WO2011133923A1 - Modulation of lactosylceramide synthase (lcs) expression - Google Patents

Abstract

Provided herein are methods, compounds, and compositions for reducing expression of lactosylceramide synthase (LCS) mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for preventing or decreasing airway inflammation and/or airway hyperresponsiveness in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of airway inflammation or airway hyperresponsiveness, or a symptom thereof.

Description

MODULATION OF LACTOSYLCERAMIDE SYNTHASE (LCS) EXPRESSION

Sequence Listing

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0127WOSEQ.txt, created on April 22, 2011 which is 109 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

Field of the Invention

Provided herein are methods, compounds, and compositions for reducing expression of

LCS mRNA and protein in an animal. Also, provided herein are methods, compounds, and compositions having a LCS inhibitor for reducing LCS related diseases or conditions in an animal. Such methods, compounds, and compositions are useful, for example, to treat, prevent, delay, decrease or ameliorate any one or more airway inflammation or airway

hyperresponsiveness (AHR), or a symptom thereof, in an animal.

Background

Mast cells and granulocytes represent the key effector cells in allergy and asthma

(Bischoff, S.C. Nat. Rev. Immunol. 2007 7: 93-104). Lipid rafts present in the cell membrane of mast cells and granulocytes reportedly can play an important role in modulating the activation of these cells (Draber, P. et al., Mol Immunol. 2002, 38: 1247-52), which in turn can be regulated by the composition of glycosphingolipids (GSL) in the rafts' membrane (Mishra, S. et al., J

Neurochem. 2007, 1: 135-42).

Lactosylceramide synthase (also known as LCS, B4GALT6 or UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 6) catalyzes the transfer of galactose from UDP- Galactose to glucosylceramide to form lactosylceramide. Lactosylceramide is a

glycosphingolipid present in many mammalian cells and is the precursor for the synthesis of several classes of glycosphingolipids (Takizawa et al., Biochim Biophys Acta. 1999,

1438(2):301-4). Accordingly, the potential role of LCS in allergy and asthma makes it an attractive target for investigation in airway inflammation and/or airway hyperresponsiveness.

Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of LCS expression. Antisense oligonucleotides targeted to a number of targets including, but not limited to p38 alpha MAP kinase (US Patent Publication No. 20040171566, incorporated by reference); the CD28 receptor ligands B7-1 and B7-2 (US Patent Publication 20040235164, incorporated by reference);

intracellular adhesion molecule (ICAM) (WO 2004/108945, incorporated by reference); and adenosine A} receptor (Nyce and Metzger, Nature, 1997, 555:721-725) have been tested for their ability to inhibit pulmonary inflammation and airway hyperresponsiveness (AHR) in mouse, rabbit, and/or monkey models of asthma when delivered by inhalation. However, treatment with any ASO targeted to any inflammatory mediator involved in pulmonary inflammation is not always effective at reducing AHR and/or pulmonary inflammation. Antisense oligonucleotides targeted to Jun N-terminal Kinase (JNK-1) found to decrease target expression in vitro were tested in a mouse model of asthma. Treatment with each of two different antisense

oligonucleotides targeted to JNK-1 were not effective at reducing methacholine induced AHR, eosinophil recruitment, or mucus production at any of the antisense oligonucleotide doses tested (Zhang, J.P. et al., Clin Exp Immunol. 2000. 122: 20-27).

Although previous antisense inhibition of various targets has not been always effective in reducing pulmonary inflammation and airway hyperresponsiveness, the potential role of LCS in allergy and asthma makes it an attractive antisense target for investigation in airway inflammation and/or airway hyperresponsiveness. Summary of the Invention

Provided herein are compounds useful for modulating gene expression and associated pathways via antisense mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy.

Provided herein are methods, compounds, and compositions for inhibiting or reducing expression of LCS and thereby treating, preventing, delaying, decreasing or ameliorating a LCS related disease, condition or a symptom thereof. In certain embodiments, the LCS related disease or condition is airway inflammation, airway hyperresponsiveness or a pulmonary disease.

In certain embodiments, the compounds or compositions of the invention comprise a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to LCS. The LCS target can have a sequence selected from any one of SEQ ID NOs: 1-3. The modified oligonucleotide targeting LCS can have a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of SEQ ID NOs: 1-3. The modified oligonucleotide can have a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleobases. The contiguous nucleobase portion of the modified oligonucleotide can be complementary to an equal length portion of a LCS region selected from any one of SEQ ID NOs: 1-3. The contiguous nucleobase portion of the modified oligonucleotide can be a sequence selected from any one of SEQ ID NOs: 35-113. In certain embodiments, the compound comprises a modified oligonucleotide having a gap motif with a gap segment consisting of eight to twelve linked deoxynucleosides, a 5' wing segment consisting of three to seven linked nucleosides and a 3' wing segment consisting of three to seven linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment. In certain embodiments, the compound comprises a modified oligonucleotide having at least one 2'-MOE sugar, at least one phosphorothioate linkage and at least one 5'- methylcytosine.

Certain embodiments provide a pharmaceutical composition comprising the compound of the invention, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

Certain embodiments provide a method of reducing LCS expression in an animal comprising administering to the animal a compound comprising the modified oligonucleotide targeting LCS described herein.

Certain embodiments provide a method of decreasing or ameliorating airway

inflammation, airway hyperresponsiveness or pulmonary disease in an animal comprising administering to the animal a compound comprising a modified oligonucleotide targeted to LCS described herein, wherein the modified oligonucleotide reduces LCS expression in the animal.

Certain embodiments provide a method for treating an animal with airway inflammation and/or airway hyperresponsiveness comprising: 1) identifying the animal prone to airway inflammation and/or airway hyperresponsiveness, and 2) administering to the animal a therapeutically effective amount of a compound consisting of 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to SEQ ID NO: 1 -3 as measured over the entirety of said compound. In certain embodiments, the therapeutically effective amount of the compound administered to the animal reduces airway inflammation and/or airway

hyperresponsiveness in the animal.

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

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

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

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

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

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

"3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3 '-most nucleotide of a particular antisense compound or oligonucleotide. "5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5' -most nucleotide of a particular antisense compound or oligonucleotide.

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

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

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

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

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

"Administering" means providing an agent to an animal, and includes, but is not limited to, administering by a medical professional and self-administering. Administering the antisense compound of the invention to an animal can be performed by a variety of routes including, but not limited to, intranasal, intrapulmonary and intratracheal. Devices for administration of the antisense compound include, but are not limited to, metered dose inhalers, nebulizers and colloidal dispersion systems.

"Agent" means an active substance that can provide a therapeutic benefit when administered to an animal. "First Agent" means a therapeutic compound of the invention. For example, a first agent can be an antisense oligonucleotide targeting LCS. "Second agent" means a second therapeutic compound of the invention (e.g. a second antisense oligonucleotide targeting LCS) and/or a non-LCS therapeutic compound. "Airway inflammation" means inflammation of the air passages of a subject. Airway inflammation can be present in subjects suffering from a variety of diseases including, but not limited to, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, emphysema, chronic obstructive pulmonary disease (COPD) and asthma (e.g., allergic asthma, non-allergic asthma).

"Airway hyperresponsiveness", "AHR" or "bronchial hyperresponsiveness" is a state where the airways (bronchioles) of a subject are easily triggered to spasm (constrict). Airway hyperresponsiveness can be assessed in a subject with a challenge test. For example,

administering a compound (e.g., metacholine or Wstamine) or irritant (e.g., cold air) to the subject then measuring the length of time to trigger bronchospasms, the volume of the airways or lung function (e.g. spirometry or plethysmography). Airway hyperresponsiveness can include any number of conditions, including, but not limited to, emphysema, chronic bronchitis or asthma.

