits deleted from the 5' end, or alternatively can have two or more subunits deleted from the 3' end, of the antisense compound. In certain embodiments, the deleted nucleosides can be dispersed throughout the antisense compound, for example, in an antisense compound having one or more subunits deleted from the 5' end and one or more subunits deleted from the 3' end. In certain embodiments, a shortened antisense compound targeted to a STAT3 nucleic acid can have one or more subunits deleted from the the central portion of the antisense compound.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit can be located at the 5' or 3' end or the central portion of the antisense compound. When two or more additional subunits are present, the added subunits can be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound or the central portion of the antisense compound. Alternatively, the added subunits can be dispersed throughout the antisense compound, for example, in an antisense compound having one or more subunits added to the 5' end, one or more subunits added to the 3' end and/or one or more subunits added to the central portion.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.

Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this

oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides. Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the R A strand of an RNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer can in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include 2'-MOE, and 2'-0-CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides can include those having a 4'-(CH2)n- 0-2' bridge, where n=l or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as "X-Y-Z", where "X" represents the length of the 5' wing region, "Y" represents the length of the gap region, and "Z" represents the length of the 3' wing region. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. Thus, no intervening nucleotides exist between the 5' wing segment and gap segment, or the gap segment and the 3' wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. Thus, gapmers include, but are not limited to, for example 3-10-3, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1 -18-1 , 2-10-2, 1-10-1 , 2-8-2, 6-8-6, 5-8-5, 1 -8-1 , 2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2, or 2-18-2.

In certain embodiments, the antisense compound as a "wingmer" motif, having a wing-gap or gap- wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer configurations include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or 5-13.

In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid possess a 3-10-3 gapmer motif.

In certain embodiments, an antisense compound targeted to a STAT3 nucleic acid has a gap-widened motif.

In certain embodiments, a gap-widened antisense oligonucleotide targeted to a STAT3 nucleic acid has a gap segment of thirteen 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5' wing segment of two chemically modified nucleosides and a 3' wing segment of five chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2'-sugar modification. In another embodiment, the chemical modification comprises a 2'-MOE sugar modification.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode STAT3 include, but is not limited to, the sequences listed in Table

1.

It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO can comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region can encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for STAT3 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region can encompass the sequence from a 5 ' target site of one target segment within the target region to a 3' target site of another target segment within the target region.

In certain embodiments, a "target segment" is a smaller, sub-portion of a target region within a nucleic acid. For example, a target segment can be the sequence of nucleotides of a target nucleic acid to which one or more antisense compounds are targeted. "5' target site" refers to the 5 '-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment.

Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

A target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, they can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain

embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous.

Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.

Suitable target segments can be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment can specifically exclude a certain structurally defined region such as the start codon or stop codon.

The determination of suitable target segments can include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that can hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in STAT3 mRNA levels are indicative of inhibition of STAT3 protein expression. Reductions in levels of a STAT3 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes, such as a reduction of the level of proinflammatory cytokines or glucose, can be indicative of inhibition of STAT3 mRNA and/or protein expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a

STAT3 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized. Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., 2001). In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a STAT3 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a STAT3 nucleic acid).

Non-complementary nucleobases between an antisense compound and a STAT3 nucleic acid can be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound can hybridize over one or more segments of a STAT3 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or 100% complementary to a STAT3 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) non- complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol, 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482 489). In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound can be fully complementary to a STAT3 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is "fully complementary" to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound can be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase can be at the 5' end or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases can be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they can be either contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non- complementary nucleobase(s) relative to a target nucleic acid, such as a STAT3 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a STAT3 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein can also have a defined percent identity to a particular nucleotide sequence, SEQ ED NO, or sequence of a compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases can be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ED NOs, or a portion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.

Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. Modified Intemucleoside Linkages

The naturally occurring intemucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, intemucleoside linkages are often selected over antisense compounds having naturally occurring intemucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom.

Representative phosphorus containing intemucleoside linkages include, but are not limited to,

phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid comprise one or more modified intemucleoside linkages. In certain embodiments, the modified intemucleoside linkages are phosphorothioate linkages. In certain embodiments, each intemucleoside linkage of an antisense compound is a phosphorothioate intemucleoside linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R])(R₂) (R, Ri and R are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5 '-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005- 0130923 , published on June 16, 2005) or alternatively 5 '-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein LNA is substituted with for example a 5'- methyl or a 5 '-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH₃, 2'-OCH₂CH₃, 2'-OCH₂CH₂F and 2'- 0(CH₂)₂OCH₃ substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-C C₁₀ alkyl, OCF₃, OCH₂F, 0(CH₂)₂SCH₃, 0(CH₂)₂-0-N(R_m)(R_n), 0-CH₂-C(=0)- N(R_m)(R_n), and 0-CH₂-C(=0)-N(R,)-(CH₂)₂-N(R_m)(R_n), where each R,, R_m and R_n is, independently, H or substituted or unsubstituted Ci-Cio alkyl.

