Cancer Treatment

Abstract

In certain embodiments, methods, compounds, and compositions for treating B-cell lymphoma or hepatocellular carcinoma by inhibiting expression of STAT3 mRNA or protein in an animal are provided herein. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate B-cell lymphoma or hepatocellular carcinoma.

Description

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0216USL2SEQ.txt created Mar. 12, 2013, which is 124 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

[0002] In certain embodiments, methods, compounds, and compositions for treating B-cell lymphoma by inhibiting expression of STAT3 mRNA or protein in an animal are provided herein. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate B-cell lymphoma or hepatocellular carcinoma.

BACKGROUND

[0003] 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 six distinct members of the STAT family (STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6) and several isoforms (STAT1.alpha., STAT1.beta., STAT3.alpha. and STAT3.beta.). The activities of the STATs are modulated by various cytokines and mitogenic stimuli. Binding of a cytokine to its receptor results in the activation of Janus protein tyrosine kinases (JAKs) associated with these receptors. This phosphorylates STAT, resulting in translocation to the nucleus and transcriptional activation of STAT responsive genes. Phosphorylation on a specific tyrosine residue on the STATs results in their activation, resulting in the formation of homodimers and/or heterodimers of STAT which bind to specific gene promoter sequences. Events mediated by cytokines through STAT activation include cell proliferation and differentiation and prevention of apoptosis.

[0004] The specificity of STAT activation is due to specific cytokines, i.e., each STAT is responsive to a small number of specific cytokines. Other non-cytokine signaling molecules, such as growth factors, have also been found to activate STATs. Binding of these factors to a cell surface receptor associated with protein tyrosine kinase also results in phosphorylation of STAT.

[0005] STAT3 (also acute phase response factor (APRF)), in particular, has been found to be responsive to interleukin-6 (IL-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).

[0006] 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).

[0007] Recently, STAT3 was detected in the mitochondria of transformed cells, and was shown to facilitate glycolytic and oxidative phosphorylation activities similar to that of cancer cells (Gough, D. J., et al., Science, 2009, 324, 1713-1716). The inhibition of STAT3 in the mitochondria impaired malignant transformation by activated Ras. The data confirms a Ras-mediated transformation function for STAT3 in the mitochondria in addition to its nuclear roles.

[0008] Aberrant expression of or constitutive expression of STAT3 is associated with a number of disease processes.

SUMMARY

[0009] B-cell lymphoma is a B-lymphocyte blood cell cancer that is clinically classified as either Hodgkin's lymphoma or non-Hodgkin's lymphoma. There are several types of non-Hodgkin's lymphoma, of which diffuse large B-cell lymphoma (DLBCL) is the most common type, accounting for approximately 30 percent of all lymphomas. In the United States, DLBCL affects about 7 out of 100,000 people each year.

[0010] Several embodiments provided herein relate to the discovery that inhibiting the JAK-STAT signaling pathway can be useful for treating B-cell lymphoma. In certain embodiments, antisense compounds targeting STAT3 are useful for treating B-cell lymphoma, such as DLBCL, at unexpectedly low doses for an antisense compound as a cancer therapeutic. In several embodiments, antisense compounds targeting STAT3 provided herein are administered to a subject having B-cell lymphoma at a fixed total weekly dose in the range of about 15-750 mg. In certain embodiments, antisense compounds targeting STAT3 provided herein are administered to a subject having B-cell lymphoma in the range of about 0.2 to 3.5 milligrams of the antisense compound per kilogram of the subject's body weight per week (0.2-3.5 mg/kg/wk). Such dose ranges are unexpectedly low for treating cancer. By comparison, a Phase 1 study of LY2275796, an antisense oligonucleotide targeted to cap-binding protein eukaryotic initiation factor 4E (eIF-4E), concluded that the maximum tolerable dose (MTD) and biologically effective dose (BED) of LY2275796 is 1,000 mg under a loading and maintenance dose regimen, but even at a 1,000 mg dose, no tumor response was observed. (Hong D. S. et al., Clin Cancer Res. 2011 17(20):6582-91).

DETAILED DESCRIPTION

[0011] 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 the term "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.

[0012] 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

[0013] 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 may 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.

[0014] Unless otherwise indicated, the following terms have the following meanings:

[0015] "2'-deoxynucleoside" means a nucleoside comprising 2'-H furanosyl sugar moiety, as found naturally occurring in deoxyribonucleosides (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

[0016] "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0017] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'-MOE modified sugar moiety.

[0018] "2'-substituted nucleoside" means a nucleoside comprising a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0019] "5'-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. A 5-methylcytosine is a modified nucleobase.

[0020] "About" as applied to dosing amounts means within 12% of a value. For example, if it is stated, "the dose is an amount in the range of about 15-750 mg," it is implied that the dose is an amount in the range of 13-840 mg. In another example, if it is stated that the dose is an amount of "about 50 mg," it is implied that the dose can be from 44 mg to 56 mg.

[0021] "About" as applied to activity levels 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 STAT3 levels are inhibited within a range of 63% and 77%.

[0022] "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.

[0023] "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.

[0024] "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.

[0025] "Administering" means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.

[0026] "Amelioration" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

[0027] "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.

[0028] "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.

[0029] "Antisense compound" means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, and shRNAs.

[0030] "Antisense inhibition" means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid as compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

[0031] "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.

[0032] "Bicyclic sugar" means a furosyl ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar.

[0033] "Bicyclic nucleoside" (also BNA) means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.

[0034] "Cap structure" or "terminal cap moiety" means chemical modifications, which have been incorporated at either terminus of an antisense compound.

[0035] "cEt" or "constrained ethyl" means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH.sub.3)--O-2'.

[0036] "Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH.sub.3)--O-2' bridge.

[0037] "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'-O-methoxyethyl modifications.

[0038] "Chimeric antisense compound" means an antisense compound that has at least two chemically distinct regions.

[0039] "Co-administration" means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

[0040] "Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0041] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0042] "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 may be a liquid, e.g. saline solution.

[0043] "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 may 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 may 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 may be stated as the amount of pharmaceutical agent per hour, day, week, or month. In certain embodiments, single dose means administration of one dose, and only one dose, to a subject.

[0044] "Dosage unit" means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized ISIS 481464. In certain embodiments, a dosage unit is a vial containing reconstituted ISIS 481464.

[0045] "Dosing regimen" is a combination of doses designed to achieve one or more desired effects. In certain embodiments, a dose regimen is designed to provide a therapeutic effect quickly.

[0046] "Duration" means the period of time during which an activity or event continues. For example, the duration of a loading phase is the period of time during which loading doses are administered. For example, the duration of the maintenance phase is the period of time during which maintenance doses are administered.

[0047] "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 may 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.

[0048] "First phase" means a dosing phase during which administration is initiated and steady state concentrations of pharmaceutical agents can be, but is not necessarily, achieved in a target tissue. "Second phase" means a dosing phase after the "first phase." In certain embodiments, the dose or total weekly dose of the first phase and the second phase are different.

[0049] "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 target nucleic acid is a second nucleic acid.

[0050] "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 may be referred to as the "gap" and the external regions may be referred to as the "wings."

[0051] "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.

[0052] "HCC" means hepatocellular carcinoma. It is the most common form of liver cancer and also referred to as malignant hepatoma.

[0053] "Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

[0054] "Hyperproliferative disease" means a disease characterized by rapid or excessive growth and reproduction of cells. Examples of hyperproliferative diseases include cancer, e.g., carcinomas, sarcomas, lymphomas, and leukemias as well as associated malignancies and metastases.

[0055] "Identifying an animal at risk for hyperproliferative disease" means identifying an animal having been diagnosed with a hyperproliferative disease or identifying an animal predisposed to develop a hyperproliferative disease. Individuals predisposed to develop a hyperproliferative disease include those having one or more risk factors for hyperproliferative disease including older age; history of other hyperproliferative diseases; history of tobacco use; history of exposure to sunlight and/or ionizing radiation; prior contact with certain chemicals, especially continuous contact; past or current infection with certain viruses and bacteria; prior or current use of certain hormone therapies; genetic predisposition; alcohol use; and certain lifestyle choices including poor diet, lack of physical activity, and/or being overweight. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

[0056] "Immediately adjacent" means there are no intervening elements between the immediately adjacent elements.

[0057] "Inhibiting STAT3" means reducing expression of STAT3 mRNA and/or protein levels in the presence of a STAT3 antisense compound, including a STAT3 antisense oligonucleotide, as compared to expression of STAT3 mRNA and/or protein levels in the absence of a STAT3 antisense compound, such as an antisense oligonucleotide.

[0058] "Individual" means a human or non-human animal selected for treatment or therapy.

[0059] "Intemucleoside linkage" refers to the chemical bond between nucleosides.

[0060] "ISIS 481464" means a STAT3 antisense oligonucleotide having the nucleobase sequence "CTATTTGGATGTCAGC", incorporated herein as SEQ ID NO: 12, where each internucleoside linkage is a phosphorothioate intemucleoside linkage, each cytosine is a 5-methylcytosine, and each of nucleosides 1-3 and 14-16 comprise a cEt moeity. ISIS 481464 is complementary to nucleobases 3016-3031 of the sequence of GENBANK Accession No. NM_139276.2, incorporated herein as SEQ ID NO:1.

[0061] "Linked nucleosides" means adjacent nucleosides which are bonded together.

[0062] "Loading phase" means a dosing phase during which administration is initiated and steady state concentrations of pharmaceutical agents are achieved in a target tissue. For example, a loading phase is a dosing phase during which steady state concentrations of antisense oligonucleotide are achieved in liver.

[0063] "Maintenance phase" means a dosing phase after target tissue steady state concentrations of pharmaceutical agents have been achieved. For example, a maintenance phase is a dosing phase after which steady state concentrations of antisense oligonucleotide are achieved in liver.

[0064] "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.

[0065] "Modified intemucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester intemucleoside bond).

[0066] "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).

[0067] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified intemucleoside linkage, or modified nucleobase. A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.

[0068] "Modified oligonucleotide" means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

[0069] "Modified sugar" refers to a substitution or change from a natural sugar.

[0070] "Motif" means the pattern of chemically distinct regions in an antisense compound.

[0071] "Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0072] "Natural sugar moiety" means a sugar found in DNA (2'-H) or RNA (2'-OH).

[0073] "Nucleic acid" refers to molecules composed of monomeric 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).

[0074] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0075] "Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.

[0076] "Nucleoside" means a nucleobase linked to a sugar.

[0077] "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 such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. 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(.dbd.O)--O-- or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.

[0078] "Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0079] "Off-target effect" refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

[0080] "Oligomeric compound" or "oligomer" means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

[0081] "Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

[0082] "Parenteral administration" means administration through injection (e.g., bolus 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.

[0083] "Peptide" means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.

[0084] "Pharmaceutical composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

[0085] "Pharmaceutically acceptable derivative" encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

[0086] "Pharmaceutically acceptable salts" means 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.

[0087] "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 (P.dbd.S) is a modified internucleoside linkage.

[0088] "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.

[0089] "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.

[0090] "Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.

[0091] "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 may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

[0092] "Signal Transducer and Activator of Transcription 3 nucleic acid" or "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.

[0093] "Single-stranded oligonucleotide" means an oligonucleotide which is not hybridized to a complementary strand.

[0094] "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.

[0095] "Subject" means a human selected for treatment or therapy.

[0096] "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.

[0097] "Target nucleic acid," "target RNA," "target mRNA," and "target RNA transcript" all refer to a nucleic acid capable of being targeted by antisense compounds.

[0098] "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.

[0099] "Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

[0100] "Treat" refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.

[0101] "Unmodified nucleotide" means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. .beta.-D-ribonucleosides) or a DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).

CERTAIN EMBODIMENTS

[0102] In certain aspects, there is provided a method of treating cancer in a subject which comprises administering to the subject an inhibitor of the JAK-STAT pathway. In certain embodiments the cancer is B-cell lymphoma or hepatocellular carcinoma (HCC).

[0103] In certain aspects, there is provided a method of treating B-cell lymphoma in a subject which comprises administering to the subject an inhibitor of the JAK-STAT pathway.

[0104] In certain aspects, there is provided a method of treating cancer, such as B-cell lymphoma or HCC, in a subject which comprises administering to the subject a weekly dose of an antisense compound complementary to a nucleic acid encoding human STAT3, wherein the dose comprises about 0.2 to 3.5 milligrams of the antisense compound per kilogram of the subject's body weight per week (0.2-3.5 mg/kg/wk). In certain embodiments, the dose is about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2.0 mg, about 2.1 mg, about 2.2 mg, about 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3.0 mg, about 3.1 mg, about 3.2 mg, about 3.3 mg, about 3.4 mg, or about 3.5 mg of the antisense compound per kilogram of the subject's body weight. In certain embodiments, the dose comprises about 1.5 to 3.5 milligrams of the antisense compound per kilogram of the subject's body weight (1.5-3.5 mg/kg/wk. In certain embodiments, the dose is 2.0 milligrams of the antisense compound per kilogram of the subject's body weight per week (2.0 mg/kg/wk). In certain embodiments, the dose is effective to treat cancer and acceptably tolerable. The dose can be administered for at least 1-52 weeks, at least 1-10 weeks, at least 1-7 weeks, at least 1-5 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In certain embodiments, the dose can be administered to the subject 1, 2, 3, 4, 5, 6, or 7 times per week. In certain embodiments, the dose is administered to the subject 1-6 times per week. In several embodiments, the dose can be administered 6 times during the first week and 1 time each subsequent week. In certain embodiments, the subject's body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0105] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a single dose of a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein the single dose comprises an amount of the compound in the range of about 15-250 mg. In certain embodiments, the single dose comprises an amount of the compound in the range of about 100-250 mg. In certain embodiments, the single dose is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, or about 250 mg. In certain embodiments, the dose is effective to treat cancer and acceptably tolerable.

[0106] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a total weekly dose of a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein the total weekly dose comprises an amount of the compound in the range of about 15-750 mg weekly. In certain embodiments, the total weekly dose comprises an amount of the compound in the range of about 100-750 mg weekly. In certain embodiments, the total weekly dose is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. In certain embodiments, the dose is effective to treat cancer and acceptably tolerable. The total weekly dose can be administered in 2, 3, 4, 5, 6, or 7 equal doses within a week, such that the total weekly dose does not exceed about 750 mg. In certain embodiments, the total weekly dose is administered in 3 equal doses within a week. It will be understood that the aforementioned total weekly dose ranges can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the total weekly dose by the subject's body weight, such as the subject's ideal body weight. For example, dividing the aforementioned total weekly dose by an average adult body weight of 70 kg, in certain embodiments the total weekly dose can be represented as an amount of about 15 mg/70 kg (0.2 mg/kg/wk) to 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, the total weekly dose can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0107] In certain aspects, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0108] a loading phase comprising a total weekly dose in the range of about 15-750 mg for the first 1-10 weeks, and

[0109] a maintenance phase comprising a total weekly dose in the range of 15-250 mg for at least 1 week after the loading phase.

[0110] In certain embodiments, the loading phase is 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In certain embodiments, the loading phase comprises administering the compound in 2, 3, 4, 5, 6, or 7 equal doses within a week. In certain embodiments, the loading phase comprises administering the compound in 3 equal doses within a week. In several embodiments, the total weekly dose of the antisense compound in the loading phase is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. It will be understood that the aforementioned total weekly dose ranges in the loading phase can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the total weekly dose by the subject's body weight, such as the subject's ideal body weight. For example, dividing the aforementioned total weekly dose in the loading phase by an average adult body weight of 70 kg, in certain embodiments the total weekly dose can be represented as an amount of about 15 mg/70 kg (0.2 mg/kg/wk) to 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, the total weekly dose in the loading phase can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0111] In certain embodiments, the maintenance phase comprises administering the compound in 2, 3, 4, 5, 6, or 7 equal doses within a week. In several embodiments, the total weekly dose of the antisense compound in the maintenance phase is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, or about 250 mg. In certain embodiments, the total weekly dose in the maintenance phase is administered as a single dose per week. It will be understood that the aforementioned total weekly dose ranges in the maintenance phase can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the total weekly dose by the subject's body weight, such as the subject's ideal body weight. For example, dividing the aforementioned total weekly dose by an average adult body weight of 70 kg, in certain embodiments the total weekly dose in the maintenance phase can be represented as an amount of about 15 mg/70 kg (0.2 mg/kg/wk) to 250 mg/70 kg (3.6 mg/kg/wk). In certain embodiments, the total weekly dose can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), or about 250 mg/70 kg (3.6 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0112] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0113] a loading phase about 6, 7, 8, 9, or 10 weeks, and

[0114] a maintenance phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0115] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0116] a loading phase comprising a dose in the range of about 3 to 4 mg/kg/wk for about 6, 7, 8, 9, or 10 weeks, and

[0117] a maintenance phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0118] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0119] a loading phase comprising a dose of about 3 mg/kg/wk for about 8 weeks, and

[0120] a maintenance phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0121] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0122] a first phase comprising a total weekly dose in the range of about 15-750 mg for the first 1-10 weeks, and

[0123] a second phase comprising a total weekly dose in the range of 15-250 mg for at least 1 week after the loading phase.

[0124] In certain embodiments, the first phase is 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In certain embodiments, the first phase comprises administering the compound in 2, 3, 4, 5, 6, or 7 equal doses within a week. In certain embodiments, the first phase comprises administering the compound in 3 equal doses within a week. In several embodiments, the total weekly dose of the antisense compound in the first phase is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. It will be understood that the aforementioned total weekly dose ranges in the first phase can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the total weekly dose by the subject's body weight, such as the subject's ideal body weight. For example, dividing the aforementioned total weekly dose in the first phase by an average adult body weight of 70 kg, in certain embodiments the total weekly dose can be represented as an amount of about 15 mg/70 kg (0.2 mg/kg/wk) to 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, the total weekly dose in the first phase can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0125] In certain embodiments, the second phase comprises administering the compound in 2, 3, 4, 5, 6, or 7 equal doses within a week. In several embodiments, the total weekly dose of the antisense compound in the second phase is an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, or about 250 mg. In certain embodiments, the total weekly dose in the second phase is administered as a single dose per week. It will be understood that the aforementioned total weekly dose ranges in the second phase can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the total weekly dose by the subject's body weight, such as the subject's ideal body weight. For example, dividing the aforementioned total weekly dose by an average adult body weight of 70 kg, in certain embodiments the total weekly dose in the second phase can be represented as an amount of about 15 mg/70 kg (0.2 mg/kg/wk) to 250 mg/70 kg (3.6 mg/kg/wk). In certain embodiments, the total weekly dose can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), or about 250 mg/70 kg (3.6 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0126] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0127] a first phase for about 6, 7, 8, 9, or 10 weeks, and

[0128] a second phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0129] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0130] a first phase comprising a dose in the range of about 3 to 4 mg/kg/wk for about 6, 7, 8, 9, or 10 weeks, and

[0131] a second phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0132] In certain embodiments, a method comprises administering to a subject having cancer, such as B-cell lymphoma or HCC, a pharmaceutical composition comprising an antisense compound complementary to a nucleic acid encoding human STAT3, wherein administering the antisense compound comprises:

[0133] a first phase comprising a dose of about 3 mg/kg/wk for about 8 weeks, and

[0134] a second phase comprising a dose of about 2 mg/kg/wk for at least 1 week after the loading phase. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0135] In any of the above embodiments, the B-cell lymphoma is a non-Hodgkin's B-cell lymphoma. Examples of non-Hodgkin's B-cell lymphoma of certain aspects include, but are not limited to, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. In certain embodiments, the non-Hodgkin's B-cell lymphoma is diffuse large B cell lymphoma (DLBCL).

[0136] In any of the above embodiments, the B-cell lymphoma is Hodgkin's B-cell lymphoma.

[0137] In any of the foregoing embodiments, administering the dose of the antisense compound reduces tumor size or tumor volume in the subject. In certain embodiments, administering the dose of the antisense compound prolongs survival of the subject. In certain embodiments, administering the dose of the antisense compound treats cancer, such as B-cell lymphoma, in the subject. In any of the above embodiments, the method is effective to treat cancer and acceptably tolerable in a subject.

[0138] In certain of the foregoing embodiments, the subject is identified as having cancer, such as B-cell lymphoma, prior to administering the antisense compound to the subject. In certain embodiments, the subject identified as having cancer, such as B-cell lymphoma, received or is currently receiving anti-cancer treatment, such as a first-line treatment regimen. For example, in certain embodiments the first-line treatment regimen is a combination of cyclophosphamide, hydroxydanuorubicin, oncovin (vincristine), prednisone or prednisolone (CHOP). In certain embodiments, the first-line treatment regimen is a combination of rituximab and CHOP (R-CHOP). In certain embodiments, the subject is refractory to a first-line treatment regimen such as CHOP and/or R-CHOP.

[0139] In any of the foregoing embodiments, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 3008 to 3033 of SEQ ID NO: 1, wherein the nucleobase sequence is complementary to SEQ ID NO: 1.

[0140] In any of the foregoing embodiment, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 3016 to 3031 of SEQ ID NO: 1, wherein the nucleobase sequence is complementary to SEQ ID NO: 1.

[0141] In any of the foregoing embodiments, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 6476 to 6491 of SEQ ID NO: 2, wherein the nucleobase sequence is complementary to SEQ ID NO: 2.

[0142] In any of the foregoing embodiments, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 250-286; 250-285; 264-285; 264-282; 728-745; 729-745; 729-744; 787-803; 867-883; 955-978; 1146-1170; 1896-1920; 1899-1920; 1899-1919; 1899-1918; 1899-1916; 1901-1916; 1946-1963; 1947-1963; 2155-2205; 2155-2187; 2156-2179; 2204-2221; 2681-2696; 2699-2716; 3001-3033; 3008-3033, 3010-3033, 3010-3032, 3015-3033, 3015-3032, 3015-3031, 3016-3033, 3016-3032, 3016-3033; 3452-3499; 3460-3476; 3583-3608; 3591-3616; 3595-3615; 3595-3614; 3595-3612; 3675-3706; 3713-3790; 3715-3735; 3833-3878; 3889-3932; 3977-4012; 4067-4100; 4225-4256; 4234-4252; 4235-4252; 4235-4251; 4236-4252; 4306-4341; 4431-4456; 4439-4454; 4471-4510; 4488-4505; 4530-4558; 4539-4572; 4541-4558; 4636-4801; 4782-4796; 4800-4823; 4811-4847; 4813-4859; 4813-4815; 4813-4831; 4827-4859; 4827-4844; or 4842-4859 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 1.

[0143] In any of the foregoing embodiments, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 2668-2688; 2703-2720; 5000-5021; 5001-5017; 5697-5722; 5699-5716; 6475-6490; 6475-6491; 6476-6491; 7682-7705; 8078-8097; 8079-8095; 9862-9811; 9870-9897; 9875-9893; 9875-9891; 9877-9893; 11699-11719; 12342-12366; 12345-12364; 12346-12364; 12347-12364; 12353-12380; 12357-12376; 12358-12376; 12358-12373; 12360-12376; 14128-14148; 16863-16883; 46091-46111; 50692-50709; 50693-50709; 50693-50708; 61325-61349; 66133-66157; 66136-66157; 66136-66155; 66136-66153; 66138-66153; 66184-66200; 67067-67083; 4171-74220; 74199-74220; 74202-74220; 74171-74219; 74199-74219; 74202-74219; 74171-74218; 74199-74218; 74202-74218; 74723-74768; 74764-74803; 74782-74802; 74782-74801; 74782-74800; 74782-74799; 74783-74802; 74783-74801; 74783-74800; 74783-74799; 74862-74893; 74900-74977; 74902-74922; 74902-74920; 75070-75119; 75164-75199; 75254-75287; 75412-75443; 75421-75439; 75422-75439; 75422-75438; 75423-75439; 75423-75438; 75493-75528; 75616-75643; 75626-75641; 75658-75699; 75676-75692; 75717-75745; 75726-75759; 75726-75745; 75727-75745; 75728-75745; 75831-75988; 75852-75969; 75969-75984; 75987-76056; 76000-76046; 76000-76032; 76000-76018; 76014-76046; 76014-76032; 76029-76046; or 76031-76046 of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 2.

[0144] In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 12 or consists of the sequence of SEQ ID NO: 12. In certain embodiments, the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 or 2.

[0145] In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the sequence of any of the STAT3 antisense oligonucleotides described in WO 2012/135736, which is incorporated by reference in its entirety herein.

[0146] In certain embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide. In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage. In several embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, at least one nucleoside comprises a modified sugar, such as a bicyclic sugar including, but not limited to, a 4'-CH.sub.2--O-2' bridge or a 4'-CH(CH.sub.3)--O-2' bridge. In certain embodiments, the modified sugar comprises a 2'-O(CH.sub.2).sub.2--OCH.sub.3 group. In certain embodiments, at least one nucleoside comprises a modified nucleobase, such as a 5-methylcytosine.

[0147] In certain embodiments, the modified oligonucleotide comprises:

[0148] a 5'-wing consisting of 1 to 5 linked nucleosides;

[0149] a 3'-wing consisting of 1 to 5 linked nucleosides; and

[0150] a gap between the 5'-wing and the 3'-wing consisting of 8 to 12 linked 2'-deoxynucleosides;

wherein at least one of the 5'-wing and the 3'-wing comprises at least one bicyclic nucleoside or one 2'-substituted nucleoside. In certain embodiments, the 2'-substituted nucleoside comprises a 2'-O(CH.sub.2).sub.2--OCH.sub.3 group or a 2'-O--CH.sub.3 group. In certain embodiments, the bicyclic nucleoside comprises a 4'-CH.sub.2--O-2' bridge or a 4'-CH(CH.sub.3)--O-2' bridge.

[0151] In certain embodiments, pharmaceutical compositions described herein are administered in the form of a dosage unit (e.g., injection, infusion, etc.). In certain embodiments, such pharmaceutical compositions comprise an antisense oligonucleotide in an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. It will be understood that the aforementioned amounts of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. For example, dividing the aforementioned amounts by an average adult body weight of 70 kg, in certain embodiments the dosage unit can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0152] In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464.

[0153] In certain embodiments, pharmaceutical compositions described herein comprise a dose of antisense oligonucleotide in an amount in the range of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. It will be understood that the aforementioned amounts of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. For example, dividing the aforementioned amounts by an average adult body weight of 70 kg, in certain embodiments the dose of antisense oligonucleotide can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0154] The compositions described herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions described herein. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.

[0155] Antisense oligonucleotides may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the 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.

[0156] Antisense oligonucleotides 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 oligonucleotide having terminal nucleic acid 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 Jan. 16, 2003.

Certain Treatments

[0157] In certain aspects there is provided a method of treating a subject suffering from cancer comprising administering to the subject an antisense compound complementary to human STAT3. In certain embodiments the antisense compound complementary to human STAT3 is as described herein or as disclosed in WO2012/135736.

[0158] In certain embodiments the cancer is selected from B-cell lymphoma or hepatocellularcarcinoma. In certain aspects there is provided an antisense compound complementary to human STAT3 for use in treating cancer. In certain embodiments the antisense compound complementary to human STAT3 is as described herein or as disclosed in WO2012/135736. In certain embodiments the cancer is selected from B-cell lymphoma or hepatocellularcarcinoma.

[0159] In certain aspects there is provided an antisense compound complementary to human STAT3 for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject the antisense compound in a loading phase and then a maintenance phase, wherein the loading phase involves administering a total weekly dose of the compound in the range of about 15-750 mg for the first 1-10 weeks, and the maintenance phase involves administering a total weekly dose in the range of 15-250 mg for at least 1 week after the loading phase. In certain embodiments the antisense compound complementary to human STAT3 is as described herein or as disclosed in WO2012/135736. In certain embodiments the cancer is selected from B-cell lymphoma or hepatocellularcarcinoma.

[0160] Certain aspects are directed to use of an antisense compound complementary to human STAT3 for the manufacture of a medicament for treating cancer. In certain embodiments the antisense compound complementary to human STAT3 is as described herein or as disclosed in WO2012/135736. In certain embodiments the cancer is selected from B-cell lymphoma or hepatocellularcarcinoma.

[0161] In particular embodiments of any of these aspects, the B-cell lymphoma is a non-Hodgkin's B-cell lymphoma. Examples of non-Hodgkin's B-cell lymphoma of certain aspects include, but are not limited to, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. In certain embodiments, the non-Hodgkin's B-cell lymphoma is diffuse large B cell lymphoma (DLBCL).

Certain Dosing Regimens

[0162] In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen. In certain such embodiments, the dosing regimen comprises a loading phase and a maintenance phase. In certain such embodiments, the dosing regimen is effective to treat cancer and acceptably tolerable in a subject. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464.

[0163] In certain embodiments, the loading phase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 doses.

[0164] In certain embodiments, the loading phase lasts from 1 day to 6 months. In certain embodiments a loading phase lasts 1 day, 2 days, 3, days, 4, days, 5 days, 6 days, or 7 days as measured from administration of the first dose of the loading phase to administration of the first dose of the maintenance phase. In certain embodiments a loading phase lasts 1 week, 2 weeks, 3, weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, or 26 weeks as measured from administration of the first dose of the loading phase to administration of the first dose of the maintenance phase. In certain embodiments, the loading phase lasts 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months as measured from administration of the first dose of the loading phase to administration of the first dose of the maintenance phase.

[0165] In certain embodiments, the dose administered during the loading phase is lower than the dose administered during the maintenance phase. In certain embodiments, the dose administered during the loading phase is lower than the dose administered during the maintenance phase to avoid undesired side effects. In certain embodiments, the undesired side effect is increased liver markers. In certain embodiments, the undesired side effect is increased ALT. In certain embodiments, the undesired side effect is increased AST. In certain embodiments, the undesired side effect is thrombocytopenia or neutropenia.

[0166] In certain embodiments, the dose administered during the loading phase is higher than the dose administered during the maintenance phase. In certain embodiments, the dose administered during the loading phase is higher than the dose administered during the maintenance phase to quickly achieve steady state reduction of STAT3 mRNA expression, STAT3 protein expression, and/or STAT3 activity. In certain embodiments, the dose administered during the loading phase is higher than the dose administered during the maintenance phase to avoid undesired side effects in the maintenance phase. In certain embodiments, the undesired side effect is increased liver markers. In certain embodiments, the undesired side effect is increased ALT. In certain embodiments, the undesired side effect is increased AST. In certain embodiments, the undesired side effect is thrombocytopenia or neutropenia.

