WO2008033432A2 - Immunomodulation par des ribonucléosides et des oligoribonucléotides chimiquement modifiés - Google Patents

Immunomodulation par des ribonucléosides et des oligoribonucléotides chimiquement modifiés Download PDF

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WO2008033432A2
WO2008033432A2 PCT/US2007/019873 US2007019873W WO2008033432A2 WO 2008033432 A2 WO2008033432 A2 WO 2008033432A2 US 2007019873 W US2007019873 W US 2007019873W WO 2008033432 A2 WO2008033432 A2 WO 2008033432A2
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modified
cytidine
syndrome
subject
immune
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PCT/US2007/019873
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WO2008033432A3 (fr
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Jorg Vollmer
Stefan Bauer
Grayson B. Lipford
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Coley Pharmaceutical Group, Inc.
Coley Pharmaceutical Gmbh
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Publication of WO2008033432A2 publication Critical patent/WO2008033432A2/fr
Publication of WO2008033432A3 publication Critical patent/WO2008033432A3/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates generally to the field of immunology, and more particularly to immunomodulatory molecules. More specifically the invention relates to modified ribonucleic acid (RNA) molecules, including oligoribonucleotides, with immunosuppressive activity.
  • RNA ribonucleic acid
  • TLRs Toll-like receptors
  • PRR pattern recognition receptor
  • PAMPs pathogen-associated molecular patterns
  • TLRl - TLRlO The cytoplasmic domains of the various TLRs are characterized by a Toll-interleukin 1 receptor (TIR) domain.
  • TIR Toll-interleukin 1 receptor
  • the TIR domain-containing adapter protein MyD88 has been reported to associate with TLRs and to recruit interleukin 1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to the TLRs.
  • IRAK interleukin 1 receptor-associated kinase
  • TNF tumor necrosis factor receptor-associated factor 6
  • the MyD88- dependent signaling pathway is believed to lead to activation of NF- ⁇ B transcription factors and c-Jun NH 2 terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs), critical steps in immune activation and production of inflammatory cytokines.
  • Jnk c-Jun NH 2 terminal kinase mitogen-activated protein kinases
  • TLR2 TLR2
  • Ligands for TLR2 include peptidoglycan and lipopeptides.
  • Lipopolysaccharide (LPS) is a ligand for TLR4.
  • Poltorak A et al. (1998) Science 282:2085-8; Hoshino K et al. (1999) J Immunol 162:3749-52.
  • Bacterial flagellin is a ligand for TLR5.
  • RNA unmethylated bacterial DNA and synthetic analogs thereof
  • CpG DNA CpG DNA
  • ligands for certain TLRs include certain nucleic acid molecules.
  • certain types of RNA are immunostimulatory in a sequence-independent or sequence- dependent manner. Further, it has been reported that these various immunostimulatory RNAs stimulate TLR3, TLR7, or TLR8.
  • the invention is based in part on the discovery that modifications of specific nucleotides in single stranded oligoribonucleotides (ORN) outside the immune modulatory motif can result in suppression of the immunomodulatory capacity of the ORN. It was discovered that 2'O-methyl modification of rA, rG or rU, but not rC, nucleosides within a stimulatory ORN produced a molecule having reduced immunostimulatory potential compared to unmodified versions of the same ORN. Additionally, certain 2' modified ORN (either single-stranded ORN, whole RNA or 18S rRNA) act as TLR-7, -8, or -9 antagonists by suppressing immune stimulation of ligands.
  • ORN single stranded oligoribonucleotides
  • One aspect of the invention is a method for treating autoimmune disease in a subject, comprising administering to a subject in need of such treatment an effective amount for treating autoimmune disease of a modified oligoribonucleotide having an immune modulatory motif 4 to 8 nucleotides long and including at least one 2' modification on a nucleoside 3' or 5 'of the immune modulatory motif.
  • the modification is within 9 nucleotides of the immune modulatory motif.
  • the 2' modification of the immune modulatory motif decreases immune stimulatory activity of the ORN containing the motif.
  • the autoimmune disease involves antibody-mediated or T-cell mediated immunity.
  • the autoimmune disease is selected from the group comprising scleroderma, juvenile rheumatoid arthritis, ulcerative colitis, graft versus host disease, transplanted organ rejection, asthma, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic
  • the 2' modification is on a rA, rG or rU residue. In another embodiment the 2' modification is O-methyl.