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

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

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

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

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

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

"Asthma" is a predisposition to inflammation of the lungs where airways are reversibly narrowed and which can progress into a chronic inflammation of the lungs. During asthma attacks (exacerbations of asthma), the smooth muscle cells in the airways constrict, the airways become inflamed and swollen, edema of the mucosa occurs, mucus accumulates in the bronchi and bronchioles and breathing becomes difficult. Indications of asthma can include airflow obstruction, bronchial hyperresponsiveness, and an underlying inflammation. "Allergic asthma" is asthma caused by an allergen.

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

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

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

"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'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0-methoxyethyl modifications.

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

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

"Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, complementarity between the first and second nucleic acid may be between two DNA strands, between two RNA strands, or between a DNA and an RNA strand. In certain embodiments, some of the nucleobases on one strand are matched to a complementary hydrogen bonding base on the other strand. In certain embodiments, all of the nucleobases on one strand are matched to a complementary hydrogen bonding base on the other strand. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid. In certain such embodiments, an antisense

oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.

"Constrained ethyl" or "cEt" refers to a bicyclic nucleoside having a furanosyl sugar that comprises a methyl(methyleneoxy) (4'-CH(CH₃)-0-2') bridge between the 4' and the 2' carbon atoms.

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

"COPD" or "chronic obstructive pulmonary disorder" is a disease in which the airways become narrowed, limiting the airflow into and out of the lungs and causing a shortage of breadth in the affected subject. The airway narrowing usually is poorly reversible and progressively increases. COPD includes chronic bronchitis and emphysema.

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

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

"Decreasing" airway inflammation and/or airway hyperresponsiveness means to ameliorate at least one symptom or aspect of airway inflammation or airway hyperresponsiveness. For example, increasing lung function (as assayed by spirometry or plethysmography) decreases airway inflammation and/or airway hyperresponsiveness.

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

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

"Dosage unit" means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense oligonucleotide. "Dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily

accommodated by a single injection, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month. Doses can be expressed, for example, as mg/kg.

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

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

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

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

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

"Identifying" or "selecting a subject having airway inflammation and/or airway hyperresponsiveness" means identifying or selecting a subject prone to or having been diagnosed with a airway inflammation and/or airway hyperresponsiveness; or, identifying or selecting a subject having airway inflammation and/or airway hyperresponsiveness or symptom thereof, including, but not limited to, reduced lung function, reduced lung volume, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, emphysema, chronic obstructive pulmonary disease (COPD) and asthma. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring lung function, lung volume and the like.

"Improved pulmonary outcome" means a reduction in the occurrence of adverse pulmonary events, or the risk thereof. Examples of adverse pulmonary events include, without limitation, death, airway restriction, an asthmatic attack, airway inflammation and airway hyperresponsiveness.

"Immediately adjacent" means there are no intervening elements between the

immediately adjacent elements, for example, between regions, segments, nucleotides and/or nucleosides.

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

"Induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g. denote quantitative differences between two states. For example, "an amount effective to inhibit the activity or expression of LCS" means that the level of activity or expression of LCS in a treated sample will differ from the level of LCS activity or expression in an untreated sample. Such terms are applied to, for example, levels of expression, and levels of activity.

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

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

"Intravenous administration" means administration into a vein of a subject.

"Intranasal administration" means administration into the nose of a subject.

"Intrapulmonary administration" means administration into the lungs of a subject.

"Intratracheal administration" means administration into the trachea of a subject.

"LCS" or "lactosylceramide synthase" means any nucleic acid or protein of

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

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

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

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

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

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

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

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

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

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

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

"Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage. "Natural sugar moiety" means a sugar found in DNA (2'-H) or RNA (2'-OH).

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

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

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

"Nucleoside" means a nucleobase linked to a sugar.

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

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

"Nucleotide mimetic" includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=0)-0- or other non-phosphodiester linkage).

"Oligomeric compound" or "oligomer" refers to a polymeric structure comprising two or more sub-structures and capable of hybridizing to a region of a nucleic acid molecule. In certain embodiments, oligomeric compounds are oligonucleosides. In certain embodiments, oligomeric compounds are oligonucleotides. In certain embodiments, oligomeric compounds are antisense compounds. In certain embodiments, oligomeric compounds are antisense oligonucleotides. In certain embodiments, oligomeric compounds are chimeric oligonucleotides.

"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. "Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. Admimstration can be continuous, or chronic, or short or intermittent.

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

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

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

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

"Pharmaceutically acceptable derivative" encompasses derivatives of the compounds described herein such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelled variants, conjugates, pharmaceutically acceptable salts and other derivatives known in the art.

"Pharmaceutically acceptable salts" or "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. The term "pharmaceutically acceptable salt" or "salt" includes a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. "Pharmaceutically acceptable salts" of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley- VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic aclrninistration to humans. Accordingly, in one embodiment the compounds described herein are in the form of a sodium salt.

"Phosphorothioate linkage" means a linkage between nucleosides where the

phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

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

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

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

"Pulmonary Disease" means a disease of the lung. Pulmonary disease can include, but is not limited to, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, pulmonary hyperresponsiveness, emphysema, chronic obstructive pulmonary disease (COPD) and asthma.

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

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

"Second agent" or "second therapeutic agent" means an agent that can be used in combination with a "first agent". A second therapeutic agent can be any agent that treats, prevents, delays, decreases or ameliorates airway inflammation and/or airway

hyperresponsiveness. A second therapeutic agent can include, but is not limited to, an siRNA or antisense oligonucleotide including antisense oligonucleotides targeting LCS. A second agent can also include antibodies (e.g., anti-LCS antibodies), peptide inhibitors (e.g., LCS peptide inhibitors) and anti-inflammatory agents.

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

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

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

aminotransferase levels in serum can indicate liver toxicity or liver function abnormality. For example, increased bilirubin can indicate liver toxicity or liver function abnormality.

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

"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.

"Subcutaneous administration" means administration just below the skin.

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

"Targeting" or "targeted" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

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

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

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

"Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside). Certain Embodiments

In certain embodiments, the compounds of the invention comprise an oligonucleotide. In certain embodiments, the antisense oligonucleotide can comprise 10 to 30 linked nucleosides in length targeted to LCS. The LCS target can have a sequence selected from any one of SEQ ID NOs: 1-3. In certain embodiments, the antisense oligonucleotide can consist of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NOs: 1-3. In certain embodiments, the antisense oligonucleotide can consist of 10 to 30 linked nucleosides and have a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NO: 43 or 63. In certain embodiments, the antisense

oligonucleotide can consist of 10 to 30 linked nucleosides and have a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NO: 35-113. In certain embodiments, the antisense oligonucleotide consists of any one of the nucleobase sequences recited in SEQ ID NO: 35-113. In certain embodiments, the antisense oligonucleotide consists of any one of the nucleobase sequences recited in SEQ ID NO: 43 or 63

In certain embodiments, the compound of the invention comprises a salt.

In certain embodiments, the compound of the invention comprises a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the compound of the invention is single-stranded.

In certain embodiments, the nucleobase sequence of the compound is at least 70%, 80%, 90%, 95% or 100%) complementary to any one of SEQ ID NO: 1-3 as measured over the entirety of the compound.

In certain embodiments, the compound of the invention consists of 20 linked nucleosides.

In certain embodiments, at least one internucleoside linkage of said compound is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

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

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

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

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

In certain embodiments, the compound comprises an antisense oligonucleotide consisting of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5' wing segment consisting of five linked nucleosides, the 3' wing segment consisting of five linked nucleosides, the gap segment is positioned between the 5' wing segment and the 3' wing segment and each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar and each internucleoside linkage is a phosphorothioate linkage.

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

In certain embodiments, the antisense oligonucleotide of the invention consists of 20 linked nucleosides having a nucleobase sequence 100% complementary to an equal length portion of any of SEQ ID NO: 1-3, wherein the antisense oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5' wing segment consisting of five linked nucleosides; and c) a 3' wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the antisense oligonucleotide comprises at least 8 contiguous nucleobases of a nucleobase sequence selected from a sequence recited in any of SEQ ID NOs: 35-113. In certain embodiments, the antisense oligonucleotide consists of a nucleobase sequence selected from any of SEQ ID NOs: 43 or 63.

