Abstract:
Disclosed herein are antisense compounds and methods for decreasing CD40. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to CD40 include hyperproliferative disorders, graft versus host disease (GVHD), graft rejection, asthma, airway hyperresponsiveness, chronic obstructive pulmonary disease (COPD), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis.

Description:
RELATED APPLICATIONS 
       [0001]    This application is continuation of U.S. patent application Ser. No. 12/743,797, filed Feb. 15, 2011, which is a U.S. National Phase filing under 35 U.S.C. §371 claiming priority to International Serial No. PCT/US2008/012998 filed Nov. 20, 2008, which claims priority to U.S. Provisional Application 60/989,421, filed Nov. 20, 2007, each of which is incorporated herein by reference in its entirety. 
     
    
     SEQUENCE LISTING 
       [0002]    The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0096USC1SEQ_ST25.txt, created Nov. 12, 2014, which is 72 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 
       [0003]    The present invention provides methods and compositions for lowing levels of CD40 in an animal. Such methods and compositions are useful as anti-inflammatory compounds and anti-tumor compounds. 
       BACKGROUND OF THE INVENTION 
       [0004]    The immune system serves a vital role in protecting the body against infectious agents. It is well established, however, that a number of disease states and/or disorders are a result of either abnormal or undesirable activation of immune responses. Common examples include graft versus host disease (GVHD) and graft rejection, and autoimmune linked diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis. 
         [0005]    In general, an immune response is activated as a result of either tissue injury or infection. Both cases involve the recruitment and activation of a number of immune system effector cells (e.g., B- and T-lymphocytes, macrophages, eosinophils, neutrophils) in a process coordinated through a series of complex cell-cell interactions. A typical scenario by which an immune response is mounted against a foreign protein is as follows: foreign proteins captured by antigen presenting cells (APC&#39;s) such as macrophages or dendritic cells are processed and displayed on the cell surface of the APC. Circulating T-helper cells which express an immunoglobulin that recognizes (i.e. binds) the displayed antigen undergo activation by the APC. These activated T-helpers in turn activate appropriate B-cell clones to proliferate and differentiate into plasma cells that produce and secrete humoral antibodies targeted against the foreign antigen. The secreted humoral antibodies are free to circulate and bind to any cells expressing the foreign protein on their cell surface, in effect marking the cell for destruction by other immune effector cells. In each of the stages described above, direct cell-cell contact between the involved cell types is required in order for activation to occur. (Gruss et al.,  Leuk. Lymphoma  1989, 24:393). In recent years, a number of cell surface receptors that mediate these cell-cell contact dependent activation events have been identified. Among these cell surface receptors is CD40 and its physiological ligand, CD40 Ligand (CD40L) which is also known as CD154. 
         [0006]    CD40 was first characterized as a receptor expressed on B-lymphocytes. It was later found that engagement of B-cell CD40 with CD40L expressed on activated T-cells is essential for T-cell dependent B-cell activation (i.e. proliferation, immunoglobulin secretion, and class switching). It was subsequently revealed that functional CD40 is expressed on a variety of cell types other than B-cells, including macrophages, dendritic cells, thymic epithelial cells, Langerhans cells, and endothelial cells. These studies have led to the current belief that CD40 plays a broad role in immune regulation by mediating interactions of T-cells with B-cells as well as other cell types. In support of this notion, it has been shown that stimulation of CD40 in macrophages and dendritic results is required for T-cell activation during antigen presentation. (Gruss et al.,  Leuk. Lymphoma,  1997, 24:393). Recent evidence points to a role for CD40 in tissue inflammation as well. Production of the inflammatory mediators IL-12 and nitric oxide by macrophages have been shown to be CD40 dependent. (Buhlmann and Noelle, J. Clin. Immunol., 1996, 16:83). In endothelial cells, stimulation of CD40 by CD40L has been found to induce surface expression of E-selectin, ICAM-1, and VCAM-1, promoting adhesion of leukocytes to sites of inflammation (Buhlmann and Noelle,  J. Clin. Immunol.,  1996, 16:83); Gruss et al.,  Leuk. Lymphoma,  1997, 24:393). Finally, a number of reports have documented overexpression of CD40 in epithelial and hematopoietic tumors as well as tumor infiltrating endothelial cells, indicating that CD40 may play a role in tumor growth and/or angiogenesis as well (Gruss et al.,  Leuk. Lymphoma,  1997, 24:393; Kluth et al.,  Cancer Res.,  1997, 57:891). 
         [0007]    Due to the pivotal role that CD40 plays in humoral immunity, the potential exists that therapeutic strategies aimed at downregulating CD40 or interfering with CD40 signaling may provide a novel class of agents useful in treating a number of immune associated disorders, including but not limited to graft-versus-host disease (GVHD), graft rejection, and autoimmune diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis Inhibitors of CD40 may also prove useful as anti-inflammatory compounds, and could therefore be useful as treatment for a variety of inflammatory and allergic conditions such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, autoimmune encephalomyelitis, thyroiditis, various dermatological conditions, and psoriasis. Recently, both CD40 and CD154 have been shown to be expressed on vascular endothelial cells, vascular smooth muscle cells and macrophages present in atherosclerotic plaques, suggesting that inflammation and immunity contribute to the atherogenic process. That this process involves CD40 signaling is suggested by several studies in mouse models in which disruption of CD154 (by knockout or by monoclonal antibody) reduced the progression or size of atherosclerotic lesions. (Mach et al.,  Nature,  1998, 394:200-3; Lutgens et al., 1999,  Nat Med.  5:1313-6). 
         [0008]    Finally, as more is learned of the association between CD40 overexpression and tumor growth, inhibitors of CD40 may prove useful as anti-tumor agents and inhibitors of other hyperproliferative conditions as well. 
         [0009]    Currently, there are no known therapeutic agents which effectively inhibit the synthesis of CD40. To date, strategies aimed at inhibiting CD40 function have involved the use of a variety of agents that disrupt CD40/CD40L binding. These include monoclonal antibodies directed against either CD40 or CD40L, soluble forms of CD40, and synthetic peptides derived from a second CD40 binding protein, A20. The use of neutralizing antibodies against CD40 and/or CD40L in animal models has provided evidence that inhibition of CD40 signaling would have therapeutic benefit for GVHD, allograft rejection, rheumatoid arthritis, SLE, MS, and B-cell lymphoma. (Buhlmann and Noelle,  J. Clin. Immunol,  1996, 16:83). Clinical investigations were initiated using anti-CD154 monoclonal antibody in patients with lupus nephritis. However, studies were terminated due to the development of thrombotic events. (Boumpas et al., 2003,  Arthritis Rheum.  2003, 48:719-27). 
         [0010]    Due to the problems associated with the use of large proteins as therapeutic agents, there is a long-felt need for additional agents capable of effectively inhibiting CD40 function. Antisense oligonucleotides avoid many of the pitfalls of current agents used to block CD40/CD40L interactions and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications. U.S. Pat. No. 6,197,584 (Bennett and Cowsert) discloses antisense compounds targeted to CD40. 
       SUMMARY OF THE INVENTION 
       [0011]    Provided herein are antisense compounds, compositions, and methods for the treatment and prevention of inflammatory conditions and cancer. 
         [0012]    Antisense compounds described herein may be 12 to 30 nucleobases in length targeted to a CD40 nucleic acid. In certain embodiments, the CD40 nucleic acid may be any of the sequences as set forth in GENBANK® Accession No. X60592.1, incorporated herein as SEQ ID NO: 1; GENBANK® Accession No. H50598.1, incorporated herein as SEQ ID NO: 2; GENBANK® Accession No. AA203290.1, incorporated herein as SEQ ID NO: 3; and nucleotides 9797000 to nucleotide 9813000 of GENBANK Accession No. NT 011362.9, incorporated herein as SEQ ID NO: 4, or GENBANK® Accession No. BC064518.1, incorporated herein as SEQ ID NO: 237. 
         [0013]    The antisense compound may be 12 to 30 nucleobases in length and may have a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of an intron region of the CD40 gene, selected from the following regions of SEQ ID NO: 4:
       (a) positions 11250-12685, corresponding to intron 6;   (b) positions 2943-6367, corresponding to intron 1,   (c) positions 6447-6780, corresponding to intron 2,   (d) positions 6907-7157, corresponding to intron 3,   (e) positions 7305-7673, corresponding to intron 4,   (f) positions 7768-11187, corresponding to intron 5,   (g) positions 12773-12877, corresponding to intron 7, or   (h) positions 12907-13429, corresponding to intron 8,
 
wherein the remaining part or parts of the antisense compound are at least 70% complementary to the sequence shown in SEQ ID NO: 4. Preferably, the remaining parts of the antisense compound are at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100% complementary to the sequence shown in SEQ ID NO: 4.
       
 
         [0022]    Preferably, the antisense compound may comprise at least 8 contiguous nucleobases complementary to an equal length portion of positions 12527 to 12685 of SEQ ID NO: 4, which is a region that can be either part of intron 6, or can be part of an alternative version of exon 7 when a different splice acceptor site is selected. Preferably, the antisense compound has a nucleobase sequence comprising at least 8 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 208, wherein the nucleobase sequence of the compound is at least 70% complementary to the sequence shown in SEQ ID NO: 4. Preferably, the antisense compound is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100% complementary to the sequence shown in SEQ ID NO: 4. More preferably, the antisense compound has the sequence of SEQ ID NO: 208. Even more preferably, the antisense compound is 20 nucleobases in length and consists of the nucleobase sequence of SEQ ID NO: 208. Most preferably, the antisense compound is an antisense oligonucleotide 20 nucleotides in length having the sequence of nucleotides as set forth in SEQ ID NO:208, wherein each cytosine is a 5-methylcytosine, each internucleoside linkage is a phosphorothioate linkage, nucleotides 1-5 and 16-20 are 2′-O-methoxyethyl nucleotides, and nucleotides 6-15 are 2′-deoxynucleotides; most preferably the antisense compound is ISIS 396236. 
         [0023]    In an alternative embodiment, the antisense compound may be 12 to 30 nucleobases in length and have a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of a region of the CD40 gene, corresponding to positions 13662-16001 of SEQ ID NO: 4, which forms part of exon 9 or a region 3′ to exon 9, wherein the remaining parts of the antisense compound are at least 70% complementary to the sequence shown in SEQ ID NO: 4. Preferably, the target region of the CD40 gene corresponds to positions 13877-14084, even more preferably to positions 13937-13996, of SEQ ID NO: 4. Preferably, the remaining parts of the antisense compound are at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100% complementary to the sequence shown in SEQ ID NO: 4. 
         [0024]    In yet another alternative embodiment, the antisense compound is 12 to 30 nucleobases in length and has a nucleobase sequence complementary to the sequence shown in SEQ ID NO: 1, starting at position 69 or 70 of SEQ ID NO: 1, wherein the nucleobase sequence is at least 95% complementary to the sequence shown in SEQ ID NO: 1. Preferably, the nucleobase sequence is essentially complementary to the sequence shown in SEQ ID NO: 1. More preferably, the nucleobase sequence is selected from the sequences of SEQ ID Nos: 90 and 163. Even more preferably, the antisense compound has a nucleobase sequence of SEQ ID NO: 90. Even more preferably, the antisense compound is 18 or 20 nucleobases in length and consists of the nucleobase sequence of SEQ ID NO: 90 or SEQ ID NO: 163. The antisense compound may be ISIS26163, ISIS396201 or ISIS396278. Preferably, the antisense compound is an antisense oligonucleotide 18 nucleotides in length having the sequence of nucleotides as set forth in SEQ ID NO: 90, wherein each cytosine is a 5-methylcytosine, each internucleoside linkage is a phosphorothioate linkage, nucleotides 1-4 and 15-18 are 2′-O-methoxyethyl nucleotides, and nucleotides 5 to 14 are 2′-deoxynucleotides. Most preferably, the antisense compound is ISIS26163. 
         [0025]    An antisense compound according to the invention may comprise a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 5 to 236. Preferably, the compound consists of a single-stranded modified oligonucleotide. Preferably, the nucleobase sequence of the modified oligonucleotide is 100% complementary to a nucleobase sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 237. 
         [0026]    The antisense compound may comprise linked nucleosides. Preferably, the antisense compound is an antisense oligonucleotide. 
         [0027]    The antisense compound may be a single-stranded or double-stranded oligonucleotide. Preferably, the antisense compound is a single-stranded oligonucleotide. 
         [0028]    The antisense oligonucleotide may be modified, wherein at least one internucleoside linkage is a modified internucleoside linkage. The internucleoside linkage may be a phosphorothioate internucleoside linkage. 
         [0029]    The antisense oligonucleotide may be modified, wherein at least one nucleoside comprises a modified sugar. The modified sugar may be a bicyclic sugar. Preferably, the at least one bicyclic sugar comprises a 4′-CH(CH3)-O-2′ bridge. The modified sugar may comprise a 2′-O-methoxyethyl. The antisense compound may comprise at least one tetrahydropyran modified nucleoside, wherein a tetrahydropyran ring replaces the furanose ring. Preferably, each of the at least one tetrahydropyran modified nucleoside has the structure 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein Bx is an optionally protected heterocyclic base moiety. 
         [0030]    The antisense compound may comprise a modified nucleobase. The modified nucleobase may be a 5-methylcytosine. Preferably, every cytosine is a 5-methylcytosine. 
         [0031]    The antisense compound may be a gapmer, for example an oligonucleotide comprising:
       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. Preferably, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and preferably each internucleoside linkage is a phosphorothioate linkage.       
 
         [0036]    The antisense oligonucleotide may be a 5-10-5 MOE gapmer or a 2-15-3 MOE gapmer. The antisense oligonucleotide may consist of 20 linked nucleosides. 
         [0037]    The antisense oligonucleotide may be a 4-10-4 MOE gapmer. The antisense oligonucleotide may consist of 18 linked nucleosides. 
         [0038]    Compositions described herein may comprise an oligonucleotide consisting of 12 to 30 linked nucleosides, targeted to a CD40 nucleic acid or a salt thereof and a pharmaceutically acceptable carrier or diluent. 
         [0039]    The composition may comprise a single-stranded or double-stranded oligonucleotide. 
         [0040]    Another embodiment of the invention is a pharmaceutical composition comprising an antisense compound as described above and a liposome or a lipid based delivery system. Preferably, said liposome is an amphoteric liposome. Preferably, said amphoteric liposome is formed from a lipid phase comprising an amphoteric lipid or a mixture of lipid components with amphoteric properties. Said amphoteric liposome may further comprise one or more neutral or zwitterionic lipids. More preferably, said amphoteric liposome is formed from a lipid phase comprising
       (a) about 15 mol % POPC, about 45 mol % DOPE, about 20 mol % MoChol, about 20 mol % Chems   (b) about 60 mol % POPC, about 10 mol % DOTAP, about 30 mol % Chems   (c) about 30 mol % POPC, about 10 mol % DOTAP, about 20 mol % Chems, about 40 mol % Chol   (d) about 60 mol % POPC, about 20 mol % HistChol, about 20 mol % Chol.       
 