[0167] In certain embodiments where the loading phase includes more than one dose, the doses administered during the loading phase are all the same amount as one another. In certain embodiments, the doses administered during the loading phase are not all the same amount. In certain embodiments, the doses given during the loading phase increase over time. In certain embodiments, the doses given during the loading phase decrease over time.

[0168] In certain embodiments, a loading dose is administered by parenteral administration. In certain embodiments, the parenteral administration is subcutaneous administration. In certain embodiments, the parenteral administration is intravenous infusion.

[0169] In certain embodiments, the doses administered during the loading phase are about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2.0 mg, about 2.1 mg, about 2.2 mg, about 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3.0 mg, about 3.1 mg, about 3.2 mg, about 3.3 mg, about 3.4 mg, or about 3.5 mg of the antisense compound per kilogram of the subject's body weight. In certain embodiments, the dose is 2.0 milligrams of the antisense compound per kilogram of the subject's body weight per week (2.0 mg/kg/wk). In certain embodiments, the subject's body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0170] In certain embodiments, the doses administered during the loading phase are about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, or about 750 mg. It will be understood that the aforementioned doses of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. For example, dividing the aforementioned amounts by an average adult body weight of 70 kg, in certain embodiments the doses can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), about 250 mg/70 kg (3.6 mg/kg/wk), about 275 mg/70 kg (3.9 mg/kg/wk), about 300 mg/70 kg (4.3 mg/kg/wk), about 325 mg/70 kg (4.6 mg/kg/wk), about 350 mg/70 kg (5.0 mg/kg/wk), about 375 mg/70 kg (5.4 mg/kg/wk), about 400 mg/70 kg (5.7 mg/kg/wk), about 425 mg/70 kg (6.1 mg/kg/wk), about 450 mg/70 kg (6.4 mg/kg/wk), about 475 mg/70 kg (6.8 mg/kg/wk), about 500 mg/70 kg (7.1 mg/kg/wk), about 525 mg/70 kg (7.5 mg/kg/wk), about 550 mg/70 kg (7.9 mg/kg/wk), about 575 mg/70 kg (8.2 mg/kg/wk), about 600 mg/70 kg (8.6 mg/kg/wk), about 625 mg/70 kg (8.9 mg/kg/wk), about 650 mg/70 kg (9.3 mg/kg/wk), about 675 mg/70 kg (9.6 mg/kg/wk), about 700 mg/70 kg (10.0 mg/kg/wk), about 725 mg/70 kg (10.4 mg/kg/wk), or about 750 mg/70 kg (10.7 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0171] In certain embodiments, dose, dose frequency, and duration of the loading phase may be selected to achieve a desired effect. In certain embodiments, those variables are adjusted to result in a desired concentration of pharmaceutical agent in a subject. For example, in certain embodiments, dose and dose frequency are adjusted to provide plasma concentration of a pharmaceutical agent at an amount sufficient to achieve a desired effect. In certain embodiments, the plasma concentration is maintained above the minimal effective concentration (MEC). In certain embodiments, pharmaceutical compositions described herein are administered with a dosage regimen designed to maintain a concentration above the MEC for 10-90% of the time, between 30-90% of the time, or between 50-90% of the time. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464.

[0172] In certain embodiments, doses, dose frequency, and duration of the loading phase may be selected to achieve a desired plasma trough concentration of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464. In certain embodiments, the desired plasma trough concentration is from 5-100 ng/mL. In certain embodiments, the desired plasma trough concentration is from 5-50 ng/mL. In certain embodiments, the desired plasma trough concentration is from 10-40 ng/mL. In certain embodiments, the desired plasma trough concentration is from 15-35 ng/mL. In certain embodiments, the desired plasma trough concentration is from 20-30 ng/mL.

[0173] In certain embodiments, dose, dose frequency, and duration of the loading phase may be selected to achieve a desired effect within 1 to 26 weeks. In certain embodiments, the dose is the same and the dose frequency is varied to achieve the desired effect within 1 to 26 weeks. In certain embodiments, the dose increases over time and the dose frequency remains constant. In certain embodiments, one or more doses of the loading phase are greater than one or more doses of the maintenance phase. In certain embodiments, each of the loading doses is greater than each of the maintenance doses. In certain embodiments, it is desirable to achieve a desired effect as quickly as possible. In certain embodiments, a loading phase with a high dose and/or high dose frequency may be desirable.

[0174] In certain embodiments, doses, dose frequency, and duration of the loading phase may be selected to achieve an acceptable safety profile. For example, in certain embodiments, such variables may be selected to mitigate toxicity of the pharmaceutical composition. In certain embodiments, such variables are selected to mitigate liver toxicity. In certain embodiments, such variables are selected to mitigate renal toxicity. In certain embodiments, such variables are selected to mitigate thrombocytopenia or neutropenia.

[0175] In certain embodiments, doses increase over time. In certain embodiments, one or more doses of the loading phase are lower than one or more doses of the maintenance phase. In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal. In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal, and bilirubin is elevated two or more times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal, and bilirubin elevations that do not exceed two times the upper limit of normal. In certain embodiments, when administration of a pharmaceutical composition of the invention results in ALT elevations that are above three times the upper limit of normal, the dose and/or dose frequency is adjusted to mitigate the ALT elevation.

[0176] In certain embodiments, the maintenance phase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 doses.

[0177] In certain embodiments, the maintenance phase lasts from one day to the lifetime of the subject. In certain embodiments, the maintenance phase lasts 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days as measured from administration of the last dose of the loading phase to administration of the last dose of the maintenance phase. In certain embodiments, the maintenance phase lasts 1 week, 2 weeks, 3, weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks as measured from administration of the last dose of the loading phase to administration of the last dose of the maintenance phase. In certain embodiments, the maintenance phase lasts 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months as measured from administration of the last dose of the loading phase to administration of the last dose of the maintenance phase. In certain embodiments, the maintenance phase lasts 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, or 50 years as measured from administration of the last dose of the loading phase to administration of the last dose of the maintenance phase. In certain embodiments, the maintenance phase lasts as long as the dose continues to be needed, effective, and tolerated.

[0178] In certain embodiments where the maintenance phase includes more than one dose, the doses administered during the maintenance phase are all the same as one another. In certain embodiments, the doses administered during the maintenance phase are not all the same. In certain embodiments, the doses increase over time. In certain embodiments, the doses decrease over time.

[0179] In certain embodiments, a maintenance dose is administered by parenteral administration. In certain embodiments, the parenteral administration is subcutaneous administration. In certain embodiments, the parenteral administration is intravenous infusion.

[0180] In certain embodiments, the doses during the maintenance phase are about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2.0 mg, about 2.1 mg, about 2.2 mg, about 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3.0 mg, about 3.1 mg, about 3.2 mg, about 3.3 mg, about 3.4 mg, or about 3.5 mg of the antisense compound per kilogram of the subject's body weight. In certain embodiments, the dose is 2.0 milligrams of the antisense compound per kilogram of the subject's body weight per week (2.0 mg/kg/wk). In certain embodiments, the subject's body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0181] In certain embodiments, the doses during the maintenance phase are about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, or about 250 mg. It will be understood that the aforementioned doses of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. For example, dividing the aforementioned amounts by an average adult body weight of 70 kg, in certain embodiments the doses can be represented as any of about 15 mg/70 kg (0.2 mg/kg/wk), about 20 mg/70 kg (0.3 mg/kg/wk), about 30 mg/70 kg (0.4 mg/kg/wk), about 40 mg/70 kg (0.6 mg/kg/wk), about 50 mg/70 kg (0.7 mg/kg/wk), about 75 mg/70 kg (1.1 mg/kg/wk), about 100 mg/70 kg (1.4 mg/kg/wk), about 125 mg/70 kg (1.8 mg/kg/wk), about 150 mg/70 kg (2.1 mg/kg/wk), about 175 mg/70 kg (2.5 mg/kg/wk), about 200 mg/70 kg (2.9 mg/kg/wk), about 225 mg/70 kg (3.2 mg/kg/wk), or about 250 mg/70 kg (3.6 mg/kg/wk). In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet.

[0182] In certain embodiments, doses, dose frequency, and duration of the maintenance phase may be selected to achieve a desired effect. In certain embodiments, those variables are adjusted to result in a desired concentration of pharmaceutical agent in a subject. For example, in certain embodiments, dose and dose frequency are adjusted to provide plasma concentration of a pharmaceutical agent described herein at an amount sufficient to achieve a desired effect. In certain embodiments, the plasma concentration is maintained above the minimal effective concentration (MEC). In certain embodiments, pharmaceutical compositions described herein are administered with a dosage regimen designed to maintain a concentration above the MEC for 10-90% of the time, between 30-90% of the time, or between 50-90% of the time.

[0183] In certain embodiments, doses, dose frequency, and duration of the maintenance phase may be selected to achieve a desired plasma trough concentration of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition is an antisense oligonucleotide. In certain embodiments, the desired plasma trough concentration is from 5-100 ng/mL. In certain embodiments, the desired plasma trough concentration is from 5-50 ng/mL. In certain embodiments, the desired plasma trough concentration is from 10-40 ng/mL. In certain embodiments, the desired plasma trough concentration is from 15-35 ng/mL. In certain embodiments, the desired plasma trough concentration is from 20-30 ng/mL.

[0184] In certain embodiments, doses, dose frequency, and duration of the maintenance phase may be selected to achieve a desired safety profile. For example, in certain embodiments, such variables may be selected to mitigate toxicity of the pharmaceutical composition. In certain embodiments, such variables are selected to mitigate liver toxicity. In certain embodiments, such variables are selected to mitigate renal toxicity. In certain embodiments, such variables are selected to mitigate thrombocytopenia or neutropenia. In certain embodiments, doses, dose frequency, and duration of the maintenance phase may be adjusted from time to time to achieve a desired effect. In certain embodiments, subjects are monitored for effects (therapeutic and/or toxic effects) and doses, dose frequency, and/or duration of the maintenance phase may be adjusted based on the results of such monitoring.

[0185] In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen comprising a first phase and a second phase. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464.

[0186] In certain embodiments, the first phase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 doses.

[0187] In certain embodiments, the first phase lasts from 1 day to 6 months. In certain embodiments a first phase lasts 1 day, 2 days, 3, days, 4, days, 5 days, 6 days, or 7 days as measured from administration of the first dose of the first phase to administration of the first dose of the second phase. In certain embodiments a first phase lasts 1 week, 2 weeks, 3, weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, or 26 weeks as measured from administration of the first dose of the first phase to administration of the first dose of the second phase. In certain embodiments, the first phase lasts 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months as measured from administration of the first dose of the first phase to administration of the first dose of the second phase.

[0188] In certain embodiments, the dose administered during the first phase is lower than the dose administered during the second phase. In certain embodiments, the dose administered during the first phase is lower than the dose administered during the second phase to avoid undesired side effects. In certain embodiments, the undesired side effect is increased liver markers. In certain embodiments, the undesired side effect is increased ALT. In certain embodiments, the undesired side effect is increased AST. In certain embodiments, the undesired side effect is thrombocytopenia or neutropenia.

[0189] In certain embodiments, the dose administered during the first phase is higher than the dose administered during the second phase. In certain embodiments, the dose administered during the first phase is higher than the dose administered during the second phase to quickly achieve steady state reduction of STAT3 mRNA expression, STAT3 protein expression, and/or STAT3 activity. In certain embodiments, the dose administered during the first phase is higher than the dose administered during the second phase to avoid undesired side effects in the second phase. In certain embodiments, the undesired side effect is increased liver markers. In certain embodiments, the undesired side effect is increased ALT. In certain embodiments, the undesired side effect is increased AST. In certain embodiments, the undesired side effect is thrombocytopenia or neutropenia.

[0190] In certain embodiments where the first phase includes more than one dose, the doses administered during the first phase are all the same amount as one another. In certain embodiments, the doses administered during the first phase are not all the same amount. In certain embodiments, the doses given during the first phase increase over time. In certain embodiments, the doses given during the first phase decrease over time.

[0191] In certain embodiments, a first dose is administered by parenteral administration. In certain embodiments, the parenteral administration is subcutaneous administration. In certain embodiments, the parenteral administration is intravenous infusion.

[0192] The range of dosages capable of being administered during the "first phase" and/or "second phase" are the same as can be used for the "loading phase" and "maintenance phase" referred to above. In certain embodiments, dose, dose frequency, and duration of the first phase and/or second phase may be selected to achieve a desired effect. In certain embodiments, those variables are adjusted to result in a desired concentration of pharmaceutical agent in a subject. For example, in certain embodiments, dose and dose frequency are adjusted to provide plasma concentration of a pharmaceutical agent at an amount sufficient to achieve a desired effect. In certain embodiments, the plasma concentration is maintained above the minimal effective concentration (MEC). In certain embodiments, pharmaceutical compositions described herein are administered with a dosage regimen designed to maintain a concentration above the MEC for 10-90% of the time, between 30-90% of the time, or between 50-90% of the time. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464. In certain embodiments, doses, dose frequency, and duration of the first phase and/or second phase may be selected to achieve a desired plasma trough concentration of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 12. In certain embodiments, the antisense oligonucleotide is ISIS 481464. In certain embodiments, the desired plasma trough concentration is from 5-100 ng/mL. In certain embodiments, the desired plasma trough concentration is from 5-50 ng/mL. In certain embodiments, the desired plasma trough concentration is from 10-40 ng/mL. In certain embodiments, the desired plasma trough concentration is from 15-35 ng/mL. In certain embodiments, the desired plasma trough concentration is from 20-30 ng/mL.

[0193] In certain embodiments, dose, dose frequency, and duration of the first phase and/or second phase may be selected to achieve a desired effect within 1 to 26 weeks. In certain embodiments, the dose is the same and the dose frequency is varied to achieve the desired effect within 1 to 26 weeks. In certain embodiments, the dose increases over time and the dose frequency remains constant. In certain embodiments, one or more doses of the first phase are greater than one or more doses of the second phase. In certain embodiments, each of the first doses is greater than each of the second doses. In certain embodiments, it is desirable to achieve a desired effect as quickly as possible. In certain embodiments, a first phase with a high dose and/or high dose frequency may be desirable.

[0194] In certain embodiments, doses, dose frequency, and duration of the first phase and/or second phase may be selected to achieve an acceptable safety profile. For example, in certain embodiments, such variables may be selected to mitigate toxicity of the pharmaceutical composition. In certain embodiments, such variables are selected to mitigate liver toxicity. In certain embodiments, such variables are selected to mitigate renal toxicity. In certain embodiments, such variables are selected to mitigate thrombocytopenia or neutropenia.

[0195] In certain embodiments, doses increase over time. In certain embodiments, one or more doses of the first phase are lower than one or more doses of the second phase. In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal. In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal, and bilirubin is elevated two or more times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal, and bilirubin elevations that do not exceed two times the upper limit of normal. In certain embodiments, when administration of a pharmaceutical composition of the invention results in ALT elevations that are above three times the upper limit of normal, the dose and/or dose frequency is adjusted to mitigate the ALT elevation. In certain embodiments, the second phase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 doses. In certain embodiments, the second phase lasts from one day to the lifetime of the subject. In certain embodiments, the second phase lasts 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days as measured from administration of the last dose of the first phase to administration of the last dose of the second phase. In certain embodiments, the second phase lasts 1 week, 2 weeks, 3, weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks as measured from administration of the last dose of the first phase to administration of the last dose of the second phase. In certain embodiments, the second phase lasts 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months as measured from administration of the last dose of the first phase to administration of the last dose of the second phase. In certain embodiments, the second phase lasts 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, or 50 years as measured from administration of the last dose of the first phase to administration of the last dose of the second phase. In certain embodiments, the second phase lasts as long as the dose continues to be needed, effective, and tolerated.

[0196] In certain embodiments where the second phase includes more than one dose, the doses administered during the second phase are all the same as one another. In certain embodiments, the doses administered during the second phase are not all the same. In certain embodiments, the doses increase over time. In certain embodiments, the doses decrease over time.

[0197] In certain embodiments, a second dose is administered by parenteral administration. In certain embodiments, the parenteral administration is subcutaneous administration. In certain embodiments, the parenteral administration is intravenous infusion.

Antisense Compounds

[0198] Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0199] 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.

[0200] In certain embodiments, an antisense compound targeted to a STAT3 nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a STAT3 nucleic acid is 14 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a STAT3 nucleic acid is 12 to 22 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, or 12 to 22 linked subunits, respectively. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 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 subunits 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.

[0201] In certain embodiments, antisense oligonucleotides targeted to a STAT3 nucleic acid may be shortened or truncated. For example, a single subunit may be 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 may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.

[0202] When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5' or 3' end of the antisense compound. When two or more additional subunits are present, the added subunits may 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. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.

[0203] 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.

[0204] 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.

[0205] 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

[0206] 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.

[0207] 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 may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0208] 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 may in some embodiments include R-D-ribonucleosides, .beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O--CH.sub.3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, wings may include several modified sugar moieties, including, for example 2'-MOE and constrained ethyl. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2'-MOE nucleosides, constrained ethyl nucleosides, and 2'-deoxynucleosides.

[0209] Each distinct region may comprise uniform sugar moieties, variants, or alternating sugar moieties. The wing-gap-wing motif is frequently described as "X--Y--Z", where "X" represents the length of the 5'-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. "X" and "Z" may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, "X" and "Y" may include one or more 2'-deoxynucleosides. "Y" may comprise 2'-deoxynucleosides. As used herein, a gapmer described as "X--Y--Z" has a configuration such that the gap is positioned immediately adjacent to each of the 5'-wing and the 3' wing. Thus, no intervening nucleotides exist between the 5'-wing and gap, or the gap and the 3'-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same, in other embodiments they are different. In certain embodiments, "Y" is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

[0210] In certain embodiments, gapmers provided herein include, for example, 11-mers having a motif of 1-9-1.

[0211] In certain embodiments, gapmers provided herein include, for example, 12-mers having a motif of 1-9-2, 2-9-1, or 1-10-1.

[0212] In certain embodiments, gapmers provided herein include, for example, 13-mers having a motif of 1-9-3, 2-9-2, 3-9-1, 1-10-2, or 2-10-1.

[0213] In certain embodiments, gapmers provided herein include, for example, 14-mers having a motif of 1-9-4, 2-9-3, 3-9-2, 4-9-1, 1-10-3, 2-10-2, or 3-10-1.

[0214] In certain embodiments, gapmers provided herein include, for example, 15-mers having a motif of 1-9-5, 2-9-4, 3-9-3, 4-9-2, 5-9-1, 1-10-4, 2-10-3, 3-10-2, or 4-10-1.

[0215] In certain embodiments, gapmers provided herein include, for example, 16-mers having a motif of 2-9-5, 3-9-4, 4-9-3, 5-9-2, 1-10-5, 2-10-4, 3-10-3, 4-10-2, or 5-10-1.

[0216] In certain embodiments, gapmers provided herein include, for example, 17-mers having a motif of 3-9-5, 4-9-4, 5-9-3, 2-10-5, 3-10-4, 4-10-3, or 5-10-2.

[0217] In certain embodiments, gapmers provided herein include, for example, 18-mers having a motif of 4-9-5, 5-9-4, 3-10-5, 4-10-4, or 5-10-3.

[0218] In certain embodiments, gapmers provided herein include, for example, 19-mers having a motif of 5-9-5, 4-10-5, or 5-10-4.

[0219] In certain embodiments, gapmers provided herein include, for example, 20-mers having a motif of 5-10-5.

[0220] In certain embodiments, the antisense compound has 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 provided herein 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, 5-13, 5-8, or 6-8.

[0221] In certain embodiments, antisense compound targeted to a STAT3 nucleic acid has a 2-10-2 gapmer motif.

[0222] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 3-10-3 gapmer motif.

[0223] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 5-10-5 gapmer motif.

[0224] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 1-10-5 gapmer motif.

[0225] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 3-10-4 gapmer motif.

[0226] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 2-10-4 gapmer motif.

[0227] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a 4-9-3 gapmer motif.

[0228] In certain embodiments, the antisense compound targeted to a STAT3 nucleic acid has a gap-widened motif.

[0229] In certain embodiments, the antisense compounds targeted to a STAT3 nucleic acid has any of the following sugar motifs: [0230] k-d(10)-k [0231] e-d(10)-k [0232] k-d(10)-e [0233] k-k-d(10)-k-k [0234] k-k-d(10)-e-e [0235] e-e-d(10)-k-k [0236] k-k-k-d(10)-k-k-k [0237] e-e-e-d(10)-k-k-k [0238] k-k-k-d(10)-e-e-e [0239] k-k-k-d(10)-k-k-k [0240] e-k-k-d(10)-k-k-e [0241] e-e-k-d(10)-k-k-e [0242] e-d-k-d(10)-k-k-e [0243] e-k-d(10)-k-e-k-e [0244] k-d(10)-k-e-k-e-e [0245] e-e-k-d(10)-k-e-k-e [0246] e-d-d-k-d(9)-k-k-e [0247] e-e-e-e-d(9)-k-k-e wherein, k is a constrained ethyl nucleoside, e is a 2'-MOE substituted nucleoside, and d is a 2'-deoxynucleoside.

[0248] In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J).sub.m-(B).sub.n-(J).sub.p-(B).sub.r-(A).sub.t-(D).sub.g-(A).sub.v-(B)- .sub.w-(J).sub.x-(B).sub.y-(J).sub.z [0249] wherein: [0250] each A is independently a 2'-substituted nucleoside; [0251] each B is independently a bicyclic nucleoside; [0252] each J is independently either a 2'-substituted nucleoside or a 2'-deoxynucleoside; [0253] each D is a 2'-deoxynucleoside; [0254] m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that: [0255] at least one of m, n, and r is other than 0; [0256] at least one of w and y is other than 0; [0257] the sum of m, n, p, r, and t is from 2 to 5; and [0258] the sum of v, w, x, y, and z is from 2 to 5.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

[0259] Nucleotide sequences that encode STAT3 include, without limitation, the following: GENBANK Accession No. NM_139276.2 (incorporated herein as SEQ ID NO: 1) and the complement of GENBANK Accession No. NT_010755.14 truncated from nucleotides 4185000 to U.S. Pat. No. 4,264,000 (incorporated herein as SEQ ID NO: 2).

[0260] It is understood that the sequence set forth in each SEQ ID NO contained herein is independent of any modification to a sugar moiety, an intemucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may 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.

[0261] In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may 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 may 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 same target region.

[0262] 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.

[0263] A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may 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 preceeding 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.

[0264] Suitable target segments may 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 may specifically exclude a certain structurally defined region such as the start codon or stop codon.

[0265] The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

[0266] There may 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 expression. Reductions in levels of a STAT3 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of STAT3 expression. In certain embodiments, reduced cellular growth, reduced tumor growth, and reduced tumor volume can be indicative of inhibition of STAT3 expression. In certain embodiments, amelioration of symptoms associated with cancer can be indicative of inhibition of STAT3 expression. In certain embodiments, reduction of cachexia is indicative of inhibition of STAT3 expression. In certain embodiments, reduction of cancer markers can be indicative of inhibition of STAT3 expression.

Hybridization

[0267] 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.

[0268] 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.

[0269] Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a STAT3 nucleic acid.

Complementarity

[0270] 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).

[0271] Non-complementary nucleobases between an antisense compound and a STAT3 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may 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).

[0272] In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 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.

[0273] 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 noncomplementary nucleobases may 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 four noncomplementary 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).

[0274] 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 may 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 may or may not 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.

[0275] The location of a non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be 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.

[0276] In certain embodiments, antisense compounds that are, or are up to 11, 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.

[0277] In certain embodiments, antisense compounds that are, or are up to 11, 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.

[0278] 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 9 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 an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 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 a 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

[0279] The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or 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 may 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.

[0280] 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 ID NOs, or a portion thereof, disclosed herein.

[0281] In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

[0282] In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

[0283] 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 intemucleoside linkages of the oligonucleotide.

[0284] 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.

[0285] Chemically modified nucleosides may 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 Internucleoside Linkages

[0286] The naturally occuring 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.

[0287] Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside 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.

[0288] In certain embodiments, antisense compounds targeted to a STAT3 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

[0289] Antisense compounds provided herein 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 a chemically modified ribofuranose ring moiety. 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(R1)(R)2 (R.dbd.H, C.sub.1-C.sub.12 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 Aug. 21, 2008 for other disclosed 5', 2'-bis substituted nucleosides), 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 Jun. 16, 2005), or, alternatively, 5'-substitution of a BNA (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).

[0290] Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3, and 2'-O(CH.sub.2).sub.2OCH.sub.3 substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2--O--N(Rm)(Rn), and O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl.

[0291] 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 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4' to 2' bicyclic nucleoside. Examples of such 4' to 2' bicyclic nucleosides, include, but are not limited to, one of the formulae: 4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' and 4'-CH(CH.sub.2OCH.sub.3)--O-2', and analogs thereof (see, U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2', and analogs thereof (see, published PCT International Application WO2009/006478, published Jan. 8, 2009); 4'-CH.sub.2--N(OCH.sub.3)-2', and analogs thereof (see, published PCT International Application WO2008/150729, published Dec. 11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see, published U.S. Patent Application US2004/0171570, published Sep. 2, 2004); 4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, Chattopadhyaya, et al., J Org. Chem., 2009, 74, 118-134); and 4'-CH.sub.2--C(.dbd.CH.sub.2)-2', and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008). Also see, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. 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., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos U.S. Pat. Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCT International applications WO 2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; and U.S. Patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Application Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example .alpha.-L-ribofuranose and 3-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

[0292] 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.sub.a)(R.sub.b)].sub.n--, --C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--, and --N(R.sub.a)--;

[0293] wherein:

[0294] x is 0, 1, or 2;

[0295] n is 1, 2, 3, or 4;

[0296] each R.sub.a and R.sub.b is, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl (S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and

[0297] each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.

[0298] In certain embodiments, the bridge of a bicyclic sugar moiety is, --[C(R.sub.a)(R.sub.b)].sub.n--, --[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O-- or, --C(R.sub.aR.sub.b)--O--N(R)--. In certain embodiments, the bridge is 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R)-2', and 4'-CH.sub.2--N(R)--O-2'-, wherein each R is, independently, H, a protecting group, or C.sub.1-C.sub.12 alkyl.

[0299] 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 .alpha.-L configuration or in the .beta.-D configuration. Previously, .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0300] In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) .alpha.-L-Methyleneoxy (4'-CH.sub.2--O-2') BNA, (B) .beta.-D-Methyleneoxy (4'-CH.sub.2--O-2') BNA, (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy (4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino (4'-CH.sub.2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH.sub.3)--O-2') BNA, (G) methylene-thio (4'-CH.sub.2--S-2') BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene carbocyclic (4'-(CH.sub.2).sub.3-2') BNA as depicted below.

##STR00001## ##STR00002##

wherein Bx is the base moiety and R is, independently, H, a protecting group or C.sub.1-C.sub.12 alkyl.

[0301] In certain embodiments, bicyclic nucleoside having Formula I:

##STR00003##

wherein:

[0302] Bx is a heterocyclic base moiety;

[0303] -Q.sub.a-Q.sub.b-Q.sub.c- is --CH.sub.2--N(R.sub.c)--CH.sub.2--, --C(.dbd.O)--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--, --CH.sub.2--N(R.sub.c)--O--, or --N(R.sub.c)--O--CH.sub.2;

[0304] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting group; and

[0305] T.sub.a and T.sub.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.

[0306] In certain embodiments, bicyclic nucleoside having Formula II:

##STR00004##

wherein:

[0307] Bx is a heterocyclic base moiety;

[0308] T.sub.a and T.sub.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 10 medium;

[0309] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio.

[0310] In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ.sub.c, NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and NJ.sub.eC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d, and J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.

[0311] In certain embodiments, bicyclic nucleoside having Formula III:

##STR00005##

wherein:

[0312] Bx is a heterocyclic base moiety;

[0313] T.sub.a and T.sub.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;

[0314] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl, or substituted acyl (C(.dbd.O)--).

[0315] In certain embodiments, bicyclic nucleoside having Formula IV:

##STR00006##

wherein:

[0316] Bx is a heterocyclic base moiety;

[0317] T.sub.a and T.sub.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;

[0318] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted C.sub.2-C.sub.6 alkynyl;

[0319] each q.sub.a, q.sub.b, q.sub.c and q.sub.d is, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6 aminoalkyl, or substituted C.sub.1-C.sub.6 aminoalkyl;

[0320] In certain embodiments, bicyclic nucleoside having Formula V:

##STR00007##

wherein:

[0321] Bx is a heterocyclic base moiety;

[0322] T.sub.a and T.sub.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;

[0323] q.sub.a, q.sub.b, q.sub.e and q.sub.f are each, independently, hydrogen, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy, substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j, C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;

[0324] or q.sub.e and q.sub.f together are .dbd.C(q.sub.g)(q.sub.h);

[0325] q.sub.g and q are each, independently, H, halogen, C.sub.1-C.sub.12 alkyl, or substituted C.sub.1-C.sub.12 alkyl.

[0326] The synthesis and preparation of the methyleneoxy (4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

[0327] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA, methyleneoxy (4'-CH.sub.2--O-2') BNA, and 2'-thio-BNAs, have also been prepared (see, e.g., 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 (see, e.g., 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 (see, e.g., 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.

[0328] In certain embodiments, bicyclic nucleoside having Formula VI:

##STR00008##

wherein:

[0329] Bx is a heterocyclic base moiety;

[0330] T.sub.a and T.sub.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;

[0331] each q.sub.i, q.sub.j, q.sub.k and q.sub.l is, independently, H, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j, C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k, or N(H)C(.dbd.S)NJ.sub.jJ.sub.k; and

[0332] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are .dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q are each, independently, H, halogen, C.sub.1-C.sub.12 alkyl, or substituted C.sub.1-C.sub.12 alkyl.

[0333] One carbocyclic bicyclic nucleoside having a 4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog, bridge 4'-CH.dbd.CH--CH.sub.2-2', have been described (see, e.g., 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 (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0334] 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 the 2' carbon atom and the 4' carbon atom.

[0335] As used herein, "monocylic 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.

[0336] 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: O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m are from 1 to about 10. Other 2'-substituent groups can also be selected from: C.sub.1-C.sub.12 alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH.sub.3; OCN; Cl; Br; CN; CF.sub.3; OCF.sub.3; SOCH.sub.3; SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving pharmacokinetic properties; and 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 (see, e.g., 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 O-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 (see, e.g., Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0337] 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, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), or those compounds having Formula X:

##STR00009##

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

[0338] Bx is a heterocyclic base moiety;

[0339] T.sub.3 and T.sub.4 are each, independently, an intemucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;

[0340] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted C.sub.2-C.sub.6 alkynyl; and

[0341] one of R.sub.1 and R.sub.2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and CN, wherein X is O, S, or NJ.sub.1, and each J.sub.1, J.sub.2, and J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.