As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH₂)-0-2' (LNA); 4'-(CH₂)-S-2'; 4'-(CH₂)₂-0-2' (ENA); 4'-CH(CH₃)-0-2' and 4'-CH(CH₂OCH₃)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH₃)(CH₃)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH₂-N(OCH₃)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH₂-0-N(CH₃)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH₂-N(R)-0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH₂-C(H)(CH₃)-2' (see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134); and 4'-CH₂-C- (=CH₂)-2' (and analogs thereof see published International Application WO 2008/154401 , published on December 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al, Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al, Pro Natl. Acad. Sci. U. S. A., 2000, 97, 5633-5638; Kumar et al, Bioorg. Med. Chem.

Lett., 1998, 8, 2219-2222; Singh et al, J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al, J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al, Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7; and Orum et al, Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461 ; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Serial Nos.

60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from - [C(R_a)(R_b)]_n-, -C(R_a)=C(R_b)-, -C(R_a)=N-, -C(=0)-, -C(=NR_a)-, -C(=S)-, -0-, -Si(R_a)₂-, -S(=0)_x-, and -N(R_a)-; wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_a and R_b is, independently, H, a protecting group, hydroxyl, Ci-C_]2 alkyl, substituted C1-C12 alkyl, C₂-Ci₂ alkenyl, substituted C₂-Ci₂ alkenyl, C₂-Q₂ alkynyl, substituted C₂-Ci₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJi, NJ]J₂, SJ_]; N₃, COOJi, acyl (C(=0)- H), substituted acyl, CN, sulfonyl (S(=0)₂-Ji), or sulfoxyl (S(=O)-J ; and

each Ji and J₂ is, independently, H, C_rCi₂ alkyl, substituted C Ci₂ alkyl, C₂-Ci₂ alkenyl, substituted C₂-C|2 alkenyl, C₂-C_]2 alkynyl, substituted C₂-C_]2 alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(=0)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical,

aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(R_a)(R_b)]„-, -[C(R_a)(Rb)]_n-0-, -C(R_aR_b)-N(R)-0- or-C(R_aR_b)-0-N(R)-. In certain embodiments, the bridge is 4'-CH₂-2', 4'-(CH2)2-2', 4'- 4'-CH₂-0-2', 4'-(CH₂)₂-0-2', 4'-CH₂-0-N(R)-2' and 4'-CH₂-N(R)-0-2'- wherein each R is, independently, H, a protecting group or C Gi₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the a-L configuration or in the β- D configuration. Previously, a-L-methyleneoxy (4'-CH₂-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365- 6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy (4'-CH₂-0-2') BNA , (B) β-D-methyleneoxy (4'-CH₂-0-2') BNA , (C) ethyleneoxy (4'-(CH₂)₂-0-2') BNA , (D) aminooxy (4'-CH₂-0-N(R)-2') BNA, (E) oxyamino (4'-CH₂-N(R)-0-2') BNA, and (F)

methyl(methyleneoxy) (4'-CH(CH₃)-0-2') BNA, (G) methylene-thio (4'-CH₂-S-2') BNA, (H) methylene- amino (4'-CH₂-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH₂-CH(CH₃)-2') BNA, and (J) propylene carbocyclic (4'-(CH₂)3-2') BNA as depicted below.

wherein Bx is the base moiety and R is independently H, a protecting group or C₁-C₁₂ alkyl.

diments, bicyclic nucleosides are provided having Formula Γ.

wherein:

Bx is a heterocyclic base moiety;

-Q_a-Q_b-Q_c- is -CH₂-N(R_C)-CH₂-, -C(=0)-N(R_c)-CH₂-, -(¾-0-Ν^)-, -CH₂-N(R_e)-0- or -Ν(¾)-0-

CH₂;

R_c is C₁-C₁₂ alkyl or an amino protecting group; and

T_a and T_b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_a is C_rC₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_c, NJ_cJ_d, SJ_C, N3, OC(=X)J_c, and NJ_eC(=X)NJ_cJ_d, wherein each J_c, J_d and J_e is, independently, H, C C₆ alkyl, or substituted C C6 alkyl and X is O or NJ_C.

In certain embodiments, bicyclic nucleosides are provided having Formula III:

wherein:

Bx is a heterocyclic base moiety;

Z_b is C₁-C6 alkyl, C₂-C₆ alkenyl, C₂-C alkynyl, substituted C, -C₆ alkyl, substituted C₂-C6 alkenyl, substituted C₂-C6 alkynyl or substituted acyl (C(=0)-).

In certa eosides are provided having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

Rd is Ci-Ce alkyl, substituted C_rC₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_a, q_b, q_c and q_d is, independently, H, halogen, Ci-Ce alkyl, substituted C -Ce alkyl, C₂-Cg alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C C₆ alkoxyl, substituted C C₆ alkoxyl, acyl, substituted acyl, C C₆ aminoalkyl or substituted Ci-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having Formula V:

wherein:

Bx is a heterocyclic base moiety;

q_a, _b, ¾_e and q_f are each, independently, hydrogen, halogen, Q-Cn alkyl, substituted Q-Ci₂ alkyl, C₂- Ci2 alkenyl, substituted C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C_]2 alkynyl, C_rCi₂ alkoxy, substituted C C₁₂ alkoxy, OJ_j, SJ_j, SOJ_j, S0₂J_j; NJ_jJ_k, N₃, CN, C(=0)OJ_j, C(=0)NJ_jJ_k, C(=0)J_j; 0-C(=0)NJ_jJ_k,

N(H)C(=NH)NJ_jJ_k, N(H)C(=0)NJ_jJ_k or N(H)C(=S)NJ_jJ_k;

or q_e and q_f together are =C(q_g)(q_h);

q_g and q_h are each, independently, H, halogen, C]-Ci₂ alkyl or substituted Q-C12 alkyl.