         [0045]    A further embodiment of the invention is an antisense compound or composition as described above for medical use. Yet a further embodiment of the invention is an antisense compound or a composition as described above for the treatment of cancer or an inflammatory or immune associated condition. The treatment may further comprise administering a second drug, which may be administered separately or concomitantly with the antisense compound of the invention. 
         [0046]    Methods described herein may comprise administering to an animal an antisense compound as described above, preferably an antisense compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a CD40 nucleic acid, or a composition comprising said antisense compound. Preferably, the animal is a human. 
         [0047]    Administration of the antisense compound and/or the second drug may be by parenteral administration, topical administration, oral administration or aerosol administration. Parenteral administration may be any of subcutaneous or intravenous administration. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0048]    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. 
         [0049]    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 in their entirety for any purpose. 
       DEFINITIONS 
       [0050]    Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference in their entirety. 
         [0051]    Unless otherwise indicated, the following terms have the following meanings: 
         [0052]    “2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH 2 ) 2 —OCH 3 ) refers to an O-methoxy-ethyl modification of the 2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar. 
         [0053]    “2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety. 
         [0054]    “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase. 
         [0055]    “Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits. 
         [0056]    “Active pharmaceutical ingredient” means the substance or substances in a pharmaceutical composition that provides a desired effect. 
         [0057]    “Active target region” means a target region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels. 
         [0058]    “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. 
         [0059]    “Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering. 
         [0060]    “Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. 
         [0061]    “Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound. 
         [0062]    “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. 
         [0063]    “Bicyclic sugar” means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar. 
         [0064]    “Bicyclic nucleic acid” or “BNA” or “bicyclic nucleoside” or “bicyclic nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system. As used herein, unless otherwise indicated, the term “methyleneoxy BNA” alone refers to β-D-methyleneoxy BNA. 
         [0065]    “Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound. 
         [0066]    “Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits. A “gapmer” means an antisense compound in which an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having one or more nucleotides that are chemically distinct from the nucleosides of the internal region. A “gap segment” means the plurality of nucleotides that make up the internal region of a gapmer. A “wing segment” means the external region of a gapmer. 
         [0067]    “Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially. 
         [0068]    “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. 
         [0069]    “Comply” means the adherence with a recommended therapy by a individual. 
         [0070]    “Contiguous nucleobases” means nucleobases immediately adjacent to each other. 
         [0071]    “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected the diluent may be a liquid, e.g. saline solution. 
         [0072]    “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in 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. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in a individual. In other embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month. 
         [0073]    “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. 
         [0074]    “Duration” means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered. 
         [0075]    “Efficacy” means the ability to produce a desired effect. 
         [0076]    “CD40 nucleic acid” means any nucleic acid encoding CD40. For example, in certain embodiments, a CD40 nucleic acid includes, without limitation, a DNA sequence encoding CD40, an RNA sequence transcribed from DNA encoding CD40, and an mRNA sequence encoding CD40. “CD40 mRNA” means an mRNA encoding a CD40 protein. 
         [0077]    “Fully complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid. In certain such embodiments, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid. 
         [0078]    “Gap-widened” means an antisense compound has a gap segment of 12 or more contiguous 2′-deoxyribonucleotides positioned between and immediately adjacent to 5′ and 3′ wing segments having from one to six nucleotides having modified sugar moieties. “Immediately adjacent” means there are no intervening nucleotides between the immediately adjacent elements. 
         [0079]    “Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain such embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target 
         [0080]    “Individual” means a human or non-human animal selected for treatment or therapy. 
         [0081]    “Individual compliance” means adherence to a recommended or prescribed therapy by a individual. 
         [0082]    “Injection site reaction” means inflammation or abnormal redness of skin at a site of injection in a individual. 
         [0083]    “Internucleoside linkage” refers to the chemical bond between nucleosides. 
         [0084]    “Linked nucleosides” means adjacent nucleosides which are bonded together. 
         [0085]    “Modified internucleoside linkage” refers to a substitution and/or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond). 
         [0086]    “Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase. 
         [0087]    “Modified sugar” refers to a substitution and/or any change from a natural sugar. 
         [0088]    “Modified nucleobase” means 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). 
         [0089]    “Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleotide having, independently, a modified sugar moiety or modified nucleobase. 
         [0090]    “Modified sugar moiety” means a sugar moiety having any substitution and/or change from a natural sugar moiety. 
         [0091]    “Motif” means the pattern of unmodified and modified nucleosides in an antisense compound. 
         [0092]    “Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage. 
         [0093]    “Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH). 
         [0094]    “Non-complementary nucleobase” or “mismatch” means a nucleobase of a first nucleic acid that is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid. 
         [0095]    “Nucleoside” means a nucleobase linked to a sugar. 
         [0096]    As used herein the term “nucleoside mimetic” is intended to include those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units. 
         [0097]    “Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid. 
         [0098]    “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification. 
         [0099]    “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside. 
         [0100]    The term “nucleotide mimetic” is intended to include 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(═O)—O— or other non-phosphodiester linkage). 
         [0101]    “Oligomeric compound” means a polymer or oligomer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule. 
         [0102]    “Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom. 
         [0103]    “Oligonucleotide” means a polymer or oligomer of linked nucleosides each of which can be modified or unmodified, independent one from another. 
         [0104]    “Parenteral administration,” means administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration. 
         [0105]    “Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to a individual. For example, in certain embodiments, an antisense oligonucleotide targeted to CD40 is pharmaceutical agent. 
         [0106]    “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto. 
         [0107]    “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more antisense oligonucleotides or a combination of antisense oligonucleotides and non-antisense active agents and a sterile aqueous solution or other pharmaceutically acceptable additive. 
         [0108]    “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. 
         [0109]    “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. 
         [0110]    “Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. 
         [0111]    “Recommended therapy” means a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease. 
         [0112]    “Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality. 
         [0113]    “Single-stranded modified oligonucleotide” means a modified oligonucleotide which is not hybridized to a complementary strand. 
         [0114]    “Specifically hybridizable” means an antisense compound that hybridizes to a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. 
         [0115]    The term “sugar surrogate” overlaps with the slightly broader term “nucleoside mimetic” but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. 
         [0116]    “Stringent hybridization conditions” means conditions under which a nucleic acid molecule, such as an antisense compound, will hybridize to a target nucleic acid sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. In the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. 
         [0117]    “Subcutaneous administration” means administration just below the skin. “Intravenous administration” means administration into a vein. 
         [0118]    “Targeted” or “targeted to” means having a nucleobase sequence that will allow specific hybridization of an antisense compound to a target nucleic acid to induce a desired effect. In certain embodiments, a desired effect is reduction of a target nucleic acid. In certain such embodiments, a desired effect is reduction of a CD40 mRNA. 
         [0119]    “Targeting” 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. 
         [0120]    “Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds. Target nucleic acids may include, but are not limited to, DNA, RNA (including, but not limited to pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target, and also miRNA. 
         [0121]    “Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted. 
         [0122]    “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. 
         [0123]    “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. 
         [0124]    “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). 
       Antisense Compounds 
       [0125]    Antisense compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. 
         [0126]    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. 
         [0127]    In certain embodiments, an antisense compound targeted to a CD40 nucleic acid is 12 to 30 subunits in length. In other words, antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides. 
         [0128]    In certain embodiments, a shortened or truncated antisense compound targeted to a CD40 nucleic acid has a single subunit deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a CD40 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end. 
         [0129]    When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two are more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end. 
         [0130]    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. 
         [0131]    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. 
         [0132]    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. 
         [0133]    Bhanot et al. (PCT/US2007/068401) provided short antisense compounds, including compounds comprising chemically-modified high-affinity monomers 8 to 16 monomers in length. These short antisense compounds were shown to be useful for reducing target nucleic acids and/or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Short antisense compounds were effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing. 
       Antisense Compound Motifs 
       [0134]    In certain embodiments, antisense compounds targeted to a CD40 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. 
         [0135]    Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex. 
         [0136]    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 a preferred embodiment, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH 3 , among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH 2 )n-O-2′ bridge, where n=1 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. 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, 25, 30 or more nucleotides. Thus, gapmers of the present invention include, but are not limited to, for example 5-10-5, 4-8-4, 4-10-4, 2-15-3, 4-12-3, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1 or 2-8-2. 
         [0137]    In some embodiments, the antisense compound as a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X—Y or Y—Z configuration as described above for the gapmer configuration. Thus, wingmer configurations of the present invention 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 or 8-2. 
         [0138]    In one embodiment, antisense compounds targeted to a CD40 nucleic acid possess a 5-10-5 gapmer motif. 
         [0139]    In some embodiments, an antisense compound targeted to a CD40 nucleic acid has a gap-widened motif. In other embodiments, an antisense oligonucleotide targeted to a CD40 nucleic acid has a gap-widened motif. 
         [0140]    In one embodiment, a gap-widened antisense oligonucleotide targeted to a CD40 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotides positioned between wing segments of three chemically modified nucleosides. In one embodiment, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification. 
       Target Nucleic Acids, Target Regions and Nucleotide Sequences 
       [0141]    Nucleotide sequences that encode CD40 include, without limitation, the following: 
         [0142]    GENBANK Accession No. X60592.1, first deposited with GENBANK® on Apr. 21, 1993 and incorporated herein as SEQ ID NO: 1; GENBANK® Accession No. H50598.1, first deposited with GENBANK® on Sep. 19, 1995, and incorporated herein as SEQ ID NO: 2; GENBANK Accession No. AA203290.1, first deposited with GENBANK® on Jan. 25, 1997, and incorporated herein as SEQ ID NO: 3; and nucleotides 9797000 to 9813000 of GENBANK Accession No. NT — 011362.9, first deposited with GENBANK® on Nov. 29, 2000, and incorporated herein as SEQ ID NO: 4, and GENBANK® Accession No. BC064518.1, incorporated herein as SEQ ID NO: 237. 
         [0143]    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 may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif. 
         [0144]    In one embodiment, a target region is a structurally defined region of the nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for CD40 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In other embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the target region. 
         [0145]    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 other 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. 
         [0146]    A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In one embodiment, target segments within a target region are separated by no more than about 300 nucleotides. In other embodiments, target segments within a target region are separated by no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid. In another embodiment, target segments within a target region are separated by no more than about 5 nucleotides on the target nucleic acid. In additional embodiments, target segments are contiguous. 
         [0147]    Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, or an exon. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon. 
         [0148]    The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences). There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In one embodiment, reductions in CD40 mRNA levels are indicative of inhibition of CD40 expression. Reductions in levels of a CD40 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of CD40 expression. For example, changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides, or metals is indicative of inhibition of CD40 expression. Reduction of eosinophils is indicative of inhibition of CD40 expression. Measurements of cellular status which include pH, stage of cell cycle, intake or excretion of biological indicators by the cell are also endpoints of interest. 
         [0149]    Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the CD40 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells. 
       Genomic Structure, Exons and Introns 
       [0150]    Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA. 
         [0151]    It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. 
         [0152]    Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant. 
       Hybridization 
       [0153]    In some embodiments, hybridization occurs between an antisense compound disclosed herein and a CD40 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. 
         [0154]    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. 
         [0155]    Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In one embodiment, the antisense compounds provided herein are specifically hybridizable with a CD40 nucleic acid. 
       Complementarity 
       [0156]    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 CD40 nucleic acid). 
         [0157]    Non-complementary nucleobases between an antisense compound and a CD40 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a CD40 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). 
         [0158]    In some embodiments, the antisense compounds provided herein are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% complementary to a CD40 nucleic acid. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods. 
         [0159]    For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 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). 
         [0160]    In other embodiments, the antisense compounds provided herein are fully complementary (i.e., 100% complementary) to a target nucleic acid. For example, an antisense compound may be fully complementary to a CD40 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. 
         [0161]    The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide. 
         [0162]    In one embodiment, antisense compounds up to 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 CD40 nucleic acid. 
         [0163]    In another embodiment, antisense compounds up to 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 CD40 nucleic acid. 
         [0164]    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 one embodiment, the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment. In another embodiment, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In yet another embodiment, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values. 
       Identity 
       [0165]    The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared. 
         [0166]    In one embodiment, the antisense compounds 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 
       [0167]    A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. 
         [0168]    Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. 
         [0169]    Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. 
       Modified Internucleoside Linkages 
       [0170]    The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside 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. 
         [0171]    Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphonoacetates, phosphoramidate, and phosphorothioates or phosphorodithioates. Internucleoside linkages that do not have a phosphorus atom include, amongst others, methylene(methylimino) or MMI linkages, morpholino linkages or amide linkages. In peptide nucleic acids (PNA) the sugar backbone is replaced with an amide containing backbone. 
         [0172]    Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known. 
         [0173]    Representative United States patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697; 5,625,050 and U.S. Pat. No. 6,693,187, each of which is herein incorporated by reference. 
         [0174]    Representative United States patents that teach the preparation of non-phosphorous-containing linkages include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference. 
         [0175]    In one embodiment, antisense compounds targeted to a CD40 nucleic acid comprise one or more modified internucleoside linkages. In some embodiments, the modified internucleoside linkages are phosphorothioate linkages. In other embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage. 
       Modified Sugar Moieties 
       [0176]    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 are modified by modification of the ribofuranose ring. Such modifications include without limitation, addition of substituent groups, bridging of non-geminal ring atoms to form a bicyclic nucleic acid (BNA), as in locked nucleic acids (LNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 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 Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group). 
         [0177]    Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl(R or S), 4′-S, 2′-F, 2′-OCH 3  (known as 2′-OMe) and 2′-O(CH 2 ) 2 OCH 3  (known as 2′MOE) substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, 0-allyl, 0-C1-C10 alkyl, OCF 3 , O—CH 2 CH 2 CH 2 NH 2 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —O—N(Rm)(Rn) such as 2′-dimethylaminooxyethoxy (2′-O—(CH 2 ) 2 ON(CH 3 ) 2  or 2′-DMAOE), O(CH 2 ) 2 —O—(CH 2 ) 2 —N(Rm)(Rn) such as 2′-dimethylaminoethoxyethoxy (2′-O—(CH 2 ) 2 —O—(CH 2 ) 2 —N(CH 3 ) 2  or 2′-DMAEOE and O—CH 2 —C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. 
         [0178]    Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms, e.g. a 4′-(CH 2 ) n —O-2′ bridge, where n=1 or n=2. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2; 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-C(CH 3 ) 2 —O-2′ (see PCT/US2008/068922); 4′-CH(CH 3 )—O-2′ and 4′-CH(CH 2 OCH 3 )—O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH 2 —N(OCH 3 )-2′ (see PCT/US2008/064591); 4′-CH 2 —O—N(CH 3 )-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH 2 —N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH 2 —C(CH 3 )-2′ and 4′-CH 2 —C(═CH 2 )-2′ (see PCT/US2008/066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (See PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO99/14226). 
         [0179]    In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein Bx is an optionally protected heterocyclic base moiety. 
         [0180]    Many other bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example Leumann, C. J., Bioorg. Med. Chem. 10, (2002), 841-854). Such ring systems can undergo various additional substitutions to enhance activity. 
         [0181]    Methods for the preparations of modified sugars are well known to those skilled in the art. Representative United States patents that teach the preparation of modified sugars include, but are not limited to U.S. Pat. No. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446, 137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference. 
         [0182]    In certain embodiments, a 2′-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration. 
         [0183]    In certain embodiments, the bicyclic sugar moiety comprises a bridge group between the 2′ and the 4′-carbon atoms. In certain such embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. In certain embodiments, a linked biradical group is selected from —O—, —S—, —N(R1)-, —C(R1)(R2)-, —C(R1)=C(R1)-, —C(R1)=N—, —C(═NR1)-, —Si(R1)(R2)-, —S(═O) 2 —, —S(═O)—, —C(═O)— and —C(═S)—; where each R1 and R2 is, independently, H, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted hetero-cycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, substituted oxy (—O—), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O) 2 —H), substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent group is, independently, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, amino, substituted amino, acyl, substituted acyl, C1-C12 aminoalkyl, C1-C12 aminoalkoxy, substituted C1-C12 aminoalkyl, substituted C1-C12 aminoalkoxy or a protecting group. 
         [0184]    In some embodiments, the bicyclic sugar moiety is bridged between the 2′ and 4′ carbon atoms with a biradical group selected from —O—(CH 2 )p-, —O—CH 2 —, —O—CH 2 CH2-, —O—CH(alkyl)-, —NH—(CH 2 )p-, —N(alkyl)-(CH 2 )p-, —O—CH(alkyl)-, —(CH(alkyl))-(CH 2 )p-, —NH—O—(CH 2 )p-, —N(alkyl)-O—(CH 2 )p-, or —O—N(alkyl)-(CH 2 )p-, wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, p is 1, 2 or 3. 
         [0185]    In one aspect, each of said bridges is, independently, —[C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-. In another aspect, each of said bridges is, independently, 4′-(CH 2 ) 3 -2′, 4′-(CH 2 ) 2 -2′, 4′-CH 2 —O-2′, 4′-(CH 2 ) 2 —O-2′, 4′-CH 2 —O—N(R1)-2′ and 4′-CH 2 —N(R1)-O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl. 
         [0186]    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. 
         [0187]    In one embodiment, antisense compounds targeted to a CD40 nucleic acid comprise one or more nucleotides having modified sugar moieties. In a preferred embodiment, the modified sugar moiety is 2′-MOE. In other embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif. 
       Modified Nucleobases 
       [0188]    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 may 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). 
         [0189]    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—CH 3 ) 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. 
         [0190]    Heterocyclic base moieties may 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 O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 
         [0191]    Methods for the preparations of modified nucleobases are well known to those skilled in the art. Representative United States patents that teach the preparation of modified nucleobases include, but are not limited to U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941 each of which is herein incorporated by reference. 
         [0192]    In one embodiment, antisense compounds targeted to a CD40 nucleic acid comprise one or more modified nucleobases. In an additional embodiment, gap-widened antisense oligonucleotides targeted to a CD40 nucleic acid comprise one or more modified nucleobases. In some embodiments, the modified nucleobase is 5-methylcytosine. In further embodiments, each cytosine is a 5-methylcytosine. 
       Conjugated Antisense Compounds 
       [0193]    Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. 
         [0194]    Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003. 
       Compositions and Methods for Formulating Pharmaceutical Compositions 
       [0195]    Antisense oligonucleotides may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. 
         [0196]    Antisense compound targeted to a CD40 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes for example 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 CD40 nucleic acid and a pharmaceutically acceptable diluent. In one embodiment, the pharmaceutically acceptable diluent is PBS. In other embodiments, the antisense compound is an antisense oligonucleotide. 
         [0197]    Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. 
         [0198]    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. 
         [0199]    The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. 
         [0200]    Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. 
         [0201]    The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. 
         [0202]    The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. 
         [0203]    Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients. 
         [0204]    Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. 
         [0205]    Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells. 
         [0206]    Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. 
         [0207]    Other liposomes or lipid based delivery systems known in the art are described for example in WO 05/105152; WO 06/069782; Morrissey et al., Nature Biotechnology, 23(8), 1002-1007, 2005; WO 05/007196; Wheeler et al., Gene Therapy, 6(2), 271-281, 1999; WO 02/34236; Budker et al., Nature Biotechnology, 14(6), 760-764, 1996; U.S. Pat. No. 5,965,434; U.S. Pat. No. 5,635,487; Spagnou et al., Biochemistry, 43(42), 13348-13356, 2004; U.S. Pat. No. 6,756,054; WO 06/016097 and U.S. Pat. No. 5,785,992; WO 04/035523, each of which is herein incorporated by reference. 
         [0208]    In a preferred embodiment of the invention amphoteric liposomes may be used as formulations which include the inventive antisense compounds. Amphoteric liposomes are a class of liposomes having an anionic or neutral charge at pH 7.5 and a cationic charge at pH 4. Reference is made to WO 02/066012 by Panzner et al. which is incorporated herein by reference. The use, selection and manufacturing of amphoteric liposomes for the transfection of cells is further described in WO 05/094783 of Endert et al., WO 07/031333 of Panzner et al., WO 07/107304 of Panzner et al. and WO 08/043575 of Panzner et al. Amphoteric liposomes have an excellent biodistribution and are well tolerated in animals. They can encapsulate nucleic acid molecules with high efficiency. WO 06/048329 of Panzner et al., which is incorporated herein by reference in its entirety, describes pharmaceutical compositions comprising amphoteric liposomes and oligonucleotides which are adapted to target nucleic acids encoding CD40. 
         [0209]    By “amphoteric” is meant herein that the liposomes comprise charged groups of both anionic and cationic character wherein:
       (i) at least one of the charged groups has a pKa between 4 and 7.4,   (ii) the cationic charge prevails at pH 4 and   (iii) the anionic charge prevails at pH 7.4;
 