[0342] In certain embodiments, the modified THP nucleosides of Formula X are provided wherein q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t, and q.sub.u are each H. In certain embodiments, at least one of q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t, and q.sub.u is other than H. In certain embodiments, at least one of q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t and q.sub.u is methyl. In certain embodiments, THP nucleosides of Formula X are provided wherein one of R.sub.1 and R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2 is H, R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is methoxyethoxy and R.sub.2 is H.

[0343] 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.sub.1-C.sub.10 alkyl, --OCF.sub.3, O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl. 2'-modifed nucleosides may further comprise other modifications, for example, at other positions of the sugar and/or at the nucleobase.

[0344] As used herein, "2'-F" refers to a sugar comprising a fluoro group at the 2' position.

[0345] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl" each refers to a nucleoside comprising a sugar comprising an --OCH.sub.3 group at the 2' position of the sugar ring.

[0346] 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).

[0347] 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, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

[0348] Methods for the preparations of modified sugars are well known to those skilled in the art.

[0349] 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.

[0350] In certain embodiments, antisense compounds comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleotides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a cEt. In certain embodiments, the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

[0351] Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances 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.

[0352] 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 diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). 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.

[0353] Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other 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.

[0354] 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

[0355] Antisense compounds may 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.

[0356] 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 acid 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 Jan. 16, 2003.

Certain Antisense Compounds

[0357] In certain embodiments, antisense compounds useful for treating B-cell lymphoma at the doses and dosing regimens described above include any of the antisense oligonucleotides described in WO 2012/135736, which is incorporated by reference in its entirety herein. Examples of antisense compounds described in WO2012/135736 suitable for treating B-cell lymphoma include, but are not limited to, those described in Tables 1& 2 below:

TABLE-US-00001 TABLE 1 cEt and MOE chimeric antisense oligonucleotides targeted to STAT3 (SEQ ID NO: 1) Human Human SEQ ISIS Start Stop Wing ID NO Site Site Sequence Motif Chem NO 481355 322 337 ACTGCCGCAGCTCCAT 3-10-3 cEt 3 481597 731 744 GAGATTCTCTACCA 2-10-2 cEt 4 481374 788 803 AGATCTTGCATGTCTC 3-10-3 cEt 5 481390 1305 1320 ATAATTCAACTCAGGG 3-10-3 cEt 6 481420 1948 1963 ACTTTTTCACAAGGTC 3-10-3 cEt 7 481431 2206 2221 CCATGATCTTATAGCC 3-10-3 cEt 8 481453 2681 2696 GATAGCAGAAGTAGGA 3-10-3 cEt 9 481463 3001 3016 CAAGGTTAAAAAGTGC 3-10-3 cEt 10 481688 3002 3015 AAGGTTAAAAAGTG 2-10-2 cEt 11 481464 3016 3031 CTATTTGGATGTCAGC 3-10-3 cEt 12 481689 3017 3030 TATTTGGATGTCAG 2-10-2 cEt 13 481465 3032 3047 TAGATAGTCCTATCTT 3-10-3 cEt 14 481690 3033 3046 AGATAGTCCTATCT 2-10-2 cEt 15 481466 3047 3062 AAGAAACCTAGGGCTT 3-10-3 cEt 16 481691 3048 3061 AGAAACCTAGGGCT 2-10-2 cEt 17 481467 3097 3112 GCTGATACAGTGTTTT 3-10-3 cEt 18 481692 3098 3111 CTGATACAGTGTTT 2-10-2 cEt 19 481468 3112 3127 ATACAGAAAGGCTATG 3-10-3 cEt 20 481693 3113 3126 TACAGAAAGGCTAT 2-10-2 cEt 21 481469 3127 3142 GCTTAAGTTTCTTAAA 3-10-3 cEt 22 481694 3128 3141 CTTAAGTTTCTTAA 2-10-2 cEt 23 481470 3461 3476 AGCACCAAGGAGGCTG 3-10-3 cEt 24 481695 3462 3475 GCACCAAGGAGGCT 2-10-2 cEt 25 481471 3476 3491 AAGCTGAATGCTTAAA 3-10-3 cEt 26 481696 3477 3490 AGCTGAATGCTTAA 2-10-2 cEt 27 481472 3491 3506 TTACCAGCCTGAAGGA 3-10-3 cEt 28 481697 3492 3505 TACCAGCCTGAAGG 2-10-2 cEt 29 481473 3506 3521 CAGGGATTATATAAAT 3-10-3 cEt 30 481698 3507 3520 AGGGATTATATAAA 2-10-2 cEt 31 481474 3521 3536 ACCTGAAGCCCGTTTC 3-10-3 cEt 32 481699 3522 3535 CCTGAAGCCCGTTT 2-10-2 cEt 33 481475 3536 3551 TGTCTTAAGGGTTTGA 3-10-3 cEt 34 481700 3537 3550 GTCTTAAGGGTTTG 2-10-2 cEt 35 481476 3551 3566 GGTTGCAGCTTCAGAT 3-10-3 cEt 36 481701 3552 3565 GTTGCAGCTTCAGA 2-10-2 cEt 37 481477 3567 3582 TCAACACCAAAGGCCA 3-10-3 cEt 38 481702 3568 3581 CAACACCAAAGGCC 2-10-2 cEt 39 481478 3585 3600 TCCTTAAACCTTCCTA 3-10-3 cEt 40 481703 3586 3599 CCTTAAACCTTCCT 2-10-2 cEt 41 481479 3600 3615 AAAATGCTTAGATTCT 3-10-3 cEt 42 481704 3601 3614 AAATGCTTAGATTC 2-10-2 cEt 43 481480 3628 3643 AAATAAGTCTATTTAT 3-10-3 cEt 44 481705 3629 3642 AATAAGTCTATTTA 2-10-2 cEt 45 481481 3648 3663 GGCCAATACATTACAA 3-10-3 cEt 46 481706 3649 3662 GCCAATACATTACA 2-10-2 cEt 47 481482 3670 3685 TGCCCAGCCTTACTCA 3-10-3 cEt 48 481707 3671 3684 GCCCAGCCTTACTC 2-10-2 cEt 49 481483 3685 3700 GTTGTAAGCACCCTCT 3-10-3 cEt 50 481708 3686 3699 TTGTAAGCACCCTC 2-10-2 cEt 51 481484 3700 3715 AGAAAGGGAGTCAAGG 3-10-3 cEt 52 481709 3701 3714 GAAAGGGAGTCAAG 2-10-2 cEt 53 481485 3717 3732 GCAGATCAAGTCCAGG 3-10-3 cEt 54 481710 3718 3731 CAGATCAAGTCCAG 2-10-2 cEt 55 481486 3730 3745 AGCCTCTGAAACAGCA 3-10-3 cEt 56 481711 3731 3744 GCCTCTGAAACAGC 2-10-2 cEt 57 481487 3746 3761 CCCACAGAAACAACCT 3-10-3 cEt 58 481712 3747 3760 CCACAGAAACAACC 2-10-2 cEt 59 481488 3761 3776 AGCCCTGATAAGGCAC 3-10-3 cEt 60 481713 3762 3775 GCCCTGATAAGGCA 2-10-2 cEt 61 481489 3776 3791 AATCAGAAGTATCCCA 3-10-3 cEt 62 481714 3777 3790 ATCAGAAGTATCCC 2-10-2 cEt 63 481490 3833 3848 GCCTCTAGCAGGATCA 3-10-3 cEt 64 481715 3834 3847 CCTCTAGCAGGATC 2-10-2 cEt 65 481491 3848 3863 CACGCAAGGAGACATG 3-10-3 cEt 66 481716 3849 3862 ACGCAAGGAGACAT 2-10-2 cEt 67 481492 3863 3878 TGAGGGACCTTTAGAC 3-10-3 cEt 68 481717 3864 3877 GAGGGACCTTTAGA 2-10-2 cEt 69 481493 3886 3901 CAGGATTCCTAAAACA 3-10-3 cEt 70 481718 3887 3900 AGGATTCCTAAAAC 2-10-2 cEt 71 481494 3901 3916 ATGAGGTCCTGAGACC 3-10-3 cEt 72 481719 3902 3915 TGAGGTCCTGAGAC 2-10-2 cEt 73 481495 3940 3955 CATCATGTCCAACCTG 3-10-3 cEt 74 481720 3941 3954 ATCATGTCCAACCT 2-10-2 cEt 75 481496 3955 3970 GGGCCCCATAGTGTGC 3-10-3 cEt 76 481721 3956 3969 GGCCCCATAGTGTG 2-10-2 cEt 77 481497 3977 3992 AGCTCAACCAGACACG 3-10-3 cEt 78 481722 3978 3991 GCTCAACCAGACAC 2-10-2 cEt 79 481498 3992 4007 GAACCATATTCCCTGA 3-10-3 cEt 80 481723 3993 4006 AACCATATTCCCTG 2-10-2 cEt 81 481499 4007 4022 CAAGAAACTGGCTAAG 3-10-3 cEt 82 481724 4008 4021 AAGAAACTGGCTAA 2-10-2 cEt 83 481500 4022 4037 GCCACTGGATATCACC 3-10-3 cEt 84 481501 4048 4063 AACTGAATGAAGACGC 3-10-3 cEt 85 481523 4489 4504 GCTTATTATGTACTGA 3-10-3 cEt 86 481748 4490 4503 CTTATTATGTACTG 2-10-2 cEt 87 481524 4530 4545 GCCCAAGTCTCACCTT 3-10-3 cEt 88 481749 4531 4544 CCCAAGTCTCACCT 2-10-2 cEt 89 481525 4541 4556 CCCAATGGTAAGCCCA 3-10-3 cEt 90 481750 4542 4555 CCAATGGTAAGCCC 2-10-2 cEt 91 481526 4543 4558 AACCCAATGGTAAGCC 3-10-3 cEt 92 481751 4544 4557 ACCCAATGGTAAGC 2-10-2 cEt 93 481527 4560 4575 TAGGTCCCTATGATTT 3-10-3 cEt 94 481752 4561 4574 AGGTCCCTATGATT 2-10-2 cEt 95 481528 4579 4594 AAGCCCTGAACCCTCG 3-10-3 cEt 96 481753 4580 4593 AGCCCTGAACCCTC 2-10-2 cEt 97 481529 4615 4630 CCTAAGGCCATGAACT 3-10-3 cEt 98 481754 4616 4629 CTAAGGCCATGAAC 2-10-2 cEt 99 481530 4630 4645 ACCAGATACATGCTAC 3-10-3 cEt 100 481755 4631 4644 CCAGATACATGCTA 2-10-2 cEt 101 481531 4646 4661 TACAATCAGAGTTAAG 3-10-3 cEt 102 481756 4647 4660 ACAATCAGAGTTAA 2-10-2 cEt 103 481532 4664 4679 TCCTCTCAGAACTTTT 3-10-3 cEt 104 481757 4665 4678 CCTCTCAGAACTTT 2-10-2 cEt 105 481533 4666 4681 GCTCCTCTCAGAACTT 3-10-3 cEt 106 481758 4667 4680 CTCCTCTCAGAACT 2-10-2 cEt 107 481534 4693 4708 TTCTTTAATGGGCCAC 3-10-3 cEt 108 481759 4694 4707 TCTTTAATGGGCCA 2-10-2 cEt 109 481535 4767 4782 ACGGGATTCCCTCGGC 3-10-3 cEt 110 481760 4768 4781 CGGGATTCCCTCGG 2-10-2 cEt 111 481536 4782 4797 GTAGGTAAGCAACCCA 3-10-3 cEt 112 481761 4783 4796 TAGGTAAGCAACCC 2-10-2 cEt 113 481537 4830 4845 GAATTTGAATGCAGTG 3-10-3 cEt 114 481762 4831 4844 AATTTGAATGCAGT 2-10-2 cEt 115 481538 4844 4859 TGAAGTACACATTGGA 3-10-3 cEt 116 481763 4845 4858 GAAGTACACATTGG 2-10-2 cEt 117 481539 4860 4875 ATAAATTTTTACACTA 3-10-3 cEt 118 481764 4861 4874 TAAATTTTTACACT 2-10-2 cEt 119 481765 4869 4882 CAATAATATAAATT 2-10-2 cEt 120 481541 4934 4949 CTGGAAGTTAAAGTAG 3-10-3 cEt 121 481766 4935 4948 TGGAAGTTAAAGTA 2-10-2 cEt 122

TABLE-US-00002 TABLE 2 Chimeric antisense oligonucleotides targeted to STAT3 (SEQ ID NO: 2) Human Human SEQ Start Stop Se- ID Site Site ISIS No quence Chemistry NO 5701 5716 GTACTCTT 529962 e-e-e-d(10)- 123 TCAGTGGT k-k-k 74784 74799 ATGCTTAG 529979 k-k-k-d(10)- 124 ATTCTCCT e-e-e 74905 74920 AGCAGATC 529982 k-k-k-d(10)- 125 AAGTCCAG e-e-e 75423 75438 AGGTGTTC 529983 k-k-k-d(10)- 126 CCATACGC e-e-e 75424 75439 TAGGTGTT 529984 k-k-k-d(10)- 127 CCCATACG e-e-e 5701 5716 GTACTCTT 529999 k-k-k-d(10)- 123 TCAGTGGT e-e-e 9878 9893 GGTTCCTC 530006 k-k-k-d(10)- 128 CTGTTGGC e-e-e 12361 12376 GGTTCCTC 530006 k-k-k-d(10)- 128 CTGTTGGC e-e-e 74783 74799 ATGCTTAGA 530020 e-e-k-d(10)- 129 TTCTCCTT k-e-k-e

Certain Combination Therapies

[0358] In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions provided herein. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a synergistic effect.

[0359] In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared separately. In certain embodiments, one or more other pharmaceutical agents include all-trans retinoic acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, 20 etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine, or vinorelbine. In certain embodiments, one or more other pharmaceutical agents include a combination of cyclophosphamide, hydroxydanuorubicin, oncovin (vincristine), prednisone or prednisolone (CHOP). In certain embodiments, one or more other pharmaceutical agents include a combination of rituximab and CHOP (R-CHOP). In certain embodiments, one or more other pharmaceutical agents include another antisense oligonucleotide. In certain embodiments, another antisense oligonucleotide is a second STAT3 antisense oligonucleotide.

[0360] In certain embodiments, one or more other pharmaceutical agents include molecular targeted therapies. In certain embodiments, the molecular targeted therapy is an EGFR inhibitor, a mTOR inhibitor, a HER2 inhibitor, or a VEGF/VEGFR inhibitor. In certain embodiments, EGFR inhibitors include gefitinib, erlotinib, lapatinib, cetuximab, panitumumbo. In certain embodiments, mTOR inhibitors include everolimus and temsirolimus. In certain embodiments, HER2 inhibitors include trastuzumab and lapatinib. In certain embodiments, VEGF/VEGFR inhibitors include pazopanib, bevacizumab, sunitinib, and sorafenib.

[0361] In certain embodiments, one more pharmaceutical compositions provided herein are administered with radiation therapy. In certain embodiments, one or more pharmaceutical compositions are administered at the same time as radiation therapy. In certain embodiments, one or more pharmaceutical compositions are administered before radiation therapy. In certain embodiments, one or more pharmaceutical compositions are administered after radiation therapy. In certain embodiments, one or more pharmaceutical compositions are administered at various time points throughout a radiation therapy regimen.

[0362] In certain embodiments, radiation therapy is useful for inhibiting tumor growth. In certain embodiments, radiation therapy is useful for increasing overall survival. In certain embodiments, radiation therapy used in conjunction with administration of one or more pharmaceuticals provided herein is advantageous over using either therapy alone because both radiation therapy and administration with one or more pharmaceuticals can be limited to achieve effective antiproliferative response with limited toxicity.

[0363] In certain embodiments, a physician designs a therapy regimen including both radiation therapy and administration of one more pharmaceutical compositions provided herein. In certain embodiments, a physician designs a therapy regimen including radiation therapy, administration of one or more pharmaceutical compositions provided herein, and administration of one or more other chemotherapeutic agents.

EXAMPLES

Non-Limiting Disclosure and Incorporation by Reference

[0364] 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 certain embodiments 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.

Example 1: Phase 1, Open-Label, Study for Treating a Patient Having Advanced B Cell Lymphoma with a STAT3 Antisense Oligonucleotide

[0365] The effect of intravenous infusion of the STAT3 antisense oligonucleotide, ISIS 481464, in patients with advanced B cell lymphomas was studied. Patients with diffuse large B-cell lymphomas (DLBCL) were recruited for this study.

[0366] The criteria for patient inclusion with respect to their tumor status was that the tumors should be relapsed or refractory to at least one prior anti-cancer systemic therapy, and/or for which no standard therapy exists; that their disease should be measurable or evaluable, according to RECIST version 1.1 for solid tumors, or according to the International Workshop Response Criteria for Non-Hodgkin's Lymphoma for NHL tumors (Cheson, B. D. et al., J. Clin. Oncol. 1999, 17: 1244; Cheson, B. D. et al., J. Clin. Oncol. 2007, 25(5):579-86), or according to appropriate criteria for other advanced cancers. RECIST (Response Evaluation Criteria in Solid Tumors) is an internationally accepted set of guidelines used in clinical trials for solid tumor disease.

[0367] One patient fitting the criteria above is a 63 year old female with DLBCL designated herein as Patient #1001. Prior to commencing therapy, Patient #1001 showed multiple areas of hypermetabolic adenopathy, both above and below the diaphragm, including the supraclavicular, left paratracheal, right internal mammary, pericardial, left intra-mammary, pre-hepatic, retroperitoneal, and mesenteric regions. In addition, the patient suffered from fatigue, nausea, night sweats, shortness of breath on exertion, and peripheral neuropathy. The patient also noted 5-6 days of right-sided abdominal fullness and associated pain. Patient therapy was commenced with a treatment period comprising administration during a first phase of 3 loading doses of ISIS 481464: a 3-hr intravenous infusion of 2 mg/kg ideal body weight of ISIS 481464 administered on days 1, 3, and 5 of cycle 0. The ideal body weight was determined using the Devine formula (Pai, M. P. and Paloucek, F. P. Ann. Pharmacol. 2000. 34: 1066-1069): for men (in kg)=50+2.3 kg/inch over 5 feet; for women (in kg)=45.5+2.3 kg/inch over 5 feet. Treatment was then continued in a second phase by once-weekly administrations (Cycle 1 and beyond) of 2 mg/kg ideal body weight of ISIS 481464 until disease progression, unacceptable toxicity, or patient discontinuation for any other reason occurred. Disease assessments were performed at the end of even cycles.

[0368] Tumor lesions were evaluated on each even-numbered cycle, starting with Cycle 2, day 15, by positron emission tomography (PET) scan. According to RECIST guidelines, a complete tumor response is achieved when all target lesions have disappeared. Partial response is achieved when the sum of the diameters of all tumor lesions is reduced at least 30% compared to the sum of the tumor lesion diameters at pre-dose. The sum of the lesion diameters, if any, was calculated, per RECIST guidelines (Eisenhauer, E. A. et al., Eur. J. Cancer 45: 228-247, 2009).

[0369] After 28 days of treatment with ISIS 481464, the patient reported reduced fatigue and night sweats, and was tolerating the treatment well.

[0370] After 49 days of treatment with ISIS 481464, a PET scan was performed and revealed a 55% reduction in tumor size. Tumors were reduced in all compartments, but most notably, in the supraclavicular, paratracheal, pericardial, and mesenteric regions.

[0371] After 91 days of treatment with ISIS 481464, Patient #1001 had a second PET scan and the partial response observed in the first scan was found to be maintained at a 55% reduction in tumor size.

[0372] After 133 days of treatment with ISIS 481464, Patient #1001 had a third PET scan and the partial response was found to be maintained at a 55% reduction in tumor size.

[0373] After 162 days of treatment with ISIS 481464, further treatment was paused for a month during which Patient #1001 had a fourth PET scan, and the partial response was maintained at a 55% reduction in tumor size. Patient #1001 is scheduled for further scans.

Example 2: Phase 1, Open-Label, Study for Treating a Patient Having Advanced/Metastatic Hepatocellular Carcinoma with a STAT3 Antisense Oligonucleotide

[0374] The effect of intravenous infusion of the STAT3 antisense oligonucleotide, ISIS 481464, in patients with advanced/metastatic hepatocellular carcinoma is being studied in an on-going clinical trial.

[0375] In the study described in this protocol, AZD9150 will be administered to patients with advanced/metastatic hepatocellular carcinoma at a starting dose of 1 mg/kg intravenously 3.times. during week 1 followed by 1.times. weekly and dose intensity will be escalated or de-escalated in subsequent cohorts through modification of unit dose administered and/or interval of administration to determine a maximum tolerated dose and recommended phase II dose in patients with advanced/metastatic hepatocellular carcinoma (HCC).

[0376] Following the dose escalation phase of the study additional patients will be enrolled to a dose expansion phase to explore further the safety, tolerability, pharmacokinetics and biological activity at selected dose(s)/schedules. Patients included in the study are relapsed, refractory, intolerant or unlikely to benefit from first-line systemic therapy (sorafenib).

[0377] To date, the 1 mg/kg and 1.5 mg/kg cohorts have completed. From the 1 mg/kg cohort 4 patients remain on study with stable disease in excess of 3 months. Stable disease has also been seen in 1.5 mg/kg cohort. These patients and future patients will be monitored further for clinical activity as the trial progresses.

