WO2010108035A1 - Composés et procédés de modulation d'effets toxiques et pro-inflammatoires - Google Patents

Composés et procédés de modulation d'effets toxiques et pro-inflammatoires Download PDF

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WO2010108035A1
WO2010108035A1 PCT/US2010/027863 US2010027863W WO2010108035A1 WO 2010108035 A1 WO2010108035 A1 WO 2010108035A1 US 2010027863 W US2010027863 W US 2010027863W WO 2010108035 A1 WO2010108035 A1 WO 2010108035A1
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oligomeric compound
compound
interferon
oligonucleotide
oligomeric
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PCT/US2010/027863
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English (en)
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Sebastien Burel
Scott Henry
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Isis Pharmaceuticals, Inc.
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Publication of WO2010108035A1 publication Critical patent/WO2010108035A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • Disclosed herein are methods of modulating a toxic and/or proinflammatory effect of an oligomeric compound in an animal.
  • IFNs interferons
  • Type I IFNs include multiple subtypes including IFN-alpha and IFN-beta.
  • Type I IFNs are produced by all nucleated cells in response to virus infection, hi comparison, type II IFN (IFN-gamma) are predominately made by T lymphocytes and natural killer ONfK) cells in response to T cell receptor (TCR) and natural killer (NK) cell receptor signals (Pichlmair, Immunity, 2007, 27, 370-383).
  • TCR T cell receptor
  • NK natural killer
  • IFN-alpha and IFN-beta bind to a common cellular receptor, type I interferon receptor (IFNAR).
  • Type II IFN binds to a different receptor, interferon gamma receptor (IFNGR), however, both share downstream signaling molecules and regulate many of the same genes.
  • IFNAR and IFNGR like other cytokine receptors, are coupled to a Janus-family tyrosine kinase, which in turn phosphorylates signal-transducing activators of transcriptions (STATs). The binding of phosphorylated STAT proteins to the promoters of several genes induces the synthesis of host-cell proteins that contribute to the inhibition of viral replication.
  • oligo-adnylate synthetase which polymerizes ATP into a series of 2'-5' linked oligomers. These activate an endoribonuclease that then degrades viral RNA.
  • a second protein activated enzyme phosphorylates the eukaryotic protein synthesis inhibition factor eIF-2, thereby inhibiting translation and thus contributing to the inhibition of viral replication.
  • Another interferon-inducible protein, Mx is known to be required for cellular resistance to influenza virus replication. (Janeway, C. et al. (1999). Immuno Biology, 4th Ed. (London: Current Biology Publications).
  • INF-alpha and INF-beta have three major functions. First, they induce resistance to viral replication by activating cellular genes that destroy mRNA and inhibit the translation of viral and some host proteins. Second, they induce major histocompatibility complex (MHC) class I expression in most uninfected cells in the body, which enhances resistance to NK cells and makes cells newly infected by virus more susceptible to killing by CD8 cytotoxic T cells. Third, they activate NK cells, which then kill virus-infected cells selectively. INF-gamma activates macrophages increasing release of inflammatory mediators (Janeway, C. et al. (1999) Immuno Biology, 4th Ed. (London: Current Biology Publications).
  • MHC major histocompatibility complex
  • Disclosed herein are methods of ameliorating an undesired toxic effect of a cytotoxic oligomeric compound in a cell or animal. Also disclosed herein are methods for inducing a desired effect in a cell or animal.
  • the cell may be in vitro or in an animal.
  • the animal may be a mammal.
  • the mammal may be a mouse and/or a human.
  • the oligomeric compound may comprise at least one oligonucleotide consisting of 8 to 30, 8 to 22, 20 to 30, or 12 to 24 linked nucleosides.
  • the at least one oligonucleotide may be single-stranded or double-stranded.
  • the at least one oligonucleotide may comprise one or more modified nucleosides.
  • the at least one oligonucleotide may be fully modified.
  • the at least one modified nucleoside may be any of MOE, 2'-F, BNA, 2'OMe, sugar mimetic.
  • the at least one oligonucleotide may be a gapmer.
  • An interferon modulator is any agent that changes, for example, expression, activity or processing either directly or indirectly of a component of the interferon pathway or mediator of an interferon response.
  • An interferon modulator can include, but is not limited to, proteins, peptides, polypeptides, antibodies, antisense compounds, including oligonucleotides and antisense oligonucleotides, ssRNA, dsRNA molecules, ribozymes, triple helix molecules, siRNAs, and small molecule modulators.
  • the interferon modulator can modulate a component of the interferon pathway. Such component can be, but is not limited to, IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • the interferon modulator modulates a component of the type I interferon pathway. In another embodiment, the interferon modulator modulates a pattern recognition receptor that signals through a type- 1 IFN pathway or an adaptor protein essential to pattern recognition receptor singaling.
  • Pattern recognition receptors can include, but are not limited to, type- 1 IFN induced and dsRNA-activated kinase (PKR), melanoma differentiation-associated gene 5 (MD A-5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activator of IRFs (DAI) and Toll-like receptor 3 (TLR3).
  • PPKR type- 1 IFN induced and dsRNA-activated kinase
  • MD A-5 melanoma differentiation-associated gene 5
  • RIG-I retinoic acid-inducible gene I
  • DAI DNA-dependent activator of IRFs
  • TLR3 Toll-like receptor 3
  • the interferon modulator modulates IPS-I, RIG-I, or MD A-5.
  • the interferon modulator can be an interferon inhibitor, hi one embodiment, the interferon inhibitor inhibits a type 1 interferon response.
  • the interferon inhibitor is an IPS-I inhibitor, RIG-I inhibitor, or MD A-5 inhibitor.
  • the interferon inhibitor ameliorates the undesired toxic effect of a cytotoxic oligomeric compound. In another embodiment, the interferon inhibitor inhibits an antiviral response. In another embodiment, the interferon inhibitor is an oligomeric compound.
  • An oligomeric compound may comprise an oligonucleotide which is an antisense compound.
  • the antisense compound may be complementary to a target nucleic acid.
  • the target nucleic acid may be any of a target mRNA, a target pre-mRNA, a target microRNA, a target pdRNA, and a target non-coding RNA.
  • the antisense compound may be complementary to a target viral nucleic acid.
  • the antisense compound may be complementary to an HCV nucleic acid.
  • the oligomeric compound may comprise an oligonucleotide which is not an antisense compound.
  • the oligomeric compound may comprise a conjugate.
  • the oligomeric compound and the interferon modulator are linked. Also disclosed herein are methods of co-administering an oligomeric compound and an interferon modulator to an animal.
  • the animal may be a mammal.
  • the mammal may be a mouse and/or a human.
  • the oligomeric compound and the interferon modulator may be administered together or separately.
  • the oligomeric compound and the interferon modulator may be administered at the same time or different times.
  • the oligomeric compound and the interferon modulator may be administered by the same route of administration or different routes of administration.
  • methods for ameliorating an undesired toxic effect of a cytotoxic oligomeric compound in an animal comprising co-administering to the animal a cytotoxic oligomeric compound and an interferon inhibitor and thereby ameliorating the toxic effect of the cytotoxic oligomeric compound are described.
  • methods for inducing a desired toxic effect in an animal comprising administering to the animal a cytotoxic oligomeric compound.
  • methods for inducing a desired toxic effect in an animal comprising co-administering to the animal a cytotoxic oligomeric compound and an interferon activator and thereby inducing a toxic effect in the animal are described.
  • methods for amplifying a desired toxic effect of a cytotoxic oligomeric compound in an animal comprising co-administering to the animal a cytotoxic oligomeric compound and an interferon activator and thereby amplifying the toxic effect of the cytotoxic oligomeric compound are described.
  • methods for increasing the activity of an interferon activator in an animal comprising co-administering to the animal an interferon activator and a cytotoxic oligomeric compound, thereby increasing the activity of the interferon activator are described.
  • compositions comprising an oligomeric compound and an interferon modulator.
  • the oligomeric compound and the interferon modulator may be linked or they may be separate molecules.
  • the oligomeric compound may be a cytotoxic oligomeric compound.
  • the interferon modulator may be an interferon inhibitor or activator.
  • the composition may comprise a pharmaceutical carrier.
  • compositions comprising an oligomeric compound and an anti-cancer agent.
  • the present invention provides methods of inducing an inflammatory response in an animal comprising administering to the animal an oligomeric compound capable of activating at least one member of the type 1 interferon pathway; and thereby inducing an inflammatory response in the animal.
  • the member of the type 1 interferon pathway is selected from among: Rig-I, MDA5, and IPS-I.
  • oligomeric compound is capable of activating MDA5.
  • the activation of MDA5 activates IPS-I.
  • the oligomeric compound activates Rig-I.
  • the activation of Rig-I activates IPS-I.
  • the invention provides methods of selecting a pro-inflammatory oligomeric compound comprising identifying oligomeric compounds capable of activation a member of the type 1 interferon pathway and testing the compound in an animal.
  • the member of the type 1 interferon pathway is selected from among: Rig-I, MDA5, and IPS-I.
  • the oligomeric compound is capable of activating MDA5.
  • the activation of MDA5 activates IPS-I.
  • the oligomeric compound activates Rig-I.
  • the activation of Rig-I activates IPS-I .
  • the oligomeric compound comprises an oligonucleotide consisting of 12-30 linked nucleosides and optionally comprises one or more conjugates.
  • the oligomeric compound comprises an oligonucleotide consisting of 8-30 linked nucleosides and optionally comprises one or more conjugates. In certain embodiments, the oligomeric compound comprises an oligonucleotide consisting of 8 to 30 linked nucleosides. In certain embodiments, the oligonucleotide consists of 8-22 linked nucleosides. In certain embodiments, the oligonucleotide consists of 20-30 linked nucleosides. In certain embodiments, the oligonucleotide consists of 12-24 linked nucleosides.
  • the oligomeric compound comprises a conjugate. In certain embodiments, the oligomeric compound is single-stranded. In certain embodiments, the oligomeric compound is double-stranded. In certain embodiments, the oligomeric compound comprises an oligonucleotide comprising one or more modified nucleosides selected from among: MOE, 2'-F, BNA, 2'OMe, and tetrahydropyran. In certain embodiments, the oligomeric compound comprises an oligonucleotide that is a gapmer. In certain embodiments, the oligomeric compound comprises an oligonucleotide that is fully modified. In certain embodiments, the oligomeric compound comprises an oligonucleotide having an alternating chemical motif. In certain embodiments, the oligomeric compound comprises an oligonucleotide having at least four separate regions.
  • the oligomeric compound comprises an oligonucleotide having a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO: 1 (147420). In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 20 contiguous nucleobases of SEQ ID NO: 1.
  • the oligomeric compound comprises a cytotoxic sequence.
  • the oligomeric compound is an antisense compound.
  • the antisense compound is complementary to a target nucleic acid selected from among: a target mRNA, a target pre-mRNA, a target microRNA, a target pdRNA, and a target non-coding RNA.
  • the antisense compound is complementary to a target viral nucleic acid.
  • the antisense compound is complementary to an HCV nucleic acid.
  • the compound is administered a mammal.
  • the mammal is a human.
  • the invention provides co-administering an antiviral compound.
  • the antiviral compound is an anti-HCV compound.
  • the invention provides detecting activation of the interferon type 1 pathway. In certain embodiments, the invention provides detecting activation of rig-I, MDA-5, and/or IPS-I.
  • the invention provides methods comprising co-administering to an animal an oligomeric compound and an interferon modulator.
  • the oligomeric compound and the interferon modulator are administered together.
  • the oligomeric compound and the interferon modulator are administered separately.
  • the oligomeric compound and the interferon modulator are administered at the same time.
  • the oligomeric compound and the interferon modulator are administered at different times.
  • the oligomeric compound and the interferon modulator are administered by the same route of administration.
  • the oligomeric compound and the interferon modulator are administered by different routes of administration.