whereby the liposomes have an isoelectric point of zero net charge between pH 4 and pH 7.4. Amphoteric character, by this definition, is different from “zwitterionic character”, because zwitterions do not have a pK in the range mentioned above. In consequence, zwitterions are essentially neutral over a range of pH values. Phosphatidylcholine or phosphatidylethanolamines, for example, are neutral lipids with zwitterionic character.
       
 
         [0213]    Amphoteric liposomes may be formed from a lipid phase comprising an amphoteric lipid. In some embodiments said lipid phase may comprise 5 to 30 mol.% of said amphoteric lipid, preferably 10 to 25 mol.%. 
         [0214]    Suitable amphoteric lipids are disclosed in WO 02/066489 and WO 03/070735 by Panzner et al. Preferably, said amphoteric lipid is selected from the group consisting of HistChol, HistDG, isoHistSuccDG, Acylcarnosin and HCChol. (A glossary of such abbreviated forms of the names of the lipids referred to herein is included below for ease of reference. A number of such abbreviations are those that are commonly used by those skilled in the art.) 
         [0215]    Alternatively, said amphoteric liposomes may be formed from a lipid phase comprising a mixture of lipid components with amphoteric properties. Such amphoteric liposomes may be formed from pH-responsive anionic and/or cationic components, as disclosed for example in WO 02/066012. Cationic lipids sensitive to pH are disclosed in WO 02/066489 and WO 03/070220 and in the references made therein, in particular in Budker, et al. 1996, Nat Biotechnol. 14(6):760-4, and can be used in combination with constitutively charged anionic lipids or with anionic lipids that are sensitive to pH. 
         [0216]    Alternatively, the cationic charge may be introduced from constitutively charged lipids that are known to those skilled in the art in combination with a pH sensitive anionic lipid. Combinations of constitutively charged anionic and cationic lipids, e.g. DOTAP and DPPG, are not preferred. Thus, in some presently preferred embodiments of the invention, said mixture of lipid components may comprise (i) a stable cationic lipid and a chargeable anionic lipid, (ii) a chargeable cationic lipid and chargeable anionic lipid or (iii) a stable anionic lipid and a chargeable cationic lipid. 
         [0217]    Preferred cationic components include DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18) 2 Gly +  N,N-dioctadecylamido-glycin, CTAB, CPyC, DODAP and DOEPC. 
         [0218]    Preferred anionic lipids for use with the invention include DOGSucc, POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and CetylP. 
         [0219]    Preferably, such an amphoteric mixture of lipids does not constitute more than about 70 mol.% of the lipid phase. In some embodiments, said mixture may constitute not more than 50 mol.% of the lipid phase; preferably said lipid phase comprises about 20 to about 40 mol.% of such a mixture. 
         [0220]    In some embodiments, said lipid phase may further comprise a neutral lipid, preferably a neutral phospholipid, such as a phosphatidylcholine. Presently preferred phosphatidylcholines include POPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC, DSPC and DOPC. More preferably, said phosphatidylcholine comprises POPC, non-hydrogenated soy bean PC or non-hydrogenated egg PC. 
         [0221]    The lipid phase may comprise at least 15 mol.% of said phosphatidylcholine, preferably at least 20 mol.%. In some embodiments, said lipid phase may comprise no less than about 25 mol.% phosphatidylcholine. Alternatively, said lipid phase may comprise no less than about 40 mol.% phosphatidylcholine. 
         [0222]    A presently preferred formulation in accordance with the present invention comprises a liposome having about 60 mol.% POPC, about 10 mol.% DOTAP and about 30 mol.% CHEMS. 
         [0223]    In some embodiments said neutral lipid may comprise a phosphatidylethanolamine or a mixture of phosphatidylcholine and phosphatidylethanolamine. Said neutral phosphatidylcholines or phosphatidylethanolamines or mixtures of the two may be present in the lipid phase in the molar amount (mol.%) not constituted by the other components of the lipid phase, but to at least 20 mol.% (the total for the lipid phase being 100 mol.%). 
         [0224]    Preferred phosphatidylethanolamines include DOPE, DMPE and DPPE. 
         [0225]    In some embodiments said neutral lipid may comprise POPC and DOPE. 
         [0226]    Advantageously, said lipid phase may comprise a mixture of anionic and cationic lipids with amphoteric properties, phosphatidylcholine and phosphatidylethanolamine. Amphoteric liposomes formed from such a lipid phase may be serum-stable and therefore suitable for systemic delivery. Preferably said lipid phase comprises MoChol as a cationic lipid and CHEMS or DMG-Succ as an anionic lipid. 
         [0227]    Further presently preferred amphoteric liposomes for use as formulations which include antisense compounds of the present invention may be selected from the group consisting of:
       (a) about 15 mol.% POPC, about 45 mol.% DOPE, about 20 mol.% MoChol and about 20 mol.% CHEMS;   (b) about 10 mol.% POPC, about 30 mol.% DOPE, about 30 mol.% MoChol and about 30 mol.% CHEMS;   (c) about 10 mol.% POPC, about 30 mol.% DOPE, about 20 mol.% MoChol and about 40 mol.% CHEMS;   (d) about 6 mol.% POPC, about 24 mol.% DOPE, about 47 mol.% MoChol and about 23 mol.% CHEMS.       
 
         [0232]    Alternatively, said lipid phase may comprise a mixture of anionic and cationic lipids with amphoteric properties a neutral phosphatidylcholine and cholesterol. Such liposomes may also be serum-stable. In some embodiments, said lipid phase may comprise from 30 mol.% to 50 mol.% cholesterol, preferably from about 35 mol.% to about 45 mol.%. Alternatively, said lipid phase may comprise phosphatidylcholine and from 10 mol.% to 25 mol.% cholesterol, preferably from about 15 mol.% to about 25 mol.%. 
         [0233]    A presently preferred formulation comprises 10 to 25 mol.% amphoteric lipid, e.g. HistChol, HistDG or Acylcarnosin, 15 to 25 mol.% cholesterol and the remainder being POPC, soy bean PC, egg PC, DMPC, DPPC or DOPC, preferably POPC; for example about 60 mol.% POPC, about 20 mol.% HistChol and about 20 mol.% Chol. 
         [0234]    Another presently preferred formulation in accordance with the present invention comprises a liposome including a mix of lipid components with amphoteric properties and having about 30 mol.% POPC, about 10 mol.% DOTAP, about 20 mol.% CHEMS and about 40 mol.% Chol. 
         [0235]    The amphoteric liposomes may have a size in the range 50 to 500 nm, preferably 100 to 500 nm, more preferably 150 and 300 nm. 
         [0236]    The amphoteric liposome formulations of the present invention may be formulated for use as a colloid in a suitable pharmacologically acceptable vehicle. Vehicles such as water, saline, phosphate buffered saline and the like are well known to those skilled in the art for this purpose. 
         [0237]    In some embodiments, the amphoteric liposome formulations of the present invention may be administered at a physiological pH of between about 7 and about 8. To this end, the formulation comprising the antisense compound, excipient and vehicle may be formulated to have a pH in this range. 
         [0238]    The amphoteric liposome formulations of the invention may be manufactured using suitable methods that are known to those skilled in the art. Such methods include, but are not limited to, extrusion through membranes of defined pore size, injection of lipid solutions in ethanol into a water phase containing the cargo to be encapsulated, or high pressure homogenisation. 
         [0239]    A solution of the oligonucleotide may be contacted with said excipient at a neutral pH, thereby resulting in volume inclusion of a certain percentage of the solution. An high concentrations of the excipient, ranging from about 50 mM to about 150 mM, is preferred to achieve substantial encapsulation of the active agent. 
         [0240]    Amphoteric liposomes used as formulations in accordance with the present invention offer the distinct advantage of binding oligonucleotides at or below their isoelectric point, thereby concentrating said active agent at the liposome surface. This process is described in more detail in WO 02/066012. 
         [0241]    Irrespective of the actual production process used to make the amphoteric liposome formulations, in some embodiments, non-encapsulated oligonucleotide may be removed from the liposomes after the initial production step in which the liposomes are formed as tight containers. Again, the technical literature and the references included herein describe such methodology in detail and suitable process steps may include, but are not limited to, size exclusion chromatography, sedimentation, dialysis, ultrafiltration and diafiltration. 
         [0242]    However, the removal of any non-encapsulated oligonucleotide is not required for performance of the invention, and in some embodiments the composition may comprise free as well as entrapped drug. 
         [0243]    In some aspects of the invention the amphoteric liposome formulations which include the inventive antisense compounds may be used as pharmaceutical compositions for the prevention or treatment of an inflammatory, immune or autoimmune disorder of a human or non-human animal such as graft rejection, graft-versus-host disease, multiple sclerosis, systemic lupus erythematosous, rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis or thyroiditis, Morbus Crohn and Colitis ulcerosa. 
       Glossary of Common Abbreviated Lipid Names 
       [0000]    
       
         DMPC Dimyristoylphosphatidylcholine 
         DPPC Dipalmitoylphosphatidylcholine 
         DSPC Distearoylphosphatidylcholine 
         POPC Palmitoyl-oleoylphosphatidylcholine 
         DOPC Dioleoylphosphatidylcholine 
         DOPE Dioleoylphosphatidylethanolamine 
         DMPE Dimyristoylphosphatidylethanolamine 
         DPPE Dipalmitoylphosphatidylethanolamine 
         DOPG Dioleoylphosphatidylglycerol 
         POPG Palmitoyl-oleoylphosphatidylglycerol 
         DMPG Dimyristoylphosphatidylglycerol 
         DPPG Dipalmitoylphosphatidylglycerol 
         DMPS Dimyristoylphosphatidylserine 
         DPPS Dipalmitoylphosphatidylserine 
         DOPS Dioleoylphosphatidylserine 
         POPS Palmitoyl-oleoylphosphatidylserine 
         DMPA Dimyristoylphosphatidic acid 
         DPPA Dipalmitoylphosphatidic acid 
         DOPA Dioleoylphosphatidic acid 
         POPA Palmitoyl-oleoylphosphatidic acid 
         CHEMS Cholesterolhemisuccinate 
         DC-Chol 3-β-[N—(N′,N′-dimethylethane) carbamoyl] cholesterol 
         CetylP Cetylphosphate 
         DODAP (1,2)-dioleoyloxypropyl)-N,N-dimethylammonium chloride 
         DOEPC 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine 
         DAC-Chol 3-β-[N—(N,N′-dimethylethane) carbamoyl] cholesterol 
         TC-Chol 3-β-[N—(N′,N′,N′-trimethylaminoethane) carbamoyl] cholesterol 
         DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchlorid) (Lipofectin®) 
         DOGS ((C18) 2 GlySper3 + ) N,N-dioctadecylamido-glycyl-spermine (Transfectam®) 
         CTAB Cetyl-trimethylammoniumbromide, 
         CPyC Cetyl-pyridiniumchloride 
         DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt 
         DMTAP (1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt 
         DPTAP (1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt 
         DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride) 
         DORIE (1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide) 
         DDAB Dimethyldioctadecylammonium bromide 
         DPIM 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole 
         CHIM Histaminyl-Cholesterolcarbamate 
         MoChol 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate 
         HisChol Histaminyl-Cholesterolhemisuccinate. 
         HCChol Nα-Histidinyl-Cholesterolcarbamate 
         HistChol Nα-Histidinyl-Cholesterol-hemisuccinate. 
         AC Acylcarnosine, Stearyl- &amp; Palmitoylcarnosine 
         HistDG 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl-hemisuccinate, &amp; Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives 
         IsoHistSuccDG 1,2-Dipalmitoylglycerol-Oa-Histidinyl-Nα-hemisuccinat, &amp; Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives 
         DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate &amp; Distearoyl-, Dimyristoyl- Dioleoyl- or palmitoyl-oleoylderivatives 
       
     
         [0291]    The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. 
         [0292]    One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration. 
         [0293]    Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG); cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA) or amphoteric lipids or lipid mixtures wherein a mixture of cationic and anionic lipids displays amphoteric properties. For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety. 
         [0294]    Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety. 
         [0295]    Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may 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. 
         [0296]    Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other active agents which function by a non-antisense mechanism, such as for example chemotherapeutic agents or antiinflammatory drugs. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). 
         [0297]    Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially. 
         [0298]    In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially. 
       Cell Culture and Antisense Compounds Treatment 
       [0299]    The effects of antisense compounds on the level, activity or expression of CD40 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cells are cultured according to the vendor&#39;s instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, HuVEC cells, T24, A549, and primary hepatocytes. 
       In Vitro Testing of Antisense Oligonucleotides 
       [0300]    Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds. 
         [0301]    In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture. 
         [0302]    One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.) 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. 
         [0303]    Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) 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. 
         [0304]    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. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. 
         [0305]    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. 
       RNA Isolation 
       [0306]    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, Calif.) according to the manufacturer&#39;s recommended protocols. 
       Analysis of Inhibition of Target Levels or Expression 
       [0307]    Inhibition of levels or expression of a CD40 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative 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, Calif. and used according to manufacturer&#39;s instructions. 
       Quantitative Real-Time PCR Analysis of Target RNA Levels 
       [0308]    Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;s instructions. Methods of quantitative real-time PCR are well known in the art. 
         [0309]    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, Calif.). RT, real-time-PCR reactions are carried out by methods well known to those skilled in the art. 
         [0310]    Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as GAPDH or by quantifying total RNA using RIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). 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 (Invetrogen, Inc. Eugene, Oreg.). 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. 
         [0311]    Probes and primers are designed to hybridize to a CD40 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, Calif.). 
       Analysis of Protein Levels 
       [0312]    Antisense inhibition of CD40 nucleic acids can be assessed by measuring CD40 protein levels. Protein levels of CD40 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, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of human and rat CD40 are commercially available. 
       In Vivo Testing of Antisense Compounds 
       [0313]    Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of CD40 and produce phenotypic changes, such as changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, nucleic acids, hormones, cytokines, and eosinophils. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes pulmonary administration, aerosol administration, topical administration, and parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in CD40 nucleic acid expression are measured. 
       Certain Indications 
       [0314]    In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has an inflammatory or hyperproliferative disorder. In certain embodiments the invention provides methods for prophylactically reducing CD40 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a CD40 nucleic acid. 
         [0315]    In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a CD40 nucleic acid is accompanied by monitoring of eosinophils in an individual, to determine an individual&#39;s response to administration of the antisense compound. An individual&#39;s response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention. 
         [0316]    In one embodiment, administration of an antisense compound targeted to a CD40 nucleic acid results in reduction of CD40 expression by at least 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 some embodiments, administration of a CD40 antisense compound increases the measure by at least 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 some embodiments, administration of a CD40 antisense compound decreases the measure by at least 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. 
         [0317]    In certain embodiments pharmaceutical composition comprising an antisense compound targeted to CD40 is used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory condition or a hyperproliferative disorder. 
         [0318]    The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. 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. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years. 
       Certain Combination Therapies 
       [0319]    In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately. 
         [0320]    In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include steroids and/or chemotherapeutic agents. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to prednisone, corticosteroids, and paclitaxel. In certain such embodiments, the agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered agent is the same as the dose that would be administered if the agent was administered alone. In certain such embodiments the dose of a co-administered agent is lower than the dose that would be administered if the agent was administered alone. In certain such embodiments the dose of a co-administered agent is greater than the dose that would be administered if the agent was administered alone. 
         [0321]    In certain embodiments, the co-administration of a second compound enhances the effect of a first compound, such that co-administration of the compounds results in an effect that is greater than the effect of administering the first compound alone. In other embodiments, the co-administration results in effects that are additive of the effects of the compounds when administered alone. In other embodiments, the co-administration results in effects that are supra-additive of the effects of the compounds when administered alone. In some embodiments, the first compound is an antisense compound. In some embodiments, the second compound is an antisense compound. 
       EXAMPLES 
     Nonlimiting Disclosure and Incorporation by Reference 
       [0322]    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 Human CD40 In Vitro 
       [0323]    Antisense oligonucleotides targeted to a CD40 nucleic acid were tested for their effects on CD40 mRNA in vitro. When cultured cells, grown in a 96-well plate, reached 80% confluency, they were treated with 150 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. CD40 mRNA levels were adjusted according to total RNA content as measured by normalization to RIBOGREEN®. Results are presented as percent inhibition of CD40, relative to untreated control cells in Table 1. 
         [0324]    The antisense oligonucleotides were designed as 18-mers with phosphorothioate backbones (internucleoside linkages) throughout. “5′ target site” indicates the 5′-most nucleotide which the antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK® Accession No X60592.1). Data are averages from three experiments. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibition of human CD40 mRNA levels by fully phosphorothioate oligodeoxynucleotides 
               