  • the nucleobase of the modified residue is selected from a group consisting of hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2- thiouracil, 4-thiouracil, 5-aminouracil, 5-(Cl-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)- alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5- fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Cl-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkyny
  • the immune modulatory motif has a base sequence selected from 5' U U G U 3', 5' C/U U G/U U 3 1 , 5 1 R U R G Y 3', 5' G U U G B 3 1 , 5' G U G U G/U 3', 5' G/C U A/C G G C A C 3', and N-U-Rl -R2, where C/U is cytosine (C) or uracil (U), G/U is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G/C is G or C, A/C is adenine (A) or C, N is a ribonucleoside and N does not include a U, and wherein at least one of Rl and R2 is adenosine (A) or cytosine (C) or derivatives thereof and wherein R is not U unless N-U-Rl- R2 includes at least two A
  • the subject is a subject having autoimmune disease. In another embodiment the subject is a subject at risk of developing autoimmune disease.
  • the modified oligoribonucleotide is single stranded and the oligoribonucleotide sequence is not complementary to a coding sequence in the target cell. In one embodiment the modified oligoribonucleotide comprises at least one T modified nucleoside in the immune modulatory motif.
  • Another aspect of the invention is a method for treating an inflammatory disorder in a subject, comprising administering to a subject in need of such treatment an effective amount for treating an inflammatory disorder of a modified oligoribonucleotide having an immune modulatory motif 4 to 8 nucleotides long and including at least one 2' modification on a nucleoside 3' or 5 'of the immune modulatory motif.
  • the subject is a subject at risk of developing an"inflammatory disorder.
  • the inflammatory disorder is sepsis.
  • the inflammatory disorder is an infection.
  • the 2' modification is on a rA, rG or rU residue.
  • the T modification is O-methyl.
  • the nucleobase of the modified residue is selected from a group consisting of hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2 thiouracil, 4 thiouracil, 5 aminouracil, 5- (Cl-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5 (hydroxymethyl)uracil, 5 chlorouracil, 5 fluorouracil, 5 bromouracil, 5 hydroxycytosine, 5- (Cl-C6)-alkylcytosine, 5 methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)- alkynylcytosine, 5 chlorocytosine, 5 fluorocytosine, 5 bromocytosine, N2 dimethylguan
  • the immune modulatory motif has a base sequence selected from 5' U U G U 3', 5" C/U U G/U U 3 1 , 5 1 R U R G Y 3 1 , 5 1 G U U G B 3 ⁇ 5' G U G U GAJ 3 1 , 5 1 G/C U A/C G G C A C 3', and N-U-Rl -R2, where C/U is cytosine (C) or uracil (U), G/U is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G/C is G or C, A/C is adenine (A) or C, N is a ribonucleoside and N does not include a U, and where at least one of Rl and R2 is adenosine (A) or cytosine (C) or derivatives thereof and wherein R is not U unless N-U-Rl -R2 includes at least two A.
  • compositions comprising a modified oligoribonucleotide, wherein the modified oligoribonucleotide contains at least one 2 'modification on a residue 3' or 5'of an immune modulatory motif, wherein the 2' modification is on a rA, rG or rU residue.
  • the T modification is an O- methyl.
  • the nucleobase of the modified residue is selected from a group consisting of hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2 thiouracil, 4 thiouracil, 5 aminouracil, 5-(Cl-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5 (hydroxymethyl)uracil, 5 chlorouracil, 5 fluorouracil, 5 bromouracil, 5 hydroxycytosine, 5-(Cl-C6)-alkylcytosine, 5 methyl cytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5 chlorocytosine, 5 fluorocytosine, 5 bromocytosine, N2 dimethylguan
  • the modified oligoribonucleotide has a backbone modification.
  • the backbone modification is a phosphorothioate modification.
  • the modified oligoribonucleotide is between 10 and 30 nucleotides in length.
  • the modified oligoribonucleotide contains at least two modified residues.
  • the modified oligoribonucleotide contains at least three modified residues.
  • the immune modulatory motif has a base sequence selected from 5' U U G U 3 ⁇ 5' C/U U G/U U 3', 5 1 R U R G Y 3', 5' G U U G B 3', 5 1 G U G U G/U 3 ⁇ 5' G/C U A/C G G C A C 3', and N-U-Rl -R2, where C/U is cytosine (C) or uracil (U), G/U is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G/C is G or C, A/C is adenine (A) or C, N is a ribonucleoside and N does not include a U, and where at least one of Rl and R2 is Adenosine (A) or Cytosine or derivatives thereof and wherein R is not U unless N-U-Rl -R2 includes at least two A.