The synthesis and preparation of the methyleneoxy (4'-CH₂-0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4'-CH₂-0-2') BNA and 2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett. , 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226 ). Furthermore, synthesis of 2'-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

ic nucleosides are provided having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

each q_i} ¾, q_k and qi is, independently, H, halogen, C]-Ci₂ alkyl, substituted Q-Ci₂ alkyl, C₂-Ci₂ alkenyl, substituted C₂-C_]2 alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C₁₂ alkynyl, Ci-Ci₂ alkoxyl, substituted C_r Ci2 alkoxyl, OJ_j, SJ_j, SOJ_j; S0₂J_j, NJ_jJ_k, N₃, CN, C(=0)OJ_j; C(=0)NJ_jJ_k, C(=0)J_j; 0-C(=0)NJ_jJ_k,

N(H)C(=NH)NJ_jJ_k, N(H)C(=0)NJ_jJ_korN(H)C(=S)NJ_jJ_k; and

qj and q_j or qi and q_k together are =C(q_g)(q_h), wherein q_g and q_h are each, independently, H, halogen,

Ci-C_]2 alkyl or substituted C Ci₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4'-(CH₂)₃-2' bridge and the alkenyl analog bridge 4'- CH=CH-CH₂-2' have been described (Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731 -7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al, J. Am. Chem. Soc, 2007, 129(26), 8362-8379).

As used herein, "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.

As used herein, "monocytic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, "2 '-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to: 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nNH₂, 0(CH₂)„CH₃, 0(CH₂)_nF, 0(CH₂)_nONH₂,

OCH₂C(=0)N(H)CH₃, and 0(CH₂)_nON[(CH₂)„CH₃]₂, where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: -C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, F, CF₃, OCF_3> SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and 0-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et ai., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al, Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854), fluoro HNA (F-HNA) or those compounds having Formula VII:

VII wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_a and T_b is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_a and T_b is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group; qi, q.2, < i, q4, 5, q6 and q₇ are each independently, H, C C₆ alkyl, substituted Ci-C₆ alkyl, C₂-Q alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of Ri and R₂ is selected from hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy, NJ]J₂, SJi, N₃, OC(=X)J],

and CN, wherein X is O, S or NJ, and each J_{] }} J₂ and J₃ is, independently, H or C,-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein qi, q₂, q3_> q<i, q5_> q6 and q₇ are each H. In certain embodiments, at least one of qi, q₂, q₃, q4, qs, qe and q₇ is other than H. In certain embodiments, at least one of qi, q₂, q₃, q , qs, qe and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R₂ is fluoro. In certain embodiments, Ri is fluoro and R₂ is H; Ri is methoxy and R₂ is H, and R] is methoxyethoxy and R₂ is H.

As used herein, "2'-modified" or "2 '-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2'substituents, such as allyl, amino, azido, thio, O-allyl, O-C, -C₁₀ alkyl, -OCF₃, 0-(CH₂)₂-0-CH₃, 2'-0(CH₂)₂SCH₃, 0-(CH₂)₂-0- N(R_m)(R_n), or 0-CH₂-C(=0)-N(R_m)(R_n), where each R_m and R_n is, independently, H or substituted or unsubstituted C Cio alkyl. 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.

As used herein, "2'-OMe" or "2'-OCH₃" or "2'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH₃ group at the 2' position of the sugar ring.

As used herein, "MOE" or "2'-MOE" or "2'-OCH₂CH₂OCH₃" or "2'-0-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH₂CH₂OCH₃ group at the 2' position of the sugar ring.

As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides.

In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem. , 2002, 10, 841 -854).

Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target. In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH₃)-0-2') bridging group. In certain embodiments, the (4'- CH(CH₃)-0-2') modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5- methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2

aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides targeted to a STAT3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine. Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides can be admixed with pharmaceutically acceptable active or inert substance for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound targeted to a STAT3 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable carrier or excipient.

In certain embodiments, the "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and can be selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatimzed maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with nucleic acids, suitable for parenteral or non-parenteral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) or sterile water.

PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a STAT3 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or an oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acids from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5 '-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on

January 16, 2003.