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

Certain embodiments provide a method of reducing LCS expression in an animal comprising administering to the animal a compound of the invention 10 to 30 linked nucleosides in length targeted to LCS.

Certain embodiments provide a method of preventing, decreasing or ameliorating airway inflammation and/or airway hyperresponsiveness in an animal comprising administering to the animal a compound 10 to 30 linked nucleosides in length targeted to LCS, thereby preventing, decreasing or ameliorating the airway inflammation and/or airway hyperresponsiveness in the animal. In certain embodiments, the compound is administered prophylactically.

Certain embodiments provide a method for treating an animal with airway inflammation and/or airway hyperresponsiveness comprising: a) identifying said animal prone to or with airway inflammation and/or airway hyperresponsiveness, and b) administering to said animal a therapeutically effective amount of a compound consisting of 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NO: 1-3 as measured over the entirety of said compound, thereby treating the animal with airway inflammation and/or airway hyperresponsiveness. In certain embodiments, the therapeutically effective amount of the compound administered to the animal reduces the airway inflammation and/or airway

hyperresponsiveness in the animal.

Certain embodiments of the invention provide a method for treating airway inflammation and/or airway hyperresponsiveness in an animal comprising administering to the animal a therapeutically effective amount of the compound of the invention. The compound consists of 20 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NO: 1-3, wherein the compound comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5' wing segment consisting of five linked nucleosides; and c) a 3' wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5- methylcytosine.

In certain embodiments the compound of the invention is an antisense oligonucleotide.

In certain embodiments the airway inflammation and/or airway hyperresponsiveness includes, but is not limited to, pulmonary disease, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, emphysema, chronic obstructive pulmonary disease (COPD) and asthma (e.g., allergic asthma, non-allergic asthma).

In certain embodiments, one or more symptoms or indications of airway inflammation or airway hyperresponsiveness, can be independently reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% by administering the compound of the invention to a subject. Administering the compound of the invention can result in decreased airway constriction and/or improved air flow through a subject's airways.

In certain embodiments, the animal is a human.

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

concomitantly.

In certain embodiments, the second agent is an anti-inflammation medication. Anti- inflammatory medications can include steroids, NSAIDS (non-steroidal anti-inflammatory drugs), COX inhibitors, antihistamines and the like. In certain embodiments, the second agent can be an asthma medication such as an anti-inflammatory drug, a bronchodilator (e.g., beta-2 agonists (LAB A2), theophylline, ipratropium), a leukotriene modifier, Cromolyn, nedocromil, a decongestant and immunotherapy.

In certain embodiments, the compound is aerosolized and administered by inhalation to the animal. In certain embodiments, the compound is administered intranasally, intrapulmonarily or intratracheally. Adminstration of the compound can be by any device including, but not limited to, a metered dose inhaler, nebulizer or colloidal dispersion system.

Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing one or more of airway inflammation or airway hyperresponsiveness or a symptom or indication thereof. In certain embodiments, airway inflammation or airway hyperresponsiveness can be pulmonary disease, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, emphysema, chronic obstructive pulmonary disease (COPD) and asthma.

Certain embodiments provide the use of a compound as described herein for treating, ameliorating, delaying or preventing one or more of airway inflammation or airway

hyperresponsiveness (AHR) or a symptom or indication thereof. In certain embodiments, airway inflammation or airway hyperresponsiveness can be pulmonary disease, chronic bronchitis, pulmonary fibrosis, pulmonary inflammation, emphysema, chronic obstructive pulmonary disease (COPD) and asthma.

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

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense

oligonucleotides, and siRNAs. An oligomeric compound can be "antisense" to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

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

oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain embodiments, an antisense compound targeted to LCS nucleic acid is 10 to 30 nucleotides in length. In other words, antisense compounds are from 10 to 30 linked

nucleobases. In other embodiments, the antisense compound comprises a modified

oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleobases. In certain such embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked nucleobases in length, or a range defined by any two of the above values.

In certain embodiments, the antisense compound comprises a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide can have a single nucleoside deleted from the 5' end (5' truncation), central portion or alternatively from the 3' end (3' truncation). A shortened or truncated oligonucleotide can have two or more nucleosides deleted from the 5' end, two or more nucleosides deleted from the central portion or alternatively can have two or more nucleosides deleted from the 3' end. In certain embodiments, the deleted nucleosides can be dispersed throughout the modified oligonucleotide, for example, in an antisense compound having one or more nucleoside deleted from the 5' end, one or more nucleoside deleted from the 3' end and/or one or more nucleoside deleted from the central portion of the antisense compound.

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

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

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

Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

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

Antisense Compound Motifs

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

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

Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer can in some embodiments include β-D-ribonucleosides, β-D- deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include 2'- MOE, and 2'-0-Ο¼, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides can include those having a 4'-(CH2)n-0-2' bridge, where n=l or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as "X-Y-Z", where "X" represents the length of the 5' wing region, "Y" represents the length of the gap region, and "Z" represents the length of the 3' wing region. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. Thus, no intervening nucleotides exist between the 5' wing segment and gap segment, or the gap segment and the 3' wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. Thus, gapmers include, but are not limited to, for example 5-10-5, 4- 8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, 5-8-5, 1- 8-1, 2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2 or 2-18-2.

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

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

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

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode LCS include, without limitation, the following: the murine sequence as set forth in GenBank Accession No. NM_019737.1 (incorporated herein as SEQ ID NO: 1) and GenBank Accession No. NT_039674.7 truncated from nucleotides 17841000 to 17907000 (incorporated herein as SEQ ID NO: 2) or the human sequence as set forth in GenBank Accession No. NM 004775.2 (incorporated herein as SEQ ID NO: 3). It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO can comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

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

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

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

A target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, they can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.

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

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

There can be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in LCS mRNA levels are indicative of inhibition of LCS protein expression.

Reductions in levels of LCS protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes, such as a reduction of the level of airway constriction or an increase in airway flow, can be indicative of inhibition of LCS mRNA and/or protein expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a LCS nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence- dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3^r Ed., 2001). In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a LCS nucleic acid.

Complementarity

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

An antisense compound can hybridize over one or more segments of a LCS nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

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

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases can be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) 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). In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound can be fully complementary to a LCS nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound can be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

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

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16,

17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a LCS nucleic acid, or specified portion thereof.

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

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

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

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

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the intemucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to

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

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

The naturally occurring intemucleoside linkage of RNA and DNA is a 3' to 5'

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

Oligonucleotides having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing linkages are well known. In certain embodiments, antisense compounds targeted to a LCS 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

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

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

As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4*-(CH₂)-0-2* (LNA); 4^,-(CH₂)-S-2'; 4^,-(CH₂)₂-0-2' (ENA); 4'-CH(CH₃)- 0-2' and 4'-CH(CH₂OCH₃)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH₃)(CH₃)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4·-ΟΗ2-Ν(ΟΟΗ₃)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH₂-0- N(CH₃)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH₂-N(R)-0-2', wherein R is H, Ci-C₁₂ alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH₂-C(H)(CH₃)-2' (see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134); and ^-CBb-C^CH^)-^ (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al, 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, 2007, 129(26) 8362-8379; Elayadi et al, Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7; and Orum et al, Curr. Opinion Mol Ther., 2001, 3, 239-243; U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281 ; U.S. Patent Serial Nos.

60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO

2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).