Sequence CWU 1

1

12914978DNAHomo sapiens 1ggtttccgga gctgcggcgg cgcagactgg gagggggagc cgggggttcc gacgtcgcag 60ccgagggaac aagccccaac cggatcctgg acaggcaccc cggcttggcg ctgtctctcc 120ccctcggctc ggagaggccc ttcggcctga gggagcctcg ccgcccgtcc ccggcacacg 180cgcagccccg gcctctcggc ctctgccgga gaaacagttg ggacccctga ttttagcagg 240atggcccaat ggaatcagct acagcagctt gacacacggt acctggagca gctccatcag 300ctctacagtg acagcttccc aatggagctg cggcagtttc tggccccttg gattgagagt 360caagattggg catatgcggc cagcaaagaa tcacatgcca ctttggtgtt tcataatctc 420ctgggagaga ttgaccagca gtatagccgc ttcctgcaag agtcgaatgt tctctatcag 480cacaatctac gaagaatcaa gcagtttctt cagagcaggt atcttgagaa gccaatggag 540attgcccgga ttgtggcccg gtgcctgtgg gaagaatcac gccttctaca gactgcagcc 600actgcggccc agcaaggggg ccaggccaac caccccacag cagccgtggt gacggagaag 660cagcagatgc tggagcagca ccttcaggat gtccggaaga gagtgcagga tctagaacag 720aaaatgaaag tggtagagaa tctccaggat gactttgatt tcaactataa aaccctcaag 780agtcaaggag acatgcaaga tctgaatgga aacaaccagt cagtgaccag gcagaagatg 840cagcagctgg aacagatgct cactgcgctg gaccagatgc ggagaagcat cgtgagtgag 900ctggcggggc ttttgtcagc gatggagtac gtgcagaaaa ctctcacgga cgaggagctg 960gctgactgga agaggcggca acagattgcc tgcattggag gcccgcccaa catctgccta 1020gatcggctag aaaactggat aacgtcatta gcagaatctc aacttcagac ccgtcaacaa 1080attaagaaac tggaggagtt gcagcaaaaa gtttcctaca aaggggaccc cattgtacag 1140caccggccga tgctggagga gagaatcgtg gagctgttta gaaacttaat gaaaagtgcc 1200tttgtggtgg agcggcagcc ctgcatgccc atgcatcctg accggcccct cgtcatcaag 1260accggcgtcc agttcactac taaagtcagg ttgctggtca aattccctga gttgaattat 1320cagcttaaaa ttaaagtgtg cattgacaaa gactctgggg acgttgcagc tctcagagga 1380tcccggaaat ttaacattct gggcacaaac acaaaagtga tgaacatgga agaatccaac 1440aacggcagcc tctctgcaga attcaaacac ttgaccctga gggagcagag atgtgggaat 1500gggggccgag ccaattgtga tgcttccctg attgtgactg aggagctgca cctgatcacc 1560tttgagaccg aggtgtatca ccaaggcctc aagattgacc tagagaccca ctccttgcca 1620gttgtggtga tctccaacat ctgtcagatg ccaaatgcct gggcgtccat cctgtggtac 1680aacatgctga ccaacaatcc caagaatgta aactttttta ccaagccccc aattggaacc 1740tgggatcaag tggccgaggt cctgagctgg cagttctcct ccaccaccaa gcgaggactg 1800agcatcgagc agctgactac actggcagag aaactcttgg gacctggtgt gaattattca 1860gggtgtcaga tcacatgggc taaattttgc aaagaaaaca tggctggcaa gggcttctcc 1920ttctgggtct ggctggacaa tatcattgac cttgtgaaaa agtacatcct ggccctttgg 1980aacgaagggt acatcatggg ctttatcagt aaggagcggg agcgggccat cttgagcact 2040aagcctccag gcaccttcct gctaagattc agtgaaagca gcaaagaagg aggcgtcact 2100ttcacttggg tggagaagga catcagcggt aagacccaga tccagtccgt ggaaccatac 2160acaaagcagc agctgaacaa catgtcattt gctgaaatca tcatgggcta taagatcatg 2220gatgctacca atatcctggt gtctccactg gtctatctct atcctgacat tcccaaggag 2280gaggcattcg gaaagtattg tcggccagag agccaggagc atcctgaagc tgacccaggt 2340agcgctgccc catacctgaa gaccaagttt atctgtgtga caccaacgac ctgcagcaat 2400accattgacc tgccgatgtc cccccgcact ttagattcat tgatgcagtt tggaaataat 2460ggtgaaggtg ctgaaccctc agcaggaggg cagtttgagt ccctcacctt tgacatggag 2520ttgacctcgg agtgcgctac ctcccccatg tgaggagctg agaacggaag ctgcagaaag 2580atacgactga ggcgcctacc tgcattctgc cacccctcac acagccaaac cccagatcat 2640ctgaaactac taactttgtg gttccagatt ttttttaatc tcctacttct gctatctttg 2700agcaatctgg gcacttttaa aaatagagaa atgagtgaat gtgggtgatc tgcttttatc 2760taaatgcaaa taaggatgtg ttctctgaga cccatgatca ggggatgtgg cggggggtgg 2820ctagagggag aaaaaggaaa tgtcttgtgt tgttttgttc ccctgccctc ctttctcagc 2880agctttttgt tattgttgtt gttgttctta gacaagtgcc tcctggtgcc tgcggcatcc 2940ttctgcctgt ttctgtaagc aaatgccaca ggccacctat agctacatac tcctggcatt 3000gcacttttta accttgctga catccaaata gaagatagga ctatctaagc cctaggtttc 3060tttttaaatt aagaaataat aacaattaaa gggcaaaaaa cactgtatca gcatagcctt 3120tctgtattta agaaacttaa gcagccgggc atggtggctc acgcctgtaa tcccagcact 3180ttgggaggcc gaggcggatc ataaggtcag gagatcaaga ccatcctggc taacacggtg 3240aaaccccgtc tctactaaaa gtacaaaaaa ttagctgggt gtggtggtgg gcgcctgtag 3300tcccagctac tcgggaggct gaggcaggag aatcgcttga acctgagagg cggaggttgc 3360agtgagccaa aattgcacca ctgcacactg cactccatcc tgggcgacag tctgagactc 3420tgtctcaaaa aaaaaaaaaa aaaaaagaaa cttcagttaa cagcctcctt ggtgctttaa 3480gcattcagct tccttcaggc tggtaattta tataatccct gaaacgggct tcaggtcaaa 3540cccttaagac atctgaagct gcaacctggc ctttggtgtt gaaataggaa ggtttaagga 3600gaatctaagc attttagact tttttttata aatagactta ttttcctttg taatgtattg 3660gccttttagt gagtaaggct gggcagaggg tgcttacaac cttgactccc tttctccctg 3720gacttgatct gctgtttcag aggctaggtt gtttctgtgg gtgccttatc agggctggga 3780tacttctgat tctggcttcc ttcctgcccc accctcccga ccccagtccc cctgatcctg 3840ctagaggcat gtctccttgc gtgtctaaag gtccctcatc ctgtttgttt taggaatcct 3900ggtctcagga cctcatggaa gaagaggggg agagagttac aggttggaca tgatgcacac 3960tatggggccc cagcgacgtg tctggttgag ctcagggaat atggttctta gccagtttct 4020tggtgatatc cagtggcact tgtaatggcg tcttcattca gttcatgcag ggcaaaggct 4080tactgataaa cttgagtctg ccctcgtatg agggtgtata cctggcctcc ctctgaggct 4140ggtgactcct ccctgctggg gccccacagg tgaggcagaa cagctagagg gcctccccgc 4200ctgcccgcct tggctggcta gctcgcctct cctgtgcgta tgggaacacc tagcacgtgc 4260tggatgggct gcctctgact cagaggcatg gccggatttg gcaactcaaa accaccttgc 4320ctcagctgat cagagtttct gtggaattct gtttgttaaa tcaaattagc tggtctctga 4380attaaggggg agacgacctt ctctaagatg aacagggttc gccccagtcc tcctgcctgg 4440agacagttga tgtgtcatgc agagctctta cttctccagc aacactcttc agtacataat 4500aagcttaact gataaacaga atatttagaa aggtgagact tgggcttacc attgggttta 4560aatcataggg acctagggcg agggttcagg gcttctctgg agcagatatt gtcaagttca 4620tggccttagg tagcatgtat ctggtcttaa ctctgattgt agcaaaagtt ctgagaggag 4680ctgagccctg ttgtggccca ttaaagaaca gggtcctcag gccctgcccg cttcctgtcc 4740actgccccct ccccatcccc agcccagccg agggaatccc gtgggttgct tacctaccta 4800taaggtggtt tataagctgc tgtcctggcc actgcattca aattccaatg tgtacttcat 4860agtgtaaaaa tttatattat tgtgaggttt tttgtctttt tttttttttt ttttttttgg 4920tatattgctg tatctacttt aacttccaga aataaacgtt atataggaac cgtaaaaa 4978279001DNAHomo sapiens 2ataaaaatta aaaaccctga tagtatcagc acatacacag aaatcactcc attatgcaaa 60gttcatcctc tattatgaaa ggcaaaatgt ctacatttcc tatcaaccac tggcttcaat 120tcagtaaaac ttgcatacca agtaggcaag gtggaaaaga aaaaggcaga acatttcatg 180tatttcaatt cagacgcata aaaatgtcaa gccctacacg ttatcagctt tcgtatacac 240cgtcttctgc attcgcctgt acgggccaat gggctagctg gtcggcgttt gatgcttgaa 300gtgatggaac ggagtacggg gttaaatcca ctaccctctc cccacgcact ctagtaatta 360ctctatttcc acgtcatgtt tccgggtgtg tgtgtccctg ctcacgcaga aactgaagtt 420caaagcaggc ggagtcaccc atgttctttt tgttgtcccc agaacccaat tcaggagttg 480ggtccccaga ggatctggag atacctgggg actatctaac tagctgattc ccgcgtggta 540agaggctctc aacctcgcca ccacgtggtg ccaagggccg ggaaaaggga gagcgggcag 600gagggagctg tatcaggggc atttaaagtg ccttgacgtc acgcactgcc aggaactcag 660ctgagttttc agcaggacat tccggtcatc ttccctccct ccccccgggc ttctgtgccc 720aagtcctcgg ctcttccctc gctgtggcgg agggaggagc accgaactgt cggaacagcc 780agcacagggg cgtatcagtc tcctcttggc tccgcccttt ctcctagctg ctctcctcat 840tggtcagtgg gcggggcttc ggctgtaccg cacacgcact gggacctctg ggtggccgaa 900cgagctggcc tttcatgaat tatgcatgac ggcgtgcctc ggccaggctg gggctgggcg 960aggattggct gaaggggctg taattcagcg gtttccggag ctgcggcggc gcagactggg 1020agggggagcc gggggttccg acgtcgcagc cgagggaaca agccccaacc ggatcctgga 1080caggcacccc ggcttggcgc tgtctctccc cctcggctcg gagaggccct tcggcctgag 1140ggagcctcgc cgcccgtccc cggcacacgc gcagccccgg cctctcggcc tctgccggag 1200aaacaggtga agggggtgca gggtggggcc gttggggagg cctggggacc cgggggctcc 1260gcagcggcag ggggcctctg ggaccttggg gatgttgtga tggacgctgc agtggggccg 1320ggagagatga agagacgcgg agggtcgccc tgagggaaga ctcttcggga tgacaggagc 1380gggcctcgga agggactcgg ggcgctggag ggaagtttcg ttcttcggag aaacagaacg 1440cgctcgaggg ggcaccgtgg ggcgagggcg cactcggttg cggcggcagg agtgagggac 1500agtcccccga tttcctgctc cctggggccc tggggacgtt ccggccaccg gagcgactgt 1560cacgccgacg gggatcaccg gcgcgagtgg ggggtcggaa agcgcctcct ccccgcccgg 1620tcggcggctc ccgctgagcc acttcctccg cttgccctgt tcccgctcct tcaggagaca 1680gctgtgccct tttggaggca ggaataggtg tgtctgtcgc ctgcagcctt acgggctggc 1740tggtcgtggg taggctttat tgcataagaa tcaagtttcc tgtagggaaa ttgacagacc 1800ggtactcttt ctaaattccc tcgcatcttt ttctaggtta aattatgctc cccccacgtc 1860cccgccttgt aaaaaagaga aaaaaagaca aaataaaatc cccatcaacc cgtcaagcca 1920gctctagaga gagaaataaa cctcttgaca ttgtcctttt ccaaatacct ggtaaagtcg 1980gccagaagat aaataattga gccattgcat ttactggatt gtggtgttgc ttaattgcat 2040aggacggaat gaaccaattg agagtgggag ttttctgtct cagagccaag atcttgggta 2100aatgcagagg agagggaaac aaagacaggc tggccttgaa aaaaccatgt gtgcaaactt 2160tacatgcatt tggggggtgt ggttgcactg aagttaacaa gattcaaacc gtcgcccaag 2220ttggtatttc catgtttggt acacatcact ctgtgccata tcaggtcgtt gttaagtgtg 2280gtgacaaaat cagtggttag tcattttttt aattaaaaat gtgtatagtg tgtacctgct 2340ggtcttactg tatgtgcaac taaaggttta catagtctgt gtatgggttg taaatttttg 2400gctggctgtg ctgataaagc attgggcttg aataaagcaa agcagaaaat catctcaatc 2460ttttatatgt ggatttagac tgtgttatga cttggttcag ccagttttct atcttatttt 2520atattaaata tgtctgtgtt ctctgagtca gcacatttat ttccttatta catgttccag 2580acaggagtgc tagcccagtt tttgttcagt ttgcacagtg ggatggggaa acaagtctgg 2640aatttaaaaa aaaatgtttt agaggttgga gccttgattt tagtctctat attagcacat 2700ccatcacaaa gaaccattag taaattcatg aatcttttgt tttttatgta gttcatttga 2760gaagaataat cacttagaaa tatccacagt gccaggcatg gtggtgcaca cctctgatcc 2820cagctaattg aaggctgagg tgggaggatt ccttgagtcc aggagttgag tctggtctgg 2880gcaacatggt gagaggccag gaattgggtc tagagtctag tctaagcacc ataatgagaa 2940cccatcttta agaaagaaag aaaggaaagg aggaagaaag gaaagaaaaa gaaataccca 3000cagcacagtt atgaattaac ccacaaagga cttgtgaggt gggtagttca cataacaatt 3060accctaatat cgtagataag aaaattgagg ccaaaggatc aagacacttg gccaacgcag 3120cagagtgcca tagtggtgga atttgtgcct ccttctgtat attttgtgaa aagtatcagt 3180gaaattcttt tttttttttt ttttgagtca gagtcttgct ctgttgccca ggctagagtg 3240cagtggcgca atcttggctc actgcaacct ctgcctcctg ggttcaagcg attctcctgc 3300ctcagcctcc caagtagctg ggactacagg cgtgcgccac cacgcccagc taatttttgt 3360atttttagta gagaccgggg ttttaccata ttggccaggc tggtcttgaa ctcctgacct 3420tgtgatttgc ccacctctat ctcccaaagt gctgggatta caggtgtgag ccaccgcgcc 3480cagtaagtat cagtgaaatt ctaacatata tctgaacagt aaaataccac caataggctg 3540aaagacttca tgggaggtaa atattcaata aacaggtgaa aaaagaaata caaatggagc 3600ttgcttagat tatttttcta attgctatgt ctaacttggg aagtgaggaa ctgtttttgg 3660tcagcataat ttaccatcag aatttagcta tttactaatg aaaagaaata ctaatctagg 3720tttgttttag attaaggaca gtcatgacct aaatgtcatt taaaccagag tgcattgtgg 3780cttgatcagt ggtcatttct gtctctagaa agttgcttta acttctctgc ctctacgtgt 3840ctcttgacat tcagatatga ggtggggtag aggtggtgac caactttcca gacgcctgag 3900tccaaacctt cttagcttat ggttttctta ggtgatgtgc aaatcaacaa atatatactt 3960tttttttttt ttttttgagt tggagttgca ctctatcacc caggctggag tgcagtggca 4020tgatcgtggc tcactgcaac ctcctcctcc cgggttcaag tgattctcgc acctcagcct 4080cctgagtagc tgggattaca ggtgcccgcc actacgcccg gctaattttt gtatttttag 4140tagagataag gtttcactat attgaccagg ctggtctcaa actcctgacc tcaagagatc 4200tgcccacctc agcctcccaa agtgctggga ttacaggcgt gaaccacctt gcctggccaa 4260catatatata ccttttgcaa ctttgtcaga gttgctatga agaataagtt gtatcttgtt 4320cacagaaatt gcagtctact gggggagctg ataaatgttt taaccatcca atgtaacatg 4380ttgtcatcaa agagatggtg agactttaca cttgtgctaa caaggtagct gttctacata 4440aaagaacata cagtacagat gtagaacttt tctgttatca tagaacgttc tattggacag 4500tgctaggctg aatgctacag atcttcagag aaaggagagg ttatgaggcc tggagttgtc 4560tagaaagtct ttttgccaaa gagggatttc aactgggtcc caaataatgg gtggaatttg 4620ataggtgtaa agaatttgcg gtggtttatg cctgtaatcc cagcactttg ggaggctgag 4680gcaggaggat tgcttgagcc caggagtttg agaccagctt gggcaacgtg gtaaaactcc 4740ctctccccta aaaataaaaa aaattagcca ggcctggtgg cgtggacctg tagtcccagc 4800tactggtgag actaaggtgg gaggatcacc caagccccgg gggttaaggc tgcagtgagc 4860cgtgatcccg ccaccgcact ccagcctggg tgacagagtg agaccctgtc tccaaaaaaa 4920aaaaaaattc ctggtagccc ggtagactag gagggtaagt aggggagaag tgattactta 4980caaaagacat tgaatacagg accaaggaat ttcagttctg ttcttttgta ggggaagctt 5040ttaaaacttt cggggcgccg ggcgcggtgg ctcacgcctg taatcccagc actttgggag 5100gcccagacgc gcggatcacg aggccaggag atcgagacca tcctggctaa cacggtgaaa 5160ccccatctct actaaaaata caaaaaaaag tagccgggcg ttgtggcggg cgtctgtagt 5220cccagctact cgggaggctg aggcaggaga agagcgtgaa ctcgggaggc ggagcttgca 5280gtgagccgat atcgcaccac tgcactccag cctgggcgac agagcgagac tccgtctcaa 5340aaaaaaaaaa aataaataaa taaataaata aataaaactt tggagccgaa gcactgatgt 5400ttaatcatag agtgcttact atgtgttagg cacaggcctg attgcctgat gctggttaat 5460ttgtacaaag taaatcagtg catatgccct ctgccctagg ggagttatta actggagtct 5520gacattgtac aaaggtaggt atcctgacta gtttgatttg gtactttggg tgaaaaaagt 5580atagtgtgct taagtgcaga agtgtttttt gaggattttt gattggatac aaaccaccac 5640tcatatttta tgtctttggc acttaaaaat ttcaccataa cttttgagtc atttataaaa 5700accactgaaa gagtacttga gggacatccc cgaatcctga agaacttctg gtgttctgga 5760gcagcctcag tgagatccag gaggatggca ttgctgggct ggcccagccc ttattgatta 5820tggtgtaaag aattaatatg gtggttatat actctttgtt agacaccttg gcttacaaga 5880cgtaagcgta aagtgtagtg cgctttagtc agtatggcca catggtcctt tggtggtaaa 5940ttgtttgaga tgcctccagt ttttaaaagg agtagcatat cgggccagga gcagtggctc 6000atgcctataa tcccagcact ttggaaggcc gaggcaagag gattgcttga gcccaggagt 6060tcaagaccag cctgggcaac atagtgagac cactttgttt ctttaaaaaa aaaaaaaagg 6120caaaaacagg ctgggcatgg tggctgatgc ctgtaatccc agcgctttgt gaggcagagg 6180tgagcggatc acttgaggtc aggagtttga gaccagcctg gccaacatgg taaaaccccg 6240tctctactaa aaatacaaaa attagccagg tgtggtggca cacgcctgta gttccagcta 6300ctctggaggc tgagccagga gaattgcttg aacctgggag gtggaggctg cagtgagcca 6360agatcctgcc actgcactcc agactggggg acagagtgag acattctgac agtgctacac 6420tgaatgctac atgtcttcag aggaaggaga ggttatgagg cctgggaata acatatggaa 6480gaatgaattt ctgttatggt cagttctcat ttgtcatgtt aggattactg caactcttac 6540ccagccgggt gtggtggctc atgcctgtaa ttccagcact ttgggaggct gtgggcggat 6600cacgaggtca ggagatcgag accatcctgg ctaacacggt gaaactccgc ctctactaaa 6660aatacaaaaa attagcccag cgtggtggca gacgcctgta gtcccagcta ctcaggaggc 6720tgaggcagga gaatggcatg agtcctggag gcggagcttg cagtgagctg agatcgtgcc 6780actgcactcc agcctgggca acagagtggg actccatctc aaaaaaaaaa gaaaaaaaaa 6840aggattaccg caactcttta attcagatca gcaaacatgt tgagagccag gtattgcgtc 6900aggcaggatc caaggataat gaaatattgt ccgttttcat gaaactggag atgttgcagg 6960gaccgaggtg tgtgctatgc cagtatggaa gtaggacagg ggagacgaca gggcagtgag 7020tggttcaaga ctctggctct gaagtcaaac agatctggga ctgaatcctg gatctgccac 7080ttcctagtca gaatctgagc ctctattttc ttatctgtaa aagaagatta taacagtgct 7140tatcttgtag gtactgttga cgattcaata agataatgtg gataaaatgc ttagcatagt 7200gcctggcaca tagtaagagc tcggtaaatc taagttctta ctaaatatcc aagaaaagag 7260attaattctt ttcaggagtg agagaaagtc atcattattg aggggcttta tcagatggga 7320acacctgaat agggttttat aggatgaata ggaattcttt ccacgaagtt gcgttacaaa 7380aagttgcatt caaggctgaa ggaacatgag ggtgcagagg cttaaaacag ccttgtgtgt 7440tcagggagct ataagtagaa gttcttaatt taggagaact aaaccaaggg gaaaggaggc 7500caaggaacca cagttcttat cccttttctg ttaataattg ggtttaaatg tcattaaaat 7560aagttatttt gtccttttta gaaaagtaat aacatgctat tataaaaaaa aagacttgta 7620ggaatataaa atgtgtgttt tacatgtatc ctgttaattg acttgctttt attcagattt 7680tttgcagccc tttctgttta ccaggttatc ttggagacat atttattcca aattcctttt 7740tttttttttt tttgagatgg agtctcgctc tgtcgcccag gctggagtgc agtggcgcta 7800tcttggctca ttgcaagctc cgcctcccgg gttcacgcca ctctcctgcc tcagcctccc 7860gagtagctgg gactacaggc gcccgccacc acgcccagct aatgtttttt tttttatatt 7920tttagtagcg acagggtttc accgtgttag ccaggatggt ctcaatctcc tcacattgtg 7980atccgcctgc ctcggcctcc caaagtgctg ggattacagg cgtgagccag cacgcctggc 8040cttccaaatt ccttttaaca gcctagcaaa agaataataa ggaaggtaaa tctgccccta 8100caagaaaata atgcttcgac gatccggctt tccttcctgc tacccccagc cataagaata 8160aatgaccttg ctcatcactg aaattttacc tgacctttga atttttaact gcgtcagcca 8220aagaacttat attttgagta ttcctaaggt gattgctatt gtagttttga aacacttggt 8280tggtatgttt gagggtttca tggtccaaag ttactatagc agttaaaaga gtggactatc 8340aggtcagacc tattgggctt taatcccagt tctgccttct cttagacctt gggcctgttg 8400ttttcacttc tctggttttc agtttctctg tccacaattg tggaaacgag gtccacttgt 8460agagtaattg agaggatgaa gcaagatgat gcatatcaag tactttgcat agtgccgggc 8520agacaggtaa cattcaagtg ctaataatta ctattattac tatttatttt ttgagacagg 8580ttctcactct gtcacctagg ctggagtgca gcggtgagat cacagctcat gacagccttg 8640acctcctagg ctcaagtgat cctcctgcct cagccttcgg ggtagctggg gctacaggtg 8700tgtgctacca ccctcagcta attttctaat ttttttgagt caggatctcg tcacgttgcc 8760taggctgaat tactcttatt aaaaactata atatcaggcc gagtgcggtg gctcacgcct 8820gtaatcccag cactttggga ggccaaggcg ggtggatcac ctgaggtcag gagttcaaga 8880ccagcctgcc caacagagtg agaccccccc cgtctctact aaaaatataa aaattagcca 8940gttgtggtgg tgggcacctg taatcccagc tactcgggag gctgaggcag gataatcgct 9000tgaacccggg aggcggaggt tgcggtgaac cgagatcgtg ccactgcact acagcctggg 9060tgacagagtg agactctgtc tcaaaaaaac cgaaaaacaa aaagcataat tagggtggta 9120acgcttatac ataggggcag gtggaataaa acataattag gaggtcgggc atggtggctc 9180acgcctgtaa ttccagcact ttgggaggcc gaggcgggtc aggagttcaa gaccagcctg 9240cccaacatag tgagaccccg tctctactaa aaatataaaa tttagcctgt tgtggtggcg 9300ggtgcctgta gtcccagcta cccgggaggc tgaggcagga gaattgcttt tgaacccagg 9360aggtgggggt tgcagtgagc tgagatcgcg ccgctgcact ccagcctggg agacagagca 9420agactccgtc acaaaaacaa aaaacaaaaa actgtcatat caaaaactaa actaaaatgg 9480taatatctgt tagatattac aaagtcaggc aaattatgat tcatggcagc cactaatgac 9540ccaaaggaga gaaagaataa ttagcagatt ctaacctaat gggaaaaaaa ctaaatgaat 9600agggatgggg gacttacatt ctgttagagg aaattgaggc tgtcatataa aaggaatagg 9660taaggcaaac tgtaaattcc tgtttacaca aatgcccttc tgataaatct ctgcattgcc 9720cacagtccat gattacctct cccttatttt aagtaatatt taacacatta aaaatggatt 9780accacccaag gaattgctcc cgacccagaa agtgcaggta gtgttgaagg tttgagggga 9840agaggaatga ttagagttgg ttgtgtctca ggaagaagcc aacaggagga accttatttt 9900gagtcaggta aagaaggtgg gagtgaggag gcatcccggt ggccaggtat gaagctggga 9960gctgattgct gcacattact cagctgaatt aaatgtgccc tcacatctgt gtgtgtgcgt 10020acatgcaaat gtacatgtgt