Cell culture and antisense compounds treatment

The effects of antisense compounds on the level, activity or expression of STAT3 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commercial vendors (e.g. American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In vitro testing of antisense oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which modified appropriately for treatment with other antisense compounds. In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60- 80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes

LIPOFECT AMINE 2000® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with

LIPOFECT AMINE 2000® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECT AMINE® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a Cytofectin® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Oligofectamine™ (Invitrogen Life Technologies, Carlsbad, CA). Antisense oligonucleotide is mixed with Oligofectamine™ in Opti-MEM™-l reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of oligonucleotide with an Oligofectamine™ to oligonucleotide ratio of approximately 0.2 to 0.8 μΤ per 100 nM.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis, IN). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL of serum-free RPMI to achieve the desired concentration of oligonucleotide with a FuGENE 6 to oligomeric compound ratio of 1 to 4 of FuGENE 6 per 100 nM.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, New York, 2001). Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000® (Invitrogen, Carlsbad, CA), Lipofectin® (Invitrogen, Carlsbad, CA) or Cytofectin™ (Genlantis, San Diego, CA).

Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.

Analysis of inhibition of target levels or expression

Inhibition of levels or expression of a STAT3 nucleic acid can be assayed in a variety of ways known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive realtime PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). RT and real-time-PCR reactions are carried out by methods well known to those skilled in the art. Gene (or RNA) target quantities obtained by real time PCR can be normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RJBOGREEN® (Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using REBOGREEN® RNA quantification reagent (Invitrogen, Inc. Carlsbad, CA). Methods of RNA

quantification by REBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368- 374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN® fluorescence.

Probes and primers are designed to hybridize to a STAT3 nucleic acid. Methods for designing real- time PCR probes and primers are well known in the art, and can include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, CA).

Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH or Cyclophilin A, genes whose expression are constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, OR). GAPDH or Cyclophilin A expression can be quantified by RT, real-time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, OR).

Presented in Table 2 are primers and probes used to measure GAPDH or Cyclophilin A expression in the cell types described herein. The PCR probes have JOE or FAM covalently linked to the 5' end and TAMRA or MGB covalently linked to the 3' end, where JOE or FAM is the fluorescent reporter dye and TAMRA or MGB is the quencher dye. In some cell types, primers and probe designed to a sequence from a different species are used to measure expression. For example, a human GAPDH primer and probe set can be used to measure GAPDH expression in monkey-derived cells and cell lines.

Table 2

GAPDH primers and probes for use in real-time PCR

Sequence SEQ ID

Target Name Species Sequence (5' to 3¹)

Description NO

GAPDH Human Probe TGGAATCATATTGGAACATG 27

GAPDH Mouse Forward Primer GGCAAATTCAACGGCACAGT 28

GAPDH Mouse Reverse Primer GGGTCTCGCTCCTGGAAGAT 29

GAPDH Mouse Probe AAGGCCGAGAATGGGAAGCTTGTCATC 30

GAPDH Rat Forward Primer TGTTCTAGAGACAGCCGCATCTT 31

GAPDH Rat Reverse Primer CACCGACCTTCACCATCTTGT 32

GAPDH Rat Probe TTGTGCAGTGCCAGCCTCGTCTCA 33

Cyclophilin A Human Forward Primer TGCTGGACCCAACACAAATG 34

Cyclophilin A Human Reverse Primer TGCCATCCAACCACTCAGTC 35

Cyclophilin A Human Probe TTCCCAGTTTTTCATCTGCACTGCCA 36

Cyclophilin A Human Forward Primer GACGGCGAGCCCTTGG 37

Cyclophilin A Human Reverse Primer TGCTGTCTTTGGGACCTTGTC 38

Cyclophilin A Human Probe CCGCGTCTCCTTTGAGCTGTTTGC 39

Cyclophilin A Human Forward Primer GCCATGGAGCGCTTTGG 40

Cyclophilin A Human Reverse Primer TCCACAGTCAGCAATGGTGATC 41

Cyclophilin A Human Probe TCCAGGAATGGCAAGACCAGCAAGA 42

Cyclophilin A Mouse Forward Primer TCGCCGCTTGCTGCA 43

Cyclophilin A Mouse Reverse Primer ATCGGCCGTGATGTCGA 44

Cyclophilin A Mouse Probe CCATGGTCAACCCCACCGTGTTC 45

Cyclophilin A Rat Forward Primer CCCACCGTGTTCTTCGACA 46

Cyclophilin A Rat Reverse Primer AAACAGCTCGAAGCAGACGC 47

Cyclophilin A Rat Probe CACGGCTGATGGCGAGCCC 48

Probes and primers for use in real-time PCR are designed to hybridize to target-specific sequences. The target-specific PCR probes can have FAM covalently linked to the 5' end and TAMRA or MGB covalently linked to the 3' end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.

Analysis of Protein Levels

Antisense inhibition of STAT3 nucleic acids can be assessed by measuring STAT3 protein levels. Protein levels of STAT3 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS) (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

In vivo testing of antisense compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of STAT3 and produce phenotypic changes. Testing can be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as topical, intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from tissue and changes in STAT3 nucleic acid expression are measured. Changes in STAT3 protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments the invention provides methods for prophylactically reducing STAT3 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a

therapeutically effective amount of an antisense compound targeted to a STAT3 nucleic acid.