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

wherein: x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_a and ¾ is, independently, H, a protecting group, hydroxyl, Q\-C_\i alkyl, substituted C1-C12 alkyl, C₂-C₁₂ alkenyl, substituted C2-Q2 alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂o aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJi, NJ^, SJ_ls N₃, COOJ_l5 acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)₂-J , or sulfoxyl (S(=0 Ji); and

each Ji and J₂ is, independently, H, Ci-Cn alkyl, substituted Ci-Cu alkyl, C2-Q2 alkenyl, substituted C2-C₁₂ alkenyl, C2-Ci2 alkynyl, substituted C2-Q2 alkynyl, C5-C20 aryl, substituted C5-C2₀ aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C -Cn aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(R_a)(R_b)]_n-,

-[C(R_a)(R_b)]n-0-, -C(R_aR )-N(R)-0- or -C(R_aR_b)-0-N(R)-. In certain embodiments, the bridge is 4*-CH₂-2', 4'-(CH2)2-2', 4'-(CH₂)₃-2', 4'-CH₂-0-2', 4'-(CH₂)2-0-2^*, 4'-CH₂-0-N(R)-2' and 4*-CH₂- N(R)-0-2'- wherein each Ris, independently, H, a protecting group or Ci-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric

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

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L- methyleneoxy (4'-CH₂-0-2') BNA , (B) β-D-methyleneoxy (4'-CH₂-0-2') BNA , (C) ethyleneoxy (4'-(CH₂)₂-0-2') BNA , (D) aminooxy (4'-CH₂-0-N(R)-2') BNA, (E) oxyamino (4'-CH₂-N(R)-0-2') BNA, and (F) methyl(methyleneoxy) (4'-CH(CH₃)-0-2') BNA, (G) methylene-thio (4'-CH₂-S-2') BNA, (H) methylene-amino (4' -CH₂-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH₂-CH(CH₃)-2') BNA, and (J) propylene carbocyclic (4'-(CH₂)₃-2') BNA as depicted below.

(A) (B) (C)

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

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

wherein:

Bx is a heterocyclic base moiety;

-Q_a-Q_b-Qc- is -CH₂-N(Rc)-CH₂-,

-CH₂-0-N(Rc)-, -CH₂-N(Rc)-0- or

Rc is Ci-Cn alkyl or an amino protecting group; and

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

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

wherein:

Bx is a heterocyclic base moiety;

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

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

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

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

wherein:

Bx is a heterocyclic base moiety;

T_a and Tb 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;

Zb is C C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted Q-Ce alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(=0)-).

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

wherein:

Bx is a heterocyclic base moiety;

T_a and Tb 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;

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

each q_a, qb, q_c and qa is, independently, H, halogen, C C6 alkyl, substituted Q-C6 alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C6 alkynyl,

alkoxyl, substituted C C₆ alkoxyl, acyl, substituted acyl, Ci-C₆ aminoalkyl or substituted C_\-Ce aminoalkyl;

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

wherein:

Bx is a heterocyclic base moiety;

T_a and Tb 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;

qa, qb, qe and qf are each, independently, hydrogen, halogen, CrC₁₂ alkyl, substituted d- C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C1-C12 alkoxy, substituted C C₁₂ alkoxy, OJj, SJj, SOJj, S0₂J_j, NJjJ_k, N₃, CN, C(=0)OJ_j5 C(=0)NJjJ_k, C(=0)Jj, 0-C(=0)NJ_jJ_k, N(H)C(=NH)NJ_jJ_k, N(H)C(=0)NJ_jJ_k or (H)C(=S)NJ_jJ_k; or q_e and qf together are =C(qg)(qh); q_g and q_h are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl. The synthesis and preparation of the methyleneoxy (4'-CH₂-0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their

oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

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

l certain embodiments, bicyclic nucleosides are provided having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_a and Tb 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; each qj, oj, q_k and qi is, independently, H, halogen, Ci-Cn alkyl, substituted C1-C12 alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, CpC₁₂ alkoxyl, substituted Q-C^ alkoxyl, OJ_j, SJj, SOJ_j, S0₂J_j, NJ_jJ_k, N₃, CN, C(=0)OJ_j, C(=0)NJ_jJ_k, C(=0)J_j, 0-C(=0)NJ_jJ_k, N(H)C(=NH)NJ_jJ_k, N(H)C(=0)NJ_jJ_k orN(H)C(=S)NJ_jJ_k; and

qi and qj or ¾ and q_k together are -C(q_g)(q_h), wherein q_g and q_h are each, independently, H, halogen, C!-C₁₂ alkyl or substituted Ci-C₁₂ alkyl. One carbocyclic bicyclic nucleoside having a 4'-(CH2)₃-2' bridge and the alkenyl analog bridge 4'-CH=CH-CH2-2' have been described (Freier et ah, Nucleic Acids Research, 1997, 25 22), 4429-4443 and Albaek et ah, J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et ah, J. Am. Chem. Soc, 2007, 129(26), 8362- 8379).

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

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.

As used herein, "2 '-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to:

0[(CH₂)_nO]_mCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)„F, 0(CH₂)_nONH₂, OCH₂C(=0)N(H)CH₃, and 0(CH₂)_nON[(CH₂)_nCH₃]2, where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: Ci-C alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, F, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2'-MOE side chain (Baker et ah, 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 (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et ah, 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).

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

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

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_a and T_b is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_a and T_b is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;

qi, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C_\-Ce alkyl, substituted Ci-C₆ alkyl,

C₂-C6 alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of P and R₂ is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ_!J₂, SJj, N₃, OC(=X)J_l5 OC(=X)NJ_!J₂, NJ3C(=X)NJ_!J₂ and CN, wherein X is O, S or NJj and each Ji, J₂ and J₃ is, independently, H or C C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q_ls q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of qi, q₂, q₃, q4, qs, q6 and q₇ is other than H. In certain embodiments, at least one of q_ls q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R₂ is fluoro. In certain embodiments, is fluoro and R₂ is H; R_\ is methoxy and R₂ is H, and R_\ is H and R₂ is methoxyethoxy.

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-Q-Cio alkyl, -OCF₃, 0-(CH₂)₂-0-CH₃, 2'-0(CH₂)₂SCH₃, 0-(CH₂)₂-0-N(R_m)(R_n), or 0-CH₂-C(O)- N(R_m)(R_n), where each R_m and R_n is, independently, H or substituted or unsubstituted C_\-C_\o alkyl. 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

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

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

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

As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

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

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

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

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

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

Additional unmodified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil

(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.

In certain embodiments, antisense compounds targeted to a LCS nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense

oligonucleotides targeted to a LCS nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

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

Antisense compound targeted to a LCS nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier.

In certain embodiments, the "pharmaceutical carrier" or "excipient" is a

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

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

hydroxymethylcellulose, polyvinylpyrrolidone and the like.

A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). 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 LCS nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

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

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

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic 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 January 16, 2003.

Cell culture and antisense compounds treatment

The effects of antisense compounds on the level, activity or expression of LCS nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commercial vendors (e.g. American Type Culture Collection, Manassus, VA; Zen- Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g.

Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, 3T3, 3T3-L1, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In vitro testing of antisense oligonucleotides

Described herein are methods for treatment of cells with antisense compounds such as oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN^® (Invitrogen, Carlsbad, CA).

Antisense oligonucleotides are mixed with LIPOFECTIN^® in OPTI-MEM^® 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000^® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000^® in OPTI-MEM^® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE^® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

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

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Oligofectamine™ (Invitrogen Life Technologies, Carlsbad, CA). Antisense oligonucleotide is mixed with Oligofectamine™ in Opti-MEM™-l reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of oligonucleotide with an Oligofectamine™ to oligonucleotide ratio of approximately 0.2 to 0.8 μΐ, per 100 nM.

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

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

Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor laboratory Press, Cold Spring Harbor, New York. 2001). In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000^®, Lipofectin or Cytofectin. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

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

Analysis of inhibition of target levels or expression

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

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitative real-time PCR using the ABI PRISM^® 7600, 7700, or 7900 Sequence Detection System (PE-Applied

Biosystems, Foster City, CA) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). RT and real-time-PCR reactions are carried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR can be normalized using either the expression level of a gene whose expression is constant, such as GAPDH or cyclophilin A, or by quantifying total RNA using RIBOGREEN^® (Invitrogen, Inc. Carlsbad, CA).

Cyclophilin A or GAPDH expression is quantified by real time PCR, by being run

simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN^® RNA quantification reagent. Methods of RNA quantification by RIBOGREEN^® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR^® 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN^® fluorescence.