  • the modified oligoribonucleotide is single stranded and wherein the oligoribonucleotide sequence is not complementary to a coding sequence in the target cell.
  • the modified oligoribonucleotide comprises at least one 2' modified nucleoside in the immune modulatory motif
  • Another aspect of the invention is a method for suppressing an immune response in a subject by administering to a subject in need of such treatment any one of the modified oligoribonucleotides of the invention.
  • the immune response is an RNA- mediated immune response.
  • the immune response is a DNA- mediated immune response.
  • the subject has an autoimmune disease.
  • the subject is at risk of developing an autoimmune disease.
  • the subject has an inflammatory disorder.
  • the suppression of the immune response comprises suppression of TLR8 signaling.
  • the suppression of the immune response comprises suppression of TLR7 signaling.
  • the suppression of the immune response comprises suppression of TLR9 signaling.
  • the suppression of the immune response comprises suppression of activation of antigen-presenting cells, B cells, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), monocytes, monocyte-derived cells, eosinophils, or neutrophils.
  • the subject is administered a TLR ligand.
  • the TLR ligand is a CpG oligonucleotide.
  • the ligand is an immune stimulatory RNA.
  • the modified oligoribonucleotide is single stranded and wherein the oligoribonucleotide sequence is not complementary to a coding sequence in the target cell.
  • the ligand is a small molecule.
  • Another aspect of the invention is a method of inhibiting an RNA-mediated immune response in a subject, comprising administering to a subject in need of such treatment a 2'- modif ⁇ ed cytidine.
  • the 2'-modified cytidine is 2'-O-methyl cytidine.
  • the 2'-modif ⁇ ed cytidine is 2'-O-alkyl cytidine.
  • the 2'-O-alkyl-modification is 2'-O-ethyl, 2'-O-propyl or 2'-O-butyl.
  • the 2'-modified nucleoside is a 2'-0, 4'-C-alkylen-bridged nucleoside, e.g. 2'-O, 4'-C-methylen- bridged cytidine (LNA analogue of cytidine) or 2'-O, 4'-C-ethylen-bridged cytidine.
  • the 2'-O-alkyl-modification contains at least one unsaturated carbon-carbon linkage, e.g. 2'-O-allyl or 2'-O-propinyl.
  • the 2'-O-alkyl-modification is 2'-O-(2-methoxyethyl).
  • the subject is a subject having or at risk of having a condition selected from the group comprising scleroderma, juvenile rheumatoid arthritis, ulcerative colitis, graft versus host disease, transplanted organ rejection, asthma, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST ⁇ syndrome, cold agglutinin disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis,
  • Another aspect of the invention is a method of inhibiting a DNA-mediated immune response in a subject, comprising administering to a subject in need of such treatment a 2'- modified cytidine.
  • the 2'-modif ⁇ ed cytidine is 2'-O-methyl cytidine.
  • the 2'-modified cytidine is 2'-O-alkyl cytidine.
  • the 2'-O-alkyl-modification is 2'-O-ethyl, 2'-O-propyl or 2'-O-butyl.
  • the 2'-modif ⁇ ed nucleoside is a 2'-0, 4'-C-alkylen-bridged nucleoside, e.g. 2'-0, 4'-C-methylen- bridged cytidine (LNA analogue of cytidine) or 2'-O, 4'-C-ethylen-bridged cytidine.
  • the 2'-O-alkyl-modif ⁇ cation contains at least one unsaturated carbon-carbon linkage, e.g. 2'-O-allyl or 2'-O-propinyl. In one.
  • the 2'-O-alkyl-modification is 2'-O-(2-methoxyethyl).
  • the subject is a subject having or at risk of having a condition selected from the group comprising scleroderma, juvenile rheumatoid arthritis, ulcerative colitis, graft versus host disease, transplanted organ rejection, asthma, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia,
  • Another aspect of the invention is a method of treating an autoimmune response in a subject, comprising * administering to a subject in need of such treatment a 2'-modified cytidine.
  • the 2'-modified cytidine is 2'-O-methyl cytidine.
  • the 2'-modified cytidine is 2'-O-alkyl cytidine.