  • the oligomeric compound comprises an oligonucleotide consisting of 8 to 30 linked nucleosides. In certain embodiments, the at least one oligonucleotide consists of 8-22 linked nucleosides. In certain embodiments, the at least one oligonucleotide consists of 20-30 linked nucleosides. In certain embodiments, the at least one oligonucleotide consists of 12-24 linked nucleosides. In certain embodiments, the oligomeric compound comprises a conjugate.
  • the oligomeric compound and the interferon modulator are linked together.
  • the oligomeric compound is single-stranded.
  • the oligomeric compound is double-stranded.
  • the oligomeric compound comprises at least one oligonucleotide comprising one or more modified nucleosides selected from among: MOE, 2'-F, BNA, 2'OMe.
  • the oligomeric compound comprises at least one oligonucleotide that is a gapmer.
  • the oligomeric compound comprises at least one oligonucleotide that is fully modified.
  • the oligomeric compound comprises at least one oligonucleotide having an alternating chemical motif.
  • the oligomeric compound comprises at least one oligonucleotide having at least four separate regions.
  • the oligomeric compound comprises an oligonucleotide having a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO: 1 (147420). In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 20 contiguous nucleobases of SEQ IDNO: 1.
  • the interferon modulator reduces the inflammatory activity of the oligomeric compound. In certain embodiments, the interferon modulator increases the inflammatory activity of the oligomeric compound. In certain embodiments, the oligomeric compound is a Rig-1 dependent proinflammatory compound. In certain embodiments, the oligomeric compound is an MDA-5 dependent proinflammatory compound. In certain embodiments, the oligomeric compound is an IPS-I dependent proinflammatory compound. In certain embodiments, the oligomeric compound is an IPS-I dependent proinflammatory compound and Rig-I independent proinflammatory compound. In certain embodiments, the oligomeric compound comprises an oligonucleotide having a nucleobase sequence that is not complimentary to SEQ ID NO: 1.
  • the oligomeric compound comprises an oligonucleotide comprising at least one bicyclic nucleoside.
  • nucleoside refers to a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups. Nucleosides may be modified with any of a variety of substituents.
  • modified nucleoside refers to a nucleoside comprising at least one modification compared to naturally occurring RNA or DNA nucleosides. Such modification may be at the sugar moiety and/or at the nucleobases.
  • 2 '-modified or “2 '-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH.
  • oligomeric compounds comprise a 2 'modified monomer that does not have the formula 2'-O(CH2)nH, wherein n is one to six. In certain embodiments, oligomeric compounds comprise a 2'modified monomer that does not have the formula 2'-OCH3. In certain embodiments, oligomeric compounds comprise a 2'modified monomer that does not have the formula or, in the alternative, 2'-O(CH2)2OCH3.
  • 2'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.
  • 2'-0Me refers to a nucleoside comprising a sugar comprising an O- Methyl group at the 2' position.
  • 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.
  • methyleneoxy BNA alone refers to ⁇ -D- methyleneoxy BNA.
  • MOE refers to a nucleoside comprising a sugar comprising a 2'-O- methoxyethyl substituent.
  • nucleobase refers to the base portion of a nucleoside or nucleotide.
  • a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.
  • oligomeric compound refers to a polymeric structure comprising two or more sub-structures and capable of hybridizing to a region of a nucleic acid molecule.
  • oligomeric compounds are oligonucleosides.
  • oligomeric compounds are oligonucleotides.
  • oligomeric compounds are antisense compounds.
  • oligomeric compounds are antidote compounds, hi certain embodiments, oligomeric compounds comprise conjugate groups.
  • double-stranded region refers to a region of an oligomeric compound or oligonucleotide that is hybridized to its complement.
  • double-stranded oligomeric compound or “double stranded oligonucleotide” refer to an oligomeric compound or oligonucleotide comprising two separate complementary strands hybridized to one another.
  • the two separate strands of a double stranded oligomeric compound may or may not be the same length as one another and may include regions of non-complementarity, provided the two strands remain bound to one another.
  • hair-pin refers to an oligomeric compound or oligonucleotide having sufficient self complementarity to allow formation of one or more double-stranded regions within the compound by the compound or oligonucleotide folding on itself under physiologically relevant conditions.
  • single-stranded oligomeric compound or “single-stranded oligonucleotide” refer to oligomeric compounds or oligonucleotides that are not hybridized to a separate complementary strand and that are not hair-pin compounds. Single-stranded oligomeric compounds or oligonucleotides may subsequently hybridize with a complementary nucleic acid to form a double stranded compound.
  • cytotoxic oligomeric compound refers to an oligomeric compound that, when contacted with a cell results in a cytotoxic response in the cell.
  • cytotoxic oligomeric compounds have a TC 50 less than 1 mM, 0.5 mM, 0.1 mM, 0.5 mM, 0.02 mM, 1.0 ⁇ M, 5.0 ⁇ M, 1.0 ⁇ M, or 0.5 ⁇ M in a standard assay.
  • proinflammatory oligomeric compound refers to an oligomeric compound that, when administered to an animal results in inflammation. In certain embodiments, such inflammation is measured or detected by assessing cytokine amount relative to a control animal to which the oligomeric compound was not administered.
  • immunological oligomeric compound refers to an oligomeric compound that, when administered to an animal stimulates the animal's immune system.
  • oligonucleoside refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
  • oligonucleotide refers to an oligomeric compound comprising a plurality of linked nucleosides and/or nucleotides, hi certain embodiments, one or more nucleosides of an oligonucleotide is modified.
  • an oligonucleotide comprises ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA).
  • oligonucleotides are composed of naturally- and/or non-naturally-occurring nucleobases, sugars and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.
  • modified oligonucleotide refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • nucleoside linkage refers to a covalent linkage between adjacent nucleosides.
  • naturally occurring internucleoside linkage refers to a 3' to 5' phosphodiester linkage.
  • antisense compound refers to an oligomeric compound that is at least partially complementary to a target nucleic acid molecule to which it hybridizes, hi certain embodiments, an antisense compound modulates (increases or decreases) expression or amount of a target nucleic acid. In certain embodiments, an antisense compound alters splicing of a target pre-mRNA resulting in a different splice variant.
  • Antisense compounds include, but are not limited to, compounds that are oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. Consequently, while all antisense compounds are oligomeric compounds, not all oligomeric compounds are antisense compounds.
  • antisense oligonucleotide refers to an antisense compound that is an oligonucleotide.
  • antisense activity refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, such activity may be an increase or decrease in an amount of a nucleic acid or protein. In certain embodiments, such activity may be a change in the ratio of splice variants of a nucleic acid or protein. Detection and/or measuring of antisense activity may be direct or indirect.
  • antisense activity is assessed by detecting and or measuring the amount of target protein or the relative amounts of splice variants of a target protein. In certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids and/or cleaved target nucleic acids and/or alternatively spliced target nucleic acids.
  • detecting antisense activity or “measuring antisense activity” refer to that a test for detecting or measuring antisense activity is performed on a particular sample and compared to that of a control sample. Such detection and/or measuring may include values of zero. Thus, if a test for detection of antisense activity results in a finding of no antisense activity (antisense activity of zero), the step of "detecting antisense activity" has nevertheless been performed.
  • target nucleic acid refers to any nucleic acid molecule the expression or activity of which is capable of being modulated by an antisense compound.
  • Target nucleic acids include, but are not limited to, RNA (including, but not limited to pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, and also cDNA derived from such RNA, and miRNA.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
  • target mRNA refers to a pre-selected RNA molecule that encodes a protein.
  • target pre-mRNA refers to a pre-selected RNA transcript that has not been fully processed into mRNA.
  • pre-RNA includes one or more intron.
  • target microRNA refers to a pre-selected non-coding RNA molecule about 18-30 nucleobases in length that modulates expression of one or more proteins.
  • target pdRNA refers to refers to a pre-selected RNA molecule that interacts with one or more promoter to modulate transcription.
  • target non-coding RNA refers to a pre-selected RNA molecule that is not translated to generate a protein. Certain non-coding RNA are involved in regulation of expression.
  • target viral nucleic acid refers to a pre-selected nucleic acid (RNA or DNA) associated with a virus.
  • RNA or DNA a pre-selected nucleic acid associated with a virus.
  • viral nucleic acid includes nucleic acids that constitute the viral genome as well as transcripts (including reverse-transcripts and RNA transcribed from RNA) of those nucleic acids, whether or not produced by the host cellular machinery.
  • viral nucleic acids also include host nucleic acids that are recruited by a virus upon viral infection.
  • target HCV nucleic acid refers to a pre-selected nucleic acid associated with HCV.
  • nucleic acids associated with HCV include all or portions of the HCV genome as well as host nucleic acid molecules that facilitate HCV replication (e.g., nucleic acids encoding host proteases that cleave an HCV polyprotein, or host microRNAs that modulate cellular functions to facilitate viral replication).
  • targeting refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule.
  • nucleobase complementarity refers to a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (U).
  • complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
  • nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
  • the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
  • non-complementary nucleobase refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
  • an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target.
  • antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target).
  • the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches.
  • the remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases).
  • One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid.
  • hybridization refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid or an antidote to its antisense compound). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases).
  • the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds.
  • the natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.
  • oligomeric compound specifically hybridizes to more than one target site.
  • modulation refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation.
  • modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
  • modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice- variant present compared to conditions that were not perturbed.
  • chemical motif refers to the pattern of unmodified and modified nucleotides in an oligomeric compound.
  • gapmer refers to a chimeric oligomeric compound comprising a central region (a "gap") and a region on either side of the central region (the “wings”), wherein the gap comprises at least one modification that is different from that of each wing.
  • modifications include nucleobase, monomelic linkage, and sugar modifications as well as the absence of modification (unmodified).
  • the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap.
  • each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification.
  • nucleotides in the gap and the nucleotides in the wings all comprise high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in the wings.
  • the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other.
  • nucleotides in the gap are unmodified and nucleotides in the wings are modified.
  • the modification(s) in each wing are the same.
  • the modification(s) in one wing are different from the modification(s) in the other wing.
  • oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
  • cytotoxic sequence refers to a sequence that induces cytotoxicity in cells or a pro-inflammatory response or increases the likelihood that an oligomeric compound will have cytotoxic or pro-inflammatory properties.
  • viral sequence portion refers to a sequence portion having at least partial identity with a sequence from a virus. In certain embodiments, a viral sequence portion has 90%, 95%, or 100% identity with a portion of viral nucleic acid of the same length. In certain embodiments, a viral sequence portion is 3, 4, 5, 6 or more nucleobases in length.
  • component of the interferon pathway refers to a cellular molecule that modulates or is modulated by the interferon activation or suppression.
  • activation refers to the ability of a compound to induce or increase the activity of a molecule or function and includes direct and indirect activation.
  • inhibitor refers to the ability of a compound to suppress or decrease the activity of a molecule or function and includes direct and indirect inhibition.
  • interferon inhibitor refers to a molecule that inhibits one or more effect of interferon activation.
  • an interferon inhibitor may interact with cellular interferon to reduce its activity, or an interferon inhibitor may affect other members of the interferon pathway.
  • an interferon inhibitor is an antisense compound targeted to interferon or another component of the interferon pathway.
  • interferon activator refers to a molecule that acivates one or more effect of the interferon pathway, hi certain instances, interferon itself is an interferon activator. In certain instances, interferon activators stimulate production or activity of other components of the interferon pathway. Certain components of the interferon pathway may exert a negative influence on interferon activity. Since inhibition of the amount or activity of such negative components results in an increase in interferon activation, inhibitors of negative components are interferon activators.
  • inflammation inducing refers to the ability of a compound to induce inflammation in an animal.
  • immunological refers to the ability of a compound to stimulate an immune response.
  • linked together refers to covalently linked.
  • pharmaceutically acceptable salts refers to salts of active compounds that retain the desired biological activity of the active compound and do not impart undesired toxicological effects thereto.
  • cap structure or “terminal cap moiety” refers to chemical modifications incorporated at either terminus of an antisense compound.