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

Presented in Table 1 are primers and probes used to measure GAPDH or Cyclophilin A expression in the cell types described herein. The PCR probes have JOE or FAM covalently linked to the 5' end and TAMRA or MGB covalently linked to the 3' end, where JOE or FAM is the fluorescent reporter dye and TAMRA or MGB is the quencher dye. In some cell types, primers and probe designed to a sequence from a different species are used to measure expression. For example, a human GAPDH primer and probe set can be used to measure GAPDH expression in monkey-derived cells and cell lines.

Table 1

GAPDH and Cyclophilin A primers and probes for use in real-time PCR

Analysis of Protein Levels

Antisense inhibition of LCS nucleic acids can be assessed by measuring LCS protein levels. Protein levels of LCS can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. In vivo testing of antisense compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of LCS and produce phenotypic changes. Testing can be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides can be formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline or other aqueous solution.

Administration routes of the antisense compound such as an antisense oligonucleotide to a subject can include parenteral routes of administration such as inhaled, intranasal, intrapulmonary or intratracheal. Oligonucleotides can be delivered using devices such as nebulizers, dry powder inhalers, metered dose inhalers or colloidal dispersion systems. Calculation of antisense oligonucleotide dosage and dosing frequency depends upon factors such as route of administration and animal body weight.

Following a period of treatment with antisense oligonucleotides, RNA is isolated from tissue and changes in LCS nucleic acid expression are measured. Changes in LCS protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has airway inflammation and/or airway hyperresponsiveness.

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

In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to a LCS nucleic acid is accompanied by monitoring of LCS levels or markers of airway inflammation, airway hyperresponsiveness or other disease process associated with the expression of LCS, to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targeted to a LCS nucleic acid results in reduction of LCS expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to LCS are used for the preparation of a medicament for treating a patient suffering or susceptible to airway inflammation and/or airway hyperresponsiveness.

In certain embodiments, the methods described herein include administering an antisense compound comprising a modified oligonucleotide having an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobase portion.

Administration

In certain embodiments, the antisense compounds and compositions as described herein are administered parenterally.

In certain embodiments, parenteral administration is inhalation, intranasal, intrapulmonary or intratracheal. Administration can be chronic or continuous or short or intermittent. In certain embodiments, the antisense compound is aerosolized for administration to a subject. In certain embodiments, pharmaceutical agents are delivered with a device such as a nebulizer, an inhaler (e.g., dry powder or metered dose) or colloidal dispersion system.

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

Dosing

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

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

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Dosing is also dependent on drug potency and metabolism. In certain embodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, or within a range of 0.00 lmg to 600mg, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.0 ^g to lOOmg per kg of body weight or from 0.00 lmg to 600mg, once or more daily, to once every 20 years.

Certain Combination Therapies

In certain embodiments, a first agent comprising the antisense compound of the invention is co-administered with one or more secondary agents. In certain embodiments, such second agents are designed to treat the same airway inflammation and/or airway hyperresponsiveness as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder, or condition as the first agent described herein. In certain

embodiments, such second agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, second agents are co- administered with the first agent to treat an undesired effect of the first or second agent. In certain embodiments, second agents are co-administered with the first agent to produce a combinational effect. In certain embodiments, second agents are co-administered with the first agent to produce a synergistic effect. In certain embodiments, the co-administration of the first and second agents permits use of lower dosages than would be required to achieve a therapeutic or prophylactic effect if the agents were administered as independent therapy.

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

In certain embodiments, second agents include, but are not limited to, anti-inflammation drugs. Anti-inflammatory drugs can include steroids, NSAIDS (non-steroidal anti-inflammatory drugs), COX inhibitors, antihistamines and the like. In certain embodiments, the second agent can be an asthma drug such as an anti-inflammatory drug, a bronchodilator (e.g., beta-2 agonists (LABA2), theophylline, ipratropium), a leukotriene modifier, Cromolyn, nedocromil, a decongestant and immunotherapy.

EXAMPLES

Non-limiting disclosure and incorporation by reference

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

Example 1: Antisense inhibition of murine lactosylceramide synthase (LCS) in b.END cells

Antisense oligonucleotides targeted to a murine LCS nucleic acid were tested for their effect on LCS RNA transcript in vitro. Cultured b.END cells at a density of 4,000 cells per well were transfected using Cytofectin™ reagent (Genlantis, San Diego, CA) with 90 nM antisense oligonucleotide. After approximately 24 hours, RNA was isolated from the cells and LCS RNA transcript levels were measured by quantitative real-time PCR with murine primer probe set

RTS3038 (forward sequence ACAGCGGCTGGAATTTGC, designated herein as SEQ ID NO: 32; reverse sequence GCATCGCACGATTGAATGG, designated herein as SEQ ID NO: 33; probe sequence TTTTATGTCATCGAACAGACCGGCACACX, where X is the fluorophore, designated herein as SEQ ID NO: 34). LCS RNA transcript levels were adjusted according to total RNA content, as measured by RJBOGREEN^®. Results are presented as percent inhibition of LCS relative to untreated control cells.

The antisense oligonucleotides in Tables 2 and 3 are 5-10-5 gapmers, where the gap segment comprises ten 2'-deoxynucleosides and each wing segment comprises five 2'-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. 'Target start site' indicates the 5 '-most nucleotide to which the antisense oligonucleotide is targeted. 'Target stop site' indicates the 3 '-most nucleotide to which the antisense oligonucleotide is targeted. All the antisense oligonucleotides listed in Table 2 target SEQ ID NO: 1 (GENBANK Accession No. NM_019737.1). All the antisense oligonucleotides listed in Table 3 target SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_039674.7 truncated from nucleotides 17841000 to 17907000).

The murine oligonucleotides of Tables 2 and 3 may also be cross-reactive with human gene sequences. The greater the complementarity between the murine oligonucleotide and the human sequence, the more likely the murine oligonucleotide can cross-react with the human sequence. The murine oligonucleotides in Tables 2 and 3 were compared to SEQ ID NO: 3 (GENBANK Accession No. NM_004775.2). "Human Target start site" indicates the 5'-most nucleotide to which the gapmer is targeted in the human gene sequence. 'Mismatches' indicate the number of nucleobases by which the murine oligonucleotide is mismatched with a human gene sequence with "n/a" indicating more than 3 mismatches.

Table 2

Inhibition of murine LCS RNA transcript in b.END cells by 5-10-5 gapmers targeting SEQ ID