atgagttagt tggaggggta gacctttatt ttcctgtcct 10080gtaactttcc tttgcaaact aatctgtatt cagaacagtg ttgcagttaa gaaccaccca 10140gcttgtccat gaaacaggtt ctctcacccc atctccccag ttttagagaa ggcaggaaag 10200aaaaggcagt gcttttcttt tttcctggcc gtatgcgggg caggaagaag ccagcagagc 10260ttgaaagaga aagtaaacct tctgggaaat aaacggcttg gcttccctat tgtggaggag 10320gagtgcaaat tattaggggg atgtttgggt agtttttgta gaagccattt ctgaaaactg 10380atttggatta gtgaaggtaa gcccaattta ggaaaaccct gcccagtctg gtgtcagcca 10440cctgtttccc gctttgtttg attgatttga ttagtttgtg gtattctgac ctctcatttt 10500tattacaaga gttggaagat ttgagtctga acttgagcac ctgcttcggt gaaagcttcc 10560taaaatgcat gttttttcac attttttctc atgttcattt tgttttgctt tttagcaaac 10620actttttctg acagaatcta aaagcattag acttttcttg ttttcccctt ctctccccac 10680aatgtaatct tgaaaaccca aatgttagct gtgtaaatta cctctcccgt aaaccaaaca 10740aagtgcaata ttgcattgag ttagcattga aatagtcggc ctttgaattt ttttctactt 10800gtggtttaga cataataaat atttcatctc agactgactt tctcgacaaa tcagttttgc 10860atttgggcct cttttcatca gtatgtttag ggaaagcaca tttattgaaa cattaaccaa 10920aatgaaacat aattaggagg ccgggagcga tggctcacgc ctgtaatccc agcactttgg 10980gagaccaagg catgtggatt gcttgaggtc aggagttcaa gaccatcctt gccgacttgg 11040tgaaatcctg tttctactga aaatacaaaa aactagctgg gtgtggtgac gcgtgcctgt 11100aatcccagct actctggagg ctaaagcaag agaatcgctt gaacctggga ggcagaggtt 11160gcagtgagtc gagatcgtgc cactgcactc cagcctgggc aacagagact ccgtctcaaa 11220caaccaaaaa aacaaaaaca agcataatta gggtggtaac gcttatacat aggggcaggt 11280ggaataattg aagcattctg gagccagaaa taatcaactg attaagaata atctggctgg 11340gtgcggtggc tcacgcctgt aatcccagct actcaggagg ctgaggcagg agaatcgctt 11400gaacctggga ggtggaggtt gcagtgagcc gagatcgcgc cattgcactc cagcctgggc 11460tatggagcaa gactccatct caaaaaaaaa aaaaaaaaaa atcctgtttc tgcagaaata 11520tcccaggtgt cctgggtcag cagtgcccca tagattccac ggacgtttac cctaagtttt 11580ccaatgggag ttcatacctc tatacccagt gagaatattt tctgagtaat gggaatgaga 11640ttggagatgt agggtagaga agatccatac agtctttggg ttaaactttt tcctctttgc 11700ctaggaaaga ttaatgctaa tcttaaccac agatttgtag taagaatgta tcagttttgt 11760cattcagttc tagactccag ttttctttat tgtaatacca atattttaga gtaaattttg 11820aaatgaatca gtacaaaaga tatgtagtaa gtggaaagtt agtccgcacc ttatccttgg 11880gactctttcc cagggacagc tagttaccta ctatttatct ctcctgagtt acttcatatg 11940tatgcatgca aacatgttat tctctgggtg ttgttccttc catatatagc agcaaataca 12000ccaaactctg tattttgctt tttgtcactt tatcttagag aatactcaat gcaaatacat 12060gtgtatatac ctcatgttta aaaaatctac atagtaaaat tagccaggca tggtagtgtg 12120tgcctgtaat cccagctact cgggaggctg tggtgggaga atcacttgaa ccctgagatc 12180acaccactgg actccagcct gggccacaga gcaagattct gtctcaaaaa acaaaaacaa 12240aaacaaaaaa actacagagt agtattctag gctatgcata tcataaattt gatttcctaa 12300tgataggcat agatgatttg cctgggcggc aaattagcgt tggctgtgtc tcaggaagaa 12360gccaacagga ggaaccttat tttgagtcag gttccaaaga cagaaacatt gtctgacatt 12420tgtttttggg cttatatgaa taaatctgta cacatatatt tttaatgttt taatcgtaat 12480atgtatacta tttggaaatg tggcttttta gttaacagag tgcatgtttt accccattgc 12540acttaaacat taacttgggg ataattaaat gagtctgtca cttggacagg caggaattgt 12600accccccaca aacccataaa ccgccaattt tttttttttg agacagagcc tcattctgtt 12660gcccaggctg gagtgcagtg gtgcgatctg ggcccactgt aagctcagcc tcccgggttc 12720atgccattct cctgcctcag cctcccaagt agctgggact acaggcgccc gtcacaatgc 12780ccggctaatt ttttgtattt ttagtagagt cggggtttca ccatgttagc caggatggtc 12840tctatctcct gaccttgtga tccgcccgct ttggcctccc aaagtgctgg aattacaggt 12900gtgagccacc gcacctggcc ggtttttttt tttttttttg agatggagtc ttgctctgtt 12960gccaggctgg agtgcaatgg catgatctcc gctcactgca acctccacct cccgggttca 13020agtgattctc ctgcctcagc ctcctgagta gctgggacta caggcgtgtg ccaccacgca 13080cagctaattt ttgtaatttt agtagagatg gggtttcatt aataatcatt aatattagac 13140aactgtcaga ctcacagtgg tggatacaaa ctttctcaaa ttctgatttt tactctaaag 13200ctcaaatttt atcattggca acaaatattg tcagttgctt tccctgaaca gacagcttcc 13260cttctttcat ttttgagaaa atatctgcca gtatcccagt tggtttatca atcattcttt 13320ctcttttttt ttttgagacg gagtctcact ctgtcaccca ggctggagtg cagtggcatg 13380atctcggctc actgcaacct ccacctccca ggttccagca attctcctgc ctcagcctcc 13440cgagtagctg ggattacagg ggctagcagc cacacctggc taatttttgc atttttagta 13500gagacagggt tttaccatgt tggccaggct gatcttgaac tcctgacctc atgatatgcc 13560caccttggcc tcccaaagtg ctgggattac aggtgtgagc cattgcgccc ggctctatta 13620tttcttttct ttctttcttt ttcttttttt tttttgagat ggagtttcgc tcttgttgcc 13680caggctggag tgcaatggcg cgatctcggc tcaccacaac ctccgcctcc cgaattcaag 13740tgattctctt gcctaagcct cccgagtagc tgggattaca ggcatgtgcc accacacccg 13800tctagttttg tatttttatt agagatgggg gtttctccat gttggtcagg ctggtctcga 13860actcccaacc tcaggagatc tgcctgcctc agcctcccaa agtactggga ttacagtttt 13920gagccacctg acccggtttg cttattattt cttttaaatt taaaaaataa taaataaagg 13980ggccatgaga gcgaagagtt tgagaaaggt tggtctaaag gttttaacat aagaatccct 14040gggttatttg cttaaaaaga agaaagaatc tatggatctg cctgagaggg tctgatgtag 14100tttatctggg gtcatcctca caggcatagc agatattctg attcagatgg tccttggtcc 14160ttagtttgag aaatgtggct ttacaaggcc catagaatat aaagtcttct ttggattagt 14220gaagtcatgt ccacagggtt tagaaaatgt ttttgtttta gagataaagg taagtggaag 14280agtagacatg tagtgaatga gggaaaatgt tttagagatt tctttttatt ctgtttactc 14340ttcttggtat gcacgtacct gaatattaag gatattttat gaagtcatga cattaccaga 14400ttaatgttgg ttttgtttta aggtactttc tgactgctgg ggttaattcc tacagacgat 14460tctggtaaag aatagccttt aagttttaaa agtgttgact tatttcagat gtcttaataa 14520agttaacttc cagttattac atgtaacgta tataaagctc tcattttcct ttattctcgt 14580taattgtttg cataacaaat tcaaagggaa atttgcttgg cagagatcag atagcagaga 14640tgagatttaa aaacaggtaa tttggctact agcctgggag tttgaagatt ccaagtttgc 14700atccatgtgt agtcacttaa catttctgtc cttatctgta aatgggaata acacctactt 14760gatagggttg ttacattatc ttggccacct caggttctct ttggctgagt gattgactgg 14820aaaacgcaat gtgaattcat gcttcagact gggttctttt tttttttttt ttttgagatg 14880gagtttcact cttattgccc aggctggagt gcaatggcac gatctcagct cactgcaacc 14940tctgcctccc aggttcaagc gattctcctg cctcaggctc ccgagtagct gggattacag 15000gcatgcacca ccatgcctgg ctaatttttt tgtattttta gtagagacgg ggtttcactg 15060tgttggtcag actggtttca aactcctgac ctcaggtgat ccacctgctt cagtctccca 15120aagtgctggg attacaggca tgagccaccg cacccagccc aggctaggtt ctatatgggt 15180gtgcttttta gaatttagat catgggctat ccccaacaca aactggataa tgtttctttc 15240tagattctct ctaagcgtgt attctctttc tttcctaggc acagccacca cttcacttac 15300attgtgggat tataatttca tgagtagtgg aatttcctta accttctctt gtgtgggagc 15360tgaaggacaa aatgagatat tctctgaaga gtggttacat catgcaaaac tatgatgtgt 15420aatgaggtca cttagttttc taagtacatt atacattttg ataagatttt catagaaaag 15480cttgtctcct tggggagatc actcatcttc catcttgact attatttaaa ctttatgggt 15540cagatttatc tttttaaaaa cttaaccata aagctcaatt aatttttttt tttttttttt 15600gagacggagt ctcgctctgt tgcccaggct ggagtgtagt ggcgcgatct cggctcactg 15660caagctctgc ctcccaggtt catgccattc tcctgcctca gcctcctgac tagatgggac 15720tacaggcgcc cgccacgatg cccggctaat tttttgtatt tttagtagag acggggtttc 15780accgtgttag gatggtctcg atctcctgac ctcgtgatcc acccgcctcg gcctcccaaa 15840gtgctgggat tacaagcgtg agccaccgcg cccggctcaa ttaatatatt ttaaaaatta 15900atagacttta ttatttttat tttattttat ttttgaggca gagtctcgct ctgtcaccca 15960ggctgagtgc agtggtgtga tcttggctca ctgcaaactc cacctcccgg gctcaagtga 16020ttctcctgcc tcagcctcct aagtagctag gattacaggt gcctgccacc atacccggct 16080agtttttgta attttagtag atacgtgttt tctttctttt cttttctttt ttttgagatg 16140gagtttcact ctttttgccc aggctggagt gcaatggcat gatctcggct cactgcaacc 16200tccgcctccc aggttcaagt gattctcctg cctcagcctc ccaagtagct gagattatag 16260ttgtctgcca ccacgcctgg ctaatttttt gtatgtttga tagagacagg gtttcactat 16320gttagccagg atgtctcgat ctcttgacct cgtgatccgc ctgccttggc ctcccaaagt 16380gctgggatta caggcgtgag ccactgcggc cagtctagac tttatttttt aaagcagtgt 16440tagttttaca gaaaaattat gtggaaagta cagagagttt ccatataccc cttactttct 16500cccacaactt ctattattaa catcttgcat tagtatagta cgtcccttac aactaatgaa 16560ccaactcgat acattattat taaccaaatt cctgagttta ttttatttct atttttattt 16620tattattatt attttttaga ggtagggtct cactgtgttg tccaggccag gttgcagtgg 16680catcatcata gcttgctata gcctgaaact cctgggctca agcaatcctc ctgcctcagt 16740ctcccaaagt gttggaatta caggtgtgag ccactctgtc cagcctgaag tccatagttt 16800acattacatt tcactctgtt gagcattcta tggattttga caaatgtgtg atgatgtata 16860tttgccagta cacaattata taaaatagtt ttactgccct agaaaccccc tgtgctccac 16920ctattcattc ctctgctgaa ccactggcaa ccactgatct tttataatat ctccatagtt 16980ttgtcttttc cagaatgtca tatagttgga catacagtgt gtagcctttt cagattggct 17040tctttcagta aatgatatgc atttcaggtt tcttcatgtt tttttgtggc ttgataggtt 17100gtttcttttc attggtgagt aatactctat tgtatggata taccacatgt tgtttatcaa 17160acattcacct gaaggataga catcttggtt gcttccaagt ttgagcagtt atgaataaag 17220ctgctataaa cattccagtg caggactttt cacctcctct ggataaatat caaggagtgc 17280aattgctaga tcatatggta agagtatgtt tagttttgta agaagctatc aaactatatt 17340caaagtgact gtaccattat acattcccat cagcagtgag tgagagttcc tgttactcca 17400catcttcacc agcatttagt ggtgtcagtg ttttggattt tagccatttt aatgggtgta 17460taatggtata cctattaaaa ttggtttttt ttggagacag agtttcacag tttcactctt 17520gttgccctgg ctggagtgca atggcgcaat ctcggctcac tgcagcctcc gcctcccagt 17580ttcaagtgat tctcctgcct cagcctccca agtagctggg attacaggtg cacgccacca 17640tgttctgcta atttttttgt attttagtag agatggggtt tcactgtgtt acccaggctg 17700gtcttgaact cctgagctca ggtaatccac ctgcctcagc ttcccaaagt gttaggatta 17760caggcatgag ccaccgcacc tggcctcaat tttttttttt ttttttttga gacagagttt 17820tgctcctgtt gaccaggctg gagtgcagtg gcacaatctc ggctcactgc aacctccgcc 17880tcctgagttc aagcgattct cctgccacag cctcctgagt agctgggatt ataggcgccc 17940gccactacgc ctggctaatt tttttttttt ttttaattag agacgaggtt tctccatgtt 18000ggtcaggctg gtcttgaact ccccgttctc aggtgatccg cctgcctcag cctcccaaag 18060tgctgagatt acaggtgtga gccaccgtgc cccgcctgtt ttggctttta ctgtgaagac 18120gtgttagccg ctgtgatgac tagcaagtgt ggccctccac ccagtcgctc tgggctccca 18180gctcctgcat cctgctgcaa acttgacatc ttccctcaag taacttgtag ttgtctcctg 18240tctacttgcc caaaatataa ctcttaaact tttctctctg caagtttgtg cctctctccc 18300tgtctgactt ccccatctaa ataaatggta gaccaccatc tactcctttg tgcaagccag 18360aaatctagga atcatcctta aattccctgt tctgtcttat ctctgctttc attcaaagca 18420tcagcaaatc ctgttggttc tacctctgaa gttttctcaa atactgttac ttgactcatc 18480ctgacttttg tttctgcttt atgttaggct aaatgccctg aaaactcttt tgtacaaaac 18540acctagaaat actggataaa ctgggcttaa cagggaggcc cggtgtggtg gctcacgcct 18600gtaatcccag aactttggga ggccaaggtg ggtggatcac ctgaggtcag gagttccaga 18660ccagcctggc caatacgtag tgaaacccca cctctactaa aaaaaaaaaa aaaaattagc 18720tgggtgttgt ggtgcacacc tgtaggtggt gcatgcttga acttgggagg cggaggttgc 18780agcgagctga gatcgcgcca ctgcacttca gcctgggtga cagagcagga ttctgtctct 18840taaaaaaaaa aacaaaaaaa gaaaaacagg aaaatcttca gaagcaaaaa ccaaacaatc 18900tcaccaaaga aatgagaaga tggctgggcg cggtggctca cgcctgtaat cccagcactt 18960tgggaggccg aggcgggcag atcacccgag atgggcagat cacccgaggt caggaattcg 19020agaccagcct ggccaatatg gtgaaacccc gtctctgcta aaaatacaaa aattagccag 19080gtgtggtggc aggcgcctgt aatcccagct actcaggagg ctgaggcagg agaatcgctt 19140gaacctggga ggcggaggtt gcagtgagcc gagatcatgc cactgtactc tagcctggac 19200gacagagcaa gactctgtct caaaaaaaaa aaaggctggg tgtggtggct catgcctata 19260atcctagcac tttgggaggc caaggtgggc ggatcacttg aggccaggtg aacatggcga 19320aaccccatct ctactaaaaa tactaaagtt agctgggcat ggtggtgggt gcctgtaatc 19380ccagctactc gggaggcgag gcaggagaat cgcttgaacc aggaggtgga ggttacagtg 19440aaccgagatc tcgccaccgc actctagtct gggcgacaga gcaagactcc gtctcaaaaa 19500acaacaacaa aaaaccaaca catggccaaa gtgcagtgac ttacatctgt ataatcccaa 19560tgttttggga ggctgaggca ggaggatcgc ttgagtccag gaatttgaga ccagcctggg 19620caacatagac ctcatcacca aaaaaaaaat attttttaat tagctgggtt tggcagcatg 19680tacctgtagt cctagctact caggaggctg aggtgggagg atcacttagg cccaggagtt 19740tgatagttcg aggttatagt gagctatgat cctgccactg cactccagcc tgggccacag 19800agtgagaccc tgtctcttag aaacaaaaca aaacaaaaaa aagaaactga attaaaaaca 19860acaagaacaa aaatgctgct ttttgttatt gagttgtagc ccaagtttct tgagggtaaa 19920gcattgaaaa gcaggcagta atagatttgc tgtttaaaga gatttacttg cagcactatt 19980cacaatagca aagacatgga atcaacctaa atgcccatca gtgacaaatt ggataaagaa 20040aatgtggtac atacactgtg gaatactatg cagccataaa aaacaacgag atcatgtttt 20100tgtttgtttg tttgtttgtt tgtttttgag atggagtctt gctctattgc ccaggctgga 20160atgcaggtgg cacgatttca gctcactgca acctccgcct cccaggttca agcaattctc 20220tgcctcagcc tcccgagtag ctgggattac aggtgccctc caccatgcct ggctaatttt 20280tgtatttcta gtagagatgg ggtttcaccg tgttgggcag gctgttcttg aactcctgac 20340ctcatgatcc tcccacctcg gcctcccaaa gtgccgggat tacgtgtgag ccaccgtgct 20400cggctgagat catgtttttg caggaacatg gatggagctg gaggctatta tccttagcaa 20460agtaatgcag gaacagaaaa ccgaagacca cgtgttctca cttataagtg ggagctaaat 20520gataaggact tgtgaacaca aagaaggaaa ccacagatac tggggtttac ttgagggtgg 20580agagtgggag gagggagagg aacagaaaag ataactattg ggtattgggc ttaatactta 20640atattttatc aaaataagct gtacaacaaa cccctctgac atgagtttac ctatataaca 20700aacttgcacg tgtaacccca aacctaaaat aaaagttaaa aaaaaaaaaa aaggctggtt 20760gcattgggag gctgaggcag gcagagcact tgaggccagg aattcgagac cagcttggct 20820aacgtggaga aaccctgtct ctactaaaaa ttcaaaaatt agccaggtgt ggtggtgcat 20880gcctgcagtc ccagctacca gggaggctga ggcaggagaa ttgcttgaac tcaggaggca 20940gaggttgcag tgagctgaga ttgcaccact gcattccagc ctgggcgaca gggcgagacc 21000ttgtctcaaa aaacaaaaca aaacaaaaca aaaacctgtc actttgggaa tatctcaaac 21060ctagtcatcc aagtggttgt acgattttag tgtctgcata tcaatattta gtgtgatcta 21120ctttcttaga ttctcaaata ctgccaatgg gcacatgtca tgaaataatg tcttttagag 21180gacaagagag tgctaaagtc tcattattgc agtttaagaa aaacaattct gtaacagttt 21240aactttatag gaaatgcctt ttgtttattt attttttttc ttttgaggct tagattttta 21300tttttatgtt tttagagatg gggtcttcct atgttaccca ggctggcctt gaattcctgg 21360gctcaagtga tcttcctgct tcagcctcct gagtagctgg gactagacgt ccactactgc 21420tcctggctgg aagtttagat tttaatttaa actcttctat tgggaaactt tgtatgtttg 21480ctttaccact taacatttgc atgcattatt gtacctattg tctcctactt aaggaagggc 21540agtttatgct gttatatgaa gtgaattaac ctcctatggt acttcagttt tctctatgct 21600aaaagtgtgt tctagatttt tgaaaaactt acttaatttt cattcattta ttcaaatatt 21660tgagcattct gtagttgctg gggaaatagc agtgaactga agaatgtctt tgttcttatg 21720gggcttaagt tcctagttga tcatattgga aggagataca tgaaaaaaga aatatatgaa 21780caatggaggg cgatgagtac tgtaaaggag aattcagcag gggagatgtt gctgttttag 21840atagaggggt gtcaagagac attgtgcaga gacctgaacg aagtgaggga gcaagccatg 21900gagatatcta gggaaagagc ctatcaggtg gagagaagag tcctagggca gaaacgggca 21960aggtgtgttc caggagcaga gaggggacag ctgtgagcaa ggggagagtt gtagggaagg 22020aggcaaagag agacatctgg ggcaaaatgg attgactggt gggccgtggt aggactttgg 22080attttttcct gagtgggttt tgagcagggg aatgaaatga tctgactctg gttttttttt 22140tttttggaga caaaatcttg ctctgttgcc gaggctgaag tgcagtggcg caatctcggc 22200tcattgcaac atctacttcc tgggttcaag ctatgctcct gcctcagcct cccgagtagc 22260taggattaca ggcttgggcc accatgccgg cgaatttctg tttttatttt tattttttat 22320ttatttttat gtttatgttt tttgagacgg agtctcgctg tgtcacccag gctggagtgc 22380agtggcgcga tctcagctca ctgcaacctc tgcctccccg gttcaagcaa cttctcctgc 22440ctcagcctcc cgagtagctg agattacagg cgcctgccac tacacctggc taatttttgt 22500atttttagta gaaacgggat ttcaccttgt tggccaggct ggtctcgaac tcctgacctt 22560aatttatctg ctcgccttgg cctcccaaag tgctgggatg acaggtttga gccaccgtgc 22620cagccaggac tcttattttg aaaggatctg taatgtggag aatagaaggt agagggacaa 22680ggatgaaagc atccaggcca gttagcctag tccagctatc taggtaagag atgctggtgg 22740cctggattaa ggctgcgtca gtgggaggtt gtgagaaagg ctcaccttcc tttttttttt 22800tttttttttt ttttgagaca ggatcttact ctgtctccca ggctggagtg cagtggtgca 22860atctcagctt actacaacct ccgcctcctg ggctcaagtg atacccccac ctcagcctcc 22920caagtagctg ggatcacagg cttgcgccac tatatccggc taatttttgt atatttcgta 22980gagacagggt tttgccatgt tgcctaggct ggtctcaaac tcctgagctc aagtgatcca 23040cccgcctcag cctcctaaag tgctgggatt ataggcctga gccattgtgc ccggtcactt 23100ccagattttg aagacagagc caacaggatt tgttaatgga ttaggtgtgg caggaggagg 23160gggaggaaga gagagagaga ctggagttga agttaaggct catttcaagg tttttagcct 23220caacatgtgc aggaatggag ttgtcacttg ctagaatggg ggagactgga ggagaagccg 23280gctgggagag gtttttaatg aaggggttgg ctttggatac attaagtttg acatgcattt 23340tagacatcca ggtggagata ttgaagaggc agttggctat aagtgtctga tgttcatatt 23400agcggatggg gctagagaca taaatttgag aattgtcagt gtataaacgt tgttttgaaa 23460gaaagtgggg ctgaataatt tagaaaggag tgcatagaga aaataagttt actattaaaa 23520tagctttaac aggccgggca cggtggctca tgcctgtaat cccagcactt tgggaggctg 23580gggtgggcag atcaaaaggt caggagtttg agaccagcct ggccaatatg gtgaaaccct 23640gtctctactg aaaatacaaa aattagccag gcgttgtacc gggcacctgt agtcccagct 23700acttgggagg ttgaggcagg agaatcactt caacccggga ggtggaggtt gcagtgagcc 23760aagatcacgc cactgcactc catcctgggc aacagagcaa gactccgtct caaaaaaaaa 23820aacaaaaaaa aacaaaaaaa aaaaacttta acagcaaagc ctcttccttt aaaattatga 23880atttttttct tatggaagtt ggactctttc attattaagt ctacattcaa tcactatgtt 23940agtaaaaatg ttgttctagt tgccgaatgc aataaaccag ctcagactta gtggcctaaa 24000gcagcaatca tttgactatg ttcgaagatg ccgtgggcag gaatttagat aacagcaggg 24060atggcttgtc tttgctctgc gatgtctgag gtctcactga gaaaactcaa gcggctgggg 24120gtaataatca tctggaattt tctttactcc tgtatctgat gtctgggctg cgatgactca 24180aaggctgatt tcagctgaga ctgtagacca cgtgcctact tgtggcctcc ccttttgcct 24240tgggtttctc acagaatgtg gctggttctg gagaatgaga cttccaatga aatcaggtgg 24300aaatgacatc tcgccgcttt cagcatgctc tattggttgg aacagttatg gacttagcta 24360gattcaaagg aagggaacaa agaccccctc ctctcagaga gtggggcata atgagagaat 24420ttagggccat gttatccaac caccacaaat gccttctgaa tttgaggttc tgcctcaaaa 24480gttcatagtt cctttgactg aaggacttct atatatccaa gcatcgtcag ccccaggtat 24540attgttccat gtaagtgacc aggactacct tagtatttcg tatagggaaa gtgacctgaa 24600taaatttgag aaaagaatct tccttctctc cagtaagcac tgaggtaagc attgagccat 24660attataggtt tatgactttg agactcagaa atttaaattc ttggccaggc gcagtggctc 24720acgcctgtaa ccccaacact ttgggaggcc aaggcaggca gatcacttga ggtcaggagt 24780ttgagaccaa cctggccaaa atggtgaaac tccatctcta cgaaaaatac aaaaattagc 24840caggtgtggt ggcgggcacc tgtaatccca gctacttggg aggctgaggt aagagaatgg 24900cttaagttct ctttatctgc tttatttcag ttgcctctct tagatgaata ttaatgactt 24960acatagcatt ttagatcagt ggatgttttt gtgattcttt tatttgagct ttggccaaag 25020ataacagtac ccacaggttt tttccagcta ctcgctcttc tcccttcagt ggccctcgag 25080cctggaaaat ctgacatgac

aatgtgcttg ctcaacctac cactgttttt cttttgaaaa 25140gtttggcagc ctgtttctga ctcctatgaa ggtgaattcc tcagcattca cagtttatta 25200gaaaaatact ttgcttctct ccaaactcga aattcaagat aaccaaacct atatataggc 25260tgatctttca ggatgcagtt gtcatgttga tgccatgctt ttcagtatcg tggccatcat 25320ctgttcagta ggggaggtgt acttctgtaa tgggaggtgg tggttatgtg tgtgtgcaag 25380tgtttatttg gtgtcttaag ttagcctgtg ggaagttcta aatcaggatg gtacgtggtt 25440gccagcagag agctgctcct caagtgaagg aggtagaatc aaagccaata ggaaagagcc 25500tcagatgctt atatatgtac cgtggggatt cagagtgaaa gcagtcattg gactaggggt 25560ggggttaggg agagcctgtc tgacagacac aagaaaggga tggataacgc cacccagaga 25620aaaaagcatt ttaggcaaga acaaatatga aaaaggaaca aagtctgtgg gtggggggca 25680aggaggagat aagttgactt gaaggaagac aacacttatg aaagtcacct ggaggctggg 25740tgccatggct catgcctata atcgcagcac tttgggaggc cgaggtagga ggacaacttg 25800agcccaggag ttcgagacca tcctgggcaa catggtgaga ctgagtctct accaaaaaaa 25860aaaaaaaaag aaaattatcc agacatggtg gcatgtgcct gtaatcccag ttactcagga 25920ggctgaggtg ggagggttgc ttgagcccag gaggttgagg ctgcagtgag ctgtgatcgt 25980attattgcac tccagcctgg gtaacagagc aagaccctgt ctcaaaaaat gaaagtcatc 26040tgtaggctgg agagaggaac tggaaggggc taaagttggc tgagtagtta cagagcctga 26100gataagggta aagattttgc attggacaat gagatgttag tgtgtgtttt tgagctgggg 26160agtgctgtga ttttactctt attgaagaat cactgaagga ttattcttga atcagtgatt 26220cttgatcatt cttgaatttt tcaaacagca aaactggaag agttggccta ttcctcagaa 26280tattttctaa ttgggcgcag tgtcctcact tgggagaacc tggctacaca ctttagttgt 26340aattcactcc agtcgttcat tcattcaata cctatttttt cagcacctat tatgagccag 26400acactatgct ggatgccagg gttcagggta ggacacgcta gtgagcaaaa gccaagactc 26460ttcttgtctt catggggctt tcagtccagc atagtggtta tgagtccaag ttaatggagt 26520cacagtactt gggtgcaagt catggtgatg gtgatagaag gaaggcatgt gtgagggcca 26580gtggcaggca ggagcctggt gtttttgagg acctgaagaa ggagcagagt gagtgccagg 26640aacttagcca ccagctggta ccagccatac gagaggggca gagccagcca ggatgtcggt 26700catgctagta atgagtacaa acacttacat gctgcacgct attgggctcc tgagtgctac 26760gtgttcatta gctcgatgaa tttgtacagc aaccctgtga ggtaagcact gttctctccc 26820ctttctatag atgaggaaat taaggcacaa agaggataaa taactggcac cagctacacg 26880ctaagtgatc gaagtggtgg aaccaggatt caaatccatg ctattctgcc ttaagataac 26940aaatcttgtt ttttagccta agaacagagc agtcatcagg agggttttaa gtaggggtgt 27000ggcaagatca agtttgtgtc ttgaaaaggt ctctctaccc acagtgtgga aaatggcctg 27060gaggcaagca cacagatgtt gggagacagt taacagctct tgccatggcc ccctatgcat 27120tttggctctg atgtttctgc ctgatttttc tcttgcctct gcctcttttc ctgaggggat 27180ggcaggtttt accattcagc tggagtacaa accctgaacc ctttttggtt aaatatctac 27240ttgcttttcc tacagtatta ttttgagttg ctgtggctgt aatgtcttga gggaatcgag 27300cttgacagta atttatagaa caaacagttt ttagagactg tgtggcccaa ttgccctctc 27360aatgttggca ctcctgccat gacatttacc atgctgagca tgtgaccgcc atctgaatac 27420caaatgccac aggaacctgg gaggttgtca cttactcctc cctttctctg agtcaccttt 27480gcccttcagt cagtcaccaa gtcccatcac atgtagctct gtaatgtcac agaagatgga 27540tgtctgcctc aaaacactta caatgctgct acctaaattg ggcagccacg acctcccacc 27600aggattattg cagcctgagg gatctttttg aaatgtaaat caaactatca cttgtctgtt 27660taaagctttt caaagactta ccccattgcc cttggaagaa agtgcagata tcttgacagg 27720agagccttct ccagcctcct cttctgccgt ggtctccttg tacagtctct acagtgtact 27780gcttcattag aaccctggag attattattt gctagttctg ggctaagaac tggcacctgg 27840ctttgtagag ctcctcagga gattctgagg cgtattcaga gttgagccct gatctctgct 27900ctgatttcga ggttctcgtt atatttatta atgatcacga aaaaatttat tattattctt 27960tggcctcact ttagcatcat ctgaggaatt tttttttttt tttgacagag ttttgctctt 28020gttgcccagg ctggagtgca atggcgtgat ctcagctcac tgcaacctcc gcctcccggg 28080ttcaagagat tctcctgcct cagcctccca agtagctgaa attacaggca tccaccacca 28140tgcctgctaa tgtttttgta ttttttagta gaggtgggct ttcacagtgt tggtcaggct 28200ggttttgaac tcctgacgtc agctgatcca cccacctagg ccccccagag tgctgggatt 28260acaggtgtga gccaccgtgc ccagccgtag ctttcgaaat ttgaaacctg gtcccactgt 28320cagaggttcc aatttggcac tggtttggtt cccaggcatc tttcttgctg tatatatttt 28380ttagtgtcag ccagggtgga gacctctgta ttacttcatg gggaagaatt tgggagaaga 28440tgttgtgagg agacaggttc tagtcctaga gtgatttatc ctttctcgta cagatttcca 28500ggtatttgag gggccactct tctgtaattc atgtttttct ctcctaacct cactcctgtt 28560gcctgcatct tcttgctgag caaaatattc aaggtcttca actcctcaca ccctggttgt 28620ccctccctgg atgtgtttgg ttgttttagt gttccatttc aattttgata cacagaatta 28680gaatagcatc cagatgtggg tctgttacag ctagactact agatccttca aaatccaagt 28740actagtatgt ctattaaaat accataagat cacattggct agttacaatg gttggtttgt 28800gggttactta aaaatcaact aaaattcttt tttttttttt gagatggagt tttgctcttg 28860ttgcctaggc tggaatgcaa tgacacaatc ttggctcact gccacctctg cctcccaggt 28920tcaagcaatt cccctgcctt agcctcctga gtagctggga ttacaggcat gtgccaccat 28980ggccagctaa ttctgtattt ttagtagaga tgaggttttt ccatgttggt caggctggtc 29040tcgaactccc gacctcaggt gatccacctg cctcagcctc ccaaagtgct gggattacag 29100gcgtgagcca ctgagcctgg ccaaaattcc cactttctaa tactcctgta gtagctgggt 29160acggtgggtc acatctgtaa tcccagcact tttggaggct gaggctggag gatcgcttga 29220gcctaggagt tcgagaccag cctgggcaag atggccagac gccatctcta atttaaaaaa 29280aagaaaaaac aagactccta tagtggtgaa gaacagacat tccgaaaaca gactgtgcgt 29340tatgattcca gctccatgcc tttactacct gtgttgtgac tttggataaa tcacttaaaa 29400atcttttttt tttttttttt tttttgagac ggagtcttgc tctgccgccc aggctggagt 29460gcagtggcgc gatctcggct cactgcaagc tctgcctccc aggttcacac cattctcctg 29520cctcagcctc ccaagtagct gggactgcag gtgcccgcca ctacacctgg ctaatttttt 29580gtatttttag tagagacggg gtttcaccgt gttagccagg atggtctcga tctcctgtcc 29640tcgtgatcca cccgcctcag cctctcaaag tgttgggatt acaggcgtga gccaccgcac 29700ccggccaaat cacttaaaat tctgtgcctc agtttctcct ctgtaaagtg ggataaaaat 29760agtacctatc tgatagggtt gttacaatta tgaaatgagc aaataagtat gtcaagtgtt 29820taaaacagcg cctggcttct tgtaaaaagt gctatataaa tcatagctat aatcattact 29880tatttcgact gctctttaac caaggttctt atttttcatc tttttctttt gttttgaata 29940tcacttagtg ttttcacctt ttactctttt taggacctag agccatccta ggtgaaatac 30000gtatggagat atttgatcag gtcaccaccc agctctcctg acctcccttc tctccttaaa 30060ttaacatgcc aaatcacagc atcactgact ccttccctcc cgatatgata agagtgtgca 30120ttgaaatgca tgtattttac ttagcaggga aagctgatta gtgattatca cacttaaccc 30180ctagtgaatc tgatggatta acctgctttc caggacacta aggaaatggg tttaagataa 30240gaaatatctg gctgggtgcg gtggctttac gcctgtaatc ccagcacttt gggaggccga 30300ggtgggcaga tcacgaggtc aggtgattga gatcatcctg gctaacacga tgaaaccccc 30360tctttactaa aaatacaaaa aattagccgg gtgtggtggc gggcgcctgg agtcccagct 30420actcgggagg ctgaggcaag agaatggtgt gaacccagga ggcagagctt gcagtgagct 30480gagattgtgc ccaccgcatt ccagcctggg caacagagtg agactacatc tcaaaaaaaa 30540aaaaaaaaaa agtaagaaat gtccatgaaa gggagaccct gggggaaagg aacaataact 30600gcagctctga ggatctggca ccagcagcac cagcacagag ggatgctgta caaccattat 30660tgattttaac tttacaacag ttcttcaaag gagagagagt tccctgtttt actgaagaga 30720aagcccattt ggtagtgaaa taccattccc aaagacaaat agctaataaa tgtcaggcag 30780ggttttgcac ccaggcccat ccagctcccg tctctactgt cctttccccc acaccacact 30840gatacagagg aatgtgtctg gttggggaag tggaagtgtt cccaagtggg gaggtcatct 30900gatgcacaaa tttggtctgt tttgtgggtt ttcttgtttt agttttagtt tttgtagagc 30960tcagacctgt tcttaggcag ctttaacaat caactgtgca ctcagtaatt gacaaatcat 31020gtttgttact tttaatttag agggaattag gtttgttaag ctcttgctcc ttctttagag 31080atggggtcta gctctgtcac ccaggctgga gcgcagtggt atgatcacag ctcactgcag 31140tctcaatctg ctcaagtgat cctcctgcct cagcctccat gggactacaa gcatgggcca 31200ccatgctagg ctaattttaa aaaaattttt ttgtagaggc aaggtctcac ggtgttgccc 31260aggctggtct tgaactcctg agctcaagca atccctcttc caccttggcc tctcaaagtg 31320ctagaattat aggcatgagc caccatgcct ggcctttact tctttcatat attcaaattt 31380tgtcatatta gtagggaact ataactcaag ttttcttata gattgatgtt catttttaca 31440agcttgatcg tcattggttt ttaattttaa agcaaatcct gttatatgta attgaacatt 31500acagtaatta tagtaatttg tttcagattg ggcactcaag tgttaatatt ttgtctcttt 31560aggaaatcaa aactagattt atatatagac ttcttattgc aagtatctag tcttaaatct 31620tacaaaggta ctatttggac ttaaaactat gaaattgtgt gcttactata taagtgtact 31680tattttgagt tatgttttaa acttgaaatt ccattcttaa tgtctagagt aattatgaat 31740ggttaaatta tgaatgactc taatagttta aagctacagt atttatttat ttatttattt 31800aatttatttt ttgagatgga gtttcgctct tgtcgcccag gctggagtgt agtggcacca 31860tcttggttca ctgcaacctc tgcctcgcgg gttcaagtga ttctcctgcc tcagcctccc 31920aagtggctgg gattacaggt gtatttcacc atgcctggct aatttttgta tttttagtag 31980agacaggctt ttgccatgtt agcctggctg gtctcgaact cctgacctca ggtgacctac 32040cctcctcagc ctcccaagga ttacaagcat gagccaccac acctggccta cagtatttta 32100atgtggactc tctgtcatcc attatgctgt ttatcctgtg gtgaaaattt tatgaagatt 32160gaatgttttt ctctagcgtg aattgctttc tcttactttt ctcatttttt tccttcctaa 32220tctacttgca gatacttcag attattttta gaacgtggta tggtgagaac aaataaattg 32280gggtttccaa atcttaataa attatgtggc cctcagtggg attagcaggg ttgtattgaa 32340aacaccaata gaaacaaaat agttctttta tgcgctttaa ataaaaattt cttttcaggc 32400caggcgcagt ggctcacacc tgtaatccca gcaccctggg aggctgaggc aggcagatca 32460cctcaggtca ggagtttaag acaagcctgg ccaacatggt gaagcgccgt ctctactaaa 32520aatacaaaaa ttagccgggt atgatggcgc atgcctgtaa tcccagctac tccagaggct 32580gaggcatgag aatcacttga actcaggaga tggaggttgc agtgagctga gatggtgcca 32640ctgcactcta gcctgggcaa cagagtgaga ttctgtctca aacaacaaca acaacaataa 32700caaaacatct cttttcaggc caggtactgt ggctcacgcc tgtaatccca gcactttggg 32760aggccaaaac aggagggtcg cttgagacca ggagtttgag accagcttgg gcagctggtc 32820tctatttgaa caaacaaaca aacaaacaca atactctttt catagaaaaa tgtttactac 32880acaataaact ttaaaagaat atgcagctgt attaatgcta tgactccaat gtaaaaaaaa 32940aaaaatatat atatatatat acacacacac aaacacattc tgaaatagat ttaaaggaat 33000tacatcaaca tgtcaatttt tattttttcg gagacagggt ctcgctgtgt cacccaagct 33060ggagtacagt ggtgcaatca cagctcactg cagccttgac ttcctggcct caagtgatcc 33120tcccccctca gcctcccaaa gtgctggggt cacagaccac cacacctggc aacatgtcag 33180tttttgttct gcatagtggg atggtgggat atggatgttt ttatctttta ttttcttttt 33240tatatttttc taaattttcc acattgaaca ttattttata atctttcaaa catatctctt 33300aaaaggactg gttcctatag aattcagtgc aagaaatctt ctgtgtttct ttatactttg 33360gttgccttga tcactgggcc tttcctgaca gcaaagaaga ggttagtgta ggcagcagat 33420aaaacacagg tatgctctat ttaaaatgca tgtatttata ataaaagtat aggtggtacc 33480caaaggaaaa tgtcatgaca cattgcaaag tggaacagaa gttatcttta gatcactttc 33540tgttctggat tattgtatga gcctgatttt cgtctctctt tccgccttcc ctcaccctcg 33600ttgtaaatcc actagtgcat ggatgtgaag tacaagtctt aactttaaaa agttttatga 33660agctgtgtag taaatccctt ttgtaagtgg tcttgactgc gtttctcaat atatcttttg 33720gtttcattag attcaagtat ataaatgaga actgtaactt tggacagact ttttcagtca 33780tctttacggt aataagttcc caattagaca atagttattt gttttatgac ttgctgttgg 33840taggttatcc ccaagggact gagaaattcc tgttttgaaa agtccaaaaa gtctttgatg 33900acttgctgtt tcattttttt cttttctctt cagttataga aaacaggatt acacccacct 33960tgcctttgta cagtgcatct actatctgct gacttaacct gagtaaatgc tttgaattga 34020gccccatata atgtcctaag gcagcctata tggagtaatg aattgtcttc tctcttatgc 34080acccagagtg gtagttggca ctcaagttgt tcctcagata actttgtgtg ttctggggct 34140caatgaagta gttattaagt cacaggcttg gggagaacat tcatcctatg gcattgaatg 34200aagtgttgcc caattctaga atgtctaata aaattttttt aaaaacccac aggcttagaa 34260ttattccgta gatatgaagt aatgtagtta gaacttagtg gagttcttta gattaacttg 34320taatttgaaa aaccaaaatt gaaattgtga aataacatgg gctctttgag gtcttttcca 34380gtaaaacagt tacagtaaag ctgcttggca gtgattttcc tagacacttt ggctagtcat 34440ctcctgtgac tgctgttaat taaatatggt ttgtagctaa gcagcctgta aggagaagac 34500tatggaagta tttgcatatt ctctccttga aaatactacc tggtctttgg ctttaagtta 34560tacttttatt ttcccctgta gaataactat taaagtatta cctatggtga ttagactaag 34620aagtaaaaca tgaaatcagt cattgttggt gccctggtgc cttctttttt ttttttttga 34680gacagagtct cactctgttg cccaggctgg agtgcaatgg cacgatcttg gctcactgca 34740acctctgcct cccaggttca agcgattctc ctgcctcagc ctcccaagta gctgagacta 34800caggcgccca ccaccacgcc tggctaattt ttgaattttt agtagagaca gggtttcact 34860atattggcta ggctggtctc aaactcctga ccttgtgatc cgcccacctc agcctcccaa 34920agtgctggga ttataggtgt tagccactgt gcccagcctg gtgctttaat tttatggaaa 34980aaactactag ctggtttctg ttttaagaaa taacacaggc cgggtgccat gacttgcgct 35040tgtactccca gcagtttggg aggccgaggc gggcggatca cgaggtcagg agtttgagac 35100cagcctggcc aacatagtga aaccccgtct ctactaaaaa tacaaaaatt agccgggcgt 35160ggtggggcat gcctgtagtc ccagctactc gggaggctga ggcaggagaa tcgcttgaac 35220ctgggaggtg gaggctgcag tgagccaaga tcgccccact gcacaccagc ccgggtgaca 35280gtatttcatc tcaaaaaaaa aaaaaaaaaa aagaacacaa ttattgtact acttactagc 35340cctcctctgt ccccagctaa aaataagaac agcaacaacc aaaaaatcct tagttatgta 35400ctggaaatga attagataat tttcaataac ttacacgttt ttaggatatg ttagtttgaa 35460aatgcaaata ttcatgcatg accccagtgt taatctatga tggagcaggt atagtgggat 35520gctgtttcat gatttaattt ggaccttcag ggagtagact gtgatgcctc tgcatttgta 35580tccaagacaa ataattaaat agtctatttt tggctgggca tgatgcctca tgcctgcagt 35640cccagcactt tgggaggctg aggtgggagg atcgcttgag gccaggagtt caagatcagt 35700ctgggcaaca aaatgagacc ttgtctctac aaaaactaca aaaaattagc tgaacattgt 35760ggcttgtgcc cctagtccca gctactcagg tccctgagtt aggaggattg cttgagccca 35820ggagttggag gttacagtga tctatatttg ccactgcact ccagcctggg tgacagagag 35880agaccctgtc tcaaaaaata aagtctgttt ttaaaattaa ttttaaacac tggagtttat 35940tacaaaaagc agttggttct ttttttaaat catttttttt taggagaacc accgcttttt 36000ggctacattg tctagagtag cagtgttcaa taaaaataag atccaagtca catatgtaat 36060gttaagtttt cttttagttt ctttttcttt tcttttcttt tctcttcttt ctttctttct 36120ttcttttttt tttttttgat atgcagtctc actctgttgc ccaggctgga gtgcagtggc 36180acgatctcgg cccactgcaa cctccgcctc ccgggttcaa gcaattctcc tgcctcagcc 36240tcccgagtag ctgggactac aggcatgtgc caccataccc agctaatttt tgtattttta 36300gtagagatgg agctttgcca tgttggccag tctggtctca aactcctgac ctcgggtgat 36360ccacatgctt tggcctccct aagtgctggg attacaggca tgagccacca tgccctacca 36420atgttaagtt ttctagtagc catattaaaa gaagtaaaaa gaaatgggtg aagttaattt 36480taataatata ttttatttaa cccaatatat ctaaaatatt atcatttcaa catgaacaag 36540atactttaca ttcttttgtt tttcactaag tcctcaaaat ccagtgtgta ttttatattg 36600acagcatagt tcagtttgaa gcagccacat ttcaagtgct cagtagccac atgtggctag 36660tgactccata ctggactgtg taggtttaga gtttcagtaa atttgtatgc aatagaatct 36720acataaattg gcatattatg cagatttctt tgtatgcaca tcagttcttg catagcataa 36780gtcaggtcat gatgctttta gtctatgagg cagatttttt tttttttttt tttgagacag 36840agtctcactt ggtcacccag gctggagtgt agatgcacaa tcttggctca ctgcaacctc 36900catgtgaggc agattttaac ttggccctaa tgcaaatatt gtaagagaga tctaatggcc 36960tttgatttct tacagagggc aatcaataca tgccatggtt acaatgcttc agcatatagt 37020atgcacgtca gccactgctt ttactctggc tagtgcttag tgtacctgta ccactgccca 37080ggcagcattt gtcctgtggc aggtgaatct tagggtggaa ggtggcaagt aacattgctt 37140ttttttgaga gggagtcttg ctgtattgcc caggctggag tgcagtggtg cgatctcggc 37200tcactacaac ctccacctcc cgggttcaag tgattctcct gcctcagcct cctgagtagc 37260tgggattaca gacggccacc accatgctcg gctaattttt gtatttttag tagagacggg 37320gtttcactat gttggccagg ctggtctcga actcctgacc tcgtgatcca cccgcctcgg 37380cctcccaaag ttctgggatt acaggtgtga gccaccgtgc ccagcctaca tttttaaatt 37440aattaattat aagcaggatc tcactgtgtt ggccagactg gtcttgaact gataagagtt 37500caagaccagc ctaggcaaca tggtaaaacc ctgtctacta aaaaatacaa aaaaaaaaat 37560tagctgggca tggtggtgcg tgcctataat cccagctact tgggaggctg aggcaggaaa 37620atcgcttgaa cccgggagac tgaagttgca gtgaggtgag attgcaccac tgcactccag 37680cctaggcgat tccatctcaa aaacaataac aacaaaataa cattgttgga atatttagtt 37740aatttataga agcgtattgg cctaattggg gcaaatacct tattctgaca ttctctctat 37800ttgctttact gagctttttc accagtggaa tttaagccct tgatacatga ggagggaaaa 37860taccttggag ctgtgctgca catgtaaagt acacaggaga tttagaaaac ttcgtagcaa 37920aaaaaagagt gtaaagtatc tcattaatag tttttgtggg ctggacacgg tggctcaagc 37980ctatactctt ggcacattgg gaggctgaga tgcatgagtc taggagtttg agaccagcct 38040gggcaacaca gtaggacccc gtctctacaa aaataatcag ccagatgtgg tgcgcatctg 38100tagtcccagt tacttgagag gctgaggtgg gaggatcgtt tgagctggga agttgaggct 38160acagtgagct gtgattgaac cactgcactc cagcctgggt gacagagtgc ctgtctccaa 38220aaaataaata aataaataat aatatgtttt gtatgttcat atgttgcaat aacattttgg 38280atatattaaa tgaaataaaa tacattaaaa ttaatttcac ctgtttcttt tcttttcttt 38340tttttttttt tttttgagat ggagtctcgc tatgtcatca ggctggagtg cagtggcacg 38400atctcggctc actgcaacct cctcctcctg ggttcaagcg attcttctgc ctcagcctcc 38460ctagtagctg ggattaaagg catgtgccac cacacccagc taatttttgt atttttagta 38520gagacggggt ttcaccatat tggccaggat ggtctcgatc tcctgacctc atgatccgcc 38580tgccttggcc tcccaaagtt ctgggattac aggcgtgagc cactgcaccc agcctctttt 38640aactttttaa gtatggctac cagaaaattt aaaatgcatg tgtggcctgt attctatttc 38700tgttggatgc tgctgcctta gattattaat tattcaatgt aaagactgct gggaggtact 38760acctgcactt ccctgaatat atgcttgaga gctccaccag ccgtcttcac agtagcaaga 38820ggggtattct gagtctgtcc cccaaagagg gagggagaag tgcagccctc tcaggttctg 38880tcagaaaacc tgatcccagg ccaggcgtgg tagcttacgc ctgtaatccc agcactttgg 38940gaggttgagg caggaggatt gcttaagccc aggagttcga gaccagcctg ggcaacacag 39000tgaagaccct atctctacaa aaattttttt aaaaaaatta gccaggtgca gcaatgctgc 39060ctgtactccc agctgcttgg gaggctgagg taggaggatt gcctgagccc aggagttaga 39120ggttgcagga gttagaggtt ccacgatcgc acctttcatt ccgttacatt tgctgccttg 39180agaacagaag acctgctggt tttgttgcca gtttgctcag tcatttttat gaaaaagcca 39240gtgctaacta ggtgcttctt cgtgccttct ctgagaatca agaactctag tatgtttgcg 39300tgtgttcagt ctctcattaa atgttctcac tatcccagag aaccatctca ttggaccttg 39360gtctgtacat accttcatct ttggctctga cttgtaatta tttttagaac ttctcttttt 39420ttttttttgg agacagagtt ttgctctagt tgccagactg gaatgcagtg gcacgatctc 39480agctcacctc aacctctgcc ttccaggttc aagcaattct cctgcctcaa cctcttgagt 39540agctgtaatt acaggcatgt gccaccacgc ctggctaatt ttgtgttttt agtagagaca 39600gggtttctcc aagttggtca ggctggtctc aaactcccga cctcaggtga tctgcccgcc 39660ttggcctccc aaagtgctgg gattacaggc gtaagccact gcgcctggcc taattttaga 39720acttgttaaa acaacttggc ctctattgat atttccatga cccatgctat tcagaaagag 39780gattacaggt aattagctgg ctgggtttct cataccagag catttcactg ggatgttcct 39840gaacctggga caacttttat gcctggcatt tttctttcct tctctgttgt cccagactaa 39900gcaattttta aaatagttat tatttgttga gtaggagaat ctcaggcaga tcttcctgga 39960tcctcattta tacttttaaa cctgtagtct tggaattagt gctctgtccc ccaaccccaa 40020acatccaatt tctacatttt ggctacagta caggtttact gtgtataact aaaagggctg 40080tggaggagaa agaaaggaac cgacatttgt tgggcatctg ttatgtgcca tgcactgagc 40140tggatgctgt aggaatatct