  • amelioration refers to a lessening of at least one activity or one indicator of the severity of a condition or disease.
  • the severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
  • pharmaceutical agent refers to a substance provides a therapeutic benefit when administered to a subject.
  • terapéuticaally effective amount refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.
  • administering refers to providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
  • co-administer refers to administering more than one pharmaceutical agent to an animal.
  • the more than one agent may be administered together or separately; at the same time or different times; through the same route of administration or through different routes of administration.
  • route of administration refers to the means by which a pharmaceutical agent is administered to an animal.
  • undesired toxic effect refers to physiological responses attributable to an administration of a pharmaceutical agent other than desired effects.
  • undesired toxic effect includes, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.
  • increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
  • increased bilirubin may indicate liver toxicity or liver function abnormality.
  • a desired toxic effect refers to toxicity having a therapeutic benefit.
  • a desired toxic effect is a toxicity to cancer cells.
  • a desired toxic effect is toxicity to virally infected cells, resulting in reduced viral replication.
  • inducing refers to causing a biological effect.
  • amplifying refers to increasing a biological effect.
  • a pharmaceutical composition refers to a mixture of substances suitable for administering to an animal.
  • a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
  • animal refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • subcutaneous administration refers to administration just below the skin.
  • Intravenous administration refers to administration into a vein.
  • dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a dosage unit refers to a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial comprising lyophilized antisense oligonucleotide.
  • a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
  • active pharmaceutical ingredient refers to the substance in a pharmaceutical composition that provides a desired effect.
  • prodrug refers to 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.
  • Rig-I dependent proinflammatory compound refers to a compound that exerts a proinflammatory effect, that is at least partially dependent on Rig-1.
  • a Rig-I dependent proinflammatory compound is identified by its ability to induce a proinflammatory effect in an animal, but where that proinflammatory effect is reduced, delayed, or eliminated in an animal with reduced Rig-I activity.
  • MDA-5 dependent proinflammatory compound refers to a compound that exerts a proinflammatory effect that is at least partially dependent on MDA-5.
  • a MDA-5 dependent proinflammatory compound is identified by its ability to induce a proinflammatory effect in an animal, but where that proinflammatory effect is reduced, delayed, or eliminated in an animal with reduced IPSl activity.
  • IPSl dependent proinflammatory compound refers to a compound that exerts a proinflammatory effect, that is at least partially dependent on IPSl .
  • a IPSl dependent pro proinflammatory compound is identified by its ability to induce a proinflammatory effect in an animal, but where that proinflammatory effect is reduced, delayed, or eliminated in an animal with reduced IPSl activity.
  • LGP2 dependent proinflammatory compound refers to a compound that exerts a proinflammatory effect, that is at least partially dependent on LGP2.
  • a LGP2 dependent proinflammatory compound is identified by its ability to induce a proinflammatory effect in an animal, but where that proinflammatory effect is reduced, delayed, or eliminated in an animal with reduced LGP2 activity.
  • LGP2 is also known in the art as Dhx58.
  • 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.
  • 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).
  • sugar surrogate overlaps with the slightly broader term “nucleoside mimetic” but is intended to indicate replacement of the sugar unit (furanose ring) only.
  • 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.
  • anti-cancer agent means an agent for treating cancer.
  • the anti-cancer agent is cytotoxic.
  • the anti-cancer agent kills cancer cells.
  • 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, chloramb
  • oligomeric compounds including antisense compounds, compared to naturally occurring oligomers, such as DNA or RNA.
  • Certain such modifications alter the activity of the oligomeric compound.
  • Certain such chemical modifications can alter activity by, for example: increasing affinity of an antisense compound for its target nucleic acid, increasing its resistance to one or more nucleases, and/or altering the pharmacokinetics or tissue distribution of the oligomeric compound.
  • the use of chemistries that increase the affinity of an oligomeric compound for its target can allow for the use of shorter oligomeric compounds.
  • oligomeric compounds comprise one or more modified nucleosides.
  • modified nucleosides may include a modified sugar and/or a modified nucleobases.
  • incorporation of such modified nucleosides in an oligomeric compound result in increased affinity for a target nucleic acid and/or increased stability, including but not limited to, increased resistance to nuclease degradation, and or improved toxicity and/or uptake properties
  • the naturally occurring base portion of a nucleoside is typically a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • a phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the naturally occurring linkage or backbone of RNA and of DNA is a 3' to 5' phosphodiester linkage.
  • a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp.
  • nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art. Certain sugars
  • 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.
  • nucleosides are modified by modification of the ribofuranose ring.
  • BNA bicyclic nucleic acid
  • nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 and 2'-O(CH 2 ) 2 OCH 3 substituent groups.
  • bicyclic nucleic acids examples include without limitation nucleosides comprising a bridge between the 4 1 and the 2' ribosyl ring atoms.
  • antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4'- ⁇ -D-(CH 2 )-O-2' ( ⁇ -D-LNA); 4'-(CH 2 )-S-2'; 4'- ⁇ -L-(CH 2 )-O-2' ( ⁇ -L-LNA); 4'-(CH 2 ) 2 -O-2' (ENA); 4'-C(CH 3 ) 2 -O-2' (see PCT/US2008/068922); 4'-CH(CH 3 )- O-2' and 4'-CH(CH 2 OCH 3 )-O-2' (see U.S.
  • Patent 7,399,845, issued on July 15, 2008); 4'-CH 2 - N(OCH 3 )-2' (see PCTVUS2008/ 064591); 4'-CH 2 -O-N(CH 3 )-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH 2 -N(R)-O-2' (see U.S. Patent 7, 427, 672, issued on September 23, 2008); 4'-CH 2 -C(CH 3 )-2'and 4'-CH 2 -C( CH 2 )-2' (see PCT/US2008/ 066154); and wherein R is, independently, H, C 1 -C 12 alkyl, or a protecting group.
  • nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate.
  • 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:
  • 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 review article: Leumann, Christian J., ). Such ring systems can undergo various additional substitutions to enhance activity.
  • linking groups that link nucleosides (including modified and unmodified nucleosides) together, thereby forming an oligomeric compound.
  • the two main classes of linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-0- C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-).
  • Oligomeric compounds having non-phosphorus linking groups are referred to as oligonucleosides.
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound, hi certain embodiments, linkages having a crural atom can be prepared a racemic mixtures, as separate enantomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing linkages are well known to those skilled in the art.
  • the oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • oligomeric compounds having reactive phosphorus groups useful for forming linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
  • Methods of preparation and/or purification of precursors or oligomeric compounds are not a limitation of the compositions or methods provided herein.
  • Methods for synthesis and purification of oligomeric compounds including DNA, RNA, oligonucleotides, oligonucleosides, and antisense compounds are well known to those skilled in the art.
  • oligomeric compounds comprise a plurality of monomelic subunits linked together by linking groups.
  • Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs.
  • these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops.
  • Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
  • the present invention provides chimeric oligomeric compounds.
  • chimeric oligomeric compounds are chimeric oligonucleotides.
  • the chimeric oligonucleotides comprise differently modified nucleotides.
  • chimeric oligonucleotides are mixed-backbone antisense oligonucleotides.
  • a chimeric oligomeric compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Any combination of modifications and/or mimetic groups can comprise a chimeric oligomeric compound as described herein.
  • chimeric oligomeric compounds typically comprise at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • an additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • chimeric oligomeric compounds are gapmers.
  • a mixed-backbone oligomeric compound has one type of internucleotide linkages in one or both wings and a different type of internucleoside linkages in the gap.
  • the mixed-backbone oligonucleotide has phosphodiester linkages in the wings and phosphorothioate linkages in the gap.
  • the internucleoside linkages in a wing is different from the internucleoside linkages in the gap, the internucleoside linkage bridging that wing and the gap is the same as the internucleoside linkage in the wing.
  • the internucleoside linkage bridging that wing and the gap is the same as the internucleoside linkage in the gap.
  • the present invention provides oligomeric compounds, including antisense oligomeric compounds and antidote oligomeric compounds, of any of a variety of ranges of lengths.
  • the invention provides oligomeric compounds consisting of X-Y linked oligonucleosides, where X and Y are each independently selected from 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, and 50; provided that X ⁇ Y.
  • the invention provides oligomeric compounds comprising: 8-
  • oligomeric compounds are modified by covalent attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound.
  • conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Preferred conjugate groups amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a
  • Linking groups or bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Linking groups are useful for attachment of chemical functional groups, conjugate groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
  • the linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units.
  • bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • linking groups include, but are not limited to, substituted Cl-ClO alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206- 217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
  • Oligomeric compounds provided herein can be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • the invention is not limited by the method of antisense compound synthesis.
  • compositions and Methods for Formulating Pharmaceutical Compositions are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The method of the invention is not limited by the method of oligomer purification.
  • Oligomeric compounds 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.
  • Oligomeric compounds can be utilized in pharmaceutical compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS is a diluent suitable for use in compositions to be delivered parenterally.
  • employed in the methods described herein is a pharmaceutical composition comprising an antisense compound and/or antidote compound and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is PBS.
  • compositions comprising oligomeric compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters.
  • pharmaceutical compositions comprising oligomeric compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • 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 oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active oligomeric compound.
  • Antisense mechanisms are all those involving the hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
  • a type of antisense mechanism involving target degradation includes an RNase H.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
  • chemically-modified antisense compounds have a higher affinity for target RNAs than does non-modified DNA. In certain such embodiments, that higher affinity in turn provides increased potency allowing for the administration of lower doses of such compounds, reduced potential for toxicity and improvement in therapeutic index and decreased overall cost of therapy.
  • Antisense compounds are oligomeric compounds. Accordingly, in certain embodiments, antisense compounds comprise, for example and without limitation, any of the modifications and motifs described in the discussion above for oligomeric compounds. Antisense compounds may be single-stranded or double-stranded oligomeric compounds. In embodiments where an antisense compound is a double-stranded oligomeric compound, the two strands may have the same modifications and motifs or may have modifications and motifs that are different from one another. Certain antisense compounds and modifications and motifs useful for such compounds are known in the art.
  • a target nucleic acid is a riiRNA.
  • antisense compounds are designed to modulate that target mRNA or its expression.
  • designing an antisense compound to a target nucleic acid molecule can be a multistep process. Typically the process begins with the identification of a target protein, the activity of which is to be modulated, and then identifying the nucleic acid the expression of which yields the target protein.
  • designing of an antisense compound results in an antisense compound that is hybridizable to the targeted nucleic acid molecule.
  • the antisense compound is an antisense oligonucleotide or antisense oligonucleoside.
  • an antisense compound and a target nucleic acid are complementary to one another. In certain such embodiments, an antisense compound is perfectly complementary to a target nucleic acid. In certain embodiments, an antisense compound includes one mismatch. In certain embodiments, an antisense compound includes two mismatches, hi certain embodiments, an antisense compound includes three or more mismatches.
  • RNA to be modulated include, but are not limited to, translocation functions, which include, but are not limited to, translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, and translation of protein from the RNA.
  • RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3' maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
  • Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level.
  • modulation of expression can mean increase or decrease in target RNA or protein levels.
  • modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.
  • antisense compounds specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
  • stringent hybridization conditions or “stringent conditions” refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated.
  • Tm melting temperature
  • oligomeric compounds for example oligonucleotides, with cytotoxic properties.
  • cytotoxicity can be, for example, hepatotoxicity, renal toxicity, the inhibition of cell proliferation, or the induction of cell death.
  • methods of modulating the cytotoxic properties of oligomeric compounds by selecting certain cytotoxic sequences.
  • methods for ameliorating the effects of cytotoxic oligomeric compounds by administering a therapeutically or prophylactically effective amount of an interferon modulator.
  • oligomeric compounds that can increase or decrease cytotoxic and/or proinflammatory properties of the compound. Such methods are useful in the design of oligomeric compounds used to treat cells, tissues or subjects in order to modulate the expression of a targeted molecule.