  • the 2'-O- alkyl-modif ⁇ cation is 2'-O-ethyl, 2'-O-propyl or 2'-O-butyl.
  • the 2'- modified nucleoside is a 2'-O, 4'-C-alkylen-bridged nucleoside, e.g. 2'-O, 4'-C-methylen- r bridged cytidine (LNA analogue of cytidine) or 2'-O, 4'-C-ethylen-bridged cytidine.
  • the 2'-O-alkyl-modification contains at least one unsaturated carbon-carbon linkage, e.g. 2'-O-allyl or 2'-O-propinyl.
  • the 2'-O-alkyl-modification is 2'-O-(2-methoxyethyl).
  • the subject is a subject having or at risk of having a condition selected from the group comprising scleroderma, juvenile rheumatoid arthritis, ulcerative colitis, graft versus host disease, transplanted organ rejection, asthma, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barr ⁇ syndrome, idiopathic pulmonary fibrosis,
  • rheumatica primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomena, Reiter's syndrome, rheumatoid arthritis (RA), Sjorgen's syndrome, sarcoidosis, stiff-man syndrome, systemic lupus erythematosus (SLE), Takayasu arthritis, temporal arteritis/giant cell arteritis, uveitis, vasculitis, and vitiligo.
  • Another aspect of the invention is a method for suppressing an immune response in a subject by administering to a subject in need of such treatment any one of the 2'-modified cytidines of the invention.
  • the immune response is a RNA-mediated immune response.
  • the immune response is a DNA-mediated immune response.
  • the subject has an autoimmune disease.
  • the subject is at risk of developing an autoimmune disease.
  • the subject has an inflammatory disorder.
  • the suppression of the immune response comprises suppression of TLR8 signaling.
  • the suppression of the immune response comprises suppression of TLR7 signaling.
  • the suppression of the immune response comprises suppression of TLR9 signaling.
  • the suppression of the immune response comprises suppression of activation of antigen-presenting cells, B cells, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), monocytes, monocyte-derived cells, eosinophils, or neutrophils.
  • the subject is administered a TLR ligand.
  • the TLR ligand is a CpG oligonucleotide.
  • the ligand is an immune stimulatory RNA.
  • Another aspect of the invention is a method for stimulating an immune response, comprising administering to a subject an effective amount for stimulating an immune response in the subject of a modified oligoribonucleotide having an immune stimulatory motif 4 to 8 nucleotides long and including at least one 2' modification on a rC residue 3' or 5'of the immune stimulatory motif.
  • the immune stimulatory motif has a base sequence selected from 5' U U G U 3 1 , 5* C/U U GAJ U 3 1 , 5' R U R G Y 3', 5 1 G U U G B 3', 5' G U G U GAJ 3', 5 1 G/C U A/C G G C A C 3 1 , and N-U-Rl -R2, where CAJ is cytosine (C) or uracil (U), GAJ is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G/C is G or C, A/C is adenine (A) or C, N is a ribonucleoside and N does not include a U, and where at least one of Rl and R2 is adenosine (A) or cytosine (C) or derivatives thereof and wherein R is not U unless N-U-Rl -R2 includes at least two A.
  • CAJ
  • FIG. 1 is two graphs demonstrating that 2'-0-Me modification interferes with the stimulatory effect induced by an oligoribonucleotide.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP.
  • Supernatants (SN) were collected and cytokines measured by ELISA.
  • the assay compares an unmodified ORN (SEQ ID NO:1), a 2'-O-Me-A and U modified ORN (SEQ ID NO:9), a 2'-O-Me-U modified ORN )SEQ ID NO:2) and a 2'-O-Me-A modified ORN SEQ ID NO: 10) which all show induced induction of IFN- ⁇ (FIG. Ia) and TNF- ⁇ (FIG. Ib).
  • the x-axes are ORN concentration in ⁇ M and the y- axes are cytokine concentration in pg/ml.
  • FIG. 2 is two graphs demonstrating that 2'-O-Me modification inside the GU stimulatory RNA motif interferes with the oligoribonucleotide stimulatory effect.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP.
  • SN were collected and cytokines measured by ELISA.
  • the assay compares an unmodified ORN (SEQ ID NO: 15), FIG. 2a shows IFN- ⁇ concentration and FIG. 2b shows TNF- ⁇ concentration.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 1 unmodified ORN
  • FIG. 3 is two graphs demonstrating that 2'-O-Me modification outside of the GU stimulatory RNA motif interferes with the oligoribonucleotide stimulatory effect.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP.