NO: 1

1266 1285 421854 CCCCGCATGTAAGGCAGCTT 40 49 570 2

1280 1299 421855 TGACACTGAGGAATCCCCGC 57 50 584 2

1288 1307 421856 ACTCACATTGACACTGAGGA 69 51 592 3

1325 1344 421857 TGGAGAAGAGCTGATGGACT 40 52 629 3

1386 1405 421858 TTCCATCTGGGCTTGCAGTC 36 53 690 2

1465 1484 421859 CTGGAGCATCGGAATCAGGT 77 54 769 2

1564 1583 421860 CATGGCCTCTTTGAAACCCA 64 55 868 1

1591 1610 421861 GATCACACAGTCCCAGGCTC 62 56 895 3

1618 1637 421862 TTCAGGCAGATGATCCACAT 34 57 922 1

1628 1647 421863 TCCGATCATTTTCAGGCAGA 51 58 932 2

1638 1657 421864 CCGTAGTAGTTCCGATCATT 42 59 942 2

1673 1692 421865 GCTTCGCTGCAAAGTGCCGC 58 60 n/a n/a

1851 1870 421866 TCTGGCCGGGTTACATTGTA 54 61 1155 3

1962 1981 421867 CCATCGATGTACTGCCGCTC 68 62 1266 1

2037 2056 421868 GGCATGAGGTTTACAGATAT 59 63 1341 0

2048 2067 421869 GAGCTAATTCTGGCATGAGG 71 64 1352 1

2091 2110 421870 CTACCCCACCATGAGAATGA 16 65 n/a n/a

2118 2137 421871 GCCAAGTTCATGATCCAGCA 77 66 1422 2

2142 2161 421872 ACTTCCACGGTGAGGGCACT 59 67 n a n/a

2172 2191 421873 CCGGGACACTGAGATACACC 61 68 n/a n/a

2182 2201 421874 TTTTTACAGACCGGGACACT 42 69 n/a n/a

2231 2250 421875 GAGTGTGACGGATGGAGCCC 43 70 n/a n/a

2306 2325 421876 TCCAGTCCCAAGCAATGGGT 68 71 n/a n/a

2558 2577 421877 AGGCTTAACCTTTGTTTGGC 64 72 1865 3

2574 2593 421878 CCCTTCATTTGACACAAGGC 64 73 1881 3

2586 2605 421879 GTCGCTGACAGTCCCTTCAT 63 74 n/a n/a

2652 2671 421880 CCATGAGGCTGAAGGTCTAA 48 75 n/a n/a

2706 2725 421881 AAAGTTATGCGAGTCCTTCC 46 76 n/a n a

2772 2791 421882 GATCTGATGAGACTGCAAGC 52 77 2070 3

2777 2796 421883 GGCAAGATCTGATGAGACTG 74 78 n/a n/a

3445 3464 421884 AAAACCTCACCCGTGTGTGG 43 79 n/a n/a

3453 3472 421885 GGTCATTGAAAACCTCACCC 58 80 n/a n/a

3599 3618 421886 TAGGGTTTACTTGACAGACG 61 81 n/a n/a

3633 3652 421887 GAGCAAACTTTCTCACATCC 65 82 n/a n/a

3708 3727 421888 TTGTGGTCATCAGCATTACC 52 83 n/a n/a

3720 3739 421889 GTAGGGTTTGACTTGTGGTC 57 84 n a n/a

4029 4048 421890 TGGCACAGCTTCCCACAGCA 63 85 n/a n/a

4038 4057 421891 AATTCCACATGGCACAGCTT 50 86 n/a n/a

4072 4091 421892 CCCGCCACATTATGAAGTGT 50 87 n/a n/a

4148 4167 421893 AGGCAGATGACCAGTTCTGA 69 88 n/a n/a

4236 4255 421894 CATTCCAGATGCAACACGAA 29 89 n/a n/a

4243 4262 421895 AAGCTGACATTCCAGATGCA 61 90 n/a n/a 4306 4325 421896 ACCCAGGTCACTATTCATTA 73 91 n/a n/a

4315 4334 421897 TGGAAACATACCCAGGTCAC 66 92 n/a n/a

944 963 421898 CCCGCATCATCCGCTTGAGC 69 93 248 1

949 968 421899 GGAGACCCGCATCATCCGCT 76 94 253 2

2489 2508 421900 GTCCAATGAAGGCATAACCA 58 95 1795 3

2572 2591 421901 CTTCATTTCACACAAGGCTT 8 96 1879 1

2582 2601 421902 CTGACAGTCCCTTCATTTCA 34 97 1889 2

2919 2938 421903 TTGAGGCACAAGATCCCTGA 49 98 2222 1

Table 3

Inhibition of murine LCS RNA transcript in b.END cells by 5-10-5 gapmers targeting SEQ ID