             
          
           
               
                   
                 Target 
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Oligo 
                 SEQ 
                 Start 
                 Stop 
                 Target 
                   
                 % 
                 SEQ 
               
               
                 ID 
                 ID NO 
                 Site 
                 Site 
                 Region 
                 Sequence (5′ to 3′) 
                 Inhibition 
                 ID NO 
               
               
                   
               
             
          
           
               
                 18623 
                 1 
                  18 
                  35 
                 5′ UTR 
                 CCAGGCGGCAGGACCACT 
                 31  
                  5 
               
               
                   
               
               
                 18624 
                 1 
                  20 
                  37 
                 5′ UTR 
                 GACCAGGCGGCAGGACCA 
                 28  
                  6 
               
               
                   
               
               
                 18625 
                 1 
                  26 
                  43 
                 5′ UTR 
                 AGGTGAGACCAGGCGGCA 
                 22  
                  7 
               
               
                   
               
               
                 18626 
                 1 
                  48 
                  65 
                 AUG 
                 CAGAGGCAGACGAACCAT 
                 0 
                  8 
               
               
                   
               
               
                 18627 
                 1 
                  49 
                  66 
                 Coding 
                 GCAGAGGCAGACGAACCA 
                 0 
                  9 
               
               
                   
               
               
                 18628 
                 1 
                  73 
                  90 
                 Coding 
                 GCAAGCAGCCCCAGAGGA 
                 0 
                 10 
               
               
                   
               
               
                 18629 
                 1 
                  78 
                  95 
                 Coding 
                 GGTCAGCAAGCAGCCCCA 
                 30  
                 11 
               
               
                   
               
               
                 18630 
                 1 
                  84 
                 101 
                 Coding 
                 GACAGCGGTCAGCAAGCA 
                 0 
                 12 
               
               
                   
               
               
                 18631 
                 1 
                  88 
                 105 
                 Coding 
                 GATGGACAGCGGTCAGCA 
                 0 
                 13 
               
               
                   
               
               
                 18632 
                 1 
                  92 
                 109 
                 Coding 
                 TCTGGATGGACAGCGGTC 
                 0 
                 14 
               
               
                   
               
               
                 18633 
                 1 
                  98 
                 115 
                 Coding 
                 GGTGGTTCTGGATGGACA 
                 0 
                 15 
               
               
                   
               
               
                 18634 
                 1 
                 101 
                 118 
                 Coding 
                 GTGGGTGGTTCTGGATGG 
                 0 
                 16 
               
               
                   
               
               
                 18635 
                 1 
                 104 
                 121 
                 Coding 
                 GCAGTGGGTGGTTCTGGA 
                 0 
                 17 
               
               
                   
               
               
                 18636 
                 1 
                 152 
                 169 
                 Coding 
                 CACAAAGAACAGCACTGA 
                 0 
                 18 
               
               
                   
               
               
                 18637 
                 1 
                 156 
                 173 
                 Coding 
                 CTGGCACAAAGAACAGCA 
                 0 
                 19 
               
               
                   
               
               
                 18638 
                 1 
                 162 
                 179 
                 Coding 
                 TCCTGGCTGGCACAAAGA 
                 0 
                 20 
               
               
                   
               
               
                 18639 
                 1 
                 165 
                 182 
                 Coding 
                 CTGTCCTGGCTGGCACAA 
                 5 
                 21 
               
               
                   
               
               
                 18640 
                 1 
                 176 
                 193 
                 Coding 
                 CTCACCAGTTTCTGTCCT 
                 0 
                 22 
               
               
                   
               
               
                 18641 
                 1 
                 179 
                 196 
                 Coding 
                 TCACTCACCAGTTTCTGT 
                 0 
                 23 
               
               
                   
               
               
                 18642 
                 1 
                 185 
                 202 
                 Coding 
                 GTGCAGTCACTCACCAGT 
                 0 
                 24 
               
               
                   
               
               
                 18643 
                 1 
                 190 
                 207 
                 Coding 
                 ACTCTGTGCAGTCACTCA 
                 0 
                 25 
               
               
                   
               
               
                 18644 
                 1 
                 196 
                 213 
                 Coding 
                 CAGTGAACTCTGTGCAGT 
                 5 
                 26 
               
               
                   
               
               
                 18645 
                 1 
                 205 
                 222 
                 Coding 
                 ATTCCGTTTCAGTGAACT 
                 0 
                 27 
               
               
                   
               
               
                 18646 
                 1 
                 211 
                 228 
                 Coding 
                 GAAGGCATTCCGTTTCAG 
                 9 
                 28 
               
               
                   
               
               
                 18647 
                 1 
                 222 
                 239 
                 Coding 
                 TTCACCGCAAGGAAGGCA 
                 0 
                 29 
               
               
                   
               
               
                 18648 
                 1 
                 250 
                 267 
                 Coding 
                 CTCTGTTCCAGGTGTCTA 
                 0 
                 30 
               
               
                   
               
               
                 18649 
                 1 
                 267 
                 284 
                 Coding 
                 CTGGTGGCAGTGTGTCTC 
                 0 
                 31 
               
               
                   
               
               
                 18650 
                 1 
                 286 
                 303 
                 Coding 
                 TGGGGTCGCAGTATTTGT 
                 0 
                 32 
               
               
                   
               
               
                 18651 
                 1 
                 289 
                 306 
                 Coding 
                 GGTTGGGGTCGCAGTATT 
                 0 
                 33 
               
               
                   
               
               
                 18652 
                 1 
                 292 
                 309 
                 Coding 
                 CTAGGTTGGGGTCGCAGT 
                 0 
                 34 
               
               
                   
               
               
                 18653 
                 1 
                 318 
                 335 
                 Coding 
                 GGTGCCCTTCTGCTGGAC 
                 20  
                 35 
               
               
                   
               
               
                 18654 
                 1 
                 322 
                 339 
                 Coding 
                 CTGAGGTGCCCTTCTGCT 
                 16  
                 36 
               
               
                   
               
               
                 18655 
                 1 
                 332 
                 349 
                 Coding 
                 GTGTCTGTTTCTGAGGTG 
                 0 
                 37 
               
               
                   
               
               
                 18656 
                 1 
                 334 
                 351 
                 Coding 
                 TGGTGTCTGTTTCTGAGG 
                 0 
                 38 
               
               
                   
               
               
                 18657 
                 1 
                 345 
                 362 
                 Coding 
                 ACAGGTGCAGATGGTGTC 
                 0 
                 39 
               
               
                   
               
               
                 18658 
                 1 
                 348 
                 365 
                 Coding 
                 TTCACAGGTGCAGATGGT 
                 0 
                 40 
               
               
                   
               
               
                 18659 
                 1 
                 360 
                 377 
                 Coding 
                 GTGCCAGCCTTCTTCACA 
                 6 
                 41 
               
               
                   
               
               
                 18660 
                 1 
                 364 
                 381 
                 Coding 
                 TACAGTGCCAGCCTTCTT 
                 8 
                 42 
               
               
                   
               
               
                 18661 
                 1 
                 391 
                 408 
                 Coding 
                 GGACACAGCTCTCACAGG 
                 0 
                 43 
               
               
                   
               
               
                 18662 
                 1 
                 395 
                 412 
                 Coding 
                 TGCAGGACACAGCTCTCA 
                 0 
                 44 
               
               
                   
               
               
                 18663 
                 1 
                 401 
                 418 
                 Coding 
                 GAGCGGTGCAGGACACAG 
                 0 
                 45 
               
               
                   
               
               
                 18664 
                 1 
                 416 
                 433 
                 Coding 
                 AAGCCGGGCGAGCATGAG 
                 0 
                 46 
               
               
                   
               
               
                 18665 
                 1 
                 432 
                 449 
                 Coding 
                 AATCTGCTTGACCCCAAA 
                 6 
                 47 
               
               
                   
               
               
                 18666 
                 1 
                 446 
                 463 
                 Coding 
                 GAAACCCCTGTAGCAATC 
                 0 
                 48 
               
               
                   
               
               
                 18667 
                 1 
                 452 
                 469 
                 Coding 
                 GTATCAGAAACCCCTGTA 
                 0 
                 49 
               
               
                   
               
               
                 18668 
                 1 
                 463 
                 480 
                 Coding 
                 GCTCGCAGATGGTATCAG 
                 0 
                 50 
               
               
                   
               
               
                 18669 
                 1 
                 468 
                 485 
                 Coding 
                 GCAGGGCTCGCAGATGGT 
                 34  
                 51 
               
               
                   
               
               
                 18670 
                 1 
                 471 
                 488 
                 Coding 
                 TGGGCAGGGCTCGCAGAT 
                 0 
                 52 
               
               
                   
               
               
                 18671 
                 1 
                 474 
                 491 
                 Coding 
                 GACTGGGCAGGGCTCGCA 
                 3 
                 53 
               
               
                   
               
               
                 18672 
                 1 
                 490 
                 507 
                 Coding 
                 CATTGGAGAAGAAGCCGA 
                 0 
                 54 
               
               
                   
               
               
                 18673 
                 1 
                 497 
                 514 
                 Coding 
                 GATGACACATTGGAGAAG 
                 0 
                 55 
               
               
                   
               
               
                 18674 
                 1 
                 500 
                 517 
                 Coding 
                 GCAGATGACACATTGGAG 
                 0 
                 56 
               
               
                   
               
               
                 18675 
                 1 
                 506 
                 523 
                 Coding 
                 TCGAAAGCAGATGACACA 
                 0 
                 57 
               
               
                   
               
               
                 18676 
                 1 
                 524 
                 541 
                 Coding 
                 GTCCAAGGGTGACATTTT 
                 8 
                 58 
               
               
                   
               
               
                 18677 
                 1 
                 532 
                 549 
                 Coding 
                 CACAGCTTGTCCAAGGGT 
                 0 
                 59 
               
               
                   
               
               
                 18678 
                 1 
                 539 
                 556 
                 Coding 
                 TTGGTCTCACAGCTTGTC 
                 0 
                 60 
               
               
                   
               
               
                 18679 
                 1 
                 546 
                 563 
                 Coding 
                 CAGGTCTTTGGTCTCACA 
                 7 
                 61 
               
               
                   
               
               
                 18680 
                 1 
                 558 
                 575 
                 Coding 
                 CTGTTGCACAACCAGGTC 
                 19  
                 62 
               
               
                   
               
               
                 18681 
                 1 
                 570 
                 587 
                 Coding 
                 GTTTGTGCCTGCCTGTTG 
                 2 
                 63 
               
               
                   
               
               
                 18682 
                 1 
                 575 
                 592 
                 Coding 
                 GTCTTGTTTGTGCCTGCC 
                 0 
                 64 
               
               
                   
               
               
                 18683 
                 1 
                 590 
                 607 
                 Coding 
                 CCACAGACAACATCAGTC 
                 0 
                 65 
               
               
                   
               
               
                 18684 
                 1 
                 597 
                 614 
                 Coding 
                 CTGGGGACCACAGACAAC 
                 0 
                 66 
               
               
                   
               
               
                 18685 
                 1 
                 607 
                 624 
                 Coding 
                 TCAGCCGATCCTGGGGAC 
                 0 
                 67 
               
               
                   
               
               
                 18686 
                 1 
                 621 
                 638 
                 Coding 
                 CACCACCAGGGCTCTCAG 
                 23  
                 68 
               
               
                   
               
               
                 18687 
                 1 
                 626 
                 643 
                 Coding 
                 GGGATCACCACCAGGGCT 
                 0 
                 69 
               
               
                   
               
               
                 18688 
                 1 
                 657 
                 674 
                 Coding 
                 GAGGATGGCAAACAGGAT 
                 0 
                 70 
               
               
                   
               
               
                 18689 
                 1 
                 668 
                 685 
                 Coding 
                 ACCAGCACCAAGAGGATG 
                 0 
                 71 
               
               
                   
               
               
                 18690 
                 1 
                 679 
                 696 
                 Coding 
                 TTTTGATAAAGACCAGCA 
                 0 
                 72 
               
               
                   
               
               
                 18691 
                 1 
                 703 
                 720 
                 Coding 
                 TATTGGTTGGCTTCTTGG 
                 0 
                 73 
               
               
                   
               
               
                 18692 
                 1 
                 729 
                 746 
                 Coding 
                 GGGTTCCTGCTTGGGGTG 
                 0 
                 74 
               
               
                   
               
               
                 18693 
                 1 
                 750 
                 767 
                 Coding 
                 GTCGGGAAAATTGATCTC 
                 0 
                 75 
               
               
                   
               
               
                 18694 
                 1 
                 754 
                 771 
                 Coding 
                 GATCGTCGGGAAAATTGA 
                 0 
                 76 
               
               
                   
               
               
                 18695 
                 1 
                 765 
                 782 
                 Coding 
                 GGAGCCAGGAAGATCGTC 
                 0 
                 77 
               
               
                   
               
               
                 18696 
                 1 
                 766 
                 783 
                 Coding 
                 TGGAGCCAGGAAGATCGT 
                 0 
                 78 
               
               
                   
               
               
                 18697 
                 1 
                 780 
                 797 
                 Coding 
                 TGGAGCAGCAGTGTTGGA 
                 0 
                 79 
               
               
                   
               
               
                 18698 
                 1 
                 796 
                 813 
                 Coding 
                 GTAAAGTCTCCTGCACTG 
                 0 
                 80 
               
               
                   
               
               
                 18699 
                 1 
                 806 
                 823 
                 Coding 
                 TGGCATCCATGTAAAGTC 
                 0 
                 81 
               
               
                   
               
               
                 18700 
                 1 
                 810 
                 827 
                 Coding 
                 CGGTTGGCATCCATGTAA 
                 0 
                 82 
               
               
                   
               
               
                 18701 
                 1 
                 834 
                 851 
                 Coding 
                 CTCTTTGCCATCCTCCTG 
                 4 
                 83 
               
               
                   
               
               
                 18702 
                 1 
                 861 
                 878 
                 Coding 
                 CTGTCTCTCCTGCACTGA 
                 0 
                 84 
               
               
                   
               
               
                 18703 
                 1 
                 873 
                 890 
                 Stop 
                 GGTGCAGCCTCACTGTCT 
                 0 
                 85 
               
               
                   
               
               
                 18704 
                 1 
                 910 
                 927 
                 3′ UTR 
                 AACTGCCTGTTTGCCCAC 
                 34  
                 86 
               
               
                   
               
               
                 18705 
                 1 
                 954 
                 971 
                 3′ UTR 
                 CTTCTGCCTGCACCCCTG 
                 0 
                 87 
               
               
                   
               
               