  • SN were collected and cytokines measured by ELISA.
  • FIG. 3 a shows IFN- ⁇ concentration and FIG. 3b shows TNF- ⁇ concentration.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 4 is two graphs demonstrating that 2'-O-Me modification of rU, rG and rA, but not rC interferes with RNA-mediated immune effects.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP. SN were collected and cytokines measured by ELISA.
  • FIG. 4a shows IFN- ⁇ concentration and FIG. 4b shows TNF- ⁇ concentration.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 4 is two graphs demonstrating that 2'-O-Me modification of rU, rG and rA, but not rC interferes with RNA-mediated immune effects.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the
  • FIG. 5 is two graphs demonstrating that the lack of suppressive effect of the 2'-O-Me modification of rC is position-independent.
  • Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP.
  • SN were collected and cytokines measured by ELISA.
  • FIG. 5a shows IFN- ⁇ concentration and
  • FIG. 5b shows TNF- ⁇ concentration.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 6 is four graphs demonstrating that 2'-O-Me modified oligoribonucleotide can act as antagonist for the immune response (IFN- ⁇ and TNF- ⁇ ) induced by a stimulatory oligoribonucleotide.
  • FIG. ⁇ a and 6c Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP. SN were collected and cytokines measured by ELISA.
  • FIG. 7 is four graphs demonstrating that 2'-O-Me modified oligoribonucleotide can act as antagonist for the immune response (IL- 12 and IFN- ⁇ ) induced by a stimulatory oligoribonucleotide.
  • FIG.7a and 7c Human PBMC of three healthy blood donors were incubated for 24h with the indicated amounts of unmodified or modified oligoribonucleotide in the presence of DOTAP. SN were collected and cytokines measured by ELISA.
  • Human PBMC of three healthy blood donors were incubated for 24h with l ⁇ M ORN SEQ ID NO:1 in the presence of DOTAP, and were co-cultured with different doses of the indicated compounds (modified ORN SEQ ID NO:2, S-Class ODN SEQ ID NO:3, chloroquine).
  • SN were collected and cytokines measured by ELISA.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 8 is two graphs demonstrating that phosphorothioate 2'-modified oligoribonucleotide suppress oligoribonucleotide -mediated effects stronger than phosphodiester modified oligoribonucleotide, although suppressive phosphorothioate 2'- modified oligoribonucleotide act as suppressors of the response to both, phosphodiester and phosphorothioate RNA.
  • Human PBMC of three healthy blood donors were incubated for 24h with 0.25 ⁇ M ORN SEQ ID NO:7 (FIG. 8a, phosphorothioate) or SEQ ID NO:5 (FIG.
  • FIG. 9 is two graphs demonstrating that the suppressive effect depends on the presence of a 2' modified nucleotide.
  • FIG. 9a shows IFN- ⁇ concentration
  • FIG. 9b shows TNF- ⁇ concentration.
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 10 is a graph demonstrating that CpG ODN-mediated cytokine induction is inhibited by 2' -modified oligoribonucleotide.
  • Human PBMC of three healthy blood donors were incubated for 24h with 0.25 ⁇ M of the C-Class CpG ODN SEQ ID NO:4 in the presence of DOTAP, and were co-cultured with different doses of the indicated compounds (unmodified ORN SEQ ID NO: 16 or 2'-modif ⁇ ed ORN SEQ ID N0:2).
  • SN were collected and cytokines measured by ELISA.
  • the x-axis is ORN concentration in ⁇ M and the y-axis is cytokine IFN- ⁇ in pg/ml.
  • FIG. 10 is a graph demonstrating that CpG ODN-mediated cytokine induction is inhibited by 2' -modified oligoribonucleotide.
  • FIG. 11 is four graphs demonstrating that RNA-mediated immune effects can be inhibited by single 2' modified nucleosides, especially 2'-0-Me-C, and also 2'-0-Me-A.
  • Human PBMC of three healthy blood donors were incubated for 24h with 1 ⁇ M ORN SEQ ID NO: 1 in the presence of DOTAP, and were co-cultured with different doses of the indicated nucleosides, S-Class ODN SEQ ID NO:3 or chloroquine (CQ).
  • SN were collected and cytokines measured by ELISA; IFN- ⁇ (FIG. 9a), IFN- ⁇ (FIG. 9b), TNF- ⁇ (FIG. 9c) and IL- 12p40 (FIG. 9d).