NO: 2

Target Target SEQ Human

ISIS % MisStart Stop Sequence ID Target

No inhibition matches Site Site NO Start Site

2110 2129 421840 AAGAGGAACCAAGAGCCCTA 0 35 n/a n/a

2143 2162 421842 TCCACAACTCGCCAGGCGGC 24 37 n/a n/a

2181 2200 421843 TGTGCGCCCCGAGACCCCTG 41 38 n/a n/a

2210 2229 421844 GACACCCACGAGGACAATCC 33 39 n/a n/a

2666 2685 421845 TCCAAGAGGCGAGTCCCCGC 58 40 n/a n/a

2757 2776 421846 CCAAAGCCACGGGCCAGGCT 56 41 n/a n/a

2763 2782 421847 TGGAGGCCAAAGCCACGGGC 66 42 n/a n/a

2955 2974 421848 TCCAGGCCCTAAACTTCCAT 38 43 162 0

3008 3027 421849 TCTGCCCTGCCAGTAGCGCG 60 44 n/a n/a

3034 3053 421850 CATCCGCTTGAGCGCAGACA 52 45 241 2

3041 3060 421898 CCCGCATCATCCGCTTGAGC 69 93 248 1

3046 3065 421899 GGAGACCCGCATCATCCGCT 76 94 253 2

3140 3159 421910 CCGGGTACTTACCGATGCCT 25 99 n/a n/a

20486 20505 421852 ACAGCCTGATCATGTGTCCA 69 47 419 3

20491 20510 421853 GGTATACAGCCTGATCATGT 79 48 424 3

23365 23384 421911 CCCAGGCCCCTCATGTGGGC 15 100 n/a n/a

28464 28483 421912 GGTCACACTTACGCATGTAA 19 101 n/a n/a

33762 33781 421913 CCCTGGCAGCCCCATGGTGG 50 102 n/a n/a

41977 41996 421856 ACTCACATTGACACTGAGGA 69 51 592 3

42014 42033 421857 TGGAGAAGAGCTGATGGACT 40 52 629 3

42075 42094 421858 TTCCATCTGGGCTTGCAGTC 36 53 690 2

47633 47652 421914 ACTGCCACCTTCAAAACAGA 27 103 n/a n/a

47703 47722 421859 CTGGAGCATCGGAATCAGGT 77 54 769 2

55961 55980 421860 CATGGCCTCTTTGAAACCCA 64 55 868 1

55988 56007 421861 GATCACACAGTCCCAGGCTC 62 56 895 3

56015 56034 421862 TTCAGGCAGATGATCCACAT 34 57 922 1

56025 56044 421863 TCCGATCATTTTCAGGCAGA 51 58 932 2 56035 56054 421864 CCGTAGTAGTTCCGATCATT 42 59 942 2

56070 56089 421865 GCTTCGCTGCAAAGTGCCGC 58 60 n/a n/a

56101 56120 421915 CCAGCTACTCACATGTACAT 16 104 n/a n/a

57530 57549 421916 GTGCCGCCACTGCCAGGCCA 32 105 n/a n/a

59480 59499 421866 TCTGGCCGGGTTACATTGTA 54 61 1155 3

59556 59575 421917 TCACACTCACCGTCCTAGGA 22 106 n/a n/a

60057 60076 421867 CCATCGATGTACTGCCGCTC 68 62 1266 1

60132 60151 421868 GGCATGAGGTTTACAGATAT 59 63 1341 0

60143 60162 421869 GAGCTAATTCTGGCATGAGG 71 64 1352 1

60186 60205 421870 CTACCCCACCATGAGAATGA 16 65 n/a n/a

60213 60232 421871 GCCAAGTTCATGATCCAGCA 77 66 1422 2

60237 60256 421872 ACTTCCACGGTGAGGGCACT 59 67 n/a n/a

60267 60286 421873 CCGGGACACTGAGATACACC 61 68 n/a n/a

60277 60296 421874 TTTTTACAGACCGGGACACT 42 69 n/a n/a

60326 60345 421875 GAGTGTGACGGATGGAGCCC 43 70 n a n/a

60402 60421 421876 TCCAGTCCCAAGCAATGGGT 68 71 n/a n/a

60585 60604 421900 GTCCAATGAAGGCATAACCA 58 95 1795 3

60654 60673 421877 AGGCTTAACCTTTGTTTGGC 64 72 1865 3

60670 60689 421878 CCCTTCATTTGACACAAGGC 64 73 1881 3

60682 60701 421879 GTCGCTGACAGTCCCTTCAT 63 74 n/a n/a

60748 60767 421880 CCATGAGGCTGAAGGTCTAA 48 107 n/a n/a

60802 60821 421881 AAAGTTATGCGAGTCCTTCC 46 76 n/a n/a

60868 60887 421882 GATCTGATGAGACTGCAAGC 52 77 2070 3

60873 60892 421883 GGCAAGATCTGATGAGACTG 74 78 n/a n/a

61015 61034 421903 TTGAGGCACAAGATCCCTGA 49 98 2222 1

61541 61560 421884 AAAACCTCACCCGTGTGTGG 43 79 n/a n/a

61549 61568 421885 GGTCATTGAAAACCTCACCC 58 80 n/a n/a

61695 61714 421886 TAGGGTTTACTTGACAGACG 61 81 n/a n/a

61729 61748 421887 GAGCAAACTTTCTCACATCC 65 82 n/a n/a

61804 61823 421888 TTGTGGTCATCAGCATTACC 52 83 n/a n/a

61816 61835 421889 GTAGGGTTTGACTTGTGGTC 57 84 n/a n/a

62126 62145 421890 TGGCACAGCTTCCCACAGCA 63 85 n/a n/a

62135 62154 421891 AATTCCACATGGCACAGCTT 50 86 n/a n/a

62169 62188 421892 CCCGCCACATTATGAAGTGT 50 87 n/a n/a

62245 62264 421893 AGGCAGATGACCAGTTCTGA 69 88 n/a n/a

62333 62352 421894 CATTCCAGATGCAACACGAA 29 89 n/a n/a

62340 62359 421895 AAGCTGACATTCCAGATGCA 61 90 n/a n/a

62403 62422 421896 ACCCAGGTCACTATTCATTA 73 91 n/a n/a

62412 62431 421897 TGGAAACATACCCAGGTCAC 66 92 n/a n/a

62535 62554 421904 GACAGACACCCAGTGCAAAG 49 108 n/a n/a

62705 62724 421905 GGACCACACTAGATTTGAGA 45 109 n/a n/a

62735 62754 421906 CCCAATGTCACAAGGACCTC 42 110 n/a n/a 62883 62902 421907 GGAAGCCACCTCATTAAAAC 20 111 n/a n/a

62950 62969 421908 AGGCACCTTTACACCCTGAG 67 112 n/a n/a

62986 63005 421909 TCCCAGGGATCAAATGCAGG 74 113 n/a n/a

Example 2: Dose-dependent antisense inhibition of murine LCS in b.END cells

Several of the antisense oligonucleotides exhibiting in vitro inhibition of LCS in b.END cells (see Example 1) were tested at various doses. Cells were plated at a density of 4,000 cells per well and transfected using Cytofectin™ reagent with 10 nM, 20 nM, 40 nM, 80 nM, and 160 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and LCS transcript levels were measured by quantitative real-time PCR using primer probe set RTS3038. LCS transcript levels were normalized to total RNA content, as measured by RIBOGREEN^®. Results are presented in Table 4 as percent inhibition of LCS, relative to untreated control cells.

Table 4

Dose-dependent antisense inhibition of murine LCS in b.END cells

Example 3: In vivo antisense inhibition of murine LCS in C57BL/6 mice

Selected antisense oligonucleotides from Example 2 were evaluated for their potency and tolerability in vivo.

Treatment C57/BL6 mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow (Harlan Laboratories, Indianapolis, IN). Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

The mice were divided into seven treatment groups of 4 mice each. Six groups received subcutaneous injections of ISIS 421853, ISIS 421856, ISIS 421859, ISIS 421869, ISIS 421871, or ISIS 421893 at a dose of 25 mg/kg twice a week for 6 weeks. One group of mice received subcutaneous injections of PBS twice a week for 6 weeks. This PBS group served as the control group. Blood was withdrawn from each mouse at weeks 3 and 6 and plasma samples analyzed. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.

Inhibition of LCS RNA

RNA was isolated from liver, small intestine, heart, and white adipose tissue (WAT) for real-time PCR analysis of LCS and normalized to Cyclophilin™. As presented in Table 5, treatment with antisense oligonucleotides reduced murine LCS RNA transcript expression. The results are expressed as percent inhibition of LCS transcript, relative to the PBS control.

Table 5

Percent inhibition of murine LCS RNA transcript in C57BL/6 mice

Liver function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz NW (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, PA, 1995). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) at week 3 and week 6 were measured and the results are presented in Tables 6 and 7 expressed in IU/L. Plasma levels of bilirubin (mg dL) at week 6 were also measured using the same clinical chemistry analyzer and the results are also presented in Table 7. Most of the ISIS

oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

Table 6

Effect of antisense oligonucleotide treatment on liver transaminases of C57BL/6 mice at week 3

Table 7

Effect of antisense oligonucleotide treatment on liver transaminases and bilirubin of C57BL/6 mice at week 6

Kidney function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY) (Hosten, A.O. 'BUN and Creatinine' book chapter in Clinical Methods, Butterworth Publishers, 1990). Results are presented in Table 8, expressed in mg/dL. Table 8

Effect of antisense oligonucleotide treatment on BUN (mg/dL) in the kidney of C57BL/6

Body and organ weights

The body weights of the mice were measured pre-dose and at the end of the treatment period. The body weights are presented in Table 9, and are expressed as percent increase over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 9 as a percentage change over the respective organ weights of the PBS control.

Table 9

Change in body and organ weights of C57BL/6 mice after antisense oligonucleotide treatment

(%)

Example 4: Effect of antisense inhibition of murine LCS in a prophylactic mouse model for asthma

The effect of antisense inhibition of LCS was evaluated in an ovalbumin (OVA)-induced murine model of airway hyper-responsiveness. ISIS 421869 and ISIS 421871, which displayed significant antisense inhibition in vivo (Example 3) was used to inhibit LCS expression in this assay. The effect of antisense inhibition of LCS in preventing airway hyper-responsiveness was measured by various standard assays.

Treatment

BALB/c mice (Jackson Laboratories, ME) were used in a prophylactic model for airway hyper-responsiveness. The mice were 6-8 week old at the start of the studies.

In this model, mice were pre-sensitized by intraperitoneal injections of OV A/alum or PBS/alum on days 1 and 14 (sensitization). Mice of the treatment groups were then treated between days 28-44 by intranasal instillation of 200 μg/kg of control oligonucleotide ISIS 141923 or ISIS 421869 or ISIS 421871.

All mice groups were then challenged with OVA in PBS intranasally between days 41-44. A group of mice was treated intraperitoneally with Dexamethasone (Dex) at 2.5 mg/kg between days 34-44, which prevents mast cell degranulation and therefore served as a positive control. The treatment of the various mice groups is displayed in Table 10.

Table 10

Treatment of mice groups in a prophylactic model of airway hyper-responsiveness

Effect on methacholine-induced airway hyper-responsiveness

The predisposition of these mice to asthma was measured with a bronchial challenge test. In this test, the mice were exposed to nebulized metacholine, a drug which provokes

bronchoconstriction or narrowing of the airways. Mice sensitized to OVA, and therefore predisposed to an asthmatic reaction on exposure to OVA, would react to lower doses of metacholine. The mice were tested for bronchoconstriction in a plethysmograph.

After the treatment period and one day prior to euthanasia, mice were exposed to increasing concentrations of metacholine (Sigma-Aldrich, St Louis, MO) aerosolized in PBS. Plethysmography of these mice was then performed using a plethysmography according to the manufacturer's instructions (Buxco Research Systems, Wilmington, NC) to measure airway resistance with and without metacholine treatment. Briefly, each mouse was placed without any restraint in a plethysmograph chamber, which was equipped with a port for aerosol delivery, a water bottle port and a gas sampling port. The system involves measuring a "box flow" which is the sum of nasal and thoracic flows. Actual flows are calculated in the software, taking into account temperature, humidity, and pressure. These systems are often specified for asthma studies using the enhanced pause (Penh) index of airway hyper-reactivity as an indicator of changes in airway resistance (Buxco Research Systems reference, 2005, entitled "Respiration Measurement in the Whole Body Plethysmograph"). Enhanced Pause (Penh) parameter was measured and is shown in Table 11. The data indicates that treatment with ISIS 421869 of mice sensitized to OVA (Group 6) and treatment with ISIS 421871 of mice sensitized to OVA (Group 7) reduced the enhanced pause and therefore reduced airway resistance, comparable to mice sensitized to OVA and treated with Dex (Group 4) compared to the control group (Group 5).