                 18706 
                 1 
                 976 
                 993 
                 3′ UTR 
                 ACTGACTGGGCATAGCTC 
                 0 
                 88 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Antisense Inhibition of Human CD40 In Vitro 
       [0325]    Antisense oligonucleotides targeted to a CD40 nucleic acid were tested for their effects on CD40 mRNA in vitro. T24 cells at a density of 7000 cells per well in a 96-well plate were treated with 150 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. CD40 mRNA levels were adjusted according to GAPDH content, a housekeeping gene. Results are presented as percent inhibition of CD40, relative to untreated control cells in Table 2. 
         [0326]    The antisense oligonucleotides were designed as 4-10-4 gapmers, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides and 5-methylcytosine substitutions. The antisense oligonucleotides comprise phosphorothioate backbones (internucleoside linkages) throughout. “5′ target site” indicates the 5′-most nucleotide which the antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK® Accession No X60592.1). Data are averages from three experiments. “ND” indicates a value was not determined. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having 
               
               
                 4-10-4 MOE wings and deoxy gap 
               
             
          
           
               
                   
                 Target 
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Oligo 
                 SEQ 
                 Start 
                 Stop 
                 Target 
                   
                 % 
                 SEQ 
               
               
                 ID 
                 ID NO 
                 Site 
                 Site 
                 Region 
                 Sequence (5′ to 3′) 
                 Inhibition 
                 ID NO 
               
               
                   
               
               
                 19211 
                 1 
                  18 
                  35 
                 5′ UTR 
                 CCAGGCGGCAGGACCACT 
                 76 
                  5 
               
               
                   
               
               
                 19212 
                 1 
                  20 
                  37 
                 5′ UTR 
                 GACCAGGCGGCAGGACCA 
                 77 
                  6 
               
               
                   
               
               
                 19213 
                 1 
                  26 
                  43 
                 5′ UTR 
                 AGGTGAGACCAGGCGGCA 
                 81 
                  7 
               
               
                   
               
               
                 19214 
                 1 
                  48 
                  65 
                 AUG 
                 CAGAGGCAGACGAACCAT 
                 24 
                  8 
               
               
                   
               
               
                 19215 
                 1 
                  49 
                  66 
                 Coding 
                 GCAGAGGCAGACGAACCA 
                 46 
                  9 
               
               
                   
               
               
                 19216 
                 1 
                  73 
                  90 
                 Coding 
                 GCAAGCAGCCCCAGAGGA 
                 66 
                 10 
               
               
                   
               
               
                 19217 
                 1 
                  78 
                  95 
                 Coding 
                 GGTCAGCAAGCAGCCCCA 
                 75 
                 11 
               
               
                   
               
               
                 19218 
                 1 
                  84 
                 101 
                 Coding 
                 GACAGCGGTCAGCAAGCA 
                 67 
                 12 
               
               
                   
               
               
                 19219 
                 1 
                  88 
                 105 
                 Coding 
                 GATGGACAGCGGTCAGCA 
                 65 
                 13 
               
               
                   
               
               
                 19220 
                 1 
                  92 
                 109 
                 Coding 
                 TCTGGATGGACAGCGGTC 
                 79 
                 14 
               
               
                   
               
               
                 19221 
                 1 
                  98 
                 115 
                 Coding 
                 GGTGGTTCTGGATGGACA 
                 81 
                 15 
               
               
                   
               
               
                 19222 
                 1 
                 101 
                 118 
                 Coding 
                 GTGGGTGGTTCTGGATGG 
                 58 
                 16 
               
               
                   
               
               
                 19223 
                 1 
                 104 
                 121 
                 Coding 
                 GCAGTGGGTGGTTCTGGA 
                 74 
                 17 
               
               
                   
               
               
                 19224 
                 1 
                 152 
                 169 
                 Coding 
                 CACAAAGAACAGCACTGA 
                 40 
                 18 
               
               
                   
               
               
                 19225 
                 1 
                 156 
                 173 
                 Coding 
                 CTGGCACAAAGAACAGCA 
                 60 
                 19 
               
               
                   
               
               
                 19226 
                 1 
                 162 
                 179 
                 Coding 
                 TCCTGGCTGGCACAAAGA 
                 10 
                 20 
               
               
                   
               
               
                 19227 
                 1 
                 165 
                 182 
                 Coding 
                 CTGTCCTGGCTGGCACAA 
                 24 
                 21 
               
               
                   
               
               
                 19228 
                 1 
                 176 
                 193 
                 Coding 
                 CTCACCAGTTTCTGTCCT 
                 22 
                 22 
               
               
                   
               
               
                 19229 
                 1 
                 179 
                 196 
                 Coding 
                 TCACTCACCAGTTTCTGT 
                 41 
                 23 
               
               
                   
               
               
                 19230 
                 1 
                 185 
                 202 
                 Coding 
                 GTGCAGTCACTCACCAGT 
                 82 
                 24 
               
               
                   
               
               
                 19231 
                 1 
                 190 
                 207 
                 Coding 
                 ACTCTGTGCAGTCACTCA 
                 38 
                 25 
               
               
                   
               
               
                 19232 
                 1 
                 196 
                 213 
                 Coding 
                 CAGTGAACTCTGTGCAGT 
                 40 
                 26 
               
               
                   
               
               
                 19233 
                 1 
                 205 
                 222 
                 Coding 
                 ATTCCGTTTCAGTGAACT 
                 56 
                 27 
               
               
                   
               
               
                 19234 
                 1 
                 211 
                 228 
                 Coding 
                 GAAGGCATTCCGTTTCAG 
                 32 
                 28 
               
               
                   
               
               
                 19235 
                 1 
                 222 
                 239 
                 Coding 
                 TTCACCGCAAGGAAGGCA 
                 61 
                 29 
               
               
                   
               
               
                 19236 
                 1 
                 250 
                 267 
                 Coding 
                 CTCTGTTCCAGGTGTCTA 
                 62 
                 30 
               
               
                   
               
               
                 19237 
                 1 
                 267 
                 284 
                 Coding 
                 CTGGTGGCAGTGTGTCTC 
                 70 
                 31 
               
               
                   
               
               
                 19238 
                 1 
                 286 
                 303 
                 Coding 
                 TGGGGTCGCAGTATTTGT 
                  0 
                 32 
               
               
                   
               
               
                 19239 
                 1 
                 289 
                 306 
                 Coding 
                 GGTTGGGGTCGCAGTATT 
                 19 
                 33 
               
               
                   
               
               
                 19240 
                 1 
                 292 
                 309 
                 Coding 
                 CTAGGTTGGGGTCGCAGT 
                 36 
                 34 
               
               
                   
               
               
                 19241 
                 1 
                 318 
                 335 
                 Coding 
                 GGTGCCCTTCTGCTGGAC 
                 79 
                 35 
               
               
                   
               
               
                 19242 
                 1 
                 322 
                 339 
                 Coding 
                 CTGAGGTGCCCTTCTGCT 
                 70 
                 36 
               
               
                   
               
               
                 19243 
                 1 
                 332 
                 349 
                 Coding 
                 GTGTCTGTTTCTGAGGTG 
                 63 
                 37 
               
               
                   
               
               
                 19244 
                 1 
                 334 
                 351 
                 Coding 
                 TGGTGTCTGTTTCTGAGG 
                 43 
                 38 
               
               
                   
               
               
                 19245 
                 1 
                 345 
                 362 
                 Coding 
                 ACAGGTGCAGATGGTGTC 
                 73 
                 39 
               
               
                   
               
               
                 19246 
                 1 
                 348 
                 365 
                 Coding 
                 TTCACAGGTGCAGATGGT 
                 48 
                 40 
               
               
                   
               
               
                 19247 
                 1 
                 360 
                 377 
                 Coding 
                 GTGCCAGCCTTCTTCACA 
                 61 
                 41 
               
               
                   
               
               
                 19248 
                 1 
                 364 
                 381 
                 Coding 
                 TACAGTGCCAGCCTTCTT 
                 47 
                 42 
               
               
                   
               
               
                 19249 
                 1 
                 391 
                 408 
                 Coding 
                 GGACACAGCTCTCACAGG 
                  0 
                 43 
               
               
                   
               
               
                 19250 
                 1 
                 395 
                 412 
                 Coding 
                 TGCAGGACACAGCTCTCA 
                 52 
                 44 
               
               
                   
               
               
                 19251 
                 1 
                 401 
                 418 
                 Coding 
                 GAGCGGTGCAGGACACAG 
                 50 
                 45 
               
               
                   
               
               
                 19252 
                 1 
                 416 
                 433 
                 Coding 
                 AAGCCGGGCGAGCATGAG 
                 32 
                 46 
               
               
                   
               
               
                 19253 
                 1 
                 432 
                 449 
                 Coding 
                 AATCTGCTTGACCCCAAA 
                  0 
                 47 
               
               
                   
               
               
                 19254 
                 1 
                 446 
                 463 
                 Coding 
                 GAAACCCCTGTAGCAATC 
                  0 
                 48 
               
               
                   
               
               
                 19255 
                 1 
                 452 
                 469 
                 Coding 
                 GTATCAGAAACCCCTGTA 
                 36 
                 49 
               
               
                   
               
               
                 19256 
                 1 
                 463 
                 480 
                 Coding 
                 GCTCGCAGATGGTATCAG 
                 65 
                 50 
               
               
                   
               
               
                 19257 
                 1 
                 468 
                 485 
                 Coding 
                 GCAGGGCTCGCAGATGGT 
                 75 
                 51 
               
               
                   
               
               
                 19258 
                 1 
                 471 
                 488 
                 Coding 
                 TGGGCAGGGCTCGCAGAT 
                  0 
                 52 
               
               
                   
               
               
                 19259 
                 1 
                 474 
                 491 
                 Coding 
                 GACTGGGCAGGGCTCGCA 
                 82 
                 53 
               
               
                   
               
               
                 19260 
                 1 
                 490 
                 507 
                 Coding 
                 CATTGGAGAAGAAGCCGA 
                 41 
                 54 
               
               
                   
               
               
                 19261 
                 1 
                 497 
                 514 
                 Coding 
                 GATGACACATTGGAGAAG 
                 14 
                 55 
               
               
                   
               
               
                 19262 
                 1 
                 500 
                 517 
                 Coding 
                 GCAGATGACACATTGGAG 
                 78 
                 56 
               
               
                   
               
               
                 19263 
                 1 
                 506 
                 523 
                 Coding 
                 TCGAAAGCAGATGACACA 
                 59 
                 57 
               
               
                   
               
               
                 19264 
                 1 
                 524 
                 541 
                 Coding 
                 GTCCAAGGGTGACATTTT 
                 71 
                 58 
               
               
                   
               
               
                 19265 
                 1 
                 532 
                 549 
                 Coding 
                 CACAGCTTGTCCAAGGGT 
                  0 
                 59 
               
               
                   
               
               
                 19266 
                 1 
                 539 
                 556 
                 Coding 
                 TTGGTCTCACAGCTTGTC 
                 46 
                 60 
               
               
                   
               
               
                 19267 
                 1 
                 546 
                 563 
                 Coding 
                 CAGGTCTTTGGTCTCACA 
                 64 
                 61 
               
               
                   
               
               
                 19268 
                 1 
                 558 
                 575 
                 Coding 
                 CTGTTGCACAACCAGGTC 
                 82 
                 62 
               
               
                   
               
               
                 19269 
                 1 
                 570 
                 587 
                 Coding 
                 GTTTGTGCCTGCCTGTTG 
                 70 
                 63 
               
               
                   
               
               
                 19270 
                 1 
                 575 
                 592 
                 Coding 
                 GTCTTGTTTGTGCCTGCC 
                 69 
                 64 
               
               
                   
               
               
                 19271 
                 1 
                 590 
                 607 
                 Coding 
                 CCACAGACAACATCAGTC 
                 11 
                 65 
               
               
                   
               
               
                 19272 
                 1 
                 597 
                 614 
                 Coding 
                 CTGGGGACCACAGACAAC 
                  9 
                 66 
               
               
                   
               
               
                 19273 
                 1 
                 607 
                 624 
                 Coding 
                 TCAGCCGATCCTGGGGAC 
                  0 
                 67 
               
               
                   
               
               
                 19274 
                 1 
                 621 
                 638 
                 Coding 
                 CACCACCAGGGCTCTCAG 
                 23 
                 68 
               
               
                   
               
               
                 19275 
                 1 
                 626 
                 643 
                 Coding 
                 GGGATCACCACCAGGGCT 
                 58 
                 69 
               
               
                   
               
               
                 19276 
                 1 
                 657 
                 674 
                 Coding 
                 GAGGATGGCAAACAGGAT 
                 49 
                 70 
               
               
                   
               
               
                 19277 
                 1 
                 668 
                 685 
                 Coding 
                 ACCAGCACCAAGAGGATG 
                 ND 
                 71 
               
               
                   
               
               
                 19278 
                 1 
                 679 
                 696 
                 Coding 
                 TTTTGATAAAGACCAGCA 
                 31 
                 72 
               
               
                   
               
               
                 19279 
                 1 
                 703 
                 720 
                 Coding 
                 TATTGGTTGGCTTCTTGG 
                 49 
                 73 
               
               
                   
               
               
                 19280 
                 1 
                 729 
                 746 
                 Coding 
                 GGGTTCCTGCTTGGGGTG 
                 14 
                 74 
               
               
                   
               
               
                 19281 
                 1 
                 750 
                 767 
                 Coding 
                 GTCGGGAAAATTGATCTC 
                 55 
                 75 
               
               
                   
               
               
                 19282 
                 1 
                 754 
                 771 
                 Coding 
                 GATCGTCGGGAAAATTGA 
                  0 
                 76 
               
               
                   
               
               
                 19283 
                 1 
                 765 
                 782 
                 Coding 
                 GGAGCCAGGAAGATCGTC 
                 69 
                 77 
               
               
                   
               
               
                 19284 
                 1 
                 766 
                 783 
                 Coding 
                 TGGAGCCAGGAAGATCGT 
                 54 
                 78 
               
               
                   
               
               
                 19285 
                 1 
                 780 
                 797 
                 Coding 
                 TGGAGCAGCAGTGTTGGA 
                 15 
                 79 
               
               
                   
               
               
                 19286 
                 1 
                 796 
                 813 
                 Coding 
                 GTAAAGTCTCCTGCACTG 
                 31 
                 80 
               
               
                   
               
               
                 19287 
                 1 
                 806 
                 823 
                 Coding 
                 TGGCATCCATGTAAAGTC 
                 65 
                 81 
               
               
                   
               
               
                 19288 
                 1 
                 810 
                 827 
                 Coding 
                 CGGTTGGCATCCATGTAA 
                 34 
                 82 
               
               
                   
               
               
                 19289 
                 1 
                 834 
                 851 
                 Coding 
                 CTCTTTGCCATCCTCCTG 
                 42 
                 83 
               
               
                   
               
               
                 19290 
                 1 
                 861 
                 878 
                 Coding 
                 CTGTCTCTCCTGCACTGA 
                 26 
                 84 
               
               
                   
               
               
                 19291 
                 1 
                 873 
                 890 
                 Stop 
                 GGTGCAGCCTCACTGTCT 
                 76 
                 85 
               
               
                   
               
               
                 19292 
                 1 
                 910 
                 927 
                 3′ UTR 
                 AACTGCCTGTTTGCCCAC 
                 63 
                 86 
               
               
                   
               
               
                 19293 
                 1 
                 954 
                 971 
                 3′ UTR 
                 CTTCTGCCTGCACCCCTG 
                  0 
                 87 
               
               
                   
               
               
                 19294 
                 1 
                 976 
                 993 
                 3′ UTR 
                 ACTGACTGGGCATAGCTC 
                 12 
                 88 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Antisense Inhibition of Human CD40 
       [0327]    Antisense oligonucleotides targeted to a CD40 nucleic acid were tested for their effects on CD40 mRNA in vitro. T24 cells at a density of 7000 cells per well in a 96-well plate were treated with 100 nM of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. CD40 mRNA levels were adjusted according to GAPDH content, a housekeeping gene. Results are presented as percent inhibition of CD40, relative to untreated control cells in Table 3. 
         [0328]    The antisense oligonucleotides were designed as 4-10-4 gapmers, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides. The antisense oligonucleotides comprise phosphorothioate backbones (internucleoside linkages) and 5-methylcytosine substitutions throughout. “5′ target site” indicates the 5′-most nucleotide which the antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK Accession No. X60592.1), SEQ ID NO: 2 (GENBANK® Accession No. H50598.1), and SEQ ID NO: 3 (GENBANK® Accession No. AA203290.1). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having 
               
               
                 4-10-4 MOE wings and deoxy gap 
               
             
          
           
               
                   
                 Target 
                 Target 
                 Target 
                   
                   
                   
               
               