In certain embodiments, the individual has inflammatory disease. Inflammatory disease can include, but is not limited to, cardiovascular disease, atopic conditions, autoimmune disease, infection or cancer. Cardiovascular disease includes, but is not limited to, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease (stroke), coronary heart disease, hypertension, dyslipidemia, hyperlipidemia and hypercholesterolemia. Atopic conditions include, but are not limited to, hypersensitivities such as allergies and asthma. Autoimmune disease includes, but is not limited to, rheumatoid arthritis, lupus and multiple sclerosis. Infectious diseases include, but are not limited to, sepsis, endotoxin release, bacterial infection, viral infection and fungal infection. Cancer includes, but is not limited to, colon cancer, multiple myeloma breast carcinomas, prostate cancer, brain tumors (e.g., glioblastomas and medulloblastomas), head and neck carcinomas, melanoma, leukemias, lymphomas and chronic myelogenous leukemia.

Accordingly, provided herein are methods for ameliorating a symptom associated with inflammatory disease in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with inflammatory disease. In certain embodiments, provided is a method for reducing the severity of a symptom associated with inflammatory disease. In such embodiments, the methods comprise administering to an individual in need thereof a therapeutically effective amount of a compound targeted to a STAT3 nucleic acid.

In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to a STAT3 nucleic acid is accompanied by monitoring of STAT3 levels or CRP or cytokine levels or other disease processes associated with the expression of STAT3, to determine an individual's response to administration of the antisense compound. An individual's response to

administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targeted to a STAT3 nucleic acid results in reduction of STAT3 expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, the reduction is achieved by one or more compounds having a nucleobase sequence or portion of a nucleobase sequence complementary to the sequence recited in any of SEQ ED NOs: 1-18.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to

STAT3 are used for the preparation of a medicament for treating a patient suffering or susceptible to inflammatory disease.

Administration

The compounds or pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), intradermal (for local treatment), pulmonary, (e.g., by local inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection. The injection can be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ. In certain embodiments, formulations for injection or infusion of the compounds or compositions of the invention can include, but is not limited to, sterile aqueous solutions such as PBS or water.

In certain embodiments, formulations for topical administration of the compounds or compositions of the invention can include, but is not limited to, pharmaceutical carriers, excipients, sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the compounds or compositions in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

In certain embodiments, formulations for oral administration of the compounds or compositions of the invention can include, but is not limited to, pharmaceutical carriers, excipients, powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In certain embodiments, oral formulations are those in which compounds of the invention are administered in conjunction with one or more penetration enhancers, surfactants and chelators.

In certain embodiments, formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Certain Combination Therapies

In certain embodiments, a first agent comprising the modified oligonucleotide of the invention is coadministered with one or more secondary agents. In certain embodiments, the route of administration is the same for the first agent and second agent, while in other embodiments, the first agent and the second agent are administered by different routes.

In certain embodiments, the dosages of the first agent and the second agent are amounts that are therapeutically or prophylactically effective for each agent when administered as independent therapy.

Alternatively, the combined administration permits use of lower dosages than would be required to achieve a therapeutic or prophylactic effect if administered as independent therapy. In certain embodiments, second agents are co-administered with the first agent to produce a combinational effect. In certain embodiments, second agents are co-administered with the first agent to produce a synergistic effect. In certain embodiments, combination therapy methods are useful in decreasing one or more side effects of either the first agent or second agent.

In certain embodiments, such second agents are designed to treat the same inflammatory disease as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder, or condition as the first agent described herein.

In certain embodiments, a first agent and one or more second agents are administered at the same time. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.

In certain embodiments, second agents include, but are not limited to, an anti-inflammatory or inflammation lowering agent. The inflammation lowering agent can include, but is not limited to, a therapeutic lifestyle change, a steroid, a NSAID, an auto-immune disease drug, an anti-infective agent, a chemotherapeutic or a combination thereof.

The steroid can be a corticosteroid.

The NSAID can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like.

The auto-immune disease drug can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate, any DMARD and the like.

The anti-infection agents can be, but are not limited to, antibiotics, antifungal drugs and antiviral drugs.

The chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5- fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5- FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide) or a combination thereof.

Dosing

In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen (e.g., dose, dose frequency, and duration) wherein the dosing regimen can be selected to achieve a desired effect. The desired effect can be, for example, reduction of STAT3 or the prevention, reduction, amelioration or slowing the progression of a disease or condition associated with STAT3.

In certain embodiments, the variables of the dosing regimen are adjusted to result in a desired concentration of pharmaceutical composition in a subject. "Concentration of pharmaceutical composition" as used with regard to dose regimen can refer to the compound, oligonucleotide, or active ingredient of the pharmaceutical composition. For example, in certain embodiments, dose and dose frequency are adjusted to provide a tissue concentration or plasma concentration of a pharmaceutical composition at an amount sufficient to achieve a desired effect.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Dosing is also dependent on drug potency and metabolism. In certain embodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, or within a range of O.OOlmg to l OOmg intradermal dosing, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 g to 100 mg per kg of body weight, once or more daily, to once every 20 years or ranging from O.OOlmg to lOOmg intradermal dosing. EXAMPLES

Non-limiting disclosure and incorporation by reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

Non-limiting disclosure and incorporation by reference

Example 1 : In vivo effect of antisense inhibition of acute phase response factor, STAT3, on

inflammation in an IL-6-dependent cancer model

The effect of inhibition of STAT3 on the levels of IL-6 in the tumor microenvironment, and subsequent effect on tumor cachexia was evaluated in a murine C26 colon cancer xenograft model. C26 colon carcinoma cells were cultured in RPMI medium with 5% C(¾ at 37 C. Five million cells were subcutaneously implanted in male CD2F1 mice.