caatacctct gaggagtggg aattattatc tctattttat 40200agacaaggga atagaaatct gggagttaag taatttttta atttcacaca cttctggtag 40260ataatggatt ctagaacctg gcataatagc cacttgtcat cccagtgtaa aagagatgtg 40320tggccagatg gggtggctca catatgtaat cccagcactt tgggaagccg aggcaggagg 40380atgacttgag cccaggagtt caagaccagc ctgggcatgt tttgtttgtc tcacgaaaca 40440ttttttaaaa aatgagtgtg gcatggtgtt gtgtgcctat agtcccagct cctcgggagg 40500ctgaggtggg aggatctctt gagcccatga tcatgccatt gcactctagc ctgggccaca 40560gagcaagact ctgtcttcaa aaaataataa aaaggagctg tgattatccc aaggtgggga 40620ttgtgaatgt gtttgtattg ttctaaactg ggagaaacag gctgggtgtg ttggcttatg 40680cctgtaatct cagcactttg ggaggccaag gtgggaggat cacttgagtc caggagttca 40740aggccaccct gggcaacagg caaaaaatag agaccccatc tctatttttt aaaaataaaa 40800taaactggga gaaagaagca gggtcctccc cagagcatct ttatccctag tcacagacct 40860gacacctgtg ttgggcaatg gctacttcta gattgtttac ccctactggg acttgtggtg 40920aacatatgca cactttggtt tacagttggg acccctgatt ttagcaggat ggcccaatgg 40980aatcagctac agcagcttga cacacggtac ctggagcagc tccatcagct ctacagtgac 41040agcttcccaa tggagctgcg gcagtttctg gccccttgga ttgagagtca agattggtaa 41100gtccttctta agtgactctc caaattgtta ggtttcagtt tgagtcaaga gacatgaact 41160cttaatgtca tgccttgctg ttccattaaa aaatgtatgg gtacaggtga tggggaaaat 41220gagatcagga gataaagggg caccctttgg tcttgtaaag ccttttttat cttagaaggg 41280catgtgggca actgtctttg acacattgaa accgcctgta tggtggtgga tgtcttgaag 41340gttgatttgg acctcattta cttgggcaga tcctctatat attctgataa tccagtgatg 41400tggtagacat attttttctc tgaatgtgaa ttctgtcata gctagaactt tgggttgata 41460cttgtaattc ccctttagtt aaaggaagga gccacagggg tgtattagtc tgttctcaat 41520ttgctataaa gaaatacctg agactgggta atttataaga aaagaggttt aatcggctca 41580tagttctgca ggctatatag gaagcatagc agcatctgct gctggggagg cctcagcaag 41640cttccaatca tggcggaagg cagagaggga gcaggcaggt cacatggcca cagcaagagc 41700aagagagcaa gggggaggtg ccacacactt ttaaactatc agatctcaca agaactcact 41760gtctcgagga cagtatcaac agggatggta ttaaaccatt catgagaaac ccacccccat 41820gatccagtca ccttccacca ggccccacct caaacagtgg gggttacatt tcagtatgag 41880atttgggcag ggatgtagat ccaaactaga tcacaggata agggaagtag attccattca 41940tagagcagat aatggcacag atgtccagca actattttct tcactttaat atgctcaggc 42000tcactactga ttttggttta attcaggcca gtgttaatat gacctggttt ttccagaatg 42060catactctga tttggtgaag ggccaggagg tgattcacag atgttggaga taggccatcc 42120cagcctggga ttacttattt gtactaataa atctgaccag agttaattga gggtttaaag 42180caaaacagca tatctgtcta ctttgctcaa atattttaca aatacaacag attatgagag 42240tgggtaataa tatctggaat aattgttttt ttgttttgtg gttttttttt tttttttttt 42300gagatggagt ctggctgtag cccaggctgg agtgcagtgg tacagtctcg gctcactgca 42360cctctgcctc ttggattcaa gcgattctcc cgcctcagcc tcccgagtag ctgggattac 42420aggtgcccac caccacacct ggctaatttt ttatttttag tagagacagc gtttcaccat 42480gttggccagg ttggtctgga actcctgacc tcaggtgatc cgcctgcctc agcctcccaa 42540agtgctggga ttacaggcat gagccaccat gcctggcctg gaataattgt taataattat 42600tacattgatg gcattttatt gctgagcaag aagaatctaa catgatgaat gggttatagc 42660atcaggtttg ctttgttttt ttgttttttt cctctttctt gatggtgatt tctgtgtttg 42720tgtgtatgcg tcggcttcag agccattctt tatcattctt ccttttccta gggcatatgc 42780ggccagcaaa gaatcacatg ccactttggt gtttcataat ctcctgggag agattgacca 42840gcagtatagc cgcttcctgc aagagtcgaa tgttctctat cagcacaatc tacgaagaat 42900caagcagttt cttcaggtat gatgagaaac tgaggacaag gagaaacagg acccgcagag 42960tcgggtgtta gtgttctttc ctggaagcat ctcttttctc atttggctaa gtaacgagaa 43020tctatcttgt attttcaatc acaggagaag taattagccc tttctcaaag ctctgtatac 43080ttacccgtga gcatcattac ctgagaatca cttctcttgt cacagttgaa gtaataaagt 43140gattgttatg ttaatcatac atgttagcat gttaacgcgg tccactgata ggaagatgac 43200tctcactgtt acatgttaaa tgtttgacca taatgggata cttcttgact aagtcagtag 43260cttccctgca agaccaggat agtatactgt gtaaagactc agacaaggcc aggcatggtg 43320gctcacgcct gtaatcccaa caccttagga ggttgaggtg ggaggattgc ttgagcctgg 43380gagttttgag accagcttgg gcaacataac aagacaccat ctctacagaa atttttttta 43440aaaactagct gattgtggtg gcatgcacct gtagtcccag ctactcagaa ggctgaggtg 43500agaaaattgt ttgagcctgg gaggtcgaag ctgcaataag ccgtgattgc gccactgcac 43560tccagcctgg cggacagagt gagagccagt ctcaaaaaaa aaaaaaaaag actcaggcta 43620atgtgccttc tgttacagaa atagtaacga cctccccttc gccccccgcc gacagagagc 43680cttcacccag gctctgaagc ctttgttccg ttgtttccta gaataaatgc tttccttgat 43740gaatacatta gttttaaggt gccacagttc agtccacatc tccatggtct gctgctgatt 43800tttattctct ttctctccta cttatagagc aggtatcttg agaagccaat ggagattgcc 43860cggattgtgg cccggtgcct gtgggaagaa tcacgccttc tacagactgc agccactgcg 43920gcccaggtga gacctgagac aaaacaaatc cctggtctgg gaggaatgga aaatcaaaca 43980actttataat gagataaatt attagatcta ctaaaaaaga aggaaaagaa attaaataga 44040tcaataatca taaaaataca ttgaaaaact ctaaaaaaaa agaaagttcc accccccaaa 44100atacattgaa aaactctaaa aaaaagaaag ttccaccaaa agaatccaac agacccaatg 44160gtttaaaagt tttgttttgt tctgacaaat tttctttgtt tttctttttt tttttttctg 44220agacagagtt ttgctcttgt tacccaggct agagtgcaat ggcgcgatct tggctcactg 44280caacctccac ctccagggtt caagtgattc tcctgcctca gcctcaagag tagctgggat 44340tataggcgtg tgccaccaca cccagctaat tttgtatttt tagtagagac ggggtttctt 44400catgttggtc aggctggtct cgaactcctg acctcaggtg atccgcccgc ctcagcctcc 44460cacagtgctg ggattacagg cgtgagccac tgtgcccggc ctgttctgac aaactttcat 44520agtacagatt attccaatat cattcaaact tttccaaagt ataggaaaac aagggatgtt 44580ttcagcttat tttatgaggc tggaaaaatc ctcatatcaa aacctaaaaa acagccaggt 44640gtagtagctc acgcctgtaa tcccagcact ttgggaggct gagacgggca gattgcctga 44700gcctcaggag ttcgagacca gctggggcaa tgtagcgaga cctcatctct cttttttttt 44760ttttttgaga cagagtctct ctctgtcgtc caggctggag tgcagtggtg ccatcttagc 44820tcactgcaac ctccgcctcc caggttcaag cgattctctt gcctcagcct cccgactagc 44880tgggactaca ggtgtgtgcc accaagcctg gctaattttt tgtatttttt ttagtagaga 44940tggggtttca ccttgttagg caggatggtc ttgatctcct gacttcatga tccaccggcc 45000acagcctccc aaagtgctgg gattataggc atgagccacc acgcccagcc tttttttttt 45060ttttgagaca gagtcttgct ctgttgccag gctggagtgc agtggcgtga tctcagctca 45120ctgcaacttc tgcctcccag gttcaagcta ttcccctgcc tcagcctccc aagtagctgg 45180gactacaggc gcgcgccacc acacccagct aattttttgt gtttttagta gagatggggt 45240ttcactgtgt tagccaggat ggtctcgatc tcttgacctc gtgatccgcc cgcctcggcc 45300tcccaaagtg ctgggattac aggcgtgagc aaccgcacct ggcttaatta aggatctttc 45360taaacacaag aaagaatatt tatcagaaac caaagggagc atgatgcaca gtggtgaaac 45420actattctca gtaaaaacag caaaagataa ggatgtcttt taccattgat acttttctga 45480gggatccagc ctatgcaaaa agaaaaagaa atgagggtac aaatattgga aagcaaggga 45540cagaactctt attatttaca gatagatagg tcttcctcga agatccaaga gaaacaaaac 45600taacaataac aattggaact agcaaggttt agaaaggcca ttgtatacaa gataaatatt 45660tttagaatct gcagttcccc taatcagtag cagcagtaac ctgttagaag atgtaatgaa 45720agtaaagatc tgggccaggc acgatgtctc acgcctgtaa tccaagcact ttgggaggcc 45780aaggtgggca gatcatgagg tcaagagatt gagaccatcc tggccaacat gatgaaaccc 45840catctctact aaaaatacaa aaattagctg ggtgtggtgg tacgcgcctg tagtcccagc 45900tactcgggaa gctggggcag gagaatcgct tgaacctggg aggcggaggt tgtagtgaac 45960caagattgcg ccactgcact cctgggcgac agagcgagac tccgactgaa aaaaaaaaaa 46020aaaaaaaaag aaagatctga ttcatagtag taaaactaaa tgtatgcaat ttgcatatac 46080tattggtatg tatgggaaaa tatctggaaa cacatatact aaatcattaa agtagtcggt 46140cataggagac ttttttactt tctgtgaggg gttttaccgt ctttaatatc ctataatcag 46200ggacattttt tctttttctc cgtgaccccc tgctttttaa aaaattgtgg tgaaatacac 46260ataacattac atttcaaatt tacctttgta acctttgttt tttttttttt tttttgagac 46320agtctcactc tgtcacccag gctggagtgc agtggtgtga tcacagctca ctgcagcctc 46380aaccacctgg gccctagcga tcctcctgcc tcagccttat gagtagctgg gactacaggc 46440acatgccacc atgcccagct aatttttttt tttttttttt ttggtagaga tgggctcttg 46500ccatgtttcc caggctggtg ttgaactcct gggctcatca actgatgaga aagagctctc 46560caggcagaaa gaagatcatg ttcaaagaca gaaacagaaa tgtgtattct tgggagaagt 46620gtagaaagtt cagcatctga ttgggtcggg gaagacaagc tagtcaaggc cacatgatgt 46680tttaattagt catgcctaac agtggggccc tggaagagca gtttaccaca aggggccaac 46740tgcttcggtt tgaacccgca gccctgccac ttgctctgta accttaagta aacaattttt 46800actctctctg ttcctccaat gggagtgata acaatacctt cttcatagaa ttaattcata 46860catgtaaaat gcttagaaca gtatctgaca cataaatgca aaataattta actgctttct 46920gctgctgctg acatcactat catcaccctc accattactg taggaaatgg ggacccagtg 46980aagaattttt tttttttctt ttgagacaga gtctcactct gtcacccagg ccggagtgca 47040gtgacgcgat ttcggcccac tgcaacctct gcctctcagg ttcaagcgat tctcatgtct 47100cagcttccca agtagctggg attacaggca tgagccacca cactgggcta attttttgta 47160tttttagtga gatagggttt caccatattg gccaggctgg tctcaaactc ctgacctcag 47220gtgatccatc cacctcggcc tcccaaagtg ctgggattac aggcataagc cactgtgtcc 47280ggccctagtg aggaatttta agcagaaaac tgatatgctc aggtgtgagc gaggtggtag 47340gtaacactta ctgtgcagtg ccctgtagcc caagaggtta gcacacaggc atttgctcag 47400gcagcactag gattttctgc tgtggaaaac ctttgtattt tatcctgctc cacaagataa 47460aaataagtgg tttaagtcaa tttggataga ggctccaact taccatggga ggtaggaaag 47520ccaaagttat cccaaggatg ttttcaatcg tacggattag gggtctgcaa actgtgagcg 47580tggcccaaat ccagcctgct gcttgttttt gtaaatgagg ttttttcgga acccagccac 47640actcatttat ttatgcatta tctgtggctg ctttggtgct gcagtggcag ggctatttgt 47700ggcagggact gtatgaccca ggaaaccaaa aatatttacc ctctgtccct tagagaaaaa 47760gtttgcaacc cctgatataa agctataagt tggttatttg tggcctcaac ccaggcctca 47820ctgctatttt ttctgtttac aatacctggc atgctcttaa gtgtctagaa ttggttaaag 47880atagaagagt ggatgtaatc cctgctacca agggctgtca ggctagttgg gattataagt 47940acacaaacac tcaaagtgag aaaaacacag aaaaggatgt gtgtcatttt gtctaaggaa 48000gttgaataag atttctcagg aaaagaaaca tttgaactga atttgaaggt gagtgagttc 48060aggtgtgttt gggctgaagc ccaggccatg ctgagtggat agcgggtggg aagagagtgt 48120ggaaacacac tgcatgcagg gaagagttgg gagtctgggg tgaccaaggc acagggaggg 48180aaagttgaag ttatcaattg tgtgaaacag ctttctgtgt tggcctgaga tgtttatagc 48240tggaagcagt ggggagccaa tacagttttt tacgaaggta ttagaggtgg gtttctgtgg 48300gtgatcgtta atcatgtttt ctccctttaa gtgtagtcct gcttgagaaa tagacatgag 48360aaaggaatga aggttaaaac atcagctgta ttgttggtaa aactagaatg gaaagtgtgg 48420cttgagctgg taaccatagg ggctttccaa tgcctgtgcc ctgagttaga tcttggggta 48480gagagactgg atgtgcagag cagcaccccc accccacccc agccatccat atggagcttc 48540agctgccata gaccaacaag gcagagggat aggcctctag acctgcttct agaaaccagg 48600ctgctgctct tgcttatggt gggccctagg aaggcaagag tgagaggagg gaggcaccag 48660cttaggtgct gggttctttg aagatctgtg tgtacacaga gtctttctct ccatcttacc 48720aatcagatga gtcactgtca ctgtgggaag aagtaggggc atgggtcacc ttcccaaaac 48780ttctaagaag tttgtattct gtgggcttgg atagggacca tgggaaagga agagaatggt 48840tgcccataaa actggctgta gtgtggcctc aaacttctgg acttaaatga tcctcccacc 48900tcagcctccc aagtagctag aactacaggt atatgccacc atgcccagct agttaaaaaa 48960aaattttttt ttttttttgg ttgagatgag gtctctttct atgttccctg ggccggtctc 49020aaactcccag cctcaagtga tcctcctgcc ttggtttccc aaagtgctag gattataggt 49080gggagctacc atgcctagcc caagcctgta attttttttt tttttttttg agatggagtt 49140tcacttttgt tgctcaggct ggagtgcatg gcgcagtctt ggctcaccac aacctccacc 49200tcccgggttc aggcgattct ccttcctcag cctcccgagt agctgggatt acaggcatgc 49260accaccaagc tcagctaact ttgtattttt agtagagatg ggtttctccg tgtcggtcag 49320gctggtctca aactcctgac ctcaggtaat ctgcccacct tggcctccca aagtgctggg 49380attacaggca tcagccaccg cacctggcac gaacctgtaa tttttaagtt tcatatgcta 49440tttatttttt gttatttctt taattcattc attcatttat tcattcgaga tggggcctca 49500ctatgttgac taggctagtt ttgaactcct ggcctcaagc agtcctccca cttcagcctt 49560cccaagtgct gatattatag gtgtgagctg ctacatccag ccttctttct tctttttctt 49620tttccatgtg ctatttgaca ttttccaagg taccagcctc cccttctccc caagataata 49680tcttttaata tggaatttca tccctagggc aggacttttt ttttattatc cctcagaaat 49740atactggaca ccacgtttaa gtagacatcc aacatctgct gtcataaatt gttttgaatt 49800ttttgacata cttgcccatg aggtttttga aggcatagac catgtcttag ctgaacatgt 49860ggtctcttag tgccataaag ggggtttatg gtatgacctg tgtagtgtca cctgtgtagt 49920gacagcacca ctgcctctgt ttcccttcct cttgtgatgg cagcagcgtc tcaagccaaa 49980caagaagggt agttagggtg ggatggaagc tgggtagagg tattcctctc cccatagttc 50040tgtgttcaca tgtgcattga cctccttttt ggcagcaagg gggccaggcc aaccacccca 50100cagcagccgt ggtgacggag aagcagcaga tgctggagca gcaccttcag gatgtccgga 50160agagagtgca ggtgatgcaa gttacaagcc tcgggcaggg agctttcatt aatttttttt 50220tttttttttg agacagggtc ttgctctgcc actcaggctg ggctgcagtg gcatgatcac 50280agctcactgc agcctcgacc tctcaggccc aagcgatcct cctacctcat cctcccaagt 50340agccgggacc acaggcatgc accaccacgc ccagctaatt aaaaaaaaaa aatttgtaga 50400gatgggggtc tccctgtgtt gtccaggctg atcatgaact cctgggctca agtgatcctc 50460ccaactcagc ctctcaaagt gctggcatta caggcgtgag ccactgcacc tggccaacag 50520ggagccttct cttggggata ctgcctgcag gtcctgcatg tatctttttt gaggttttgg 50580cttcatttga attctcctca gaaactttat attttctgtt cccaaggaaa tctttcttta 50640cttctgtttt tttgtttgct tattttaaac aggatctaga acagaaaatg aaagtggtag 50700agaatctcca ggatgacttt gatttcaact ataaaaccct caagagtcaa ggaggcaagt 50760gaatattaga gatgttaaaa tctctagaaa gtgagtttgt gttgttgagt tgaaagactc 50820atttgtctta actctgttta gatcttaagg cgggcggggc gcaagggagg tacgggtcct 50880caaaggagcc tggtcattaa ggacaggagt attccctcag gtccaggagt attccctcag 50940gtccaggagt attccctcag gtcaaggagt attccctcag gtcaaggagt attccctcag 51000gtccaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51060gtccaggagt attccctcag gtccaggagt attccctcag gtcaaggagt attccctcag 51120gtccaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51180gtccaggagt attccctcag gtccaggagt attccctcag gtcaaggagt attccctcag 51240gtccaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51300gtcaaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51360gtccaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51420gtccaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51480gtcaaggagt attccctcag gtccaggagt attccctcag gtccaggagt attccctcag 51540gtccaggagt attccctcag gtcaaggagt attccctcag gtcaaggagt attccctcag 51600gtcaaggagt tttttcttcc ttcgcagaca tgcaagatct gaatggaaac aaccagtcag 51660tgaccaggca gaagatgcag cagctggaac agatgctcac tgcgctggac cagatgcgga 51720gagtaagggc ataggtcgga ccacttcccc catgtgtctc gctcacttgc gggatttcag 51780cgtcttgtgg cagaacttgc ttggtttcta agaagttcct gctctggagt tgactaaaga 51840atgtggttag agacagtctg aggaaatgtt ttctgacttt gttttggttt ccaaccagag 51900catcgtgagt gagctggcgg ggcttttgtc agcgatggag tacgtgcaga aaactctcac 51960ggacgaggag ctggctgact ggaagaggcg gcaacagatt gcctgcattg gaggcccgcc 52020caacatctgc ctagatcggc tagaaaactg gtaaaggatg aaagaagctt ttcctttctt 52080tctcgaaagc tagattgaat tctgatctta actgcaggcc cacagaattg gtactatatc 52140tccaacgtgg ggacttttcc atattcaaat ttagcccaag aattaaagtt tttactttat 52200ttcggccagg cgctgtggct cacacctgta atcccagcac tttgggagac caagatgggc 52260ggatcacttg aggtcaggag tttgagacca gcctggccaa catggtgaaa acacatctct 52320actaaaaaca taaaaaaatt agccgggcgt ggtggtgcgc acctgtagtc ccagctactc 52380tgggcggctg aggcaggaga atcacttgaa cctgggatat ggaagttgca gtgagcggag 52440atcttactac cgcacaccaa ccagcctggg agacagagtg agactccatc tcaaaaaaat 52500aaaaataaaa taaagttttt actttatttg gagaaacttt gttttaaaaa atgtatttat 52560attattatat tttaagtata ttttacttaa taattcaatt aaggcttttg gtttaactgt 52620atttaacaga tagacaaacc ttttaatttt agttatttta gtaatctaaa atgacacatg 52680ccctttttaa gggaaaaaat tcaaatacag aaaattaatc aagagaagaa aaaattttta 52740aatgaaatca tcagcagtac tagtagttaa aatttagttg atgctcaatc tagacatctg 52800tcattatgta tatacacatt atgtatatac acataaagat agaaatttat acagtttata 52860ttaggatcat ttttttttct ttttttggag tcagggtctc actgtgttac ccagtctaga 52920gtacagttat gcagtcatgg ctcactggag ccttgacctc ctgggctcag gcagtcttcc 52980caccttagcc ttctcagtag ctgggactac aggcatgcac caccacacct ggctaatttt 53040taaatttttt atagagacag ggtcttactt tgttgcctgg gctggtctca aattcctggg 53100ctcaagggat catcccactt cggcctctca aaagctctgg aattatagat gtgagctgcc 53160gtgcccagcc caggatcttc ctttatatgc ttttctgtaa tttgcacttt taccttcatc 53220cagcatatct tactgcaacc cttcctgtgc aaggccctat agtgagcatg ttgcaccagc 53280ttgccttagg agaaacttga gatacagagc ctgcactgga aatttagcgc aactctacat 53340gagaatgcct gtctattcat atcctcacta accctgagtg ttgttaattt actgaaagca 53400gttttaaatg cttcctgacc agggaacgaa gaagcttaag ttctgggaat gggaggatag 53460aagtgccaga aaagagctca ggagttcaga aatccctgca gcggtccccc tccctctcct 53520ttcactttct gtctttctgg tcttttggtc tttgttacac tagtgataaa ccatcaaaga 53580atgatggaat gatgctaact tctctctttt tttaattttt ttgagacaga gtctcactct 53640gtcacccagg ttagagtgca gtggcatgat cttggcttac tgcaacctcc tcctcccagg 53700ttcaagcgat tcttagtcac aaccttccaa gtagctggga ttacaggccc atgccaccat 53760gcctggctat ttttttgtat tttagtagat cgacctgcct cggcctctca aatttttggg 53820attacaggtg tcagccactg cacctggcct aatatctcta ttcttggaga tagatttaat 53880gagctttttc tccctctcta ttcacttatt ccttgtgcat gttatcaata ttttgaaaca 53940taatgtcatg tcctttgatc agttgaaggc tgacattgaa aaggcttatg gggattgggt 54000gttgtggctc acgcctgtaa atcccaatgc tttgggaggc agagtcggga ggatcacttg 54060aacccaggag tttgagacca gcctgggcaa caaagtgaga tcccatccct acaaaaattt 54120aaaaaactag acatgtgcca ttacacttca gcctgggtga cagagtgaga ctccatctca 54180aaaaactaaa ctaaactaaa caggcatggt ggcacacacc tatagttcta gctactcagg 54240aagctgaggt aggaggatca ctcatgtcca ggagttggag gaggcagtga gctatgatca 54300tgccattgca ctgcactagg ccacagagtg ggaccctgtc tcaaaaaaaa aaaaagaaag 54360aaagaaaaga aagggctcat gtagttcaag cccttctctt catgcaaggg gatgctaagg 54420cccatgatgg tgaagggcct ggcaaagctt gcacagatag tgtgtgacag agctggctca 54480aacccatctt tgggagctgt ctaatctctt tttctgagtc tttatgttca tagacaagtt 54540aggatgagta aagtaagtgc taaattccat atttcgtgtt ctgcatatct gggctcagat 54600gcttgtcatt ttccagtgat aactccatca atgcctccta gtggtataaa ttttaatact 54660tcttgtgtgc ccagccccct cttagaaatt tgagatttta ggaagggact agtaataaaa 54720ggtaaaataa attattttct ggccaggcat ggtggctcac acctgtaatg ccagtacttc 54780gggaggtcga ggcagatgga tcacctgagg tcaggagttc aagaccagcc tggccaacaa 54840ggcaaaatcc catctctact aaaaatgcaa aaattatccg ggagtggtgg tgggtgcctg 54900taatcccagc tacttgggag gctgaggcag gagaatcact tgaacttggg aggcggaggt 54960tgcagtgagc tgagactgtg ccactgcact ccagcctggg caacagagta agactctatc 55020tcaaaaaaaa aaaaaaaaaa aaaaaggcca ggcgcagtgg cttacacctg taatctctca 55080ggaggctcag gcaggagaat cacttgaacc cgggaaatgg aggttgcagt gagccgagat 55140tgcaccactg cactccagct caaaaaataa ataaataaat aaattatttt ctttttttat 55200ttattttttc agcatccacc