                 Oligo 
                 SEQ 
                 Start 
                 Stop 
                   
                 % 
                 SEQ 
               
               
                 ID 
                 ID NO 
                 Site 
                 Site 
                 Sequence (5′ to 3′) 
                 Inhibition 
                 ID NO 
               
               
                   
               
               
                 26162 
                 1 
                  66 
                  83 
                 GCCCCAGAGGACGCACTG 
                  0 
                  89 
               
               
                   
               
               
                 26163 
                 1 
                  70 
                  87 
                 AGCAGCCCCAGAGGACGC 
                 98 
                  90 
               
               
                   
               
               
                 26164 
                 1 
                  74 
                  91 
                 AGCAAGCAGCCCCAGAGG 
                 47 
                  91 
               
               
                   
               
               
                 26165 
                 1 
                  80 
                  97 
                 GCGGTCAGCAAGCAGCCC 
                 54 
                  92 
               
               
                   
               
               
                 26167 
                 1 
                  95 
                 112 
                 GGTTCTGGATGGACAGCG 
                 66 
                  93 
               
               
                   
               
               
                 26168 
                 1 
                 102 
                 119 
                 AGTGGGTGGTTCTGGATG 
                 26 
                  94 
               
               
                   
               
               
                 26169 
                 1 
                 141 
                 158 
                 GCACTGACTGTTTATTAG 
                 43 
                  95 
               
               
                   
               
               
                 26170 
                 1 
                 154 
                 171 
                 GGCACAAAGAACAGCACT 
                 53 
                  96 
               
               
                   
               
               
                 26171 
                 1 
                 164 
                 181 
                 TGTCCTGGCTGGCACAAA 
                 29 
                  97 
               
               
                   
               
               
                 26172 
                 1 
                 171 
                 188 
                 CAGTTTCTGTCCTGGCTG 
                 48 
                  98 
               
               
                   
               
               
                 26173 
                 1 
                 180 
                 197 
                 GTCACTCACCAGTTTCTG 
                 47 
                  99 
               
               
                   
               
               
                 26174 
                 1 
                 210 
                 227 
                 AAGGCATTCCGTTTCAGT 
                 57 
                 100 
               
               
                   
               
               
                 26175 
                 1 
                 224 
                 241 
                 CTTTCACCGCAAGGAAGG 
                 34 
                 101 
               
               
                   
               
               
                 26176 
                 1 
                 250 
                 267 
                 CTCTGTTCCAGGTGTCTA 
                 78 
                  30 
               
               
                   
               
               
                 26177 
                 1 
                 257 
                 274 
                 TGTGTCTCTCTGTTCCAG 
                 57 
                 102 
               
               
                   
               
               
                 26178 
                 1 
                 264 
                 281 
                 GTGGCAGTGTGTCTCTCT 
                  0 
                 103 
               
               
                   
               
               
                 26179 
                 1 
                 314 
                 331 
                 CCCTTCTGCTGGACCCGA 
                 58 
                 104 
               
               
                   
               
               
                 26180 
                 1 
                 321 
                 338 
                 TGAGGTGCCCTTCTGCTG 
                 69 
                 105 
               
               
                   
               
               
                 26181 
                 1 
                 329 
                 346 
                 TCTGTTTCTGAGGTGCCC 
                 44 
                 106 
               
               
                   
               
               
                 26182 
                 1 
                 336 
                 353 
                 GATGGTGTCTGTTTCTGA 
                 12 
                 107 
               
               
                   
               
               
                 26183 
                 1 
                 364 
                 381 
                 TACAGTGCCAGCCTTCTT 
                 14 
                  42 
               
               
                   
               
               
                 26184 
                 1 
                 445 
                 462 
                 AAACCCCTGTAGCAATCT 
                 15 
                 108 
               
               
                   
               
               
                 26185 
                 1 
                 460 
                 477 
                 CGCAGATGGTATCAGAAA 
                 53 
                 109 
               
               
                   
               
               
                 26186 
                 1 
                 469 
                 486 
                 GGCAGGGCTCGCAGATGG 
                 79 
                 110 
               
               
                   
               
               
                 26202 
                 1 
                 485 
                 502 
                 GAGAAGAAGCCGACTGGG 
                  0 
                 111 
               
               
                   
               
               
                 26187 
                 1 
                 487 
                 504 
                 TGGAGAAGAAGCCGACTG 
                 23 
                 112 
               
               
                   
               
               
                 26204 
                 1 
                 489 
                 506 
                 ATTGGAGAAGAAGCCGAC 
                  0 
                 113 
               
               
                   
               
               
                 26205 
                 1 
                 491 
                 508 
                 ACATTGGAGAAGAAGCCG 
                  4 
                 114 
               
               
                   
               
               
                 26206 
                 1 
                 493 
                 510 
                 ACACATTGGAGAAGAAGC 
                  0 
                 115 
               
               
                   
               
               
                 26207 
                 1 
                 495 
                 512 
                 TGACACATTGGAGAAGAA 
                 46 
                 116 
               
               
                   
               
               
                 26188 
                 1 
                 496 
                 513 
                 ATGACACATTGGAGAAGA 
                  0 
                 117 
               
               
                   
               
               
                 26208 
                 1 
                 497 
                 514 
                 GATGACACATTGGAGAAG 
                  0 
                  55 
               
               
                   
               
               
                 26189 
                 1 
                 503 
                 520 
                 AAAGCAGATGACACATTG 
                  6 
                 118 
               
               
                   
               
               
                 26209 
                 1 
                 524 
                 541 
                 GTCCAAGGGTGACATTTT 
                 53 
                  58 
               
               
                   
               
               
                 26210 
                 1 
                 545 
                 562 
                 AGGTCTTTGGTCTCACAG 
                 81 
                 119 
               
               
                   
               
               
                 26211 
                 1 
                 555 
                 572 
                 TTGCACAACCAGGTCTTT 
                 48 
                 120 
               
               
                   
               
               
                 26212 
                 1 
                 570 
                 587 
                 GTTTGTGCCTGCCTGTTG 
                 76 
                  63 
               
               
                   
               
               
                 26213 
                 1 
                 572 
                 589 
                 TTGTTTGTGCCTGCCTGT 
                 50 
                 121 
               
               
                   
               
               
                 26214 
                 1 
                 574 
                 591 
                 TCTTGTTTGTGCCTGCCT 
                 87 
                 122 
               
               
                   
               
               
                 26215 
                 1 
                 576 
                 593 
                 AGTCTTGTTTGTGCCTGC 
                 83 
                 123 
               
               
                   
               
               
                 26216 
                 1 
                 577 
                 594 
                 CAGTCTTGTTTGTGCCTG 
                 80 
                 124 
               
               
                   
               
               
                 26217 
                 1 
                 578 
                 595 
                 TCAGTCTTGTTTGTGCCT 
                 88 
                 125 
               
               
                   
               
               
                 26218 
                 1 
                 580 
                 597 
                 CATCAGTCTTGTTTGTGC 
                 52 
                 126 
               
               
                   
               
               
                 26219 
                 1 
                 590 
                 607 
                 CCACAGACAACATCAGTC 
                 16 
                  65 
               
               
                   
               
               
                 26220 
                 1 
                 592 
                 609 
                 GACCACAGACAACATCAG 
                 11 
                 127 
               
               
                   
               
               
                 26221 
                 1 
                 594 
                 611 
                 GGGACCACAGACAACATC 
                 40 
                 128 
               
               
                   
               
               
                 26222 
                 1 
                 622 
                 639 
                 TCACCACCAGGGCTCTCA 
                 37 
                 129 
               
               
                   
               
               
                 26223 
                 1 
                 624 
                 641 
                 GATCACCACCAGGGCTCT 
                 82 
                 130 
               
               
                   
               
               
                 26224 
                 1 
                 658 
                 675 
                 AGAGGATGGCAAACAGGA 
                 33 
                 131 
               
               
                   
               
               
                 26225 
                 1 
                 659 
                 676 
                 AAGAGGATGGCAAACAGG 
                  0 
                 132 
               
               
                   
               
               
                 26226 
                 1 
                 660 
                 677 
                 CAAGAGGATGGCAAACAG 
                  0 
                 133 
               
               
                   
               
               
                 26227 
                 1 
                 669 
                 686 
                 GACCAGCACCAAGAGGAT 
                 57 
                 134 
               
               
                   
               
               
                 26228 
                 1 
                 671 
                 688 
                 AAGACCAGCACCAAGAGG 
                 35 
                 135 
               
               
                   
               
               
                 26229 
                 1 
                 673 
                 690 
                 TAAAGACCAGCACCAAGA 
                 13 
                 136 
               
               
                   
               
               
                 26230 
                 1 
                 676 
                 693 
                 TGATAAAGACCAGCACCA 
                  0 
                 137 
               
               
                   
               
               
                 26231 
                 1 
                 678 
                 695 
                 TTTGATAAAGACCAGCAC 
                 26 
                 138 
               
               
                   
               
               
                 26232 
                 2 
                 375 
                 392 
                 ACTCTCTTTGCCCATCCT 
                  0 
                 139 
               
               
                   
               
               
                 26233 
                 2 
                 377 
                 394 
                 CGACTCTCTTTGCCCATC 
                 31 
                 140 
               
               
                   
               
               
                 26234 
                 2 
                 380 
                 397 
                 ATGCGACTCTCTTTGCCC 
                 12 
                 141 
               
               
                   
               
               
                 26235 
                 2 
                 382 
                 399 
                 AAATGCGACTCTCTTTGC 
                 36 
                 142 
               
               
                   
               
               
                 26236 
                 2 
                 385 
                 402 
                 CTGAAATGCGACTCTCTT 
                 51 
                 143 
               
               
                   
               
               
                 26237 
                 2 
                 387 
                 404 
                 AACTGAAATGCGACTCTC 
                  0 
                 144 
               
               
                   
               
               
                 26238 
                 2 
                 406 
                 423 
                 CTTCACTGTCTCTCCCTG 
                  0 
                 145 
               
               
                   
               
               
                 26239 
                 2 
                 407 
                 424 
                 CCTTCACTGTCTCTCCCT 
                 56 
                 146 
               
               
                   
               
               
                 26240 
                 2 
                 409 
                 426 
                 AACCTTCACTGTCTCTCC 
                  0 
                 147 
               
               
                   
               
               
                 26190 
                 3 
                 520 
                 537 
                 GATCACCACAGGCTCTCA 
                  0 
                 148 
               
               
                   
               
               
                 26191 
                 3 
                 565 
                 582 
                 TGATAAGACAGCACCAAG 
                  9 
                 149 
               
               
                   
               
               
                 26192 
                 3 
                 584 
                 601 
                 GGTAGTTCTTGCCACTTT 
                  0 
                 150 
               
               
                   
               
               
                 26193 
                 3 
                 593 
                 610 
                 GGGCCTATGGGTAGTTCT 
                  0 
                 151 
               
               
                   
               
               
                 26194 
                 3 
                 617 
                 634 
                 ATTATCTCTGGGTCTGCT 
                  9 
                 152 
               
               
                   
               
               
                 26195 
                 3 
                 646 
                 663 
                 ACTGACACATTTGAGCAG 
                  0 
                 153 
               
               
                   
               
               
                 26196 
                 3 
                 654 
                 671 
                 GACTCCCTACTGACACAT 
                  0 
                 154 
               
               
                   
               
               
                 26197 
                 3 
                 689 
                 706 
                 CAAAGAGCGGTTCTCCAC 
                  0 
                 155 
               
               
                   
               
               
                 26198 
                 3 
                 696 
                 713 
                 AATTCTCCAAAGAGCGGT 
                  0 
                 156 
               
               
                   
               
               
                 26199 
                 3 
                 728 
                 745 
                 TCTTGACATCCTTTTCAT 
                  0 
                 157 
               
               
                   
               
               
                 26200 
                 3 
                 736 
                 753 
                 CCCACCTATCTTGACATC 
                  0 
                 158 
               
               
                   
               
               
                 26201 
                 3 
                 791 
                 808 
                 AGGCCGAGAGTTCAAAAT 
                  0 
                 159 
               
               
                   
               
             
          
         
       
     
       Example 4 
     Antisense Inhibition of Human CD40 
       [0329]    Antisense oligonucleotides targeted to a CD40 nucleic acid were tested for their effects on CD40 mRNA in vitro. A549 cells at a density of 5000 cells per well in a 96-well plate were treated with 120 nM of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. CD40 primer probe set LTS37 was used to measure mRNA levels. CD40 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of CD40, relative to untreated control cells in Table 4. 
         [0330]    The antisense oligonucleotides were designed as 4-10-4 gapmers, 5-10-5 gapmers, or 2-15-3 gapmers, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides. The motif for each compound is indicated by the column labeled “motif” The antisense oligonucleotides comprise phosphorothioate backbones (internucleoside linkages) and 5-methylcytosine substitutions throughout. “5′ target site” indicates the 5′-most nucleotide which the antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK Accession No. X60592.1) or SEQ ID NO: 4 (nucleotides 9797000 to 9813000 of GENBANK Accession No. NT — 011362.9). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having 4-10-4 MOE wings 
               
               
                 and deoxy gap, 5-10-5 MOE wings and deoxy gap, and 2-15-3 MOE wings and deoxy gap 
               
             
          
           
               
                   
                 Target 
                   
                 Target 
                 Target 
                   
                   
                   
               
               
                 Oligo 
                 SEQ 
                   
                 Start 
                 Stop 
                   
                 % 
                 SEQ 
               
               
                 ID 
                 ID NO 
                 Motif 
                 Site 
                 Site 
                 Sequence (5′ to 3′) 
                 Inhibition 
                 ID NO 
               
               
                   
               
               
                  26163 
                 4 
                 4-10-4 
                 2914 
                 2931 
                 AGCAGCCCCAGAGGACGC 
                 74 
                  90 
               
               
                   
               
               
                 396243 
                 4 
                 5-10-5 
                 2728 
                 2747 
                 CCAGCAATTCACCGCGCAGG 
                  0 
                 160 
               
               
                   
               
               
                 396320 
                 4 
                 2-15-3 
                 2728 
                 2747 
                 CCAGCAATTCACCGCGCAGG 
                  0 
                 160 
               
               
                   
               
               
                 396199 
                 4 
                 5-10-5 
                 2892 
                 2911 
                 TGCAGAGGCAGACGAACCAT 
                 75 
                 161 
               
               
                   
               
               
                 396276 
                 4 
                 2-15-3 
                 2892 
                 2911 
                 TGCAGAGGCAGACGAACCAT 
                 55 
                 161 
               
               
                   
               
               
                 396200 
                 4 
                 5-10-5 
                 2904 
                 2923 
                 CAGAGGACGCACTGCAGAGG 
                 79 
                 162 
               
               
                   
               
               
                 396277 
                 4 
                 2-15-3 
                 2904 
                 2923 
                 CAGAGGACGCACTGCAGAGG 
                 69 
                 162 
               
               
                   
               
               
                 396201 
                 4 
                 5-10-5 
                 2913 
                 2932 
                 AAGCAGCCCCAGAGGACGCA 
                 76 
                 163 
               
               
                   
               
               
                 396278 
                 4 
                 2-15-3 
                 2913 
                 2932 
                 AAGCAGCCCCAGAGGACGCA 
                 78 
                 163 
               
               
                   
               
               
                 396202 
                 4 
                 5-10-5 
                 2924 
                 2943 
                 CAGCGGTCAGCAAGCAGCCC 
                 68 
                 164 
               
               
                   
               
               
                 396279 
                 4 
                 2-15-3 
                 2924 
                 2943 
                 CAGCGGTCAGCAAGCAGCCC 
                 88 
                 164 
               
               
                   
               
               
                 396244 
                 4 
                 5-10-5 
                 2928 
                 2947 
                 CTCACAGCGGTCAGCAAGCA 
                 86 
                 165 
               
               
                   
               
               
                 396321 
                 4 
                 2-15-3 
                 2928 
                 2947 
                 CTCACAGCGGTCAGCAAGCA 
                 75 
                 165 
               
               
                   
               
               
                 396245 
                 4 
                 5-10-5 
                 3349 
                 3368 
                 GCTGGCAAGGAGATGATAAC 
                 51 
                 166 
               
               
                   
               
               
                 396322 
                 4 
                 2-15-3 
                 3349 
                 3368 
                 GCTGGCAAGGAGATGATAAC 
                 54 
                 166 
               
               
                   
               
               
                 396246 
                 4 
                 5-10-5 
                 3480 
                 3499 
                 AGGTTGGAACACCCAAGATA 
                 69 
                 167 
               
               
                   
               
               
                 396323 
                 4 
                 2-15-3 
                 3480 
                 3499 
                 AGGTTGGAACACCCAAGATA 
                 78 
                 167 
               
               
                   
               
               