  • the x-axes are ORN concentration in ⁇ M and the y-axes are cytokine concentration in pg/ml.
  • FIG. 12 is a graph demonstrating that single 2' modified nucleosides do not exert an effect on LPS-mediated TNF- ⁇ induction with the exception of 2'-0-Me-A.
  • Human PBMC of three healthy blood donors were incubated for 24h with 100ng/ml LPS, and were co- cultured with different doses of the indicated nucleosides.
  • SN were collected and cytokine (TNF- ⁇ ) concentration measured by ELISA.
  • the x-axis is ORN concentration in ⁇ M and the y-axis is TNF- ⁇ concentration in pg/ml.
  • Fig. 13 is a graph demonstrating that DNA-mediated immune effects can be inhibited by single 2' modified nucleosides, especially 2'-O-Me-C.
  • Human PBMC of three healthy blood donors were incubated for 24h with 0.25 ⁇ M of the C-Class CpG ODN SEQ ID NO:4, and were co-cultured with different doses of the indicated nucleosides or chloroquine.
  • SN were collected and cytokines measured by ELISA.
  • the x-axis is ORN concentration in ⁇ M and the y-axis is IFN- ⁇ concentration in pg/ml.
  • Fig. 14 is three graphs demonstrating that 2'-O-Me-C mediates its strongest suppressive effect on CpG-mediated responses, not all RNA-dependent effects are affected by the 2'-modified nucleoside.
  • Human PBMC of three healthy blood donors were incubated for 24h either with 0.25 ⁇ M of the C-Class CpG ODN SEQ ID NO:4, 100ng/ml LPS, or l ⁇ M ORN SEQ ID NO:1 (the latter in the presence of DOTAP).
  • PBMC were co-cultured with the indicated doses of 2'-O-Me-C, S-Class ODN SEQ ID NO:3 or chloroquine.
  • SN were collected and cytokines and chemokines measured by Luminex cytokine array.
  • the x-axes represent % inhibition of the indicated ORN.
  • Figure 15 is a graph showing the effect of 2'-O-Methyl modified ORN on murine TLR7-induced activation of cytokine induction.
  • RAW264 murine macrophages were stimulated for 2Oh with l.O ⁇ M (splenocytes) or 0.25 ⁇ M (RAW264) ORN SEQ ID NO:7 complexed to DOTAP, or in the presence of the indicated concentrations of the unmodified non-stimulatory ORN SEQ ID NO: 16, or the same sequence with a single 2'-O-methyl modification (SEQ ID NO: 8), and TNF- ⁇ concentration measured.
  • the x asix is ORN concentration in ⁇ M and the y-axis is TNF- ⁇ concentration in pg/ml.
  • Figure 16 is two graphs showing the suppressive effect of 2'-O-Methyl modified ORN on on murine TLR7 -induced activation of cytokine induction.
  • Murine splenocytes were treated with stimulatory ORN SEQ ID NO:7 along with either the unmodified non- stimulatory ORN SEQ ID NO: 16, or the same sequence with a single 2'-O-methyl modification (SEQ ID NO: 8) at the concentrations indicated, and concentrations of IL- 12 ( Figure 16a) and 11-6 ( Figure 16b) in the supernatants were measured.
  • the x-axes are ORN used and the y-axes are cytokine concentration in pg/ml.
  • the invention is based in part on the discovery by the applicants that certain modifications of oligoribonucleotides (ORN) can reduce their immune stimulatory capacity.
  • Immune stimulatory oligonucleotides are known to activate signaling by any one of or any combination of toll receptors known to activate immune cells, including toll-like receptors (TLRs) 7, 8, and 9.
  • TLRs toll-like receptors
  • modifications that reduce the immune stimulatory capacity of ORN can effectively modulate their effect on the immune system.
  • such modified ORN, as well as specific 2' modified nucleosides have the ability to suppress the immune stimulatory capacity of TLR ligands, and thus to function as TLR antagonists.
  • TLRs Toll-like receptors
  • TLRl - TLRlO The cytoplasmic domains of the various TLRs are characterized by a Toll-interleukin 1 (IL-I) receptor (TIR) domain.
  • IL-I Toll-interleukin 1
  • TIR Toll-interleukin 1
  • the TIR domain-containing adapter protein MyD88 has been reported to associate with many of the TLRs and to recruit IL-I receptor-associated kinase (IRAK) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to the TLRs.