  • oligomeric compounds are provided that increase cytotoxicity and/or proinflammatory properties by the inclusion of a cytotoxic sequence that increases the probability that an oligomeric compound, such as an antisense oligonucleotide, will have cytotoxic or proinflammatory properties.
  • a cytotoxic sequence that increases the probability that an oligomeric compound, such as an antisense oligonucleotide, will have cytotoxic or proinflammatory properties.
  • ISIS 147420 is rendered non-toxic by changing a single nucleobase. For example, changing the nucleobase at position 14 of the nucleobase sequence of ISIS 147420 from a thymidine to an adenine renders the compound non-toxic.
  • oligomeric compounds are provided that increase cytotoxicity by the inclusion of three or more CT dimers.
  • cytotoxic oligomeric compound is an oligomeric compound that has a cytotoxic effect, i.e., results in a "cytotoxic response”.
  • cytotoxic oligonucleotide is an oligonucleotide that has a cytotoxic effect.
  • a cytotoxic effect or response can be either an antiproliferative effect on cell growth or caspase activation or both. Consequently, the disclosed compounds can be administered to a cell, tissue or subject to treat a hyperproliferative condition.
  • a hyperproliferative condition is a condition where cells hyperproliferate, i.e., where cells exhibit an abnormal growth rate.
  • a cytotoxic response also includes hepatoxicity.
  • a subject such as a mammal.
  • hyperproliferative conditions include cancer (e.g., small cell lung cancer and leukemia), atherosclerosis, diabetic retinopathy, psoriasis, endometriosis, macular degenerative disorders, prostate enlargement and lipomas, hi a broad embodiment, the methods comprise the step of administering to the subject having or at risk of having a disease characterized by a hyperproliferative condition a therapeutically effective amount of a cytotoxic oligomeric compound.
  • cytotoxic oligomeric compounds can include oligonucleotides, peptide nucleic acids, morpholino compounds and locked nucleic acids.
  • the administration of the cytotoxic oligomeric compound can be topical, intratracheal, intranasal, epidermal, transdermal, oral, parenteral, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular, intracranial, intrathecal, and/or intraventricular.
  • a method for activating a caspase in a cell, tissue or subject comprises contacting the cell, tissue or subject with a cytotoxic oligomeric compound to activate a caspase. Consequently, disclosed herein are methods for treating conditions related to insufficient caspase induction in a subject, such as a mammal.
  • caspase-deficient conditions include cancer, autoimmune disorders and viral infections.
  • methods for the treatment of a cell, tissue or subject are provided to reduce a cytotoxic effect caused by the introduction of a cytotoxic oligomeric compound.
  • the method comprises contacting said treated cell, tissue or subject with an aspartic protease inhibitor, such as Cathepsin D.
  • the disclosed embodiments can be used to avoid a particular cytotoxic sequence in the design of an oligomeric compound in order to allay cytotoxic effects.
  • the disclosed cytotoxic sequences can be avoided in the design of antisense compounds where the cytotoxicity has an undesired effect.
  • the cytotoxicity of an oligomeric compound containing three or more CT dimers can be reduced wherein the oligomer can potentially form a hairpin structure.
  • the cytotoxicity of an oligomeric compound is characterized by its ability to bind nucleolin or LRPPRC, or to modulate the cellular localization of nucleolin. In another aspect, the cytotoxicity is indicated by caspase activation, extracellular lactate dehydrogenase activity or chromatin condensation in cells.
  • compounds of the present invention are immunostimulatory. In certain embodiments, compounds of the present invention are pro-inflammatory. In certain embodiments, compounds of the present invention are interferon activating. Certain such compounds comprise a toxic sequence. Certain immunostimulory compounds, including immunostimulatory oligomeric compounds have been described. See for example, U.S. Patent Application No. 11/404,544, which is hereby incorporated by reference in its entirety.
  • TLR9 Toll-like receptor 9
  • MAPK mitogen activated protein kinase
  • NF nuclear factor
  • This stimulation leads to an increased production of cytokines including interferons, interleukin (IL)-6 and 12, tumor necrosis factor-alpha (TNF- ⁇ ), chemokines, costimulatory molecules, and antigen presentation on antigen presenting cells (APCs).
  • IL interleukin
  • TNF- ⁇ tumor necrosis factor-alpha
  • APCs antigen presentation on antigen presenting cells
  • CpG oligonucleotides The cellular and humoral immune responses stimulated by exposure to CpG oligonucleotides is likely a result of the relative abundance of unmethylated CpG motifs in bacteria and other pathogens, as compared to mammalian DNA, in which such motifs are rare.
  • the human immune system has apparently evolved to recognize CpG sequences as early signs of infection and to initiate an immediate immune response against pathogens without causing adverse reactions frequently seen with other proinflammatory agents.
  • CpG containing nucleic acids relying on this innate immunity, can utilze a distinct and natural pathway for immune therapy. Oligonucleotides containing CpG motifs have been developed to exploit this process for use for the prevention and treatment of infections (Dalpke et al, BioDrugs.
  • CpG proinflammatory oligomeric compounds can be tested in various immune cell assays. Such methods are described in detail in PCT Published Patent Applications PCT/US95/01570 (WO 96/02555) and PCT/US97/19791 (WO 98/18810) claiming priority to U.S. Ser. Nos. 08/386,063 and 08/960,774, filed on Feb. 7, 1995 and Oct. 30, 1997 respectively. See also, U.S. Ser. No. 11/404,544. Certain methods require the use of primary cells from either mice or humans. The effectiveness of the assay on determining the activity of a nucleic acid is dependent upon the appropriate receptors and signaling pathways being present in the cells used.
  • oligonucleotides devoid of CpG motifs are capable of eliciting a proinflammatory response in vivo and in vitro.
  • TLR9 a mouse knockout model
  • the response to these oligonucleotides is mediated by TLR9.
  • the immunostimulatory motif consists of a 5'-TC dinucleotide in a thymidine rich background, preferably about at least 35% (Vollmer et al., Immunol. 2004. 113:212-223, incorporated herein by reference). The motif was found to stimulate B-cell activation, but lacked a ThI -like cytokines and chemokines.
  • Oligonucleotides intended for antisense applications are designed to avoid CpG and other proinflammatory motifs that induce a proinflammatory response by using various chemical modifications (i.e. 2'MOE sugars and methylation of cytosine residues) (Henry et al., J Pharmacol. Expt. Thera. 2000. 292: 468-479).
  • some oligonucleotides have immnunostimulatory activity, despite having no identifiable proinflammatory motifs and/or containing modified nucleobases to reduce immnunostimulatory activity. Such activity is often not identified until the oligonucleotide is administered to an animal.
  • CpG motifs, 5'-TC motifs, and highly repetitive sequences are known and easily identifiable by their sequences. Oligonucleotides having CpG motifs are useful for their stimulation of a proinflammatory response via the TLR9 pathway, making them useful for the prevention, amelioration, and/or treatment of disease. CpG motifs are also useful as vaccine adjuvants. However, oligonucleotides including such motifs are limited in their usefulness as antisense oligonucleotides designed to act through a sequence specific, hybridization mechanism or other target specific pathway.
  • RAW 264.7 cells are a mouse macrophage cell line established from a tumor induced in a male mouse by intraperitoneal injection of Abelson Leukemia Virus (A-MuLV) (Raschkea et al., Cell. 15:261-267.1978) were used.
  • the cells are capable of antibody dependent lysis of sheep erythrocytes and tumor targets. They also express some TLRs, including TLR9 (Hatao et al., J. Leukoc. Biol. 76:904-908. 2004).
  • TLR9 nuclear Factor of Activated T- cells
  • Proinflammatory activity was determined by NFAT activation as determined by ELISA assay.
  • the cell line was found to have both positive and negative predictive value for proinflammatory activity as previously determined in mice and in primary mouse bone marrow cells.
  • the method provides an in vitro system that does not require primary cells to test for TLR9 independent proinflammatory activity of oligonucleotides. This allows for the screening of oligonucleotides intended for uses wherein proinflammatory activity is not desired, without the cost and effort required for animal testing or the preparation of primary cells.
  • the present invention includes the use of proinflammatory oligomeric compounds in a manner similar to CpG oligonucleotides for the stimulation of a proinflammatory response for the prevention, amelioration, and/or treatment of disease, or as a vaccine adjuvant.
  • the invention includes a pharmaceutical composition comprising an effective amount for stimulating an immune response of proinflammatory oligomeric compounds, and a pharmaceutically acceptable carrier.
  • Another aspect provided herein is a composition of matter, comprising a proinflammatory oligomeric compounds.
  • the proinflammatory oligomeric compounds may contain 2'-methoxyethoxy modifications.
  • the invention is a composition of a proinflammatory oligomeric compounds and an antigen.
  • compositions are a proinflammatory oligomeric compounds and an anti-microbial agent.
  • the anti-microbial agent can be selected from the group consisting of an anti- viral agent, an anti-parasitic agent, an anti-bacterial agent and an anti-fungal agent.
  • a vaccine formulation is provided.
  • the vaccine includes any of the compositions of the invention in combination with an antigen.
  • Another aspect is a method of stimulating an immune response.
  • the method involves administering a proinflammatory oligomeric compounds to a subject in an amount effective to induce an immune response.
  • the proinflammatory oligomeric compound is administered orally, locally, in a sustained release device, mucosally to a mucosal surface, systemically, parenterally, or intramuscularly.
  • the proinflammatory oligomeric compound may be delivered in an amount effective for inducing a mucosal immune response or a systemic immune response.
  • the mucosal surface is selected from the group consisting of an oral, nasal, rectal, vaginal, and ocular surface.
  • the method includes exposing the subject to an antigen wherein the immune response is an antigen-specific immune response.
  • the antigen may be encoded by a nucleic acid vector which can be delivered to the subject.
  • the disclosed nucleic acids are useful for treating cancer.
  • the proinflammatory oligomeric compounds are also useful according to other aspects of the invention in preventing cancer (e.g., reducing a risk of developing cancer) in a subject at risk of developing a cancer.
  • cancer e.g., reducing a risk of developing cancer
  • a number of cancers are well known to those skilled in the art.
  • the proinflammatory oligomeric compounds may also be used for increasing the responsiveness of a cancer cell to a cancer therapy (e.g., an anti-cancer therapy), optionally when the proinflammatory oligomeric compounds is administered in conjunction with an anti-cancer therapy.
  • the anti-cancer therapy may be a chemotherapeutic agent, a vaccine (e.g., an in vitro primed dendritic cell vaccine or a cancer antigen vaccine) or an antibody based therapy. This latter therapy may also involve administering an antibody specific for a cell surface antigen of, for example, a cancer cell, wherein the immune response results in antigen dependent cellular cytotoxicity (ADCC).
  • ADCC antigen dependent cellular cytotoxicity
  • a subject having cancer or at risk of having a cancer is administered a proinflammatory oligomeric compounds and an anti-cancer therapy.
  • the anti-cancer therapy is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine.
  • the proinflammatory oligomeric compounds may be co-administered, either simultaneously or during the same course of treatement, with interferon gamma.
  • Cancer chemotherapeutic and immunotherapeutic agents are well known to those skilled in the art.
  • the invention in other aspects relates to methods for preventing disease in a subject.
  • the method involves administering to the subject a proinflammatory oligomeric compound on a regular basis to promote immune system responsiveness to prevent disease in the subject.
  • diseases or conditions sought to be prevented using the prophylactic methods of the invention include microbial infections (e.g., sexually transmitted diseases) and anaphylactic shock from food allergies.
  • the invention is a method for inducing an innate immune response by administering to the subject a proinflammatory oligomeric compound in an amount effective for activating an innate immune response.
  • Another method involves administering to a subject having or at risk of having a viral, including, but not limited to, retroviral infection, an effective amount of proinflammatory oligomeric compounds for treating or preventing the viral or retroviral infection of any of the compositions of the invention.