caacatggtg aaaaattcct cttttcttaa tgtcactgaa 55260ctgtaaactt aagatgaaaa attgtaaatt tcatgctata tatatttcac cacaataaaa 55320aaattccttg ttcttattgt agtggtctcc atgtcttcag tatttccttc cccttctcca 55380tctcacctgt atacattcac tttggtaatt agcatctttc ttaatttatt ggcaggataa 55440cgtcattagc agaatctcaa cttcagaccc gtcaacaaat taagaaactg gaggagttgc 55500agcaaaaagt ttcctacaaa ggggacccca ttgtacagca ccggccgatg ctggaggaga 55560gaatcgtgga gctgtttaga aacttaatga aaaggtaatt tagcatcctt gtccctttcc 55620ctcatctaaa aaatacctaa agactcacgt ggtagagtga gaggcgggct gacttctggt 55680catggccgtg gcgcgtgagc ccatcttctc tttcctcagt gcctttgtgg tggagcggca 55740gccctgcatg cccatgcatc ctgaccggcc cctcgtcatc aagaccggcg tccagttcac 55800tactaaagtc aggtaggcca tgccacttcc atttccagta gagattttac tgagggacac 55860tgttagggtg agggtagagt tggtggccag ggtcattctt tccaggtgtg gtgtcacagg 55920cagtacactg ttgcggggtt gaaatttgtt gccatactat ctgcttgctc tctgattctg 55980atgtcaaaag caaaagagca gtcatctttt tgaaggtacc tgggcatatt cctatgattg 56040tagacctgga gtctcaggcc acagcttctc cttctgccca agggacaaaa taatgtcatc 56100tattttctgt tctttgaggc tactcttccc tgtggatttt aagggaaaga gtaaggctta 56160gtgatgggga agctgagagg ccccagggca ggtgggtggt gggcctgtag ggtgaggtgt 56220tactttcaca ctcaagtcag aacaggtgtg ctggggtttt gaccttctgc agcaaaattt 56280ccctcctcag aaacttagta tggtgttcgg tttcaggatt aatagaacaa aatgccagct 56340gcacagcatg tgttcctgta atatttttca ttatatggct ttgattatcc ttttgtgaat 56400ctctcacaac tttaagttgt tagttcttag atgttttctc agtacctttg gcttgaagga 56460gtgatactca tcttttgttt ttgtttgaga cagggtctca ctctcaccca ggctggtgtg 56520cagtggcatg atctcagctc actgcaacct ccatctccca ggttcaagtg attcttgtgc 56580ctcagcctcc tgagtaactg ggaatagagg tgcgtgccac cacacccggc taattttttt 56640ttttttgaga cagagtctcg ctctttcggc caggccagag tgcgtgttgc aatctcaact 56700cactgcaacc tccacctccc aggttcaagc gattctcctg ccttagcctc cctgagtagc 56760tggaccggca cactccacca tgcccggcta atttttgtat ttttagtaga gacagggttt 56820ctccatgttg gccaggctgg tctcaaaact cctgacctca gtaatccacc caccccggcc 56880tccaaaagtg ctgggattac agatgtgagc caccacgctc ggcctttttt tttttttttt 56940tttttttgag atggagtctt tctctatcac ccaggctaga gtgctgaggt gtgatctcgg 57000atcactgcag cctctgcctc ctgggttcaa gtgattctcc tgcctcagcc tcccaagtag 57060ctgggattac aggtacctgc caccatgccc ggctgatttt tgtattttta gtagagacgg 57120ggtttcacca tcttggccag gctggtctcg aactcctgac cttgtgatcc acctgccttg 57180gcctcccaaa gtgctgggat tacaggtgtg agtcaccgca cccagcccta ttttaatttt 57240tttaaagaga gagatagggg ccaggcacgg tggctctcgc ctgtaatccc agcactttgg 57300gaggccaagg tgggtggatc acctgaggtc gggagttcga gaccatcctg accaacatgg 57360agaaactctg tctctactaa aaatacaaaa ttagctgagc gtggtggcgc gcgcctgtaa 57420tcccagctac ttgagaggct gaggcaggag aatcacttga acccaggagg cggaggttgc 57480ggtgaacgga gattgcgcca ttgcactcca gcctgggtaa cgagagaaac tgtctcaaaa 57540aaaaaaaaag agaaagagag ataggatctc gctctgtcat ctaggctaga gtgcagtggc 57600atgatcatag atcactgtag ccttgaactc ctgggcacaa gtgatcctct tgcctcagcc 57660tcccgagtaa ctgcgactac aggtacatgc taccacaccc cgctaatttt taaatttttt 57720atagatgtgg gctctcactt tgttgcccag actgttatgg aactcctggg ctcaagggat 57780cctcccagct tggcctccca cagtgctgag attatagatg tgagcctgta attatagaca 57840gcttggccta tttacctgtt ggaaatgaag aattatgaat tttacatttc ttcaagaaaa 57900ggttatggga gagttactga ctttttttcc ttggattttt tctttttaaa taggttgctg 57960gtcaaattcc ctgagttgaa ttatcagctt aaaattaaag tgtgcattga caagtaagta 58020ctcctatctt agctctgttt ttcaaatgag gaatagaaaa atgagaactt tgacagacat 58080catttgaact agagactctg tctttattca gagatcttca ttttgtggac aaaagttttc 58140aaaagccttg gggtgcattg tcatttacgt gtctgaacaa agccacaaag ctgggggtac 58200agatttgatt tgtggttgct attgtgacaa ccagtccctc ttttccttgt ttagtttttt 58260acttgtacat gtcattcatg catattatat ataagactga gatcatgtgt taattaacga 58320ctgggatacg ttctgcaaaa tgtatcatta ggcaattttg ttgtgcaaat gttgtagagt 58380atatagtcct tacacaaacc tgggtggcag aacctactgc acacctacgc tatgtggcag 58440agcctactgg tcgtaggctg taaacctgta cagtatgtta ctgtgctgaa taccgtaggc 58500aattgtaaca catctcaatg aagtaggaat ttttcagctc catgataatc ttatgggacc 58560accatcatat atgcattttg ttgttgaccg aaacgtcgtt atatattctt tccatacata 58620gcatgtggaa agaatagatc tctttttttt aattgttcca cactttacca tataatggaa 58680tacgcaaaat ttcacaatac ctttcaggat gtaaaataca tatacccttt gacgacatta 58740gaaaagagaa aatgtgggcc gggcgcggtg gctcatgcct gtaatcccag cactttggga 58800ggccgaggcg ggcggatcac gaggtcagga gatcgagacc atcctgggta acacggtgaa 58860accccgtctc tactaaaaat acaaaaaaac tagctgggcg tggtggcggg cacctgtagt 58920cccagctact caggaggctg aggcaggaga atggcatgaa cctgggaggt ggagtttgca 58980gtgagccaag atcacaccac tgcactccag cctgggcgac agagactcca tctcaaaaaa 59040aaaaaaaaag aaaagaaaag agaaaatgtg gctgggcgcg gtggctcacg cctgtaatct 59100cagcactttg ggaggctgag gtgggcagat cacctgaggt cgagagttcg aaaccagccc 59160gaccaacatg gagaaacctt gtctctacta aaaatacaaa attagccagg tgtgttggcg 59220catgcccgta atcccagcta cacgggaggc tgaggcagga gaatcacttg aactcaggag 59280gtggaggttg tggtgagccg agatcacacc attgcactcc agcctgggca acaagagcga 59340aactatctca aaaaaaaaaa aagaaaaaag aaaagataaa atgcattctt atttttagtt 59400gatgtaatta tgtggaaatt tcatgaggat gcactggaaa ataatgaaat aagggagttg 59460acgaaggtgg taggtttaat aagtacatat gcaatatgaa acataggttc cccttcctat 59520ggggaggcaa ccaactgtgc ctgctacgca gaggtgttat gttgcgctga tcaactgtaa 59580ctgaatagtt taaagaaatg cccaggagca cagaggtttt ttcatgacag taaataacag 59640gtggtcaaag taggcttttt gaagaaacac agagcctatt ttattaacaa cagtctgtgt 59700tcttacagag actctgggga cgttgcagct ctcagagggt aagttcagcc tagaggcttc 59760cttttgttcc gtttaaccta acttcatcct ccggctactt ggtcacctac atagttgatt 59820gttcccctgt gattcagatc ccggaaattt aacattctgg gcacaaacac aaaagtgatg 59880aacatggaag aatccaacaa cggcagcctc tctgcagaat tcaaacactt ggtatgtggg 59940aggagctccc cttcacaaag ggcctctggc tgccggagag ggctagggag agcctcacag 60000gacacctgcc tttttctttt cttacagacc ctgagggagc agagatgtgg gaatgggggc 60060cgagccaatt gtgatgtaag ttttgttggg gatgaaagac aactggggtg ttttccttga 60120gggagagagg ggtaaagatc cttcttaatc cccagaatta gaaacatcaa cctgttcttt 60180cagctgtagt tattccaaaa agtcacttca ggccaaagtg acatgaacag aagttccatg 60240tgccatggag ctctctggct tggaacattt ccgtgaatat ctgggagttg gctcctcctt 60300aaggagaagt ggaaagtccc ttgctgagtt gttctccaca cccatgtggt ataaagcagc 60360tttccacctt gcctggggct ttccaaattc cccatccagc tcctgcggct gaccctgctt 60420ggctccattt ttagtgccct gtttttctct cccactgagg tgggatagag ggtgtaaaag 60480caacagattt gagttaaact ttaaaataaa tgaccacctt gcattagctt gcttaggaaa 60540agagtacata aaataaaatg aacaaacaaa aacccatctt gttctttatc ccccttattt 60600tctgcttttc attgattcag attattggat tcttattgtc aagaataaac tttaaacaaa 60660caaacaaaaa aaggtaaatg tgacggaagg ctagttttca gtcattttta aaaattgtga 60720tgccccgttc tttttcttac atttgtcccc tgaacaattc ttcctcttta aaatgtagca 60780gtcctagctg ggcgtgctgg ctcacacccc gtactttggg atgccaaggc aggctggtca 60840cttgaggtca ggagttcaag accagcctgg ccaacatggt gaaagcccgt ctctactaaa 60900gatacaaaaa ttagctgggt gtggtggtgc acgcctgtag tcccagttac tggggaggct 60960gaggcatgag aatcgcttga acctgagagg tggagcttgc agtgagccaa gattttgcca 61020ctgcactcta gcctgggcaa cagagtgaaa ctctgtctca aaaaaataaa taaaataaaa 61080tgtagcagtc ctttttaaaa atgtggaatt ttacttgaca gtagagtgaa gtagcctgta 61140tgcaatgata tgggaaaatg tacatgacat attaagaaaa agcaaaatgt aaaataattt 61200gaatagtatt attagtatat gtgttttaaa aatacactat actcttatgt gtattcatat 61260gtatattaag aaattctgga ggaatatacc agcagtgcta tgtgtattag tgctgctgtt 61320ggtatccatg gctattctag actgtctctg tgatatttgc attttaaact gaatatatta 61380cttttataat cagaaaaata gtattaaaaa tgaattataa tttaatttct tttttctttt 61440ttttttttga gtcggagtct cgttctatcg gattgcagtg gtgcgatctc agctcactgc 61500aacctctgcc tcctaggttc aagcgattct cctgtctcag cctcccaagt agctgggacg 61560ataggtgcat gccaccacgc ctggctaatt tttgcatttt tagtagagac agggtttcac 61620catattggtc aggctggtct tgaactcctg acctcgtgat ccacccatct cggcctccga 61680aagtgctggg attacaggca tgagccgctg tgcccagact agaattcaat ttttgagaat 61740tcattgacaa ctcttactta aaataaggtt gctgtactga tgtgagacat tgttgtagtc 61800agtttggaaa acaatttggc agtataaaaa tgaacatacc tgtaaaccaa cggtgccatt 61860cccaggattt aatagcagag aaatctttgc atatatgtcc caggagacat atataaagtg 61920gacatcagcc tgattataag ctctaaatgc aacccaaata aatacccatc aacattagaa 61980tgaatacatt atttgtggta tagacacaat ggaatactcc gcagctgtga aaaggaatac 62040actgcagata cacataacca tgtggattca tttcacatca agtgaaaagt gaatcccaaa 62100agaattcatt ggagtccata agtgtaaggt tcacaaatgt cccaaactaa acaatacctg 62160cattgcttag ataaacaaat atggtaaaac tgtaaaaaaa caaaacaaaa caagacaaaa 62220agggctagga aatgataaac ccaaaagaca aaatagcagt tatttctgag ggaggaggga 62280aggggatggg gttggggaag ggcacccaga gaattttagg agtgatggac ttttccttaa 62340attgaatggt gggttcatat tgttttgtta ttctttgtgc cttacgtatt ttacaaataa 62400ccaattggat ctatgtaata ttataataca aactgagtaa aggattaggt tgaggatcac 62460agcattggaa gttcttggtg ttgaagagag taagtgccga gcaagttgtg tccctggcag 62520tttgtttgtg accacctggt ggcttaccct tcttggtgtg gtgaggcttg gcatgtcact 62580ttccttggct gtggctgtta gtactgaatg ccattctctc tgaggaaaag tgtccttctc 62640ttttttattg attgactgat tgattgagac agagtctcac tctgtcaccc aggctggagt 62700gcagtggcgt gatctcggct cactgcatcc tctgcctcct gggttcaagc gattctcctg 62760cctcagcctc ctgagtagct gggactacag gcgcccacta ccacacccag ctaatttttg 62820tattcttagt agaaacgggg tttcaccaaa ttattggcca ggctggtctc gaactcctga 62880ccatgtgatc cacctgcctc ggcctcccaa aatgctggga ttgtaggtgt gagccatcac 62940gctcagcctt tttttttatt taatttaatt tttttttaag acagggtctc actctgtcac 63000cccagctaga gtgcagtggc acaatcatag ctcgctgcag cctccatctc ctaggctcaa 63060gccatcctcc cacctcagcc tctcgagtag ctggggctat aggtgtgcac caccacaccc 63120agctaatttt tgtatttttt gcagagatgg agttttgctg tgctgcttag actggtctcg 63180aactcctggg ctcaggcaat cctcctgcct tggcctccca aagtgctggg attacaggca 63240tgagccacca cacctggcct aagagtgtcc ttctcgttac tgtaggcttc cctgattgtg 63300actgaggagc tgcacctgat cacctttgag accgaggtgt atcaccaagg cctcaagatt 63360gacctagagg taagttctgc agcagaatcg gtgagaggct acgtacaggg gtgactcagg 63420acaaaaactt ccactgggat ttttacaaga gaaggtggaa tgattactgt ttgcttaaca 63480ctgtgtttat ttttgcttac ttttctccaa aaaaatcctt ggcatcccat ctggcaataa 63540agtcttgctt gaatgcttag aagatgtgtg tatattcagc tttcagcaaa cttgatatga 63600aaatctctat ttagaaattg attggccggg cgcggtggct cacgcctgta atcccagcac 63660tttgggaggc tgaggcgggt ggatcacgag gtcaggagtt cgagaccagc ctggccaaca 63720tgacgaaacc ccgtctctac taaaatacaa aaattagctg ggtatggtgg cggacgccta 63780taatcccagc tactcgggag gctgaggcag gagaatcact tgaacctggg aggcagaggt 63840tgcagtgagc tgagattgtg ccattgcact ccagcctggg tgacagagtg agactccgtc 63900tcaaaaaaaa aaaaaagaaa ttagaactga ctttataaag tttgggcata agagtcttag 63960cagccagtgt gtttagtata cagaaaattg tggcaatgac attctccttt cccaactttc 64020ttgattttta aattaagata tacctagaaa agcaggaatc ctggtctttg attcctgaga 64080cctccctgtt tcatgtgaag atacagcttc aagtcttgga gaatgcctcc aaggtcttaa 64140aaatggggaa tctgtggatt gtgagtcaag ctttgagcaa gtcaggtttt acaagggacc 64200ggtatattcc gactgcagcc tgagttgtgt ggccacgctg ggcattcttt ccactatgag 64260tgctcactga gctgactcac tcacactcct cgcctagagt tggcagcagg tgtggtttat 64320ggcatgtcct ttcattctga gccccgtgag atgcgggtga agagatttcc aaggctgtga 64380gagcccctct gcctccccag ctcagtcccc actccctccg cagacccact ccttgccagt 64440tgtggtgatc tccaacatct gtcagatgcc aaatgcctgg gcgtccatcc tgtggtacaa 64500catgctgacc aacaatccca aggttagtgc cccctccttt tagttggtgc cccgggatct 64560cttgcgactt aggggtacct agtatagaca atgagcacca tccctcatct aaacaagcaa 64620atgtgttctt tccaatagaa tgtaaacttt tttaccaagc ccccaattgg aacctgggat 64680caagtggccg aggtcctgag ctggcagttc tcctccacca ccaagcgagg actgagcatc 64740gagcagctga ctacactggc agagaaactc ttgggtccgc atttcacccc ttctccctcc 64800cgcccacccg cccagaaaag ggatccggcc catagggctg ttcatttggg ccatgtctac 64860tgagcattag gccatgtttc tttcctgagc aaggcgctgt gctggtgcca ggaaacaggg 64920gagttgggga gttggggtgc agagacagtt tgcagttttc agtcgaggtg atcatttttg 64980aggtgggagg tagatttctt ttctcctggt tgctgtctca ttcacccact ctatctaact 65040ttagaagatc ttttaagtgt gtgttggaag gtggcactaa aggcttgaca ttccctgtcc 65100atttttttaa taaactatag gctagttggt tttttttgtc ttattttatt tatttattta 65160tttttttgag acgagtcttg ctctgttgcc caggctggag tgcagtagtg tgatctcggc 65220tcactgcaac ctccgccttc tgggttcaag cgattcttct gcctcggcct cccgagtagc 65280tgagactaca ggtgctcacc accacgccca gctaattttt gtatttttag tagagacggg 65340gttttaccat gttggccagg atagtctccg tctcttcacc tcgtgatccg cccacctcgg 65400cctcctaaag tgctgggatt acaggcttga gccactgtgc ccagcgtagg ctagttttta 65460aaaaagaatt agtggaatat tttatgtgcc acctgggcta gaagtagctt tgttctaata 65520aagctgttgc caccaaatac acctgtctga cacccgatgt cagcttgtta gtgagtgctg 65580ctgttggttc ccagcctacc acccgaggtt gggaagagca gggggacttg ttatatcacc 65640ctccatccct gctgggctac ccagcaacac aagtgagtca aatgatggga tagtgtttgt 65700cctcatgtgc acacacacaa cagtgcctac cttcaaagat gtgaaagctg attattttgt 65760ggcccattgt gggatgaatg tgtgtgtgtt ctgttttaag aaataacctc ttgaccccaa 65820gctgaaaatg tactacttga ctctttttct ttccttcagg acctggtgtg aattattcag 65880ggtgtcagat cacatgggct aaattttgca aagtaagcaa tcttgttaaa ttctcgtggg 65940aatgggaatg ctcacctgca cggctgtcgt tgagggctct ggcttgaagg ccctgaactc 66000ttggtccagc ggccagtagg acctgcctga aggtagacgg gcctgaggat ttgggtgatg 66060cactgcaccc ctaggaaggg aagggctggg atggcagtag acttggcttt cccattactc 66120ttttctccag gaaaacatgg ctggcaaggg cttctccttc tgggtctggc tggacaatat 66180cattgacctt gtgaaaaagt acatcctggc cctttggaac gaagggtagg ttggacagag 66240tgtgcacaga tgtaaccaag tcccctgctc tcagcaagcc agtggcaggg gatggatgcc 66300ctgttagcaa taacaacatt gttcctcctc cttggctcca ggtacatcat gggctttatc 66360agtaaggagc gggagcgggc catcttgagc actaagcctc caggcacctt cctgctaaga 66420ttcagtgaaa gcagcaaaga aggaggcgtc actttcactt gggtggagaa ggacatcagc 66480ggtaagggag gctcccaccc accccacctg ctggtggctg ctgaggcctc atcactgctt 66540ctagttgcaa gcacctactg ccccctggtg ggtggagatg gccttgactc cctgtttcac 66600tcagactcgc aaaacacatt tgcgtgactt ctaaatcctt ccagctgaag gattggtttg 66660ctttgttttg cttgctccag tgactatttg ttgagaattt tgcaatttaa attgtattct 66720tcatctcttt ttctacttaa ccctgttaat atatcttacg caagtagtta tattcaagtt 66780tattttctat gacccaacta gtagcctctt cttaattaga agccagcctg aatatttcca 66840cagtgccagg ccactgaaca gggtgttcag ggtctcaaca ctagggtggc ttaagtcttt 66900tccccttcga ggaaagaaaa aatgggcagt tttctctgag atgacctagc tgtaggttcc 66960atgatctttc cttcccatgt cctgtgacag gtaagaccca gatccagtcc gtggaaccat 67020acacaaagca gcagctgaac aacatgtcat ttgctgaaat catcatgggc tataagatca 67080tggatgctac caatatcctg gtgtctccac tggtctatct ctatcctgac attcccaagg 67140aggaggcatt cggaaagtat tgtcggccag agagccagga gcatcctgaa gctgacccag 67200gtagttgttg attttccatg ttcctggcat ttaatttttg ggaaaagttg gaaattttgg 67260gatccttgga ggatagatag gcaaatgcct gaataacctg ggggataatt atttctcctt 67320atgggaaaga attgtagtga gtgcttttgt tggggtgacc gatgggattt gagaggagaa 67380tcagaatcac ttagagtagt gtagttcctg ctccacagag agtgcatgag tctaaagagg 67440ggatacagcc tgggcaatat ggtgaaacct cgtctctaca aaaaatccaa aaaaattacc 67500cggtgtggtg gcacgcattt gtagtcgtag ctacttggga ggctgaggtg ggaggatcac 67560ctgagccaag gagttcaagg ctgtagtgag cggtgatcat gccaccgcac tccagcctgg 67620ctgatagagt gagatactgt gtcaaaaaat aaaaataaag aggggatcaa tacacatacg 67680tcccccaaaa catgcctgaa acacgagaag ggaaagtgag ggcagttaac aggatgccct 67740gctggcacag tgcttcttag tagatgctag aaggtttgag gcccagattt cagcccagca 67800tatggctttt tgcctgtaac tgaaccatgt cagtgtgcca gatggtctga agaaagggtt 67860tctggaggaa attattatta gctgcatggg agtatggttt acactagagt agaagagctg 67920ggagcatcac gtttgaaggg gaagacagtg actgggtgga ggggcaaggg attagtattt 67980agagtgtgca actattgaaa ataaggtata ttttaatgtg taagaggaca tgtacttata 68040tgttatatat aaattatttt agctgggtga agtggctcat gcctatagtc ctagcacttt 68100gggaggccca ggcgggagga tcacttgagc ctgggagttt gagaacagcc tagacaacat 68160agtgagaccc tatctataca aaaataattt tttttaaatt agccacgtgt ggtggtttgt 68220gcctgtagtc ctagctactc gggaggctga ggtgggagga ttgcttaagc ccaggaggtt 68280gaggctgcag tgagccatga tcgcaccact gcactccagc ctgggtgaca gagcaagacc 68340ttgactcacc aaaaaaaaaa aaaaaaaaaa gagagagaaa ttaaaaatac tgtaatctca 68400gctgggcatg ggggttcaca cctgcagtcc tagcactttg ggaggctgaa gcaggaggat 68460cacttgaggc caggaactca agaccagcct ggcaacatag caagacccca ctacacacac 68520acacacacac acacacaaag aagagaaaga aaaaaacgaa acaaaactgt aatctctgca 68580gctgtcctca gtgtggaggg ggtagccctg tctgttcccc ttcagcactt gctgttttga 68640ctctctgggt tctttgtgca ggtcttgatg gggagtctct ggtttgccat tctttgtttg 68700atttaacttt ctgtaatcat aaagccaatg atgggctttt tttttttttt tttttttaga 68760ctaagtcttg ctctatcacc caagctggag tgcagtggca ccatctcggc tcactgcaac 68820ctccacctcc cggttcaagc aattctcctg cctcagcctc ccgagtagct gggattatgg 68880gcttgtgcca ccatgcccag ctgatttttg tattttttgt agagaaaggg tttcgccatg 68940ttggccaggc tggcctcgaa ctcctgacct caggtgatct gcccacttca gcctcccaaa 69000gtgctgggat tacaggcgtg agccactgtg cctggcctaa tgatgggctc tttaatgtga 69060tcctttaggg ttggcgcctt gccctagttg ctgttgaaaa aactattttt gtccaaatag 69120cacacacaca gaaacctacc aacttccctc ccactttttc ctaggaattc cttctgaggg 69180atttcttgag atggggcaga atggggcttg gaagagggag ttggagctaa ttgaccgttg 69240cctttctcct ttgttggggt cctgagtctt gttcctgctg taagagttac tcacttcctg 69300tctgccacct atctcccttt gcatgtgtgc ttcagttggg agatctgttt atcagcccct 69360gccacacggc tctttgttcc ttctgcagag gacgttgggg tcccacggct ggtccttttg 69420actcattttg ctttcaaggt cccacctccc agtctgaggc tgcatcctcc attaccatcg 69480cccttcctgt gggctgggag gccaggtcct ttcctgccca gcgatgtcag cgtttcctca 69540ggggccaggc actcatcagg agaaaggaac taattacttg agtaatttgc cttgccttgc 69600tgagaggagt gtgccctgag ggactccatg tgagtgtggt gacgggtgtg ggggtgtccc 69660tgtgttattt taaaatgggt gccttcagga cgatgagcat gtgaccattt cctctctatt 69720tccatcacaa gagtattatg gtatgagggt ctcaggttag attatcctcc caagactctt 69780ctctcttcct tctctactgg aagcccacat agcatttcct tatggcttga gggagaggtt 69840cggagccact tacaaattag ataaagtaca tttacaatct tgtacaaagc cacacaatga 69900agtcattttt ctcagctttt tttttttttt tttttttttt tttgagcctg agtctcgctc 69960tatcgtccag actggagtgc agtggcgcga tcttgcttca ctgaaacctc tgcctcccag 70020gttcaagaga ttctcatacc tcagcctcct gagtagctgg gattacagac atgcaccact 70080atgcctggct aatttttgga tttttagtag agaccgggtt tcaccctgtt ggccaggctg 70140gtctcgaacc cctgacctca agtgatcttc ccgcctgggc ctcccaaagt gctgggatta 70200taggtgtgag ccacagtgcc cagccttgtt tttgtttttg ttttgttttg acagtctgtc 70260actctgtcac ccaggctgga