ISIS 337332 (GAAGCCCTTGCCAGCCATGT, incorporated herein as SEQ ID NO: 49) is a chimeric antisense oligonucleotide designed as a 5-10-5 MOE gapmer targeting human STAT3 (GENBANK Accession No. NM_139276.2, incorporated herein as SEQ ID NO: 1; oligonucleotide target site starting at position 1898). The gapmer is fully cross-reactive to mouse STAT3 (GENBANK Accession No.

NM_213659.2, incorporated herein as SEQ ID NO: 11 , oligonucleotide target site starting at position 1937). The gapmer is 20 nucleotides in length, wherein the central gap segment is comprised of 10 consecutive 2'- deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleosides each. Each nucleoside in each wing segment has a 2'-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P=S) internucleoside linkages. All cytosine residues throughout the gapmer are 5'methylcytosines.

ISIS 383741 (GACTCTTGCAGGAATCGGCT, incorporated herein as SEQ ID NO: 50) is a chimeric antisense oligonucleotide designed as a 5-10-5 MOE gapmer targeting murine STAT3 (GENBANK Accession No. NM 213659.2, incorporated herein as SEQ ID NO: 11, oligonucleotide target site starting at position 484). The gapmer is 20 nucleotides in length, wherein the central gap segment is comprised of 10 consecutive 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleosides each. Each nucleoside in each wing segment has a 2'-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P=S) internucleoside linkages. All cytosine residues throughout the gapmer are 5'methylcytosines.

C26 tumor Study A: Effect of STAT3 Inhibition (ISIS 337332) on tumor cachexia and survival rate

The effect of inhibiting STAT3 with ISIS 337332 and its role in increasing the survival of the mice without adverse effects on the general health of the mice was evaluated in a tumor model.

Male CD2F1 mice were separated in groups and treated as shown in Table 3.

Table 3

Summary of Mice Study Groups

On day 4 after tumor implantation, a group of 8 CD2Flmice was injected intraperitoneally with 50 mg/kg of ISIS 337332 twice a week for a total of 7 injections. One control group of 8 mice, implanted with tumor, was injected with PBS twice a week for a total of 7 injections. Another control group of 8 mice which had not been implanted with tumor was injected with 50 mg/kg of ISIS 337332 twice a week for a total of 7 injections. The body weights and survival of the mice were monitored during the treatment period. The results are presented in Tables 4 and 5. The results demonstrate that inhibition of STAT-3 in this model significantly improves the survival rate of the mice equivalent to that of mice not implanted with tumor, and with no adverse effect on the body weight.

Table 4

Effect of Antisense Oligonucleotide Treatment on survival

Table 5

Effect of Antisense Oligonucleotide Treatment on body weights

C26 tumor Study B: Effect ofSTAT3 Inhibition (ISIS 337332) on IL-6 levels

The effect of inhibiting STAT3 with ISIS 337332 and its role in decreasing IL-6 levels was evaluated in the IL-6-dependent C26 tumor model.

Male CD2F1 mice were separated in groups and treated as shown in Table 6. Table 6

Summary of Mice Study Groups

On day 4 after tumor implantation, a group of 8 CD2Flmice was injected intraperitoneally with 50 mg/kg of ISIS 337332 twice a week for a total of 7 injections. One control group of 8 mice, implanted with tumor, was injected with PBS twice a week for a total of 7 injections. Another control group of 8 mice which had not been implanted with tumor was injected with 50 mg/kg of ISIS 337332 twice a week for a total of 7 injections. A third control group of mice, with no tumor implantation, was injected with PBS twice a week for a total of 7 injections. On day 24 after the first oligonucleotide dose, plasma was collected from all groups in heparin tubes and IL-6 levels were measured using MSD® 96-well Multi-spot® Mouse cytokine assay kit (Meso Scale Discovery, Gaithersburg, MD). The results are presented in Table 7 (expressed as pg/mL) and demonstrate that inhibition of STAT3 with ISIS 337332 significantly decreased levels of the proinflammatory cytokine, IL-6 in tumor-implanted mice.