  • the virus is caused by a hepatitis virus, HIV, hepatitis B, hepatitis C, herpes virus, or papillomavirus.
  • Another aspect is a method involves administering to a subject having or at risk of having a bacterial infection or parasitic infection an effective amount for treating or preventing the bacterial infection or parasitic infection of any of the compositions of the invention.
  • the proinflammatory oligomeric compounds of the instant invention are not necessarily species specific, the oligonucleotides may be used in animals as well as man.
  • compositions of the invention may be coadminstered, either simultaneously or in the same course of treatment, with other allergy and/or asthma medication. Such medications are well known to those skilled in the art.
  • Another method involves administering to a subject having or at risk of an immune deficiency, an effective amount of a composition of the instant invention for treating or preventing the immune deficiency.
  • Another aspect is a method for inducing a ThI immune response by administering to a subject any of the compositions of the invention in an effective amount to produce a ThI immune response, preferably without stimulating a Th2 response.
  • the disclosed proinflammatory oligomeric compounds are useful for not only their immune stimulatory properties but also in the treatment of bone and neural diseases as they are thought to act through the TREM pathways.
  • the disclosed proinflammatory oligomeric compounds are useful in some aspects of the invention as a prophylactic vaccine for the treatment of a subject at risk of developing an infection with an infectious organism or a cancer in which a specific cancer antigen has been identified or an allergy or asthma where the allergen or predisposition to asthma is known.
  • Methods of identification of individuals at risk for allergy, asthma, or cancer; or having allergy, asthma, or cancer are well known to those skilled in the art.
  • the proinflammatory oligomeric compounds can also be given without the antigen or allergen for shorter term protection against infection, allergy or cancer, and in this case repeated doses will allow longer term protection. If the antigen is an allergen and the subject develops allergic responses to that particular antigen and the subject may be exposed to the antigen, i.e., during pollen season, then that subject is at risk of exposure to the antigen.
  • the invention also encompasses the use of the proinflammatory oligomeric compounds for the treatment of a subject having an infection, an allergy, asthma, or a cancer.
  • the proinflammatory oligomeric compounds can be used with an antigen to mount an antigen specific systemic or mucosal immune response that is capable of reducing the level of or eradicating the infectious pathogen.
  • An infectious disease as used herein, is a disease arising from the presence of a foreign microorganism in the body. It is particularly important to develop effective vaccine strategies and treatments to protect the body's mucosal surfaces, which are the primary site of pathogenic entry.
  • the subject is exposed to the antigen.
  • the term exposed to refers to either the active step of contacting the subject with an antigen or the passive exposure of the subject to the antigen in vivo.
  • Methods for the active exposure of a subject to an antigen are well-known in the art.
  • an antigen is administered directly to the subject by any means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration.
  • the antigen can be administered systemically or locally. Methods for administering the antigen and the proinflammatory nucleic acid are described in more detail below.
  • a subject is passively exposed to an antigen if an antigen becomes available for exposure to the immune cells in the body.
  • a subject may be passively exposed to an antigen, for instance, by entry of a foreign pathogen into the body or by the development of a tumor cell expressing a foreign antigen on its surface.
  • the methods in which a subject is passively exposed to an antigen can be particularly dependent on timing of administration of the proinflammatory oligomeric compounds. For instance, in a subject at risk of developing a cancer or an infectious disease or an allergic or asthmatic response, the subject may be administered the proinflammatory oligomeric compounds on a regular basis when that risk is greatest, i.e., during allergy season or after exposure to a cancer causing agent.
  • An antigen as used herein is a molecule capable of provoking an immune response.
  • Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and muticellular organisms such as parasites and allergens.
  • the term antigen broadly includes any type of molecule which is recognized by a host immune system as being foreign.
  • Antigens include but are not limited to cancer antigens, microbial antigens, and allergens.
  • a cancer antigen as used herein is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule.
  • Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens.
  • Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • a microbial antigen as used herein is an antigen of a microorganism and includes but is not limited to virus (e.g., HIV, Picornavurudae, and Coronoviridae); bacteria (e.g., Gram negative and Gram positive bacteria); parasite; fungus (e.g., Chlamydia, Cryptococcus); and protist (e.g., Plasmodium, Leshmania).
  • virus e.g., HIV, Picornavurudae, and Coronoviridae
  • bacteria e.g., Gram negative and Gram positive bacteria
  • parasite e.g., Chlamydia, Cryptococcus
  • protist e.g., Plasmodium, Leshmania
  • a compound is similar to a natural microorganism antigen if it induces an immune response (humeral and/or cellular) to a natural microorganism antigen.
  • immune response humidity and/or cellular
  • Such antigens are used routinely in the art and are well known to those of ordinary skill in the art.
  • Other medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference.
  • Nonhuman vertebrates are also capable of developing infections which can be prevented or treated with the proinflammatory oligomeric compounds disclosed herein.
  • the methods of the invention are useful for treating infections of animals.
  • Many vaccines for the treatment of non-human vertebrates are disclosed in Bennett, K., Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995.
  • the term treat, treated, or treating when used with respect to an infectious disease includes a prophylactic treatment which increases the resistance of a subject (a subject at risk of infection) to infection with a pathogen or, in other words, decreases the likelihood that the subject will become infected with the pathogen as well as a treatment after the subject (a subject who has been infected) has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
  • Treatment can include administration of single or multiple doses of the compounds of the instant invention.
  • An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject.
  • the list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g., penicillin). Allergens are well known to those skilled in the art.
  • the antigen may be an antigen that is encoded by a nucleic acid vector or it may be not encoded in a nucleic acid vector. In the former case the nucleic acid vector is administered to the subject and the antigen is expressed in vivo. In the latter case the antigen may be administered directly to the subject.
  • An antigen not encoded in a nucleic acid vector as used herein refers to any type of antigen that is not a nucleic acid.
  • the antigen not encoded in a nucleic acid vector is a polypeptide. Minor modifications of the primary amino acid sequences of polypeptide antigens may also result in a polypeptide which has substantially equivalent antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as antigenicity still exists.
  • the polypeptide may be, for example, a viral polypeptide.
  • substantially purified refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification.
  • the substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non- reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide.
  • the purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis. Other types of antigens not encoded by a nucleic acid vector such as polysaccharides, small molecule, mimics etc are described above, and included within the invention.
  • the invention also utilizes polynucleotides encoding the antigenic polypeptides.
  • the antigen may be delivered to the subject in a nucleic acid molecule which encodes for the antigen such that the antigen must be expressed in vivo.
  • Such antigens delivered to the subject in a nucleic acid vector are referred to as antigens encoded by a nucleic acid vector.
  • the nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell.
  • the gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter- enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked.
  • the gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Such promoters are well known to those skilled in the art.
  • the invention is not limited by the specific gene expression cassette used.
  • the antigen nucleic acid of the invention may be delivered to the immune system alone or in association with a vector.
  • a vector is any vehicle capable of facilitating the transfer of the antigen nucleic acid to the cells of the immune system so that the antigen can be expressed and presented on the surface of the immune cell.
  • the vector generally transports the nucleic acid to the immune cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vector optionally includes at least one gene expression sequence to enhance expression of the antigen nucleic acid in immune cells.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antigen nucleic acid sequences.
  • Such vectors are well known to those skilled in the art.
  • the selection of a specific vector type is not a limitation of the instant invention.
  • Suitable viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Such viruses are well known to those skilled in the art.
  • Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Plasmids are commercially available and well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA.
  • Certain gene carrying plasmids can be delivered to the immune system using bacteria.
  • Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles.
  • the bacterial delivery vehicles can be administered to a host subject orally or by other administration means.
  • the bacteria deliver the plasmid to immune cells, e.g., B cells, dendritic cells, likely by passing through the gut barrier. High levels of immune protection have been established using this methodology.
  • immune cells e.g., B cells, dendritic cells, likely by passing through the gut barrier. High levels of immune protection have been established using this methodology.
  • Such methods of delivery are useful for the aspects of the invention utilizing systemic delivery of antigen, proinflammatory nucleic acid and/or other therapeutic agent.
  • the proinflammatory oligomeric compounds are useful as vaccine adjuvants. It was previously established that CpG oligonucleotides are excellent vaccine adjuvants. It was also demonstrated, however, that CpG oligonucleotide which are superb vaccine adjuvants in mice are not the suitable adjuvants in non-rodent animals.
  • the proinflammatory oligomeric compounds can be administered to a subject with an anti-microbial agent.
  • An anti-microbial agent refers to a naturally-occurring or synthetic compound which is capable of killing or inhibiting infectious microorganisms.
  • the type of anti-microbial agent useful according to the invention will depend upon the type of microorganism with which the subject is infected or at risk of becoming infected.
  • Anti-microbial agents include but are not limited to anti-bacterial agents, anti- viral agents, anti-fungal agents and anti-parasitic agents.
  • anti-bacterial agents kill or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds having similar functions.
  • Antibiotics are low molecular weight molecules which are produced as secondary metabolites by cells, such as microorganisms. In general, antibiotics interfere with one or more bacterial functions or structures which are specific for the microorganism and which are not present in host cells.
  • Anti-viral agents can be isolated from natural sources or synthesized and are useful for killing or inhibiting viruses.
  • Anti-fungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections.
  • Anti-parasite agents kill or inhibit parasites. Such agents are well known to those skilled in the art.
  • Antibacterial agents kill or inhibit the growth or function of bacteria.
  • a large class of antibacterial agents is antibiotics.
  • Antibiotics which are effective for killing or inhibiting a wide range of bacteria, are referred to as broad spectrum antibiotics.
  • Other types of antibiotics are predominantly effective against the bacteria of the class gram-positive or gram-negative. These types of antibiotics are referred to as narrow spectrum antibiotics.
  • Other antibiotics which are effective against a single organism or disease and not against other types of bacteria are referred to as limited spectrum antibiotics.
  • Antibacterial agents are sometimes classified based on their primary mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors. Such agents are well known to those skilled in the art.
  • Antiviral agents are compounds which prevent infection of cells by viruses or replication of the virus within the cell. There are many fewer antiviral drugs than antibacterial drugs because the process of viral replication is so closely related to DNA replication within the host cell, that non-specific antiviral agents would often be toxic to the host. There are several stages within the process of viral infection which can be blocked or inhibited by antiviral agents.
  • These stages include, attachment of the virus to the host cell (immunoglobulin or binding peptides), uncoating of the virus (e.g., amantadine), synthesis or translation of viral mRNA (e.g., interferon), replication of viral RNA or DNA (e.g., nucleoside analogues), maturation of new virus proteins (e.g., protease inhibitors), and budding and release of the virus.
  • viruses e.g., amantadine
  • synthesis or translation of viral mRNA e.g., interferon
  • replication of viral RNA or DNA e.g., nucleoside analogues
  • maturation of new virus proteins e.g., protease inhibitors
  • Anti-fungal agents are useful for the treatment and prevention of infective fungi. Antifungal agents are sometimes classified by their mechanism of action. Such agents are well known to those skilled in the art.
  • Proinflammatory oligomeric compounds can be combined with other therapeutic agents such as adjuvants to enhance immune responses.
  • the proinflammatory oligomeric compounds and other therapeutic agents may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time.
  • the other therapeutic agents are administered sequentially with one another and with proinflammatory oligomeric compounds, when the administration of the other therapeutic agents and the proinflammatory oligomeric compounds is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
  • Other therapeutic agents include but are not limited to adjuvants, cytokines, antibodies, antigens, etc.
  • the proinflammatory oligomeric compounds are useful as adjuvants for inducing a systemic immune response. Thus, either can be delivered to a subject exposed to an antigen to produce an enhanced immune response to the antigen.
  • compositions of the invention may also be administered with non-nucleic acid adjuvants.
  • a non-nucleic acid adjuvant is any molecule or compound except for the proinflammatory oligomeric compounds described herein which can stimulate the humoral and/or cellular immune response.
  • Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, immune stimulating adjuvants, and adjuvants that create a depo effect and stimulate the immune system. Such adjuvants are well known to those skiled in the art.
  • An immune stimulating adjuvant is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines. Such agents are well known to those skilled in the art.
  • the proinflammatory oligomeric compounds are also useful as mucosal adjuvants. It has previously been discovered that both systemic and mucosal immunity are induced by mucosal delivery of CpG nucleic acids.
  • the systemic immunity induced in response to CpG nucleic acids included both humoral and cell-mediated responses to specific antigens that were not capable of inducing systemic immunity when administered alone to the mucosa.
  • Mucosal adjuvants referred to as non-nucleic acid mucosal adjuvants may also be administered with proinflammatory oligomeric compounds.
  • a non-nucleic acid mucosal adjuvant as used herein is an adjuvant other than an proinflammatory oligomeric compounds that is capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with an antigen. Such agents are well known to those skilled in the art.
  • Immune responses can also be induced or augmented by the co-administration or co- linear expression of cytokines (Bueler & Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasaki et al., 1997; Tsuji et al., 1997) with the proinflammatory oligomeric compounds.
  • the cytokines can be administered directly with proinflammatory oligomeric compounds or may be administered in the form of a nucleic acid vector that encodes the cytokine, such that the cytokine can be expressed in vivo.
  • the cytokine is administered in the form of a plasmid expression vector.
  • the term cytokine is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment.
  • cytokines include, but are not limited to IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-IO, IL-12, IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-gamma (gamma-IFN), IFN-alpha, tumor necrosis factor (TNF), TGF-beta, FLT-3 ligand, and CD40 ligand.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • gamma-IFN interferon-gamma
  • TNF tumor necrosis factor
  • TGF-beta FLT-3 ligand
  • CD40 ligand CD40 ligand.
  • Cytokines play a role in directing the T cell response.
  • Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including other T cells.
  • Most mature CD4+ T helper cells express one of two cytokine profiles: ThI or Th2.
  • the ThI subset promotes delayed-type hypersensitivity, cell- mediated immunity, and immunoglobulin class switching to IgG 2a -
  • the Th2 subset induces humoral immunity by activating B cells, promoting antibody production, and inducing class switching to IgG 1 and IgE.
  • the cytokine be a ThI cytokine.
  • the proinflammatory oligomeric compounds can also be administered in conjunction with an anti-cancer therapy.
  • Anti-cancer therapies include cancer medicaments, radiation and surgical procedures.
  • a "cancer medicament” refers to an agent which is administered to a subject for the purpose of treating a cancer.
  • treating cancer includes preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth of an established cancer.
  • the cancer medicament is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer.
  • Various types of medicaments for the treatment of cancer are described herein.
  • cancer medicaments are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone therapy, and biological response modifiers. Treating cancer can require the administration of a single or multiple doses of the compound of the instant invention. Additionally, the methods of the invention are intended to embrace the use of more than one cancer medicament along with the proinflammatory oligomeric compounds. As an example, where appropriate, the proinflammatory oligomeric compounds may be administered with both a chemotherapeutic agent and an immunotherapeutic agent.
  • the cancer medicament may embrace an immunotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine all administered to one subject for the purpose of treating a subject having a cancer or at risk of developing a cancer.
  • Cancer medicaments function in a variety of ways.
  • the methods of the invention are not limited by the cancer medicament or its mechanism of action.
  • proinflammatory oligomeric compounds in conjunction with immunotherapeutic cancer medicaments such as monoclonal antibodies is able to increase long- term survival through a number of mechanisms including significant enhancement of ADCC (as discussed above), activation of natural killer (NK) cells and an increase in IFN-alpha levels.
  • the nucleic acids when used in combination with monoclonal antibodies serve to reduce the dose of the antibody required to achieve a biological result.
  • Cancer vaccines are medicaments which are intended to stimulate an endogenous immune response against cancer cells.
  • Currently produced vaccines predominantly activate the humoral immune system (i.e., the antibody dependent immune response).
  • Other vaccines currently in development are focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells.
  • Cancer vaccines generally enhance the presentation of cancer antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
  • cancer vaccines may take one of several forms, as discussed infra, their purpose is to deliver cancer antigens and/or cancer associated antigens to antigen presenting cells (APC) in order to facilitate the endogenous processing of such antigens by APC and the ultimate presentation of antigen presentation on the cell surface in the context of MHC class I molecules.
  • APC antigen presenting cells
  • the use of proinflammatory oligomeric compounds in conjunction with cancer vaccines provides an improved antigen-specific humoral and cell mediated immune response, in addition to activating NK cells and endogenous dendritic cells, and increasing IFN-alpha levels. This enhancement allows a vaccine with a reduced antigen dose to be used to achieve the same beneficial effect.
  • cancer vaccines may be used along with adjuvants, such as those described above.
  • cancer antigen and “tumor antigen” are used interchangeably to refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells.
  • Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens.
  • cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Other vaccines take the form of dendritic cells which have been exposed to cancer antigens in vitro, have processed the antigens and are able to express the cancer antigens at their cell surface in the context of MHC molecules for effective antigen presentation to other immune system cells.
  • oncogenes e.g., activated ras oncogene
  • suppressor genes e.g., mutant p53
  • fusion proteins resulting from internal deletions or chromosomal translocations e.g., those carried on RNA and DNA tumor viruses.
  • Other vaccines take the form of dendritic cells which have been exposed to cancer anti
  • chemotherapeutic agents embrace all other forms of cancer medicaments which do not fall into the categories of immunotherapeutic agents or cancer vaccines.
  • Chemotherapeutic agents as used herein encompass both chemical and biological agents and act by a number of mechanisms, both known and unknown. The mechanism of action of these agents is not a limitation of the invention.
  • proinflammatory oligomeric compounds are used as a replacement to the use of IFN-alpha therapy in the treatment of cancer.
  • IFN-alpha therapy In one embodiment, some treatment protocols call for the use of IFN-alpha. Since IFN-alpha is produced following the administration of some proinflammatory oligomeric compounds, these nucleic acids can be used to generate IFN-alpha endogenously.
  • a further embodiment includes a method for inducing antigen non-specific innate immune activation and broad spectrum resistance to infectious challenge using the proinflammatory oligomeric compounds.
  • antigen non-specific innate immune activation refers to the activation of immune cells other than B cells and for instance can include the activation of NK cells, T cells or other immune cells that can respond in an antigen independent fashion or some combination of these cells.
  • a broad spectrum resistance to infectious challenge is induced because the immune cells are in active form and are primed to respond to any invading compound or microorganism. The cells do not have to be specifically primed against a particular antigen. This is particularly useful in biowarfare, and the other circumstances such as for travelers going to areas with high incidence of infectious disease.
  • the proinflammatory oligomeric compounds may be directly administered to the subject or may be administered in conjunction with a nucleic acid delivery complex.
  • a nucleic acid delivery complex shall mean a nucleic acid molecule associated with (e.g., ionically or covalently bound to; or encapsulated within) a targeting means (e.g., a molecule that results in higher affinity binding to target cell (e.g., B cell surfaces and/or increased cellular uptake by target cells).
  • nucleic acid delivery complexes examples include nucleic acids associated with a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., a ligand recognized by target cell specific receptor).
  • a sterol e.g., cholesterol
  • a lipid e.g., a cationic lipid, virosome or liposome
  • a target cell specific binding agent e.g., a ligand recognized by target cell specific receptor
  • Delivery vehicles or delivery devices for delivering antigen and nucleic acids to surfaces have been described and are well known to those skilled in the art. Some examples are provided below in the discussion of vectors.
  • an effective amount of a proinflammatory nucleic acid refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount of a proinflammatory nucleic acid for inducing mucosal immunity is that amount necessary to cause the development of IgA in response to an antigen upon exposure to the antigen, whereas that amount required for inducing systemic immunity is that amount necessary to cause the development of IgG in response to an antigen upon exposure to the antigen.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular proinflammatory nucleic acid being administered, the antigen, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular proinflammatory nucleic acid and/or antigen and/or other therapeutic agent without necessitating undue experimentation.
  • Subject doses of the compounds described herein for mucosal or local delivery typically range from about 0.1 ug to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time there between. However, dosing may be at substantially higher or lower ranges. Determination of appropriate dosing ranges and frequency is well within the ability of those skilled in the art.
  • the therapeutically effective amount can be initially determined from animal models.
  • a therapeutically effective dose can also be determined from human data for CpG oligonucleotides which have been tested in humans (human clinical trials have been initiated) and for compounds which are known to exhibit similar pharmacological activities, such as other mucosal adjuvants, e.g., LT and other antigens for vaccination purposes, for the mucosal or local administration. Higher doses are required for parenteral administration.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • an effective amount of the proinflammatory nucleic acid can be administered to a subject by any mode that delivers the nucleic acid to the desired surface, e.g., mucosal, systemic.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Suitable routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intratracheal, inhalation, ocular, vaginal, and rectal. Such formulations are well known by those skilled in the art, as are considerations for optimal dosing routes.
  • the proinflammatory oligomeric compounds and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. Such salts are well known to those skilled in the art.
  • compositions of the invention contain an effective amount of a proinflammatory oligomeric compound and optionally antigens and/or other therapeutic agents optionally included in a pharmaceutically-acceptable carrier.
  • pharmaceutically- acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • the proinflammatory oligomeric compounds useful in the invention may be delivered in mixtures with additional adjuvant(s), other therapeutics, or antigen(s).
  • a mixture may consist of several adjuvants in addition to the proinflammatory nucleic acid or several antigens or other therapeutics.
  • the present invention provides oligomeric compounds that activate one or more interferon.
  • interferon activating compounds are also cytotoxic oligomeric compounds, hi certain embodiments, certain oligomeric compounds activate at least one interferon, but are not cytotoxic, or are only mildly cytotoxic.
  • Interferons are cytokines with diverse biological functions, including antitumor, immunomodulatory, antiviral and antiparasitic actions.
  • the IFN family includes numerous proteins and is grouped into two types: Type I and Type II. Based on their genetic similarities and differences, the type I IFNs consist primarily of IFN-alpha and IFN-beta. The type II IFNs consist of IFN-gamma.
  • IFNs act by binding to specific cell receptors, which are found on the surface of most cells, and causing the translocation to the nucleus of cytoplasmic transcription factors that enhance or suppress the expression of specific genes.
  • the products of these interferon-stimulated genes are primarily polypeptides that act as mediators of the biological activities associated with the respective IFN.
  • IFN-alpha and IFN-beta share the same receptor complex. IFN-gamma binds to a separate receptor. Nonetheless, IFN-alpha, IFN-beta, and IFN-gamma all require the presence of the cytoplasmic protein tyrosine kinase Jak 1 to enhance the expression of specific genes.
  • autoimmune diseases such as autoimmune diseases, IFN-alpha, IFN-beta, or IFN-gamma activation of interferon-stimulation genes can cause a deleterious inflammatory reaction in an individual.
  • cell based models that are sensitive to treatment with a test compound, such as an oligomeric compound.
  • the response of a test compound in the cell based model is predictive of the in vivo pro-inflammatory response.
  • the cell based model provided herein is therefore useful, for example, to screen for a test compound having a beneficial proinflammatory profile.
  • the cell based model is also useful for screening against test compounds having non-beneficial pro-inflammatory profiles. Certain methods of identifying proinflammatory oligonucleotides are described in USSN 11/404, 544 and such methods, procedures, cells, pathways, genes, proteins and related material is specifically incorporated by reference herein in its entirety.
  • the cell based models provided herein can also be used to predict a species-specific inflammatory response to a test compound.
  • species-specificity can be determined, for example, by assessing the gene expression and protein expression pattern displayed in the in vitro model.
  • Such patterns can include cytokines and chemokines expression patterns. These expression patterns provide a pro-inflammatory profile for target agent that is predictive of a species specific in vivo response.