  • IRAK IL-I receptor-associated kinase
  • TNF tumor necrosis factor receptor-associated factor 6
  • the MyD88-dependent signaling pathway is believed to lead to activation of NF- ⁇ B transcription factors and c-Jun NH 2 terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs), critical steps in immune activation and production of inflammatory cytokines.
  • Jnk c-Jun NH 2 terminal kinase mitogen-activated protein kinases
  • Ligands for TLR2 include peptidoglycan and lipopeptides. Yoshimura A et al. (1999) J Immunol 163:1-5; Yoshimura A et al. (1999) J Immunol 163:1-5; Aliprantis AO et al. (1999) Science 285:736-9. Viral-derived double-stranded RNA (dsRNA) and poly I:C, a synthetic analog of dsRNA, have been reported to be ligands of TLR3. Alexopoulou L et al. (2001) Nature 413:732-8.
  • Lipopolysaccharide is a ligand for TLR4.
  • Poltorak A et al. (1998) Science 282:2085-8; Hoshino K et al. (1999) J Immunol 162:3749-52.
  • Bacterial flagellin is a ligand for TLR5. Hayashi F et al. (2001 ) Nature
  • Certain low molecular weight synthetic compounds have also been reported to be ligands of TLR7 and TLR8.
  • Bacterial DNA has been reported to be a TLR9 ligand. Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad Sci USA 98, 9237-42.
  • TLR7 has been reported to be expressed in placenta, lung, spleen, lymph nodes, tonsil and on plasmacytoid dendritic cells (pDCs).
  • pDCs plasmacytoid dendritic cells
  • Human TLR8 has been reported to be expressed in lung, peripheral blood leukocytes (PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N et al.
  • T-cell mediated immunity involves recognition of pathogen- associated molecular patterns (PAMPs) shared in common by certain classes of molecules expressed by infectious microorganisms or foreign macromolecules. PAMPs are believed to be recognized by pattern recognition receptors (PRRs) on or in certain immune cells.
  • PAMPs pathogen-associated molecular patterns
  • PRRs pattern recognition receptors
  • Antibody-mediated immunity involves immune cell activation to produce cytokines that stimulate B cell antibody synthesis.
  • An immune response may involve activation of B cells, monocytoid dendritic cells, plasmacytoid dendritic cells, monocytes, monocyte-derived cells, and eosinophils, for example.
  • An aberrant immune response such as one involving excessive or chronic activation of immune cells, can result in detrimental conditions such as autoimmune disease and chronic inflammatory disorders.
  • suppression of activation refers to the administration of a molecule according to the invention such that activation of immune cells is reduced or eliminated.
  • the term “suppressing an immune response” refers to the administration of a molecule according to the methods of the invention such that an immune response is reduced or eliminated.
  • an "RNA-mediated immune response” is an immune response activated by an immune stimulatory RNA.
  • a "DNA-mediated immune response” is an immune response activated by immune stimulatory DNA.
  • RNA molecules that are known to have immune stimulatory effects contain a sequence motif thought to be responsible for the immune modulatory activity of the RNA.
  • a base sequence thought to activate TLR8 typically includes at least one guanosine (G) and at least one uracil (U).
  • an "immune modulatory motif is a sequence motif which confers immune modulatory activity to the molecule. The immune modulatory motif in some cases is between 4 and 8 bases long. Lipford et al. US 2003/0232074. Nucleic acid molecules containing GUU, GUG, GGU, GGG, UGG, UGU, UUG, UUU, multiples and any combinations thereof are believed to be TLR8 ligands.
  • RNA molecules may have multiple immune modulatory motifs.
  • the term "immune modulatory motif describes a sequence motif in a molecule that provides the immune modulatory activity to the molecule.
  • the immune modulatory motif itself need not be modified to produce an ORN with altered immune modulatory capacity.
  • a further surprising aspect of the invention is based on the discovery by the inventors that these modified ORN can exert an immunosuppressive effect alone or in the presence of other immune stimulatory molecules.
  • Another suprising aspect was that 2' modification of an ORN not containing an immune stimulatory motif can exert the immunosuppressive effect.
  • the term "modified ORN" refers to an ORN which includes a residue having a modification at the 2' position on a residue outside the immune modulatory motif.