                 396247 
                 4 
                 5-10-5 
                 3649 
                 3668 
                 GGAGAAACCCCTGGTTTCTC 
                 45 
                 168 
               
               
                   
               
               
                 396324 
                 4 
                 2-15-3 
                 3649 
                 3668 
                 GGAGAAACCCCTGGTTTCTC 
                 26 
                 168 
               
               
                   
               
               
                 396248 
                 4 
                 5-10-5 
                 3860 
                 3879 
                 TCATTCCTGCCCAGGCTTCA 
                 43 
                 169 
               
               
                   
               
               
                 396325 
                 4 
                 2-15-3 
                 3860 
                 3879 
                 TCATTCCTGCCCAGGCTTCA 
                 39 
                 169 
               
               
                   
               
               
                 396249 
                 4 
                 5-10-5 
                 3950 
                 3969 
                 TCAGGTGAAAGTGAAAGCTG 
                 68 
                 170 
               
               
                   
               
               
                 396326 
                 4 
                 2-15-3 
                 3950 
                 3969 
                 TCAGGTGAAAGTGAAAGCTG 
                 69 
                 170 
               
               
                   
               
               
                 396250 
                 4 
                 5-10-5 
                 4490 
                 4509 
                 TACCATCTTCAAACACATGA 
                 79 
                 171 
               
               
                   
               
               
                 396327 
                 4 
                 2-15-3 
                 4490 
                 4509 
                 TACCATCTTCAAACACATGA 
                 71 
                 171 
               
               
                   
               
               
                 396251 
                 4 
                 5-10-5 
                 4604 
                 4623 
                 TTACCCAAAATGGGAAAGGA 
                 86 
                 172 
               
               
                   
               
               
                 396328 
                 4 
                 2-15-3 
                 4604 
                 4623 
                 TTACCCAAAATGGGAAAGGA 
                 48 
                 172 
               
               
                   
               
               
                 396252 
                 4 
                 5-10-5 
                 4810 
                 4829 
                 GAAAGAATACATGTATATGG 
                 72 
                 173 
               
               
                   
               
               
                 396329 
                 4 
                 2-15-3 
                 4810 
                 4829 
                 GAAAGAATACATGTATATGG 
                 10 
                 173 
               
               
                   
               
               
                 396253 
                 4 
                 5-10-5 
                 4944 
                 4963 
                 AGAGTCAGACAGCTTTAGAC 
                 78 
                 174 
               
               
                   
               
               
                 396330 
                 4 
                 2-15-3 
                 4944 
                 4963 
                 AGAGTCAGACAGCTTTAGAC 
                 79 
                 174 
               
               
                   
               
               
                 396254 
                 4 
                 5-10-5 
                 5651 
                 5670 
                 GTACCACCCATGCTATTAAT 
                 79 
                 175 
               
               
                   
               
               
                 396331 
                 4 
                 2-15-3 
                 5651 
                 5670 
                 GTACCACCCATGCTATTAAT 
                 84 
                 175 
               
               
                   
               
               
                 396255 
                 4 
                 5-10-5 
                 5740 
                 5759 
                 ACAGTGACAGAGTCCAAATG 
                 85 
                 176 
               
               
                   
               
               
                 396332 
                 4 
                 2-15-3 
                 5740 
                 5759 
                 ACAGTGACAGAGTCCAAATG 
                 75 
                 176 
               
               
                   
               
               
                 396256 
                 4 
                 5-10-5 
                 5830 
                 5849 
                 AATGTAAAGCTGGAAGGGTA 
                 52 
                 177 
               
               
                   
               
               
                 396333 
                 4 
                 2-15-3 
                 5830 
                 5849 
                 AATGTAAAGCTGGAAGGGTA 
                 37 
                 177 
               
               
                   
               
               
                 396257 
                 4 
                 5-10-5 
                 5964 
                 5983 
                 GGGCTATGTTTAGCACTTGG 
                 79 
                 178 
               
               
                   
               
               
                 396334 
                 4 
                 2-15-3 
                 5964 
                 5983 
                 GGGCTATGTTTAGCACTTGG 
                 73 
                 178 
               
               
                   
               
               
                 396258 
                 4 
                 5-10-5 
                 6078 
                 6097 
                 GGGCTTGATGCCTGAGTCAT 
                 73 
                 179 
               
               
                   
               
               
                 396335 
                 4 
                 2-15-3 
                 6078 
                 6097 
                 GGGCTTGATGCCTGAGTCAT 
                 40 
                 179 
               
               
                   
               
               
                 396259 
                 4 
                 5-10-5 
                 6251 
                 6270 
                 TGAAGTGCAAGTCAAAACAG 
                 52 
                 180 
               
               
                   
               
               
                 396336 
                 4 
                 2-15-3 
                 6251 
                 6270 
                 TGAAGTGCAAGTCAAAACAG 
                 44 
                 180 
               
               
                   
               
               
                 396260 
                 4 
                 5-10-5 
                 6332 
                 6351 
                 GCAATTTGAAGGGATCTTGA 
                 68 
                 181 
               
               
                   
               
               
                 396337 
                 4 
                 2-15-3 
                 6332 
                 6351 
                 GCAATTTGAAGGGATCTTGA 
                 42 
                 181 
               
               
                   
               
               
                 396203 
                 4 
                 5-10-5 
                 6374 
                 6393 
                 CATGCAGTGGGTGGTTCTGG 
                 77 
                 182 
               
               
                   
               
               
                 396280 
                 4 
                 2-15-3 
                 6374 
                 6393 
                 CATGCAGTGGGTGGTTCTGG 
                 83 
                 182 
               
               
                   
               
               
                 396204 
                 4 
                 5-10-5 
                 6385 
                 6404 
                 GTTTTTCTCTGCATGCAGTG 
                 78 
                 183 
               
               
                   
               
               
                 396281 
                 4 
                 2-15-3 
                 6385 
                 6404 
                 GTTTTTCTCTGCATGCAGTG 
                 70 
                 183 
               
               
                   
               
               
                 396205 
                 4 
                 5-10-5 
                 6424 
                 6443 
                 GCTGGCACAAAGAACAGCAC 
                 61 
                 184 
               
               
                   
               
               
                 396282 
                 4 
                 2-15-3 
                 6424 
                 6443 
                 GCTGGCACAAAGAACAGCAC 
                 65 
                 184 
               
               
                   
               
               
                 396261 
                 4 
                 5-10-5 
                 6709 
                 6728 
                 CACTAACCACACAATGATCA 
                 85 
                 185 
               
               
                   
               
               
                 396338 
                 4 
                 2-15-3 
                 6709 
                 6728 
                 CACTAACCACACAATGATCA 
                 62 
                 185 
               
               
                   
               
               
                 396206 
                 4 
                 5-10-5 
                 6787 
                 6806 
                 TGTGCAGTCACTCACCAGTT 
                 83 
                 186 
               
               
                   
               
               
                 396283 
                 4 
                 2-15-3 
                 6787 
                 6806 
                 TGTGCAGTCACTCACCAGTT 
                 72 
                 186 
               
               
                   
               
               
                 396207 
                 4 
                 5-10-5 
                 6838 
                 6857 
                 GTCTAGGAATTCGCTTTCAC 
                 95 
                 187 
               
               
                   
               
               
                 396284 
                 4 
                 2-15-3 
                 6838 
                 6857 
                 GTCTAGGAATTCGCTTTCAC 
                 85 
                 187 
               
               
                   
               
               
                 396208 
                 4 
                 5-10-5 
                 6843 
                 6862 
                 CAGGTGTCTAGGAATTCGCT 
                 98 
                 188 
               
               
                   
               
               
                 396285 
                 4 
                 2-15-3 
                 6843 
                 6862 
                 CAGGTGTCTAGGAATTCGCT 
                 90 
                 188 
               
               
                   
               
               
                 396209 
                 4 
                 5-10-5 
                 6883 
                 6902 
                 GTCGCAGTATTTGTGCTGGT 
                 84 
                 189 
               
               
                   
               
               
                 396286 
                 4 
                 2-15-3 
                 6883 
                 6902 
                 GTCGCAGTATTTGTGCTGGT 
                 86 
                 189 
               
               
                   
               
               
                 396262 
                 4 
                 5-10-5 
                 7154 
                 7173 
                 ACCCGAAGCCCTAGGTCTGA 
                 92 
                 190 
               
               
                   
               
               
                 396339 
                 4 
                 2-15-3 
                 7154 
                 7173 
                 ACCCGAAGCCCTAGGTCTGA 
                 84 
                 190 
               
               
                   
               
               
                 396210 
                 4 
                 5-10-5 
                 7158 
                 7177 
                 CTGGACCCGAAGCCCTAGGT 
                 82 
                 191 
               
               
                   
               
               
                 396287 
                 4 
                 2-15-3 
                 7158 
                 7177 
                 CTGGACCCGAAGCCCTAGGT 
                 90 
                 191 
               
               
                   
               
               
                 396211 
                 4 
                 5-10-5 
                 7163 
                 7182 
                 TTCTGCTGGACCCGAAGCCC 
                 65 
                 192 
               
               
                   
               
               
                 396288 
                 4 
                 2-15-3 
                 7163 
                 7182 
                 TTCTGCTGGACCCGAAGCCC 
                 80 
                 192 
               
               
                   
               
               
                 396212 
                 4 
                 5-10-5 
                 7204 
                 7223 
                 CTTCTTCACAGGTGCAGATG 
                 79 
                 193 
               
               
                   
               
               
                 396289 
                 4 
                 2-15-3 
                 7204 
                 7223 
                 CTTCTTCACAGGTGCAGATG 
                 72 
                 193 
               
               
                   
               
               
                 396263 
                 4 
                 5-10-5 
                 7590 
                 7609 
                 AGCCAGTGGCCAGGCAGGAC 
                 70 
                 194 
               
               
                   
               
               
                 396340 
                 4 
                 2-15-3 
                 7590 
                 7609 
                 AGCCAGTGGCCAGGCAGGAC 
                 56 
                 194 
               
               
                   
               
               
                 396214 
                 4 
                 5-10-5 
                 7704 
                 7723 
                 GAAGAAGCCGACTGGGCAGG 
                 76 
                 195 
               
               
                   
               
               
                 396291 
                 4 
                 2-15-3 
                 7704 
                 7723 
                 GAAGAAGCCGACTGGGCAGG 
                 80 
                 195 
               
               
                   
               
               
                 396215 
                 4 
                 5-10-5 
                 7709 
                 7728 
                 TTGGAGAAGAAGCCGACTGG 
                 77 
                 196 
               
               
                   
               
               
                 396292 
                 4 
                 2-15-3 
                 7709 
                 7728 
                 TTGGAGAAGAAGCCGACTGG 
                 80 
                 196 
               
               
                   
               
               
                 396216 
                 4 
                 5-10-5 
                 7718 
                 7737 
                 GATGACACATTGGAGAAGAA 
                 76 
                 197 
               
               
                   
               
               
                 396293 
                 4 
                 2-15-3 
                 7718 
                 7737 
                 GATGACACATTGGAGAAGAA 
                 65 
                 197 
               
               
                   
               
               
                 396264 
                 4 
                 5-10-5 
                 7953 
                 7972 
                 TGTCTATTACCTCAAAGAGA 
                 89 
                 198 
               
               
                   
               
               
                 396341 
                 4 
                 2-15-3 
                 7953 
                 7972 
                 TGTCTATTACCTCAAAGAGA 
                 72 
                 198 
               
               
                   
               
               
                 396265 
                 4 
                 5-10-5 
                 8492 
                 8511 
                 ACAGTGTGTTCAGAGGATTG 
                 82 
                 199 
               
               
                   
               
               
                 396342 
                 4 
                 2-15-3 
                 8492 
                 8511 
                 ACAGTGTGTTCAGAGGATTG 
                 67 
                 199 
               
               
                   
               
               
                 396266 
                 4 
                 5-10-5 
                 9755 
                 9774 
                 ACAATACACTTTACATGTTT 
                 90 
                 200 
               
               
                   
               
               
                 396343 
                 4 
                 2-15-3 
                 9755 
                 9774 
                 ACAATACACTTTACATGTTT 
                 63 
                 200 
               
               
                   
               
               
                 396267 
                 4 
                 5-10-5 
                 10414  
                 10433  
                 ATTGTGTCTTTAGAACCAGA 
                 84 
                 201 
               
               
                   
               
               
                 396344 
                 4 
                 2-15-3 
                 10414  
                 10433  
                 ATTGTGTCTTTAGAACCAGA 
                 59 
                 201 
               
               
                   
               
               
                 396268 
                 4 
                 5-10-5 
                 10528  
                 10547  
                 GGGCCCTAAAGGATGTAAAA 
                 34 
                 202 
               
               
                   
               
               
                 396345 
                 4 
                 2-15-3 
                 10528  
                 10547  
                 GGGCCCTAAAGGATGTAAAA 
                 76 
                 202 
               
               
                   
               
               
                 396217 
                 4 
                 5-10-5 
                 11218  
                 11237  
                 CAGTCTTGTTTGTGCCTGCC 
                 70 
                 203 
               
               
                   
               
               
                 396294 
                 4 
                 2-15-3 
                 11218  
                 11237  
                 CAGTCTTGTTTGTGCCTGCC 
                 79 
                 203 
               
               
                   
               
               
                 396269 
                 4 
                 5-10-5 
                 11244  
                 11263  
                 TGTCCAGGACTCACCACAGA 
                 77 
                 204 
               
               
                   
               
               
                 396346 
                 4 
                 2-15-3 
                 11244  
                 11263  
                 TGTCCAGGACTCACCACAGA 
                 83 
                 204 
               
               
                   
               
               
                 396270 
                 4 
                 5-10-5 
                 11801  
                 11820  
                 TATGGCACCTTCTTAAATAT 
                 85 
                 205 
               
               
                   
               
               
                 396347 
                 4 
                 2-15-3 
                 11801  
                 11820  
                 TATGGCACCTTCTTAAATAT 
                 81 
                 205 
               
               
                   
               
               
                 396271 
                 4 
                 5-10-5 
                 12248  
                 12267  
                 TGCTTTTGGTATAGAAGAGT 
                 86 
                 206 
               
               
                   
               
               
                 396348 
                 4 
                 2-15-3 
                 12248  
                 12267  
                 TGCTTTTGGTATAGAAGAGT 
                 76 
                 206 
               
               
                   
               
               
                 396235 
                 4 
                 5-10-5 
                 12526  
                 12545  
                 AAATGTGGCTGGCAGATGTC 
                 79 
                 207 
               
               
                   
               
               
                 396312 
                 4 
                 2-15-3 
                 12526  
                 12545  
                 AAATGTGGCTGGCAGATGTC 
                 82 
                 207 
               
               
                   
               
               
                 396236 
                 4 
                 5-10-5 
                 12572  
                 12591  
                 GTCAGAGCTCATCTACATCA 
                 87 
                 208 
               
               
                   
               
               
                 396313 
                 4 
                 2-15-3 
                 12572  
                 12591  
                 GTCAGAGCTCATCTACATCA 
                 82 
                 208 
               
               
                   
               
               
                 396237 
                 4 
                 5-10-5 
                 12754  
                 12773  
                 CTGATAAAGACCAGCACCAA 
                 69 
                 209 
               
               
                   
               
               
                 396314 
                 4 
                 2-15-3 
                 12754  
                 12773  
                 CTGATAAAGACCAGCACCAA 
                 70 
                 209 
               
               
                   
               
               
                 396238 
                 4 
                 5-10-5 
                 12762  
                 12781  
                 AGGACTCACTGATAAAGACC 
                 69 
                 210 
               
               
                   
               
               
                 396315 
                 4 
                 2-15-3 
                 12762  
                 12781  
                 AGGACTCACTGATAAAGACC 
                 43 
                 210 
               
               
                   
               
               
                 396239 
                 4 
                 5-10-5 
                 12982  
                 13001  
                 CAGACTCTGAATCAGTTTTA 
                 78 
                 211 
               
               
                   
               
               
                 396316 
                 4 
                 2-15-3 
                 12982  
                 13001  
                 CAGACTCTGAATCAGTTTTA 
                 70 
                 211 
               
               
                   
               
               
                 396240 
                 4 
                 5-10-5 
                 13021  
                 13040  
                 CAGTCCCCAATTCTGCTGCC 
                 43 
                 212 
               
               
                   
               
               
                 396317 
                 4 
                 2-15-3 
                 13021  
                 13040  
                 CAGTCCCCAATTCTGCTGCC 
                 70 
                 212 
               
               
                   
               
               
                 396241 
                 4 
                 5-10-5 
                 13107  
                 13126  
                 CCAGTGTTAGGCTCTGCCAG 
                 76 
                 213 
               
               
                   
               
               
                 396318 
                 4 
                 2-15-3 
                 13107  
                 13126  
                 CCAGTGTTAGGCTCTGCCAG 
                 85 
                 213 
               