  • the in vitro models provided herein are also used to predict a cell-type specific inflammatory response to a test compound. Such cell-type specificity can be determined, for example, by assessing the gene expression and protein expression pattern displayed in one or more in vitro models.
  • Such models can include multiple different cell types and a cell-type specificity can be detected by comparing the pro-inflammatory profile of a test compound in one cell-type with the pro-inflammatory profile of the test compound in another cell-type.
  • a liver sinusoidal endothelial cell-type specificity may be identified by administering a compound to the LSEC in vitro model provided herein and comparing the resulting proinflammatory profile with the pro-inflammatory profile observed when the test compound is administered to the hepatocyte model.
  • in vitro models that recapitulate specific proinflammatory and hepatotoxic effects seen in vivo upon administration of specific oligonucleotides.
  • in vitro cultures of LSECs are capable of producing INF-beta in response to stimulation with hepatotoxic oligonucleotide ISIS 147420 but not with the mild pro-inflammatory oligonucleotide ISIS 104838.
  • LSECs are primarily responsible for the induction of type I interferon-mediated hepatotoxicity.
  • in vitro cultures of LSECs are a suitable model to study the mechanism of ASO-mediated hepatotoxcity as well as suitable to identify hepatotoxic oligonucleotides with a mechanism of action similar to ISIS 147420.
  • the present invention provides models for screening for compounds that activate one or more interferon.
  • interferon activating compounds are mildly pro-inflammatory.
  • certain compounds activate at least one interferon and are severly pro-inflammatory.
  • certain compounds activate at least one interferon and are severly pro-inflammatory and hepatotoxic.
  • Interferons are cytokines with diverse biological functions, including antitumor, immunomodulatory, antiviral and antiparasitic actions.
  • the IFN family includes numerous proteins and is grouped into two types: Type I and Type II. Based on their genetic similarities and differences, the type I IFNs consist primarily of IFN-alpha and IFN-beta.
  • the type II IFNs consist of IFN-gamma. IFNs act by binding to specific cell receptors, which are found on the surface of most cells, and causing the translocation to the nucleus of cytoplasmic transcription factors that enhance or suppress the expression of specific genes.
  • the products of these interferon-stimulated genes are primarily polypeptides that act as mediators of the biological activities associated with the respective IFN.
  • IFN-alpha and IFN-beta share the same receptor complex. IFN-gamma binds to a separate receptor. Nonetheless, IFN-alpha, IFN-beta, and IFN-gamma all require the presence of the cytoplasmic protein tyrosine kinase Jak 1 to enhance the expression of specific genes.
  • autoimmune diseases such as autoimmune diseases, IFN-alpha, IFN-beta, or IFN-gamma activation of interferon-stimulation genes can cause a deleterious inflammatory reaction in an individual.
  • IFNs interferons
  • Type I IFNs include multiple subtypes including IFN-alpha and IFN-beta. Type I IFNs are produced by all nucleated cells in response to virus infection. In comparison, type II IFN (IFN-gamma) are predominately made by T lymphocytes and natural killer (NK) cells in response to T cell receptor (TCR) and natural killer (NK) cell receptor signals (Pichlmair, Immunity, 2007, 27, 370-383). Thus, IFN-gamma appears mainly after the induction of the adaptive immune response.
  • IFN-alpha and IFN-beta bind to a common cellular receptor, type I interferon receptor (IFNAR).
  • Type II IFN binds to a different receptor, interferon gamma receptor (IFNGR), however, both share downstream signaling molecules and regulate many of the same genes.
  • IFNAR and IFNGR like other cytokine receptors, are coupled to a Janus-family tyrosine kinase, which in turn phosphorylates signal-transducing activators of transcriptions (STATs). The binding of phosphorylated STAT proteins to the promoters of several genes induces the synthesis of host-cell proteins that contribute to the inhibition of viral replication.
  • oligo-adnylate synthetase which polymerizes ATP into a series of 2 '-5' linked oligomers. These activate an endoribonuclease that then degrades viral RNA.
  • a second protein activated enzyme phosphorylates the eukaryotic protein synthesis inhibition factor eIF-2, thereby inhibiting translation and thus contributing to the inhibition of viral replication.
  • Another interferon-inducible protein, Mx is known to be required for cellular resistance to influenza virus replication. (Janeway, C. et al., (1999). Immuno Biology, 4th Ed. (London: Current Biology Publications).
  • INF-alpha and INF-beta have three major functions. First, they induce resistance to viral replication by activating cellular genes that destroy mRNA and inhibit the translation of viral and some host proteins. Second, they induce major histocompatibility complex (MHC) class I expression in most uninfected cells in the body, which enhances resistance to NK cells and makes cells newly infected by virus more susceptible to killing by CD8 cytotoxic T cells. Third, they activate NK cells, which then kill virus-infected cells selectively. INF-gamma activates macrophages increasing release of inflammatory mediators (Janeway, C. et al. (1999) Immuno Biology, 4th Ed. (London: Current Biology Publications).
  • MHC major histocompatibility complex
  • Interferon Regulatory Factor 7 is a member of the interferon regulatory transcription factor (IRF) family. IRF is involved in transcriptional activation of virus-inducible genes, including type I interferon genes. Expression of type I interferon genes in response to viral infection is regulated at the transcriptional level.
  • Interferon Induced Protein with Tertratricopeptide Repeats 2 is a protein involved in antiviral pathways.
  • Ubiquitin-Specific Protease USPl 8
  • USP 18 is involved in interferon antiviral activity.
  • Myxovirus resistance 2 is a protein upregulated by type I interferon. MX2 can also refer to the type I interferon gene that encodes the indicated protein. Any reference herein to a protein name is understood to also refer to the nucleic acid that encodes the protein and vice versa where appropriate.
  • a target cell type exists which is responsible for certain pro-inflammatory and hepatotoxic effects.
  • a target cell type that is sensitive to the administration of a target compound can be used to assess the proinflammatory and hepatotoxic properties of such target compound.
  • in vitro experiments can be performed using certain cell types known for their participation in various aspects of the innate immune response.
  • Those cells include, but are not limited to, bone marrow derived cells (macrophages, granulocytes and dendritic cell types), hematopoietic (from spleen, liver and blood), dendritic (from liver, spleen and blood), parenchymal (hepatocytes) and non-parenchymal (primarily, liver sinusoidal endothelial cells (LSECs)) cells; other cells type such as lung or kidney derived primary cells as well as established rodent or human cell lines.
  • LSECs liver sinusoidal endothelial cells
  • In vitro models prepared from any of the above identified cells may include immortalization as described in more detail herein.
  • the identity of the various cell populations can be ascertained based on cell surface antigens using flow cytometry, or by the cell's morphological characteristics. The cells can then be treated with test compound.
  • Positive controls include Lipopolysaccharide (LPS) and polyinosinic and polycytidylic acid (PoIy-IC), poly(dA-dT)poly(dt-dA) (PoIy-AT) are used as positive controls.
  • LPS activates innate immune responses through the toll like receptor 4 (TLR4) in the type I interferon pathway; PoIy-IC is recognized by TLR3 when cells are not transfected and activates a type- 1 interferon response (Beutler, 2004; Li, Chen, Kato, Gale, & Lemon, 2005).
  • RNA-helicase which is able to induce a type- 1 interferon response, recognizes PoIy-IC under transfection conditions (Kato et al., 2006).
  • PBS is used as a negative control.
  • phosphorothioate oligonucleotides containing CpG motifs are used for comparison because the mechanism of inflammation is known.
  • Enzyme linked immuno-sorbent assays were used to detect relevant cytokine or chemokine levels from cell supernatants. Relative gene expression is analyzed by qRT-PCR from the total RNA of the treated cells.
  • Short-hairpin RNA make a tight hairpin turn that can be used to specifically silence gene expression via RNA interference using a similar mechanism to siRNAs.
  • This approach determines the phenotypic gene response when the cells are treated with a test compound such as a hepatotoxic oligonucleotide.
  • Cells that show an inflammatory response to a test compound are suitable as in vitro models.
  • Such cell lines include, but are not limited to, MHT, brain endothelial (bEND) or mouse embryonic fibroblasts (MEF) as well as immortalized primary cells (such as im-LSEC).
  • a responsive cell line is identified, transduction of appropriate controls (non-targeting shRNA) and shRNA for a target gene is performed. Stable clones are selected for using puromycin, and knockdown of the target gene is validated by qRT-PCR or western blot. Once the gene is knocked-down, the cell line is treated with test compound and appropriate controls. ELISA and qRT-PCR are used to demonstrate the involvement of the specific gene target.
  • ISIS 104838 Compounds found to be mildly pro-inflammatory (PI) in vivo such as ISIS 104838 were found to upregulate several genes associated with innate immune response including but not limited to MX2, IRF7, USP 18, IFIT2 (type 1 interferon stimulated genes) in the in vitro model. This upregulation was not observed with non inflammatory oligonucleotides such as ISIS 141923. Oligonucleotides which demonstrate high pro-inflammatory effects in vivo (such as ISIS 147420) were also shown to upregulate genes involved in the innate immune response in the in vitro model similar to ISIS 104838 but to a much higher level. Unlike the mild PI oligonucleotides, high PI oligonucleotides cause increased levels of IFN- beta in the cell culture supernatant.
  • PI mildly pro-inflammatory
  • oligonucleotides Treatment of cells in vitro provided a means to identify oligonucleotides as either non-. inflammatory, mildly proinflammatory or capable to induce a type I interferon response and/or hepatotoxicity.
  • primary LSECs provide a very sensitive model by which the various categories of oligonucleotides can be identified. The level of inflammation can be assessed and categorized as mild to severe based on the amount of damage to cells and/or tissues in vivo and/or amount of cytokine expression or cytokine pathway activation relative to a saline control.
  • Immortalization of primary cells responsive to a test compound can accomplished by any known methods in the art or by methods provided herein including expression of SV40 LTag.
  • the isolation of cell for preparation of an in vitro model system is a complex process that needs to be streamlined in order to use such cells to screen test compounds.
  • the cell should be immortalized.
  • Cells can be transfected with lentiviral particles capable of inducing the expression of the SV40 LTag.
  • Expression of SV40 LTag causes the immortalization of primary cells that are otherwise unable to remain in culture for more than a few days.
  • the immortalization process allows the cells to be maintained in culture indefinitely while retaining their original cellular characteristics.
  • immortalized cell lines are preferred as they retain the ability to respond to target compound in the same way the primary cell responds.
  • immortalized cells can be used routinely for screening or mechanistic assessment purposes without the need to isolate the cells de novo from mice or other animals of interest. While the LSEC cell is specifically exemplified herein, other immortalized primary cell type should also be suitable for screening or mechanistic assessment purposes.
  • interferon-beta and other cytokines such as, but not limited to, MCP-I or IL-6 can be measured in cell culture supernatant.
  • Gene expression resulting from interferon stimulation can also be measured after RNA extraction.
  • Reporter assay systems involved transient or stable expression of reporter vector allowing the inducible expression of the secreted embryonic alkaline phosphatase (SEAP) gene.
  • SEAP embryonic alkaline phosphatase
  • the SEAP gene is cloned under the control of different promoters (Inteferon-alpha or Interferon-beta) that are activated by various transcription factors, such as IRF3, IRF7. Those transcription factors have been implicated in vivo as playing a fundamental role in the type I interferon response induced by oligonucleotides such as ISIS 147420.
  • the screening can be done as follows: ISIS-147420 modified and selected to express the reporter is transfected to im-LSEC.
  • the oligonucleotides induces expression of type I interferon through the modulation of type I interferon promoter.
  • the artificial type I interferon promoter in the reporter vector causes the production and excretion of SEAP that can be easily measure in the supernatant by a rapid colorimetric assay without the need of time consuming ELISA or RT- PCR assay.
  • oligonucleotides particularly pro-inflammatory signaling pathways that may be activated in response to such target compound.
  • certain oligonucleotides more particularly T- modified oligonucleotides, even more particularly 2'MOE modified oligonucleotides have been found to activate certain signaling pathways.