Table 7

Effects of Antisense Oligonucleotide Treatment on EL-6 levels

The mice from all groups were euthanized on day 24 and liver and tumor tissue was harvested from each group, directly lyzed and mRNA isolated for RT-PCT analysis using the murine STAT3 primer probe set, mSTAT3_LTS00664 (forward sequence CGACAGCTTCCCCATGGA, designated herein as SEQ ID NO: 51 , reverse sequence ATGCCCAGTCTTGACTCTCAATC, designated herein as SEQ ID NO: 52, probe sequence CTGCGGCAGTTCCTGGCACCTT, designated herein as SEQ ID NO: 53). The results from mice implanted with tumor and treated with ISIS 337332 are presented in Table 8, expressed as percent inhibition over the control mice implanted with tumor and injected with PBS. Table 8

Percent inhibition of STAT3 compared to the PBS control

C26 tumor Study C: Effect of STAT3 Inhibition (ISIS 337332 and ISIS 383741) on pro-inflammatory cytokine levels

The effect of inhibiting STAT3 with ISIS 337332 or ISIS 383741 and their role in decreasing levels of pro-inflammatory cytokines, such as IL-4, IL-5, IL-6, IL-Ιβ, and IFN-γ was evaluated in the Independent C26 tumor model.

Male CD2F1 mice were separated in groups and treated as shown in Table 9.

Table 9

Summary of Mice Study Groups

On day 4 after tumor implantation, a group of 15 CD2Flmice was injected intraperitoneally with 50 mg/kg of ISIS 337332 twice a week for 3 weeks. Another group of 15 CD2F1 mice, implanted with tumor, was injected intraperitoneally with 50 mg/kg of ISIS 383741 twice a week for 3 weeks. Another group of 15 CD2F1 mice, implanted with tumor, was injected intraperitoneally with PBS twice a week for 3 weeks. Three separate groups of 15 mice each, which had not been implanted with tumor, received similar treatment of ISIS 337332, ISIS 383741 or PBS injected intraperitoneally twice a week for 3 weeks.

On day 24 after the first oligonucleotide dose, plasma was collected from all groups in heparin tubes and cytokine levels were measured using MSD® 96-well Multi-spot® Mouse cytokine assay kit (Meso Scale Discovery, Gaithersburg, MD). The results are presented in Table 10 (expressed as pg/mL) and demonstrate that inhibition of STAT3 with ISIS 337332 and ISIS 383741 significantly decreased levels of proinflammatory cytokines. Table 10

Effects of Antisense Oligonucleotide Treatment on cytokine levels

The mice from all groups were euthanized on day 24 and tumor tissue was harvested. The tumor mass was dissociated into single cells which were then sorted via flow cytometry technique into CD45⁺ cells (macrophages and other leukocytes) and CD45 cells (tumor fibroblasts and stromal cells). The isolated populations of cells were lyzed and mRNA isolated for RT-PCR analysis using the murine STAT3 primer probe set, mSTAT3_LTS00664 and the murine IL-6 primer probe set mIL6_LTS00629 (forward sequence TTCCATCCAGTTGCCTTCTTG, designated herein as SEQ ID NO: 54, reverse sequence

ACAGGTCTGTTGGGAGTGGTATC, designated herein as SEQ ID NO: 55; probe sequence

TGCTGGTGACAACCACGGCCTTC, designated herein as SEQ ID NO: 56). The results are presented in Tables 11 and 12 and demonstrate the inhibition of STAT3 and decrease in IL-6 mRNA levels.

Table 11

Percent inhibition in tumor macrophages compared to the PBS control

Table 12

Percent inhibition in tumor fibroblasts/stromal cells compared to the PBS control

Example 2: Effect of antisense inhibition of human STAT3 on IL-6-induced C-reactive protein (CRP) expression in Hep3B cells

The effect of inhibition of STAT3 on the levels of CRP in Hep3B cells, with or without the addition of IL-6, was evaluated.

ISIS 455291 (CAGCAGATCAAGTCCAGGGA, incorporated herein as SEQ ID NO: 57) is a chimeric antisense oligonucleotide designed as a 5-10-5 MOE gapmer targeting human STAT3 (GENBANK Accession No. NM_139276.2, incorporated herein as SEQ ID NO: 1; oligonucleotide target site starting at position 3715). The gapmer is 20 nucleotides in length, wherein the central gap segment is comprised of 10 consecutive 2 '-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleosides each. Each nucleoside in each wing segment has a 2'-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P=S) internucleoside linkages. All cytosine residues throughout the gapmer are 5'methylcytosines.

ISIS 347526 is a control oligonucleotide with no known human target. It is designed as a 5-10-5 MOE gapmer 20 nucleotides in length, wherein the central gap segment is comprised of 10 consecutive 2'- deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleosides each. Each nucleoside in each wing segment has a 2'-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P=S) internucleoside linkages. All cytosine residues throughout the gapmer are 5'methylcytosines.