               
                   
               
               
                 396242 
                 4 
                 5-10-5 
                 13134  
                 13153  
                 GAATGCCAGGAAAGGAGTGA 
                 69 
                 214 
               
               
                   
               
               
                 396319 
                 4 
                 2-15-3 
                 13134  
                 13153  
                 GAATGCCAGGAAAGGAGTGA 
                 84 
                 214 
               
               
                   
               
               
                 396272 
                 4 
                 5-10-5 
                 13171  
                 13190  
                 CAGCCCCAAGGCCCAAAGAT 
                 48 
                 215 
               
               
                   
               
               
                 396349 
                 4 
                 2-15-3 
                 13171  
                 13190  
                 CAGCCCCAAGGCCCAAAGAT 
                 57 
                 215 
               
               
                   
               
               
                 396220 
                 4 
                 5-10-5 
                 13491  
                 13510  
                 CTGCACTGGAGCAGCAGTGT 
                 81 
                 216 
               
               
                   
               
               
                 396297 
                 4 
                 2-15-3 
                 13491  
                 13510  
                 CTGCACTGGAGCAGCAGTGT 
                 74 
                 216 
               
               
                   
               
               
                 396221 
                 4 
                 5-10-5 
                 13517  
                 13536  
                 ACCGGTTGGCATCCATGTAA 
                 61 
                 217 
               
               
                   
               
               
                 396298 
                 4 
                 2-15-3 
                 13517  
                 13536  
                 ACCGGTTGGCATCCATGTAA 
                 73 
                 217 
               
               
                   
               
               
                 396222 
                 4 
                 5-10-5 
                 13525  
                 13544  
                 CCTGGGTGACCGGTTGGCAT 
                 71 
                 218 
               
               
                   
               
               
                 396299 
                 4 
                 2-15-3 
                 13525  
                 13544  
                 CCTGGGTGACCGGTTGGCAT 
                 82 
                 218 
               
               
                   
               
               
                 396223 
                 4 
                 5-10-5 
                 13802  
                 13821  
                 CAAGTTGGGAGACTGGATGG 
                 65 
                 219 
               
               
                   
               
               
                 396300 
                 4 
                 2-15-3 
                 13802  
                 13821  
                 CAAGTTGGGAGACTGGATGG 
                 79 
                 219 
               
               
                   
               
               
                 396224 
                 4 
                 5-10-5 
                 13810  
                 13829  
                 CTTTAATACAAGTTGGGAGA 
                 68 
                 220 
               
               
                   
               
               
                 396301 
                 4 
                 2-15-3 
                 13810  
                 13829  
                 CTTTAATACAAGTTGGGAGA 
                 64 
                 220 
               
               
                   
               
               
                 396225 
                 4 
                 5-10-5 
                 13877  
                 13896  
                 TCGGAAGGTCTGGTGGATAT 
                 65 
                 221 
               
               
                   
               
               
                 396302 
                 4 
                 2-15-3 
                 13877  
                 13896  
                 TCGGAAGGTCTGGTGGATAT 
                 83 
                 221 
               
               
                   
               
               
                 396226 
                 4 
                 5-10-5 
                 13896  
                 13915  
                 TGGGCACCAAACTGCTGGAT 
                 70 
                 222 
               
               
                   
               
               
                 396303 
                 4 
                 2-15-3 
                 13896  
                 13915  
                 TGGGCACCAAACTGCTGGAT 
                 76 
                 222 
               
               
                   
               
               
                 396227 
                 4 
                 5-10-5 
                 13937  
                 13956  
                 TATGGCTTCCTGGGCGCAGG 
                 59 
                 223 
               
               
                   
               
               
                 396304 
                 4 
                 2-15-3 
                 13937  
                 13956  
                 TATGGCTTCCTGGGCGCAGG 
                 74 
                 223 
               
               
                   
               
               
                 396228 
                 4 
                 5-10-5 
                 13961  
                 13980  
                 AATGCTGCAATGGGCATCTG 
                 78 
                 224 
               
               
                   
               
               
                 396305 
                 4 
                 2-15-3 
                 13961  
                 13980  
                 AATGCTGCAATGGGCATCTG 
                 83 
                 224 
               
               
                   
               
               
                 396229 
                 4 
                 5-10-5 
                 13977  
                 13996  
                 GTTCACTATCACAAACAATG 
                 84 
                 225 
               
               
                   
               
               
                 396306 
                 4 
                 2-15-3 
                 13977  
                 13996  
                 GTTCACTATCACAAACAATG 
                 67 
                 225 
               
               
                   
               
               
                 396230 
                 4 
                 5-10-5 
                 13997  
                 14016  
                 CAGTTAAGCAGCTTCCAGTT 
                 84 
                 226 
               
               
                   
               
               
                 396307 
                 4 
                 2-15-3 
                 13997  
                 14016  
                 CAGTTAAGCAGCTTCCAGTT 
                 85 
                 226 
               
               
                   
               
               
                 396231 
                 4 
                 5-10-5 
                 14028  
                 14047  
                 AATTTTATTTAGCCAGTCTC 
                 80 
                 227 
               
               
                   
               
               
                 396308 
                 4 
                 2-15-3 
                 14028  
                 14047  
                 AATTTTATTTAGCCAGTCTC 
                 79 
                 227 
               
               
                   
               
               
                 396232 
                 4 
                 5-10-5 
                 14046  
                 14065  
                 GTTGTATAAATATATTCTAA 
                 44 
                 228 
               
               
                   
               
               
                 396309 
                 4 
                 2-15-3 
                 14046  
                 14065  
                 GTTGTATAAATATATTCTAA 
                 25 
                 228 
               
               
                   
               
               
                 396233 
                 4 
                 5-10-5 
                 14065  
                 14084  
                 ACAGTGTTTTTGAGATTCTG 
                 83 
                 229 
               
               
                   
               
               
                 396310 
                 4 
                 2-15-3 
                 14065  
                 14084  
                 ACAGTGTTTTTGAGATTCTG 
                 50 
                 229 
               
               
                   
               
               
                 396273 
                 4 
                 5-10-5 
                 14725  
                 14744  
                 CTCAGGACCCAGAGTGAGGA 
                 37 
                 230 
               
               
                   
               
               
                 396350 
                 4 
                 2-15-3 
                 14725  
                 14744  
                 CTCAGGACCCAGAGTGAGGA 
                 50 
                 230 
               
               
                   
               
               
                 396274 
                 4 
                 5-10-5 
                 15073  
                 15092  
                 TGGGTTAAACCTCACCTCGA 
                 59 
                 231 
               
               
                   
               
               
                 396351 
                 4 
                 2-15-3 
                 15073  
                 15092  
                 TGGGTTAAACCTCACCTCGA 
                 56 
                 231 
               
               
                   
               
               
                 396275 
                 4 
                 5-10-5 
                 15350  
                 15369  
                 ATTAGGTCCCAAAGTTCCCC 
                 23 
                 232 
               
               
                   
               
               
                 396352 
                 4 
                 2-15-3 
                 15350  
                 15369  
                 ATTAGGTCCCAAAGTTCCCC 
                 48 
                 232 
               
               
                   
               
               
                 396234 
                 1 
                 5-10-5 
                   42 
                   61 
                 GGCAGACGAACCATGGCGAG 
                 86 
                 233 
               
               
                   
               
               
                 396311 
                 1 
                 2-15-3 
                   42 
                   61 
                 GGCAGACGAACCATGGCGAG 
                 82 
                 233 
               
               
                   
               
               
                 396213 
                 1 
                 5-10-5 
                  435 
                  454 
                 GTAGCAATCTGCTTGACCCC 
                 82 
                 234 
               
               
                   
               
               
                 396290 
                 1 
                 2-15-3 
                  435 
                  454 
                 GTAGCAATCTGCTTGACCCC 
                 79 
                 234 
               
               
                   
               
               
                 396218 
                 1 
                 5-10-5 
                  590 
                  609 
                 GACCACAGACAACATCAGTC 
                 89 
                 235 
               
               
                   
               
               
                 396295 
                 1 
                 2-15-3 
                  590 
                  609 
                 GACCACAGACAACATCAGTC 
                 85 
                 235 
               
               
                   
               
               
                 396219 
                 1 
                 5-10-5 
                  683 
                  702 
                 CCACCTTTTTGATAAAGACC 
                 65 
                 236 
               
               
                   
               
               
                 396296 
                 1 
                 2-15-3 
                  683 
                  702 
                 CCACCTTTTTGATAAAGACC 
                 41 
                 236 
               
               
                   
               
             
          
         
       
     
       Example 5 
     Antisense Inhibition of Human CD40 in HuVEC Cells, Primer Probe Set LTS37 
       [0331]    Several antisense oligonucleotides exhibiting in vitro inhibition of CD40 (see Example 4) were tested at various doses in HuVEC cells. Cells were plated at densities of 5000 cells per well and treated with nM concentrations of antisense oligonucleotide as indicated in Table 5. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. Human CD40 primer probe set LTS37 was used to measure mRNA levels. CD40 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of CD40, relative to untreated control cells. As illustrated in Table 5, CD40 mRNA levels were reduced in a dose-dependent manner. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Antisense Inhibition of human CD40 in HuVEC cells, 
               
               
                 Primer Probe Set LTS37 
               
             
          
           
               
                 ISIS 
                 0.2344 
                 0.4688 
                 0.9375 
                 1.875 
                 3.75 
                 7.5 
                 15.0 
                 30.0 
               
               
                 No 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
               
               
                   
               
               
                 26163 
                 17 
                 35 
                 38 
                 51 
                 62 
                 67 
                 82 
                 89 
               
               
                 396236 
                 23 
                 49 
                 59 
                 77 
                 86 
                 92 
                 91 
                 89 
               
               
                 396266 
                 35 
                 45 
                 58 
                 74 
                 55 
                 72 
                 57 
                 56 
               
               
                 396307 
                 21 
                 45 
                 43 
                 56 
                 80 
                 79 
                 82 
                 82 
               
               
                 396218 
                 34 
                 47 
                 52 
                 57 
                 78 
                 82 
                 86 
                 86 
               
               
                 396279 
                 34 
                 54 
                 59 
                 49 
                 72 
                 82 
                 88 
                 87 
               
               
                 396287 
                 31 
                 48 
                 52 
                 50 
                 64 
                 77 
                 85 
                 86 
               
               
                 396264 
                 39 
                 34 
                 49 
                 56 
                 71 
                 84 
                 88 
                 86 
               
               
                   
               
             
          
         
       
     
       Example 6 
     Antisense Inhibition of Human CD40 in AGS Cells 
       [0332]    Antisense oligonucleotides exhibiting in vitro inhibition of CD40 (see Example 4) were tested at various doses in AGS cells (human adenocarcinoma cells). Antisense oligonucleotides of SEQ ID No. 90 and SEQ ID No. 208 were designed as 4-10-4 gapmers or 5-10-5 gapmers, respectively, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE or 2′OMe nucleotides. The antisense oligonucleotides comprise phosphorothioate backbones (internucleoside linkages) and 5-methylcytosine substitutions throughout. 
         [0333]    Cells were plated at densities of 5000 cells per well and treated with nM concentrations of antisense oligonucleotide as indicated in Table 6. After a treatment period of approximately 24 hours, RNA was isolated from the cells and relative CD40 mRNA expression levels were quantified by real time RT-PCR using the QuantiTect™ SYBR® Green RT-PCR kit (Qiagen). CD40 mRNA levels were adjusted according to GAPDH content, a housekeeping gene. Results are presented as percent inhibition of CD40, relative to cells treated with a scrambled control oligonucleotide 
         [0000]                            ( TCCATTTATTAGTCTAGGAA              
(5-10-5 gapmer, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides. The oligonucleotide comprises phosphorothioate backbones (internucleoside linkages) and 5-methylcytosine substitutions throughout.
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Antisense Inhibition of human CD40 in AGS cells 
               
             
          
           
               
                 ISIS 
                   
                 Wing 
                   
                   
                   
                   
               
               
                 No 
                 Motif 
                 segment 
                 12.5 nM 
                 25.0 nM 
                 50.0 nM 
                 Seq ID 
               
               
                   
               
             
          
           
               
                  26163 
                 4-10-4 
                 2′MOE 
                 80 
                 69 
                 90 
                 90 
               
               
                 396236 
                 5-10-5 
                 2′MOE 
                 83 
                 86 
                 94 
                 208 
               
               
                 — 
                 4-10-4 
                 2′OMe 
                 51 
                 54 
                 66 
                 90 
               
               
                 — 
                 5-10-5 
                 2′OMe 
                 60 
                 63 
                 69 
                 208 
               
               
                   
               
             
          
         
       
     
         [0334]    As illustrated in Table 6, CD40 mRNA levels were reduced in a dose-dependent manner. Antisense oligonucleotides comprising 2′MOE wing segments are more active than those with 2′OMe wing segments. 
       Example 7 
     Antisense Inhibition of Murine CD40 In Vitro 
       [0335]    Chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxy gap and 4-10-4 MOE wings and deoxy gap may be designed to target murine CD40. These antisense oligonucleotides can be evaluated for their ability to reduce CD40 mRNA in primary mouse hepatocytes using similar methods as described in the human in vitro study. 
         [0336]    For example, primary mouse hepatocytes may be treated with 0.2344 nM, 0.4688 nM, 0.9375 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15.0 nM, and 30.0 nM of antisense oligonucleotides for a period of approximately 24 hours. RNA can be isolated from the cells and CD40 mRNA levels can be measured by quantitative real-time PCR, as described herein. Murine CD40 primer probe sets can be used to measure mRNA levels. CD40 mRNA levels can then be adjusted according to total RNA content as measured by RIBOGREEN®. 
       Example 8 
     Antisense Inhibition of Murine CD40 In Vivo 
       [0337]    Antisense oligonucleotides showing statistically significant dose-dependent inhibition from an in vitro study can be evaluated for their ability to reduce CD40 mRNA in vivo. 
       Treatment 
       [0338]    Antisense oligonucleotide can be evaluated in Balb/c mice and compared to a control group treated with saline. Oligonucleotide or saline would be administered subcutaneously at a dose of 5 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kg twice a week for three weeks. After the treatment period, whole liver can be collected for RNA analysis and protein analysis. 
       RNA Analysis 
       [0339]    Liver RNA can be isolated for real-time PCR analysis of CD40. It is theorized that an antisense oligonucleotide showing significant dose-dependent inhibition in vitro may show significant dose-dependent inhibition in vivo. 
       Protein Analysis 
       [0340]    Liver CD40 protein may be measured by Western blot. 
       Example 9 
     Tolerability of Antisense Compounds in Rodents 
       [0341]    Male 6 week old Balb/c mice were dosed subcutaneous 2× per week for 4 weeks with 25 or 50 mg/kg of antisense oligonucleotides Isis 26163 or Isis 396236. Mice were sacrificed 2 days following last administration. Body weights of the animals were monitored throughout the study. After sacrification liver, spleen and kidney weights and liver enzymes ALT and AST from mouse plasma were determined. 
         [0342]    Compared to a saline control treatment body weights of the mice are not affected by antisense oligonucleotides Isis 26163 or Isis 396236. Liver weight and spleen weight displayed a slight increase for Isis 26163 but not for Isis 396236. LFT (liver function test) elevations were small and within the normal range of high dose mouse studies. 
       Example 10 
     Antisense Inhibition of Human CD40 In Vitro on T24 Cells—Comparative Data for ISIS 26163 and ISIS 19216 
       [0343]    Antisense oligonucleotides ISIS 26163 and ISIS 19216 targeted to a CD40 nucleic acid were tested for their effects on CD40 mRNA in vitro. The antisense oligonucleotides were designed as 4-10-4 gapmers, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides. The antisense oligonucleotides comprise phosphorothioate backbones (internucleoside linkages) and 5-methylcytosine substitutions throughout or in the wings, respectively. 
         [0344]    T24 cells at a density of 7000 cells per well in a 96-well plate were treated with 100 nM or 150 nM, respectively, of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CD40 mRNA levels were measured by quantitative real-time PCR, as described herein. CD40 mRNA levels were adjusted according to GAPDH content, a housekeeping gene. 
         [0345]    Results are presented in Table 7 as percent inhibition of CD40, relative to untreated control cells. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Oligo ID 
                 Target site 
                 nM on T24 cells 
                 % Inhibition 
                 SEQ ID No. 
               
               
                   
               
             
             
               
                 26163 
                 70-87 
                 100 
                 98 
                 90 
               
               
                 19216 
                 73-90 
                 150 
                 66 
                 10 
               
               
                   
               
             
          
         
       
     
         [0346]    Sequence ISIS 26163 shows a superior activity over ISIS 19216, which overlaps the sequence by 15 nucleobases.