  • pattern recognition receptors signaling through a type- 1 IFN pathway have been shown to be activated by specific 2'MOE oligonucleotides.
  • the pattern recognition receptors include, but are not limited to, type- 1 IFN induced and dsRNA-activated kinase (PKR), melanoma differentiation-associated gene 5 (MD A-5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activator of IRFs (DAI) and Toll-like receptor 3 (TLR3).
  • PLR type- 1 IFN induced and dsRNA-activated kinase
  • MD A-5 melanoma differentiation-associated gene 5
  • RIG-I retinoic acid-inducible gene I
  • DAI DNA-dependent activator of IRFs
  • TLR3 Toll-like receptor 3
  • the in vitro models provided herein include cell types that express one or more genes associated with a pro-inflammatory response including, but not limited to MD A-5, RIG-I, PKR, DAI, TLR3, TRIF, IPS-I .
  • Such cell types can be identified by isolating cells from STATl and IFNARl knock-out mice or any other appropriate knock-out mouse including those with one or more IFN pathway genes knocked out. Isolated cells from such animals can be treated in vitro to compare the type- 1 IFN response with in vivo findings.
  • target genes such as MDA- 5, RIG-I, PKR, DAI, TLR3, TRIF, IPS-I may be knocked-down using antisense or RNAi based mechanisms.
  • siRNA small interfering RNA molecules
  • ASOs antisense oligonucleotides
  • nucleoside sequences set forth in the sequence listing and Examples are independent of any modification to a sugar moiety, a monomelic linkage, or a nucleobase.
  • oligomeric compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
  • Oligomeric compounds described by Isis Number indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase, as indicated.
  • ISIS 147420 is a 5-10-5 MOE gapmer, wherein each internucleoside linkage is a phosphorothioate linkage, having the sequence: AATGTGCCTGCTGTCCTTGA (SEQ ID NO: 1).
  • ISIS 141923 is a mismatched control.
  • Three cohorts of twenty male 8 week old Balb/c mice were injected once, subcutaneously, with saline, 100 mg/kg of ISIS 141923, or 100 mg/kg of ISIS 147420.
  • Four animals from each cohort were sacrificed at 4 hours, 8 hours, 24 hours, 48 hours, and 72 hours.
  • Plasma was collected from each of the animals for transaminases and cytokine analysis.
  • Whole liver was collected for gene expression analysis.
  • Whole genome profiling of liver was performed at 24 hours, 48 hours, and 72 hours using Illumina beadchip array mouse-6. Liver was histologically stained (formalin fixation, H&E staining).
  • mice treated with ISIS 147420 showed high levels of transaminases (ALT&AST). No other mice showed statistically relevant increases in transaminases. Additionally, histologic stains revealed that only the mice treated with ISIS 147420 developed severe liver injury as evidenced by eosinophilic degeneration, nonparenchymal cell apoptosis, and sinusoidal RBC and leukocyte extravasation. Liver from mice treated with ISIS 141923 and saline exhibited normal liver morphology.
  • Toxicity of ISIS 147420 was tested in 129SvEv and C57BL/6 wild type and knockout mouse models.
  • Wild type 129SvEv mice were compared to Statl deficient, IFNARl deficient, and IFNGRl deficient mice.
  • 4 mice were injected with a single dose of saline, 4 mice were injected with a single 300 mg/kg dose of ISIS 141923, and 8 mice were injected with a single 300 mg/kg dose of ISIS 147420.
  • mice treated with ISIS 147420 4 were sacrificed at 72 hours and 4 were sacrificed at 96 hours. All of the mice treated with saline and ISIS 141923 were sacrificed at 72 hours.
  • Wild type C57BL/6 mice were compared to IRF-3 deficient mice and IRF-7 deficient mice.
  • 4 mice were injected with a single dose of saline, 4 mice were injected with a single 300 mg/kg dose of ISIS 141923, and 8 mice were injected with a single 300 mg/kg dose of ISIS 147420.
  • the mice treated with ISIS 147420 4 were sacrificed at 72 hours and 4 were sacrificed at 96 hours. All of the mice treated with saline and ISIS 141923 were sacrificed at 72 hours.
  • Statl knockout 129SvEv mice did not exhibit elevated plasma ALT or IFN-beta at 72 or 96 hours.
  • IFNARl knockout 129SvEv mice did not exhibit elevated plasma ALT or IFN-beta at 72 or 96 hours.
  • Wild type 129SvEv mice exhibited elevated plasma ALT and IFN-beta at 72 hours and 96 hours.
  • IRF-3 knockout C57BL/6 mice exhibited slightly elevated plasma ALT at 72 and 96 hours, but did not exhibit elevated plasma IFN-beta at 72 or 96 hours.
  • IRF3 knockout marked reduction
  • Statl knockout and IFNARl knockout complete abrogation
  • mice Five cohorts often male Balb/c mice were injected once, subcutaneously, with saline, ISIS 104838, or ISIS 147420. One cohort was treated with saline, one cohort was treated with 300 mg/kg ISIS 104838, one cohort was treated with 25 mg/kg ISIS 147420, one cohort was treated with 50 mg/kg ISIS 147420, and one cohort were treated with 100 mg/kg ISIS 147420. Five animals from each cohort were sacrificed at 24 and 48 hours. Plasma was collected from each of the animals for cytokine analysis. Plasma was analyzed for cytokine production by ELISA and RNA was isolated from livers and analyzed by RT-PCR. Cytokine analysis
  • IFN-alpha was detected in all cohorts at levels less than 10 pg/mL.
  • IFN-alpha was detected in the saline, ISIS 147420 (25 mg/kg), and ISIS 104838 (300 mg/kg) treated mice at levels less than 10 pg/mL.
  • IFN-alpha was measured at approximately 40 pg/mL in the ISIS 147420 (50 mg/kg) mice and at approximately 120 pg/mL in the ISIS 147420 (100 mg/kg) mice.
  • IFN-beta At 24 hours no statistically relevant amount of IFN-beta was detected in any of the cohorts. At 48 hours IFN-beta was detected in ISIS 147420 (50 mg/kg and 100 mg/kg) treated mice.
  • the data show presence of a type I interferon response in plasma from mice treated with ISIS 147420 at 48 hours.
  • the absence of increased IFN-gamma levels suggests absence of type II interferon response.
  • MX2, IFIT2, USPl 8, and IRF7 mRNA were detected in mice treated with ISIS 147420 (25mg/kg), ISIS 147420 (50 mg/kg), ISIS 147420 (100 mg/kg), and ISIS 104838 (300 mg/kg).
  • a statistically significant increase of MX2, IFIT2, USPl 8, and IRF7 mRNA in all cohorts was observed in comparison to saline treated mice.
  • a statistically significant increase of MX2, IFIT2, USP18, and IRF7 mRNA in ISIS 147420 50 mg/kg and 100 mg/kg was observed in comparison to mice treated with ISIS 104838. In all instances the amount of mRNA measured at 24 hours was less than the amount of mRNA measured at 48 hours.
  • mice Three cohorts of male Balb/c mice were injected once, subcutaneously, with 100 mg/kg ISIS 141923, 300 mg/kg ISIS 104838, or 100 mg/kg ISIS 147420 36 hours prior to cell collection. Separation of parenchymal and non-parenchymal cell fractions in the liver were performed via centrifugation. Whole cell fractions were analyzed by RT-PCR. Ex vivo data showed type I interferon responsive genes were upregulated in response to treatment with ISIS 147420, ISIS 104838, and ISIS 141923. Gene upregulation was greatest in mice treated with ISIS 147420, then ISIS 104838, and then ISIS 141923. Greater upregulation of type I interferon responsive genes was observed in the parenchymal fraction in comparison to the non-parenchymal fraction.
  • Example 5 In vitro treatment ofhepatocytes and LSECs with ISIS 141923, ISIS 104838, and ISIS 147420
  • Murine parenchymal and non-parenchymal cells were isolated by liver perfusion. Parenchymal cells (hepatocytes) were cultured for at least two hours, and were treated with ISIS 141923, ISIS 104838, or ISIS 147420. LSECs were isolated from the non-parenchymal fraction using a centrifugation gradient and were cultured for four days in media containing endothelial growth factor to favor their expansion prior to treatments. After an overnight treatment the supernatants were collected for cytokine profiling and RNA was isolated from the LSECs for gene expression profiling.
  • Both LSECs and hepatocytes showed changes in type I interferon stimulated gene expression when treated with ISIS 147420 and to a lesser extent when treated with ISIS 104838.
  • LSECs were significantly more responsive to ISIS 147420 and ISIS 104838 than hepatocytes. Between ISIS 147420 and ISIS 104838, LSECs were more responsive to ISIS 147420.
  • in vitro cultures of LSECs have also been shown to produce IFN- beta in the cell supernatants in response to treatment with ISIS 147420 but not ISIS 104838.
  • ISIS 147420 induces a higher level of type I interferon stimulated gene expression in hepatocytes and LSECs compared with ISIS 104838.
  • LSECs are more responsive to ISIS 147420 and ISIS 104838 stimulation than hepatocytes.
  • LSECs may be used in vitro to assess the pro-inflammatory properties of oligonucleotides in the liver.
  • mice were pretreated with antisense oligonucleotide (ASO) targeted to RIG-I or IPS-I or pretreated with ISIS 104838 (control oligonucleotide mismatched to mouse).
  • ASO antisense oligonucleotide
  • ISIS 104838 control oligonucleotide mismatched to mouse.
  • ASO was injected subcutaneously(50mg/kg/day) for three days (Ohr, 24hr and 48hr). Mice were then dosed with ISIS 147420 (100mg/kg) 48 hours after the last pretreatment dose.
  • ALT and AST were measured 72 hours and 96 hours after administration of ISIS 147420.
  • ELISA and qRT-PCR analyses were performed 96 hours after treatment with ISIS 147420.
  • Pretreatment with ASO targeting IPS-I reduced IPS-I levels (mRNA and protein) and prevented RIG-I induction caused by ISIS 147420. Pretreatment also led to an abrogation of IFN- ⁇ production and ALT/AST release. Pretreatment also resulted in normal liver morphology.
  • Pretreatment with ASO targeting RIG-I also prevented RIG-I induction caused by ISIS 147420 but failed to bring RIG-I below baseline level.
  • Pretreatment with anti-RIG-I ASO delayed IFN- ⁇ production and ALT/AST release. Without being bound to a particular theory, it is believed the RIG-I level must be reduced below baseline level (the level of RIG-I in mice treated with saline) to prevent ISIS 147420 toxicity.
  • Example 7 Pretreatment with MDA-5 Antisense Inhibits ISIS 147420 Mediated Interferon Response and Hepatotoxicity
  • mice were pretreated with antisense oligonucleotide (ASO) targeted to MDA-5 or pretreated with ISIS 104838 (control oligonucleotide mismatched to mouse).
  • ASO antisense oligonucleotide
  • ISIS 104838 control oligonucleotide mismatched to mouse.
  • ASO was injected subcutaneously (50mg/kg/day) for three days (Ohr, 24hr and 48hr). Mice were then dosed with ISIS 147420 (lOOmg/kg) 48 hours after the last pretreatment dose.
  • ALT and AST were measured 96 hours after administration of ISIS 147420.
  • qRT-PCR analysis was performed 96 hours after treatment with ISIS 147420.
  • Pretreatment with ASO targeting MDA-5 reduced MDA-5 mRNA and prevented RIG-I induction caused by ISIS 147420. Pretreatment also led to an abrogation of IFN- ⁇ production ALT/AST release.

Abstract

La présente invention concerne des composés et des procédés d'induction ou de modulation d'une réponse toxique, inflammatoire et/ou immunitaire dans une cellule, un tissu, ou un animal. L'invention concerne également des procédés d'identification de ces composés.
PCT/US2010/027863 2009-03-18 2010-03-18 Composés et procédés de modulation d'effets toxiques et pro-inflammatoires WO2010108035A1 (fr)

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