Hep3B cells were cultured on 6-well collagen-coated plates at a density of 120,000 cells/well with DMEM media and 10% FBS. The cells were transfected using lipofectin reagent 18 hours later with 50 nM oligonucleotide. After a treatment period of 4 hours, the wells were washed and fresh media was added. After 24 hrs, the media was changed to media with 1% FBS, the cells were incubated for another 16 hrs, after which the media was again changed to that with 0.1% FBS. IL-6 cytokine (R&D system: # 206-IL-OlO/CF) at a final concentration of 25 ng/mL was added to each well. The cells were harvested after 24 hrs for RNA analysis of STAT3 and CRP. The primer probe sets utilized were RTS2033 (forward' sequence

GAGGCCCGCCCAACA, designated herein as SEQ ID NO: 59; reverse sequence

TTCTGCTAATGACGTTATCCAGTTTT, designated herein as SEQ ID NO: 60; probe sequence

CTGCCTAGATCGGC, designated herein as SEQ ID NO: 61) for STAT3, and a CRP primer probe set from Applied Biosystems (Catalogue # Hs00357041_ml). The results are presented in Table 13, and demonstrate that inhibition of STAT3 by antisense oligonucleotides decreased the expression of CRP, and subsequently reduced the advent of inflammatory events in the cells. Table 13

Percent change in STAT3 mRNA and CRP mRNA expression in Hep3B cells compared to the PBS control, after IL-6 treatment

Claims

What is Claimed:

1. A method of preventing, ameliorating or treating an inflammatory disease in an animal comprising administering to the animal a compound comprising a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3, wherein the inflammatory disease is prevented, ameliorated or treated in the animal.

2. A method of reducing CRP levels in an animal comprising administering to the animal a compound comprising a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3, wherein the level of CRP is reduced in the animal.

3. A method of reducing a cytokine level in an animal comprising administering to the animal a compound comprising a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to STAT3, wherein the cytokine level is reduced in the animal.

4. The method of any one of claims 1-3, wherein the compound consists of a single-stranded modified oligonucleotide.

5. The method of any one of claims 1 -3, wherein the animal is a human.

6. The method of claim 4, wherein the modified oligonucleotide has a nucleobase sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to the sequence of any of SEQ ED NO: 1 -18 as measured over the entirety of said modified oligonucleotide.

7. The method of claim 4, wherein at least one internucleoside linkage of said modified

oligonucleotide is a modified internucleoside linkage.

8. The method of claim 7, wherein each internucleoside linkage of said modified oligonucleotide is a phosphorothioate internucleoside linkage.

9. The method of claim 4, wherein at least one nucleoside of said modified oligonucleotide comprises a modified sugar.

10. The method of claim 9, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces a furanose ring.

11. The method of claim 10, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety.

12. The method of claim 9, wherein at least one modified sugar is a bicyclic sugar.

13. The method of claim 9, wherein at least one modified sugar comprises a 2'-0-methoxyethyl or a 4'- (CH₂)_n-0-2' bridge, wherein n is 1 or 2.

14. The method of claim 9, wherein at least one modified sugar is cEt.

15. The method of claim 4, wherein at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase.

16. The method of claim 15, wherein the modified nucleobase is a 5-methylcytosine.

17. The method of claim 4, wherein the modified oligonucleotide consists of 16 linked nucleosides.

18. The method of claim 4, wherein the modified oligonucleotide comprises:

a gap segment consisting of linked deoxynucleosides;

a 5' wing segment consisting of linked nucleosides;

a 3' wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

19. The method of claim 4, wherein the modified oligonucleotide consists of 16 linked nucleosides, has a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NO: 1-18 and comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of three linked nucleosides; a 3' wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a cEt, wherein each intemucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

20. The method of claim 4, wherein the modified oligonucleotide consists of 20 linked nucleosides, has a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NO: 1-18 and comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of five linked nucleosides;

a 3' wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'MOE, wherein each intemucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

21. A method for treating an animal with inflammatory disease comprising

a. identifying said animal with inflammatory disease,

b. administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 16 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NO: 1-18 as measured over the entirety of said modified oligonucleotide,

wherein said animal with the inflammatory disease is treated.

22. The method of claim 2, wherein reducing the CRP level prevents, ameliorates or treats an inflammatory disease.

23. The method of claim 2, wherein the CRP level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

24. The method of claim 3, wherein reducing the cytokine level prevents, ameliorates or treats an inflammatory disease.

The method of claim 3, wherein the cytokine is IL-6, IL-lbeta, IL-4, IL-5 or IFN-gamma.

26. The method of claim 3, wherein the cytokine level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

27. The method of any of claims 1, 22 or 24, wherein the inflammatory disease is cardiovascular disease, autoimmune disease, infection or cancer.

28. The method of claim 27, wherein the cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia,

hyperlipidemia or hypercholesterolemia.

29. The method of claim 27, wherein the cancer is colon cancer or multiple myeloma.

30. The method of any one of claims 1-3, wherein administration comprises parenteral administration.

31. The method of any one of claims 1 -3, wherein the compound is a first agent and further comprising administering a second agent.

32. The method of claim 31 , wherein the first agent and the second agent are co-administered.

33. The method of claim 31 , wherein the second agent is an anti-inflammatory agent,

chemotherapeutic agent or anti-infection agent.

34. Use of a STAT3 inhibitor to prevent, ameliorate or treat an inflammatory disease in an animal.

35. Use of a STAT3 inhibitor to reduce CRP levels in an animal.

36. Use of a STAT3 inhibitor to reduce cytokine levels in an animal.

37. Use of the STAT3 inhibitor in any of claims 34-36, wherein the STAT3 inhibitor comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to a STAT3 sequence as shown in any of SEQ ID NO: 1-18.