gtgcagtggt gcgatctcac ctcacttcag cctctgcctc 70320ccaggttcaa gtgattctcc tgtctcagcc tcctgagtag ctgggattac aggcgtgcca 70380ccacgcccag ctatttttgt aatttcatta aagacagggt ttccccatgt tggtgaggct 70440ggtcttgaac tcctggcctc aagtgatcca cctgcttcag cctcccaaag tgcagggatt 70500acaggcatga gccactgtgc ctggcctcag ctatcttgaa tgctggagaa ttaaatcctt 70560ttctgtctag ggtgtcagct ccctaagggc tgggccaaaa cagttggatt tataagacac 70620tagagtcttg cctcagtagc tcctttgaat tctgcactga attgatcagt ttcttggccc 70680aaagtaaact cagatggcag cccaagagcc actctgcagt gccttctttc acatggtcat 70740catgctctct gatccctcag gttctgtcta agcctcatgt tttatgaccg tgctgttctc 70800agcccacctc accctgcccc atgccttctc aatggtttgt tcacctgaat tccccagatt 70860tcatgccagt atccccaagg ttccttgacc tcttggtgta agcattcagc atctaaaatt 70920cattttattc ccgtcaacgc atttctaact gtagaacaag aattataaat gacaaagctc 70980atagaaaatt ggcaccttgt cttccccctc cctcttattt tatacataaa agagaatatg 71040ggctgggcat tgtggccaag gctgggcatg atagctcata cttgtaatcc agcactttgg 71100gagggtgagg cagatggatc acctgaggtc aggagttcaa gaccagcctg gccaacatgg 71160tgaaacctca tctctactaa aattacaaaa aaaaaattag ctaggcatgg tggcagatgc 71220ctgtaatcca gctactcagg aggctgatga aggagaatca cttgaaccct ggaggcagag 71280gttgtagaga gccaagatgg cgctactgca ctccaacctg ggcgaaagag agcaagactc 71340cgtctcaaaa aaaaaaaaga caaaaattag ccaggcatgg tggtgccacc tgtagtccca 71400gctgcttggg agcctaaggc aggagaatcg ttttgacctg ggagtaggag gttgcggtaa 71460ccgagattgt gccactgcac ttgagcctgg gcaacagagt gagactctgt ctcaaaacaa 71520taagaacaac agcaacaaaa gagagagacc atgccttgct ccaggtctct tagctattga 71580agatgtacct ggacccaggt ctccggtctt ctagttgaag caattgtact gccttacaaa 71640gtcacattct ctttggtgct ttttgattga cgtatttatc caactagaaa gttactcatg 71700ccctcatcca aaaatgtggt agaggccaga ttagtgctgg taggaataag agatataacc 71760tttggctttg gaaccacaag cattagcagt ctccatgttc tttaaagact tggtgatatt 71820ggtatttagg ctggacacca tgcaaagact acacaggctc ggttcctgca tgcagagaag 71880ttatctaaga gatatgacca ggccggaata gaatgctcag accacgtgga ggctgttaaa 71940cttttacata atctagggaa agaagggaca caaggtggca ttagtctagg gtcaggtggg 72000aaaaggttat gctgaaaagt ctctgcagct caggacagct ttgtgcaaag aactgaagtt 72060cacagctgct agtgcctggg agatcaaata gtataaatga gggcagacaa ccctgagggg 72120cagatggagc tttccagaca atcttggcat gaggatgagt gagtttcaaa tcagtcctgc 72180cgaggcagat ggcttcctcc agctctgctt actgaatgcg aagtcacagt cagtaagaaa 72240actggttttc ttcttcccag gcgctgcccc atacctgaag accaagttta tctgtgtgac 72300accgtaagtg gcttcctttc cccgttttgc cttcatttct aatatcctca gttatccctg 72360ggaatgggac actgggtgag agttaatctg ccaaaggttg gaagcccctg ggctatgttt 72420agtactcaaa gtgaccttgt gtgtttaaaa agcttgagct tttatttttc tgttggagac 72480cagagtttga tggcttgtgt gtgtgtgttt tgttcttttt tttttttcca ttgtgtcttg 72540tcaacccccc gtttcccctc ctgctgcccc ccatttccta cagaacgacc tgcagcaata 72600ccattgacct gccgatgtcc ccccgcactt tagattcatt gatgcagttt ggaaataatg 72660gtgaaggtgc tgaaccctca gcaggagggc agtttggtga gtatttggtt gacagacttt 72720gtccctataa gggaagttgg tcccctttgt gtgatgctct cacatgtaca caccgagagc 72780tggtcactcg gaatggtagg agattctaga gctttgcttt ccaaaagaga tggtatgaat 72840gccacatgtg tgagtataaa tcttctagca gccacactgg aaatagacga acttaatttt 72900tacaatatat tttatttaac ccactaaatc caacatactc tcaatttaac atttcagaaa 72960aagttgaggc tgggtgagtg gctcatgcct gtaatcccag cactttggga ggccgaggtg 73020ggtggatcac ttgaggtcag gagttcgaga ccagtctgac caaaatctct aaaatataaa 73080aattagctgg gcatggtggc gcatacctgt aatcccagct actcaagaag ctgaggtggg 73140aggatcgctt gagcctggga ggtggaggtt gcagtgagca gagatcgtgc cactgcactc 73200cagcctgggc gacagagtga gactccatct caaataaaca aaactaaact aaaaagaaaa 73260agttgagacc tttttttatt cttttttttc atactaagcc tttaaaatcc agtgggcttt 73320tgacagccac agcacagctc agtttggaca aaccaaatct caaatgcttg gtggccacgt 73380gtgtctcggg gctcctgaat taaacagtag atcaagggca gaagatctca ggacagcctt 73440agagcttctg taaacatgga gctctgggaa tcagttaagg tgggaatgag aaaggaccct 73500tcccgaggca gggtcctcca gggaggaggg taaatctggc ttttctgacc atccctgggc 73560cttaaggggc aggagattgg atagcagtgg tagcctgggc cctgtcctct gaagggctgg 73620gggcgtggcc tgccagttgc agagggtgga caactgaact agttttccct gtctgtccct 73680ccagagtccc tcacctttga catggagttg acctcggagt gcgctacctc ccccatgtga 73740ggagctgaga acggaagctg cagaaagata cgactgaggc gcctacctgc attctgccac 73800ccctcacaca gccaaacccc agatcatctg aaactactaa ctttgtggtt ccagattttt 73860tttaatctcc tacttctgct atctttgagc aatctgggca cttttaaaaa tagagaaatg 73920agtgaatgtg ggtgatctgc ttttatctaa atgcaaataa ggatgtgttc tctgagaccc 73980atgatcaggg gatgtggcgg ggggtggcta gagggagaaa aaggaaatgt cttgtgttgt 74040tttgttcccc tgccctcctt tctcagcagc tttttgttat tgttgttgtt gttcttagac 74100aagtgcctcc tggtgcctgc ggcatccttc tgcctgtttc tgtaagcaaa tgccacaggc 74160cacctatagc tacatactcc tggcattgca ctttttaacc ttgctgacat ccaaatagaa 74220gataggacta tctaagccct aggtttcttt ttaaattaag aaataataac aattaaaggg 74280caaaaaacac tgtatcagca tagcctttct gtatttaaga aacttaagca gccgggcatg 74340gtggctcacg cctgtaatcc cagcactttg ggaggccgag gcggatcata aggtcaggag 74400atcaagacca tcctggctaa cacggtgaaa ccccgtctct actaaaagta caaaaaatta 74460gctgggtgtg gtggtgggcg cctgtagtcc cagctactcg ggaggctgag gcaggagaat 74520cgcttgaacc tgagaggcgg aggttgcagt gagccaaaat tgcaccactg cacactgcac 74580tccatcctgg gcgacagtct gagactctgt ctcaaaaaaa aaaaaaaaaa aaagaaactt 74640cagttaacag cctccttggt gctttaagca ttcagcttcc ttcaggctgg taatttatat 74700aatccctgaa acgggcttca ggtcaaaccc ttaagacatc tgaagctgca acctggcctt 74760tggtgttgaa ataggaaggt ttaaggagaa tctaagcatt ttagactttt ttttataaat 74820agacttattt tcctttgtaa tgtattggcc ttttagtgag taaggctggg cagagggtgc 74880ttacaacctt gactcccttt ctccctggac ttgatctgct gtttcagagg ctaggttgtt 74940tctgtgggtg ccttatcagg gctgggatac ttctgattct ggcttccttc ctgccccacc 75000ctcccgaccc cagtccccct gatcctgcta gaggcatgtc tccttgcgtg tctaaaggtc 75060cctcatcctg tttgttttag gaatcctggt ctcaggacct catggaagaa gagggggaga 75120gagttacagg ttggacatga tgcacactat ggggccccag cgacgtgtct ggttgagctc 75180agggaatatg gttcttagcc agtttcttgg tgatatccag tggcacttgt aatggcgtct 75240tcattcagtt catgcagggc aaaggcttac tgataaactt gagtctgccc tcgtatgagg 75300gtgtatacct ggcctccctc tgaggctggt gactcctccc tgctggggcc ccacaggtga 75360ggcagaacag ctagagggcc tccccgcctg cccgccttgg ctggctagct cgcctctcct 75420gtgcgtatgg gaacacctag cacgtgctgg atgggctgcc tctgactcag aggcatggcc 75480ggatttggca actcaaaacc accttgcctc agctgatcag agtttctgtg gaattctgtt 75540tgttaaatca aattagctgg tctctgaatt aagggggaga cgaccttctc taagatgaac 75600agggttcgcc ccagtcctcc tgcctggaga cagttgatgt gtcatgcaga gctcttactt 75660ctccagcaac actcttcagt acataataag cttaactgat aaacagaata tttagaaagg 75720tgagacttgg gcttaccatt gggtttaaat catagggacc tagggcgagg gttcagggct 75780tctctggagc agatattgtc aagttcatgg ccttaggtag catgtatctg gtcttaactc 75840tgattgtagc aaaagttctg agaggagctg agccctgttg tggcccatta aagaacaggg 75900tcctcaggcc ctgcccgctt cctgtccact gccccctccc catccccagc ccagccgagg 75960gaatcccgtg ggttgcttac ctacctataa ggtggtttat aagctgctgt cctggccact 76020gcattcaaat tccaatgtgt acttcatagt gtaaaaattt atattattgt gaggtttttt 76080gtcttttttt tttttttttt tttttggtat attgctgtat ctactttaac ttccagaaat 76140aaacgttata taggaaccgt ctgatagcat ggcagctctg tttggctggt ggaggcttcc 76200ttttcccctg cataagttct gagggggcct cacacacagg tggggcctgg gataagggcc 76260ggaaagggtc ttgagaggag gtggttgcct taatcccccc cgccaacccc cttatgttag 76320ccaccagccc ggaggtaagg ggtgcctgga ggagcaggag gtcaatagtc caacggcaga 76380aaggtgtcag agtggaggcc tccctccccg gccccctcct accccccaga gcggcctcgt 76440cctgtctggg gtcagataag ccacctaagc ggggtggggg gtagatactc ccaccgcacc 76500aaggcctccc cttccacagt tggctccttt atcactttcc cttcagttca cccagcgggg 76560acaacacgca gacacccggt ggtggctgca gggcccccgg cagccagcgg tgataatgca 76620gggaaaggcg ccccaacctc agctacgcgg gcgcccacag ggcttctccc cacccctaca 76680cgtgccccag cgccctggag accgcgcctg ggagctacga gcgagcacct tccctcgcag 76740agatggatca gattagcccc ctggggcggt ggcacctgcc cgtcccctcc cctcctctct 76800tctcccaccc ccgccttccc cctgctctgg gccccctgca cctccctctt ggggcccacg 76860cgccgcccct ctccctcgct tgagctctga gctcagaagg aacctgcccg caaacctggc 76920tcccagcccc gcccagggag cgcgcgtccc tgagcccaag ggccccagcg acagcaggga 76980ctggctgcag ccggcagtgc gggggtccag ccggggcgag ccggtcccgc ttgctgcacc 77040tccgcctcgc acagtcgccg gcagccagct gaaaacagcg cagccgcgtg gtgccacctg 77100ctggtctctg ggaaggagga ggatgggacg gttgttgcgg ctctgggact gcccctggcc 77160ccgagggttg agaagcatct tggcgccggg ccctcagccg ccagcttcga ggctgctggg 77220agcagcagct gctaaggaga agcctgggtc agagttctgc ccacccgcgc ccatctccat 77280catttcccca aacctcgagt cccctgcttt ttaaggaatc tgtgccccct aaactggctc 77340cttacccttc tgagaagcat tgacagtatt ccaggggtgg ccagcaccct gaaaaccaca 77400gcaaagcggg cagagttaaa atttaagact aaaaatgaca gcatcaggca tgcacgcggg 77460aaggaacaga acaaccagct ccaggggttc ccatcaccca gggaggccac caacgcccca 77520accaggagcc atagcagcaa atttatgaaa aaaatatttt attccaaaac actgtttaca 77580caaatgtgtg gtctttgtac aaagtacaaa aaaacccctc ttctcatccc cacctccctt 77640cagagaagtt taaaccttgg gctctcaatc ttccatggtc agctgcccct gactgcgagt 77700cctacagcct cagagggagg agcgtgtggg gttgggagca aggcctcacc agaacacagc 77760cagttacgca acagcgcaag gcctgctcca ggccccacct ccagcggagt caaaccagat 77820cagcttttac ccctcactcc agattccaca ggcacgcagc acatggctga tttgcagctt 77880gtctttcact gcagtttttt gtttttgttt tttccaggag gcaagaggac caaccctcca 77940agtcccgggg cccctgtcca cccaccatat cctagaccca atcttttcta cctctttgtc 78000gacaaggtta caaacagaga ggcaagcaaa gaaggctggg gcccaacgga ggggagagaa 78060tatatatccc gggaaacgtg ggcaacagca tcatagactt gaatgaagcc caaggccaag 78120cagccaagca aggactaatt cagagcagct cagaaaccct cactcaaacc ggcggccccc 78180ctgtgctaag gacatggccg ggcccagacg cgctctcata gggttcacag agagtctgga 78240gtccacgttc actaaagtat aaaatactac tcgaaaatga accccagcct cccttctgag 78300agatgaatcg gttacatcaa cacatgagga cagatgcatt tacatagacc cacacactct 78360ctctttctct ctctctgtct ctctgtctct ctctcttagg agggcaaagc ttctcactcc 78420ggagtgaaac ttcagcttac agacgttacc tgtctcccct ccccttctga atcatctcat 78480tcaaatagct gctgagcaga tgaggggccc aggaggaata ggtctggctg cattgagtgc 78540ctgcagtgac agaggctcgg tctctaccat gacagagaca gactcggcgc actcttggat 78600gccatgatct gaaaagaggc caccgctgct gcagccacat cccaggactg cacaggggag 78660gcaggcgtag ctcatgcaca aggacacaca cacacacaca cacacaagca catgcatgcc 78720tgcaaaggtg tcagtgtaca cagccttact aaactcacaa cacgaccgct tcacattgca 78780tattgtttcc aaagagaagc gtgggattca aacattcatg agagggagcc tctggcagag 78840gtgaaaagac cggcaggggg cgagaggcgg gagtcaagac tgtccattgg tcggcgtaag 78900agttcctcca cgtgcctggc cacatccatg gtctcatcca ggtcgaattc tccatcctga 78960tcgagtacat ggtcagggct ggtaaagaca gggggatgtc a 79001316DNAArtificial sequenceSynthetic oligonucleotide 3actgccgcag ctccat 16414DNAArtificial sequenceSynthetic oligonucleotide 4gagattctct acca 14516DNAArtificial sequenceSynthetic oligonucleotide 5agatcttgca tgtctc 16616DNAArtificial sequenceSynthetic oligonucleotide 6ataattcaac tcaggg 16716DNAArtificial sequenceSynthetic oligonucleotide 7actttttcac aaggtc 16816DNAArtificial sequenceSynthetic oligonucleotide 8ccatgatctt atagcc 16916DNAArtificial sequenceSynthetic oligonucleotide 9gatagcagaa gtagga 161016DNAArtificial sequenceSynthetic oligonucleotide 10caaggttaaa aagtgc 161114DNAArtificial sequenceSynthetic oligonucleotide 11aaggttaaaa agtg 141216DNAArtificial sequenceSynthetic oligonucleotide 12ctatttggat gtcagc 161314DNAArtificial sequenceSynthetic oligonucleotide 13tatttggatg tcag 141416DNAArtificial sequenceSynthetic oligonucleotide 14tagatagtcc tatctt 161514DNAArtificial sequenceSynthetic oligonucleotide 15agatagtcct atct 141616DNAArtificial sequenceSynthetic oligonucleotide 16aagaaaccta gggctt 161714DNAArtificial sequenceSynthetic oligonucleotide 17agaaacctag ggct 141816DNAArtificial sequenceSynthetic oligonucleotide 18gctgatacag tgtttt 161914DNAArtificial sequenceSynthetic oligonucleotide 19ctgatacagt gttt 142016DNAArtificial sequenceSynthetic oligonucleotide 20atacagaaag gctatg 162114DNAArtificial sequenceSynthetic oligonucleotide 21tacagaaagg ctat 142216DNAArtificial sequenceSynthetic oligonucleotide 22gcttaagttt cttaaa 162314DNAArtificial sequenceSynthetic oligonucleotide 23cttaagtttc ttaa 142416DNAArtificial sequenceSynthetic oligonucleotide 24agcaccaagg aggctg 162514DNAArtificial sequenceSynthetic oligonucleotide 25gcaccaagga ggct 142616DNAArtificial sequenceSynthetic oligonucleotide 26aagctgaatg cttaaa 162714DNAArtificial sequenceSynthetic oligonucleotide 27agctgaatgc ttaa 142816DNAArtificial sequenceSynthetic oligonucleotide 28ttaccagcct gaagga 162914DNAArtificial sequenceSynthetic oligonucleotide 29taccagcctg aagg 143016DNAArtificial sequenceSynthetic oligonucleotide 30cagggattat ataaat 163114DNAArtificial sequenceSynthetic oligonucleotide 31agggattata taaa 143216DNAArtificial sequenceSynthetic oligonucleotide 32acctgaagcc cgtttc 163314DNAArtificial sequenceSynthetic oligonucleotide 33cctgaagccc gttt 143416DNAArtificial sequenceSynthetic oligonucleotide 34tgtcttaagg gtttga 163514DNAArtificial sequenceSynthetic oligonucleotide 35gtcttaaggg tttg 143616DNAArtificial sequenceSynthetic oligonucleotide 36ggttgcagct tcagat 163714DNAArtificial sequenceSynthetic oligonucleotide 37gttgcagctt caga 143816DNAArtificial sequenceSynthetic oligonucleotide 38tcaacaccaa aggcca 163914DNAArtificial sequenceSynthetic oligonucleotide 39caacaccaaa ggcc 144016DNAArtificial sequenceSynthetic oligonucleotide 40tccttaaacc ttccta 164114DNAArtificial sequenceSynthetic oligonucleotide 41ccttaaacct tcct 144216DNAArtificial sequenceSynthetic oligonucleotide 42aaaatgctta gattct 164314DNAArtificial sequenceSynthetic oligonucleotide 43aaatgcttag attc 144416DNAArtificial sequenceSynthetic oligonucleotide 44aaataagtct atttat 164514DNAArtificial sequenceSynthetic oligonucleotide 45aataagtcta ttta 144616DNAArtificial sequenceSynthetic oligonucleotide 46ggccaataca ttacaa 164714DNAArtificial sequenceSynthetic oligonucleotide 47gccaatacat taca 144816DNAArtificial sequenceSynthetic oligonucleotide 48tgcccagcct tactca 164914DNAArtificial sequenceSynthetic oligonucleotide 49gcccagcctt actc 145016DNAArtificial sequenceSynthetic oligonucleotide 50gttgtaagca ccctct 165114DNAArtificial sequenceSynthetic oligonucleotide 51ttgtaagcac cctc 145216DNAArtificial sequenceSynthetic oligonucleotide 52agaaagggag tcaagg 165314DNAArtificial sequenceSynthetic oligonucleotide 53gaaagggagt caag 145416DNAArtificial sequenceSynthetic oligonucleotide 54gcagatcaag tccagg 165514DNAArtificial sequenceSynthetic oligonucleotide 55cagatcaagt ccag 145616DNAArtificial sequenceSynthetic oligonucleotide 56agcctctgaa acagca 165714DNAArtificial sequenceSynthetic oligonucleotide 57gcctctgaaa cagc 145816DNAArtificial sequenceSynthetic oligonucleotide 58cccacagaaa caacct 165914DNAArtificial sequenceSynthetic oligonucleotide 59ccacagaaac aacc 146016DNAArtificial sequenceSynthetic oligonucleotide 60agccctgata aggcac 166114DNAArtificial sequenceSynthetic oligonucleotide 61gccctgataa ggca 146216DNAArtificial sequenceSynthetic oligonucleotide 62aatcagaagt atccca 166314DNAArtificial

sequenceSynthetic oligonucleotide 63atcagaagta tccc 146416DNAArtificial sequenceSynthetic oligonucleotide 64gcctctagca ggatca 166514DNAArtificial sequenceSynthetic oligonucleotide 65cctctagcag gatc 146616DNAArtificial sequenceSynthetic oligonucleotide 66cacgcaagga gacatg 166714DNAArtificial sequenceSynthetic oligonucleotide 67acgcaaggag acat 146816DNAArtificial sequenceSynthetic oligonucleotide 68tgagggacct ttagac 166914DNAArtificial sequenceSynthetic oligonucleotide 69gagggacctt taga 147016DNAArtificial sequenceSynthetic oligonucleotide 70caggattcct aaaaca 167114DNAArtificial sequenceSynthetic oligonucleotide 71aggattccta aaac 147216DNAArtificial sequenceSynthetic oligonucleotide 72atgaggtcct gagacc 167314DNAArtificial sequenceSynthetic oligonucleotide 73tgaggtcctg agac 147416DNAArtificial sequenceSynthetic oligonucleotide 74catcatgtcc aacctg 167514DNAArtificial sequenceSynthetic oligonucleotide 75atcatgtcca acct 147616DNAArtificial sequenceSynthetic oligonucleotide 76gggccccata gtgtgc 167714DNAArtificial sequenceSynthetic oligonucleotide 77ggccccatag tgtg 147816DNAArtificial sequenceSynthetic oligonucleotide 78agctcaacca gacacg 167914DNAArtificial sequenceSynthetic oligonucleotide 79gctcaaccag acac 148016DNAArtificial sequenceSynthetic oligonucleotide 80gaaccatatt ccctga 168114DNAArtificial sequenceSynthetic oligonucleotide 81aaccatattc cctg 148216DNAArtificial sequenceSynthetic oligonucleotide 82caagaaactg gctaag 168314DNAArtificial sequenceSynthetic oligonucleotide 83aagaaactgg ctaa 148416DNAArtificial sequenceSynthetic oligonucleotide 84gccactggat atcacc 168516DNAArtificial sequenceSynthetic oligonucleotide 85aactgaatga agacgc 168616DNAArtificial sequenceSynthetic oligonucleotide 86gcttattatg tactga 168714DNAArtificial sequenceSynthetic oligonucleotide 87cttattatgt actg 148816DNAArtificial sequenceSynthetic oligonucleotide 88gcccaagtct cacctt 168914DNAArtificial sequenceSynthetic oligonucleotide 89cccaagtctc acct 149016DNAArtificial sequenceSynthetic oligonucleotide 90cccaatggta agccca 169114DNAArtificial sequenceSynthetic oligonucleotide 91ccaatggtaa gccc 149216DNAArtificial sequenceSynthetic oligonucleotide 92aacccaatgg taagcc 169314DNAArtificial sequenceSynthetic oligonucleotide 93acccaatggt aagc 149416DNAArtificial sequenceSynthetic oligonucleotide 94taggtcccta tgattt 169514DNAArtificial sequenceSynthetic oligonucleotide 95aggtccctat gatt 149616DNAArtificial sequenceSynthetic oligonucleotide 96aagccctgaa ccctcg 169714DNAArtificial sequenceSynthetic oligonucleotide 97agccctgaac cctc 149816DNAArtificial sequenceSynthetic oligonucleotide 98cctaaggcca tgaact 169914DNAArtificial sequenceSynthetic oligonucleotide 99ctaaggccat gaac 1410016DNAArtificial sequenceSynthetic oligonucleotide 100accagataca tgctac 1610114DNAArtificial sequenceSynthetic oligonucleotide 101ccagatacat gcta 1410216DNAArtificial sequenceSynthetic oligonucleotide 102tacaatcaga gttaag 1610314DNAArtificial sequenceSynthetic oligonucleotide 103acaatcagag ttaa 1410416DNAArtificial sequenceSynthetic oligonucleotide 104tcctctcaga actttt 1610514DNAArtificial sequenceSynthetic oligonucleotide 105cctctcagaa cttt 1410616DNAArtificial sequenceSynthetic oligonucleotide 106gctcctctca gaactt 1610714DNAArtificial sequenceSynthetic oligonucleotide 107ctcctctcag aact 1410816DNAArtificial sequenceSynthetic oligonucleotide 108ttctttaatg ggccac 1610914DNAArtificial sequenceSynthetic oligonucleotide 109tctttaatgg gcca 1411016DNAArtificial sequenceSynthetic oligonucleotide 110acgggattcc ctcggc 1611114DNAArtificial sequenceSynthetic oligonucleotide 111cgggattccc tcgg 1411216DNAArtificial sequenceSynthetic oligonucleotide 112gtaggtaagc aaccca 1611314DNAArtificial sequenceSynthetic oligonucleotide 113taggtaagca accc 1411416DNAArtificial sequenceSynthetic oligonucleotide 114gaatttgaat gcagtg 1611514DNAArtificial sequenceSynthetic oligonucleotide 115aatttgaatg cagt 1411616DNAArtificial sequenceSynthetic oligonucleotide 116tgaagtacac attgga 1611714DNAArtificial sequenceSynthetic oligonucleotide 117gaagtacaca ttgg 1411816DNAArtificial sequenceSynthetic oligonucleotide 118ataaattttt acacta 1611914DNAArtificial sequenceSynthetic oligonucleotide 119taaattttta cact 1412014DNAArtificial sequenceSynthetic oligonucleotide 120caataatata aatt 1412116DNAArtificial sequenceSynthetic oligonucleotide 121ctggaagtta aagtag 1612214DNAArtificial sequenceSynthetic oligonucleotide 122tggaagttaa agta 1412316DNAArtificial sequenceSynthetic oligonucleotide 123gtactctttc agtggt 1612416DNAArtificial sequenceSynthetic oligonucleotide 124atgcttagat tctcct 1612516DNAArtificial sequenceSynthetic oligonucleotide 125agcagatcaa gtccag 1612616DNAArtificial sequenceSynthetic oligonucleotide 126aggtgttccc atacgc 1612716DNAArtificial sequenceSynthetic oligonucleotide 127taggtgttcc catacg 1612816DNAArtificial sequenceSynthetic oligonucleotide 128ggttcctcct gttggc 1612917DNAArtificial sequenceSynthetic oligonucleotide 129atgcttagat tctcctt 17

Claims

1. A method of treating cancer in a subject comprising administering to the subject a weekly dose of about 1.5 to 3.5 milligrams of a sodium salt of a single-stranded modified oligonucleotide per kilogram of the subject's body weight per week (1.5-3.5 mg/kg/wk), wherein the modified oligonucleotide consists of 16 linked nucleosides, has a nucleobase sequence consisting of the nucleobase sequence of SEQ ID NO: 12, and has: a gap segment consisting of ten linked 2'-deoxynucleosides; a 5' wing segment consisting of 3 linked nucleosides; and a 3' wing segment consisting of 3 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 constrained ethyl nucleoside; wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is 5-methylcytosine.

2. A method of treating cancer in a subject comprising administering to the subject about 15 milligrams to 250 milligrams per week of a sodium salt of a single-stranded modified oligonucleotide consisting of 16 linked nucleosides and having a nucleobase sequence consisting of the nucleobase sequence of SEQ ID NO: 12, wherein the modified oligonucleotide has: a gap segment consisting of ten linked 2'-deoxynucleosides; a 5' wing segment consisting of 3 linked nucleosides; and a 3' wing segment consisting of 3 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 constrained ethyl nucleoside; wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is 5-methylcytosine.

3. (canceled)

4. The method of claim 1, wherein 3.0 milligrams of the sodium salt of the single-stranded modified oligonucleotide is administered to the subject per kilogram of the subject's body weight per week (3.0 mg/kg/wk).

5-10. (canceled)

11. The method of claim 1, wherein the cancer is B-cell lymphoma or hepatocellular carcinoma (HCC).

12. The method of claim 11, wherein the B-cell lymphoma is a non-Hodgkin's B-cell lymphoma.

13. The method of claim 12, wherein the non-Hodgkin's B-cell lymphoma is selected from the group consisting of: diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis.

14. The method of claim 12, wherein the non-Hodgkin's B-cell lymphoma is diffuse large B cell lymphoma (DLBCL).

15-31. (canceled)

32. The method of claim 2, wherein the cancer is B-cell lymphoma or hepatocellular carcinoma (HCC).

33. The method of claim 32, wherein the B-cell lymphoma is a non-Hodgkin's B-cell lymphoma.

34. The method of claim 33, wherein the non-Hodgkin's B-cell lymphoma is selected from the group consisting of: diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis.

35. The method of claim 33, wherein the non-Hodgkin's B-cell lymphoma is diffuse large B cell lymphoma (DLBCL).

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