# Patent

## Title
Modulation of CD40 expression

## 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.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C §371 national phase application of international application serial no. PCT/US2008/012998, filed on Nov. 20, 2008 , which is a non-provisional of and claims priority to U.S. patent application Ser. No. 60/989421, filed on Nov. 20, 2007, the disclosure of each of which is incorporated herein by reference in its entirety.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 33841-513SEQLIST.txt, created Nov. 19, 2008, which is 67 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. This sequence listing is identical to the sequence listing filed on Nov. 20, 2007, with the exception of the addition of SEQ ID NO: 237.
FIELD OF THE INVENTION
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
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.
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'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.
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).
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).
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.
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).
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
Provided herein are antisense compounds, compositions, and methods for the treatment and prevention of inflammatory conditions and cancer.
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.
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.
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.
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.
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.
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.
The antisense compound may comprise linked nucleosides. Preferably, the antisense compound is an antisense oligonucleotide.
The antisense compound may be a single-stranded or double-stranded oligonucleotide. Preferably, the antisense compound is a single-stranded oligonucleotide.
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.
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




wherein Bx is an optionally protected heterocyclic base moiety.

The antisense compound may comprise a modified nucleobase. The modified nucleobase may be a 5-methylcytosine. Preferably, every cytosine is a 5-methylcytosine.
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.
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.
The antisense oligonucleotide may be a 4-10-4 MOE gapmer. The antisense oligonucleotide may consist of 18 linked nucleosides.
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.
The composition may comprise a single-stranded or double-stranded oligonucleotide.
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.
    
    


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.
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.
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
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
Definitions
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques 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.
Unless otherwise indicated, the following terms have the following meanings:
“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2—OCH3) 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.
“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.
“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.
“Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.
“Active pharmaceutical ingredient” means the substance or substances in a pharmaceutical composition that provides a desired effect.
“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.
“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.
“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
“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.
“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.
“Bicyclic sugar” means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleic acid” or “BNA” 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.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“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.
“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.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
“Comply” means the adherence with a recommended therapy by a individual.
“Contiguous nucleobases” means nucleobases immediately adjacent to each other.
“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.
“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.
“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.
“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.
“Efficacy” means the ability to produce a desired effect.
“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.
“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.
“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.
“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
“Individual” means a human or non-human animal selected for treatment or therapy.
“Individual compliance” means adherence to a recommended or prescribed therapy by a individual.
“Injection site reaction” means inflammation or abnormal redness of skin at a site of injection in a individual.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Linked nucleosides” means adjacent nucleosides which are bonded together.
“Modified internucleoside linkage” refers to a substitution and/or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
“Modified sugar” refers to a substitution and/or any change from a natural sugar.
“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).
“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.
“Modified sugar moiety” means a sugar moiety having any substitution and/or change from a natural sugar moiety.
“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).
“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.
“Nucleoside” means a nucleobase linked to a sugar.
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.
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
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).
“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.
“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
“Oligonucleotide” means a polymer or oligomer of linked nucleosides each of which can be modified or unmodified, independent one from another.
“Parenteral administration,” means administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
“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.
“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.
“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.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
“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.
“Recommended therapy” means a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease.
“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.
“Single-stranded modified oligonucleotide” means a modified oligonucleotide which is not hybridized to a complementary strand.
“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.
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.
“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.
“Subcutaneous administration” means administration just below the skin. “Intravenous administration” means administration into a vein.
“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.
“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.
“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.
“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
“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.
“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
“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
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.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to 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.
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.
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.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
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
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.
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.
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—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH2)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.
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.
In one embodiment, antisense compounds targeted to a CD40 nucleic acid possess a 5-10-5 gapmer motif.
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.
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
Nucleotide sequences that encode CD40 include, without limitation, the following: 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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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
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.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In one embodiment, the antisense compounds provided herein are specifically hybridizable with a CD40 nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a CD40 nucleic acid).
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).
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. 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).
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.
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.
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.
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.
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
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.
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
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides 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
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.
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.
Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
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.
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.
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
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).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3 (known as 2′-OMe) and 2′-O(CH2)2OCH3 (known as 2′MOE) substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O—CH2CH2CH2NH2, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn) such as 2′-dimethylaminooxyethoxy (2′-O—(CH2)2ON(CH3)2 or 2′-DMAOE), O(CH2)2—O—(CH2)2—N(Rm)(Rn) such as 2′-dimethylaminoethoxyethoxy (2′-O—(CH2)2—O—(CH2)2—N(CH3)2 or 2′-DMAEOE and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
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′-(CH2)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′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)—O-2′ (LNA); 4′-(CH2)2—O-2′ (ENA); 4′-C(CH3)2—O-2′ (see PCT/US2008/068922); 4′-CH(CH3)—O-2′ and 4′CH(CH2OCH3)—O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH2—N(OCH3)-2′ (see PCT/US2008/064591); 4′-CH2—O—N(CH3)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(CH3)-2′ and 4′-CH2—C(═CH2)-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).
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:




wherein Bx is an optionally protected heterocyclic base moiety.

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.
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.
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.
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.
In some embodiments, the bicyclic sugar moiety is bridged between the 2′ and 4′ carbon atoms with a biradical group selected from —O—(CH2)p-, —O—CH2—, —O—CH2CH2, —O—CH(alkyl)-, —NH—(CH2)p-, —N(alkyl)-(CH2)p-, —O—CH(alkyl)-, —(CH(alkyl))-(CH2)p-, —NH—O—(CH2)p-, —N(alkyl)-O—(CH2)p-, or —O—N(alkyl)-(CH2)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.
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′-(CH2)3-2′,4′-(CH2)2-2′,4′-CH2—O-2′,4′-(CH2)2—O-2′,4′-CH2—O—N(R1)-2′ and 4′-CH2—N(R1)-O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In 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
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).
Additional unmodified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties 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.
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.
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
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Ended et al., WO 07/031,333 of Panzner et al., WO 07/107,304 of Panzner et al. and WO 08/043,575 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.
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.
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. %.
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.)
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.
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.
Preferred cationic components include DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol, DPIM, CHIM, DOME, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18)2Gly+ N,N-dioctadecylamido-glycin, CTAB, CPyC, DODAP and DOEPC.
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.
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.
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.
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.
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.
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. %).
Preferred phosphatidylethanolamines include DOPE, DMPE and DPPE.
In some embodiments said neutral lipid may comprise POPC and DOPE.
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.
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.
    
    


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. %.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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


    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)2GlySper3+) 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
    His Chol Histaminyl-Cholesterolhemisuccinate.
    HCChol Nα-Histidinyl-Cholesterolcarbamate
    HistChol Nα-Histidinyl-Cholesterol-hemisuccinate.
    AC Acylcarnosine, Stearyl- & Palmitoylcarnosine
    HistDG 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl-hemisuccinate, & Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives
    IsoHistSuccDG 1,2-Dipalmitoylglycerol-Oα-Histidinyl-Nα-hemisuccinat, & Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives
    DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate & Distearoyl-, Dimyristoyl-Dioleoyl- or palmitoyl-oleoylderivatives


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.
One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
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.
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. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.
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.
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).
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.
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
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 commercial 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'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
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
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.
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.
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.
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
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's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
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's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
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's instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carried out by methods well known to those skilled in the art.
Gene (or RNA) target quantities obtained by real time PCR 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.
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
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
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
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.
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's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
In 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.
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.
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
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.
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.
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
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
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.
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.







TABLE 1







Inhibition of human CD40 mRNA levels by fully phosphorothioate oligodeoxynucleotides















Target









SEQ
Target
Target



SEQ


Oligo
ID
Start
Stop
Target

%
ID


ID
NO
Site
Site
Region
Sequence (5′ to 3′)
Inhibition
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
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.
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.