Table 11

Effect of antisense inhibition on enhanced pause in a prophylactic model of airway hyper- responsiveness

Example 5: Tissue distribution of LCS

The tissue distribution of LCS was determined by RT-PCR analysis of the various tissues extracted from C57/BL6 mice which had received no treatment. LCS mRNA levels were measured by the primer probe set RTS3038. The RNA expression levels are expressed as the ratio of 1 / 2^AX (where X is the number of PCR cycles required to obtain 50% expression of LCS mRNA) and the total RNA of the tissue, as measured by RIBOGREEN. The results are presented in Table 12. Table 12

Tissue distribution of LCS mRNA

Claims

What is claimed is:

1. A method of reducing lactosylceramide synthase (LCS) expression in an animal comprising administering to the animal a oligonucleotide comprising an antisense compound 10 to 30 linked nucleosides in length targeted to LCS, wherein expression of LCS is reduced in the animal.

2. A method for preventing or decreasing airway inflammation or airway hyperresponsiveness in an animal, comprising prophylactically administering to the animal an effective amount of a compound comprising an antisense oligonucleotide 10 to 30 linked nucleobases in length targeted to lactosylceramide synthase (LCS), wherein airway inflammation or airway hyperresponsiveness is prevented or decreased in the animal.

3. The method of any of claims 1-2, wherein the antisense oligonucleotide has a nucleobase sequence at least 90% complementary to SEQ ID NO: 3 as measured over the entirety of said antisense compound.

4. The method of any of claims 1-2, wherein the antisense oligonucleotide has a nucleobase sequence at least 95% complementary to SEQ ID NO: 3 as measured over the entirety of said antisense compound.

5. The method of any of claims 1-2, wherein the antisense oligonucleotide has a nucleobase sequence at least 100% complementary to SEQ ID NO: 3 as measured over the entirety of said antisense compound.

6. The method of any of claims 1-2, wherein the antisense oligonucleotide consists of a single-stranded oligonucleotide.

7. The method of any of claims 1-2, wherein at least one internucleoside linkage of said antisense oligonucleotide is a modified internucleoside linkage.

8. The method of any of claims 1-2, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

9. The method of any of claims 1-2, wherein at least one nucleoside of said antisense oligonucleotide comprises a modified sugar.

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

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

wherein Bx is an optionally protected heterocyclic base moiety.

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

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

14. The method of any of claims 1-2, wherein at least one nucleoside of said antisense oligonucleotide comprises a modified nucleobase.

15. The method of claim 14, wherein the modified nucleobase is a 5-methylcytosine.

16. The method of any of claims 1-2, wherein the antisense oligonucleotide consists of 20 linked nucleosides.

17. The method of any of claims 1-2, wherein the antisense oligonucleotide comprises: a. a gap segment consisting of linked deoxynucleosides; b. a 5' wing segment consisting of linked nucleosides;

c. a 3' wing segment consisting of linked nucleosides;

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

18. The method of any of claims 1 -2, wherein the antisense oligonucleotide consists of 20 linked nucleosides, has a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from any one of SEQ ID NOs: 35-113 and comprises:

a. a gap segment consisting of ten linked deoxynucleosides;

b. a 5' wing segment consisting of five linked nucleosides;

c. a 3' wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5'- methylcytosine.

19. The method of any one of claims 1-2, wherein the antisense oligonucleotide is a first agent and further comprising administering a second agent.

20. The method of claim 19, wherein the second agent is an asthma medication.

21. The method of any of claims 1-2, wherein said antisense oligonucleotide is aerosolized and inhaled by said animal.

22. The method of any of claims 1-2, wherein said antisense oligonucleotide is administered intranasally, intrapulmonarily or intratracheally.

23. The method of any of claims 1-2, wherein the administration is by a metered dose inhaler, nebulizer or colloidal dispersion system.

24. The method of claim 2, wherein said airway inflammation or airway hyperresponsiveness is associated with asthma.

25. The method of claim 24, the asthma is allergic asthma.

26. The method of claim 2, wherein the airway inflammation or airway hyperresponsiveness is lung inflammation.

27. The method of claim 2, wherein said airway inflammation or airway hyperresponsiveness is associated with chronic obstructive pulmonary disorder (COPD).

28. The method of any of claims 1-2, wherein said animal is a human.

29. The method of any of claims 1-2, wherein the antisense oligonucleotide is administered with a pharmaceutically acceptable carrier or diluent.

30. A method for treating airway inflammation or airway hyperresponsiveness in an animal comprising

a. identifying said animal prone to airway inflammation or airway hyperresponsiveness, b. administering to said animal a therapeutically effective amount of an antisense

oligonucleotide consisting of 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to SEQ ID NO: 3 as measured over the entirety of said antisense oligonucleotide,

wherein airway inflammation or airway hyperresponsiveness is treated in the animal.

31. A method for treating airway inflammation or airway hyperresponsiveness in an animal comprising administering to said animal a therapeutically effective amount of an antisense oligonucleotide consisting of 20 linked nucleosides, and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from any one of SEQ ID NOs: 35-113 and comprising:

a. a gap segment consisting of ten linked deoxynucleosides;

b. a 5' wing segment consisting of five linked nucleosides;

c. a 3' wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, wherein each cytosine is a 5'- methylcytosine, and wherein administration of the antisense oligonucleotide treats airway inflammation or airway hyperresponsiveness in the animal.

32. A compound comprising an antisense oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence complementary to an equal length portion of SEQ ID NO: 3.

33. The compound of claim 32, wherein the nucleobase sequence is at least 95%

complementary to SEQ ID NO: 3.

34. The compound of claim 32, wherein the nucleobase sequence is 100% complementary to SEQ ID NO: 3.

35. The compound of claim 32, wherein the compound is single-stranded.

36. The compound of claim 32, wherein at least one internucleoside linkage is a modified internucleoside linkage.

37. The compound of claim 32, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

38. The compound of claim 32, wherein at least one nucleoside comprises a modified sugar.

39. The compound of claim 38, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces a furanose ring.

40. The compound of claim 39, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety.

41. The compound of claim 38, wherein at least one modified sugar is a bicyclic sugar.

42. The compound of claim 38, wherein at least one modified sugar comprises a 2'-0- methoxyethyl or a 4'- (CH₂)_n-0-2' bridge, wherein n is 1 or 2.

43. The compound of claim 32, wherein at least one nucleoside comprises a modified nucleobase.

44. The compound of claim 43, wherein the modified nucleobase is a 5-methylcytosine.

45. The compound of claim 32, wherein the antisense oligonucleotide comprises:

a gap segment consisting of linked deoxynucleosides;

a 5' wing segment consisting of linked nucleosides;

a 3 ' wing segment consisting of linked nucleosides;

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

46. The compound of claim 32, wherein the antisense oligonucleotide consists of 20 linked nucleosides and comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of five linked nucleosides;

a 3' wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein each intemucleoside linkage is a phosphorothioate linkage.

47. The compound of claim 32, wherein the antisense oligonucleotide consists of 20 linked nucleosides.

48. The compound of any of claims 32-47, wherein the nucleobase sequence comprises at least 8 contiguous nucleobases of any one of SEQ ID NOs: 35-113.

49. The compound of any of claims 32-47, wherein the nucelobase sequence consists of any one of the nucleobase sequences recited in SEQ ID NOs: 35-113.

50. A pharmaceutical composition comprising the compound of any of claims 32-49, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

51. An antisense oligonucleotide consisting of 20 linked nucleosides and having a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 3, wherein the antisense compound comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of five linked nucleosides;

a 3' wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein each intemucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.

52. The antisense oligonucleotide of claim 51 comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from a sequence recited in any one of SEQ ID NOs: 35-113.

53. The antisense oligonucleotide of claim 52 consisting of a nucleobase sequence selected from any one of SEQ ID NOs: 35-113.