TABLE 2







Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having


4-10-4 MOE wings and deoxy gap















Target









SEQ
Target
Target



SEQ



ID
Start
Stop
Target

%
ID


OligoID
NO
Site
Site
Region
Sequence (5′ to 3′)
Inhibition
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
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.
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).







TABLE 3







Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having


4-10-4 MOE wings and deoxy gap














Target








SEQ
Target
Target


SEQ


Oligo
ID
Start
Stop

%
ID


ID
NO
Site
Site
Sequence (5′ to 3′)
Inhibition
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
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.
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).







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 ID

Start
Stop

%
SEQ ID


ID
NO
Motif
Site
Site
Sequence (5′ to 3′)
Inhibition
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
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.







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
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.
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 QuantiTectTM 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 (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.







TABLE 6







Antisense Inhibition of human CD40 in AGS cells













ISIS

Wing
12.5
25.0
50.0



No
Motif
segment
nM
nM
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









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
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.
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
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
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
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
Liver CD40 protein may be measured by Western blot.
Example 9
Tolerability of Antisense Compounds in Rodents
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.
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 ISIS19216
Antisense oligonucleotides ISIS 26163 and ISIS19216 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.
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.
Results are presented in Table 7 as percent inhibition of CD40, relative to untreated control cells.











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









Sequence ISIS 26163 shows a superior activity over ISIS19216, which overlaps the sequence by 15 nucleobases.

## Claims

1. A modified antisense compound 12 to 30 nucleobases in length and having a nucleobase sequence that is at least 90% complementary to an equal length portion of the human CD40 gene but not to other sequences throughout the human genome, 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;
(h) positions 12907-13429, corresponding to intron 8; and
(i) positions 13662-16001, which forms part of exon 9 or a region 3′ to exon 9.

2. The antisense compound of claim 1, wherein the nucleobase sequence is at least 90% complementary to an equal length portion of positions 12527-12685 of SEQ ID NO: 4.

3. The antisense compound of claim 2, having 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 95% complementary to the sequence shown in SEQ ID NO: 4.

4. A modified antisense compound 20 nucleobases in length and consisting of the nucleobase sequence of SEQ ID NO: 208.

5. The antisense compound of claim 1, wherein said antisense compound is an antisense oligonucleotide.

6. The antisense compound of claim 5, wherein at least one internucleoside linkage is a modified internucleoside linkage.

7. The antisense compound of claim 6, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

8. The antisense compound of claim 5, wherein at least one nucleoside comprises a modified sugar.

9. The antisense compound of claim 8, wherein at least one modified sugar is a bicyclic sugar.

10. The antisense compound of claim 9, wherein the at least one bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.

11. The antisense compound of claim 8, wherein at least one modified sugar comprises a 2′-O-methoxyethyl.

12. The antisense compound of claim 1, wherein at least one said nucleobase is a modified nucleobase.

13. The antisense compound of claim 12, wherein the modified nucleobase is a 5-methylcytosine.

14. The antisense compound of claim 1, wherein the compound is 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.

15. The antisense compound of claim 14, wherein the oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar;
and wherein each internucleoside linkage of said antisense compound is a phosphorothioate linkage.

16. The antisense compound of claim 14, wherein the oligonucleotide comprises:
a gap segment consisting of fifteen linked deoxynucleosides;
a 5′ wing segment consisting of two linked nucleosides;
a 3′ wing segment consisting of three linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar;
and wherein each internucleoside linkage of said antisense compound is a phosphorothioate linkage.

17. The antisense compound of claim 15 or 16, wherein every cytosine is a 5-methylcytosine.

18. An antisense oligonucleotide 20 nucleobases in length having the sequence of nucleobases 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.

19. A composition comprising an antisense compound of claim 1 or 18 or a salt thereof and a pharmaceutically acceptable carrier or diluent.

20. A method comprising administering to an animal an antisense compound of claim 1 or an oligonucleotide of claim 18.