  • the modified ORN of the invention may be modified such that one ⁇ -ribose unit may be replaced by a modified sugar unit.
  • a "2' modification" on an ORN is one in which the ribose on a residue of the ORN has been modified at the 2' position.
  • the modified sugar unit is for example selected from ⁇ -D-ribose, ⁇ -D-ribose, ⁇ -L-ribose (as in 'Spiegelmers'), ⁇ -L-ribose, 2'-amino-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-(Cl- C6)alkyl-ribose, 2'-O-(Cl-C6)alkyl-ribose, 2'-O-methylribose, 2'-O-(C2-C6)alkenyl-ribose, 2'-[O-(Cl-C6)alkyl-O-(Cl-C6)alkyl]-ribose, LNA and ⁇ -LNA (Nielsen P et al.
  • a "2'-O methyl" modification refers to a modified sugar unit with an O-methyl group at the T position.
  • the suppressive effect of nucleotide modifications in a stimulatory ORN can be attributed to modification of specific ribonucleosides. It was found by the inventors that modification of ribose at the 2' position outside the modulatory motif of the ORN causes a decrease in its immune modulatory activity.
  • a "decrease in immune modulatory activity” refers to a reduction or elimination of the ability of the molecule to stimulate an immune response as compared to the same molecule without the modification.
  • the term "stimulate an immune response” refers to any increase in immune parameter, such as, for example, activation of a B or T cell or other immune cell or induction in one or more cytokine levels.
  • This modification has been shown to be effective for reducing immunomodulatory capacity of the ORN when the modification occurs on rA, rG, or rU, as measured by a suppression of production of cytokines (IFN- ⁇ , TNF- ⁇ , and IFN- ⁇ ; see Examples).
  • the T modification not only results in suppression of immune modulatory effects of the modified ORN, but has also been found to suppress the immune stimulatory effects of a TLR ligand when added as an inhibitory or antagonistic ORN.
  • the 2' modification has been found to suppress DNA-mediated effects when added as an inhibitory or antagonistic ORN to stimulatory CpG ODN molecules.
  • the modified ORN of the invention comprise 2 or more modified residues.
  • the modified ORN of the invention comprise 3-10 modified residues.
  • the modified ORN are not designed to comprise a sequence complementary to that of a coding sequence in a human cell, and are therefore not considered to be antisense ORN or silencing RNA (siRNA).
  • siRNA silencing RNA
  • An ORN which is "not complementary” is one that does not comprise a sequence capable of hybridizing strongly with one particular coding region in the target cell. Therefore, administration of an ORN which is not complementary as used herein will not result in gene silencing, especially as the ORN described in this invention are single-stranded compared to the double-stranded molecules used as silencing RNAs.
  • the oligonucleotide may have other carbohydrate backbone modifications and replacements, such as peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), and oligonucleotides having backbone sections with alkyl linkers or amino linkers.
  • the alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or a racemic mixture.
  • the term "phosphorothioate backbone” refers to a stabilized sugar phosphate backbone of a nucleic acid molecule in which a non-bridging phosphate oxygen is replaced by sulfur at least one internucleotide linkage. In one embodiment non-bridging phosphate oxygen is replaced by sulfur at each and every internucleotide linkage.
  • a ⁇ -ribose unit or a ⁇ -D-2'-deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is for example selected from ⁇ -D-ribose, ⁇ -D-2'- deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-O-(Ci-C 6 )alkyl-ribose, 2'-O-methylribose, 2'-O-(C 2 -C 6 )alkenyl-ribose, 2 1 -[O-(C 1 -C 6 )alkyl-O-(Ci-C 6 )alkyl]-ribose, 2 1 - fNH 2 -2'-deoxyribose, ⁇ -D-xylo-furanose, ⁇ -arabinofuranose, 2,4-dideoxy- ⁇ -D
  • the sugar is 2'-O- methylribose.
  • the modified ORN of the invention is between 10 and 30 nucleotides in length. In some embodiments the modified ORN is between 10 and 50 nucleotides in length. In some embodiments the modified ORN of the invention is between 10 and 100 nucleotides in length. In some embodiments the modified ORN have a backbone that may be stabilized. In one embodiment the backbone is a sugar phosphate backbone that includes at least one phosphorothioate internucleotide linkage. In one embodiment the backbone is completely phosphorothioate.
  • the invention provides a method for treating a condition associated with aberrant immune stimulation in a subject.
  • the term "treat” as used in reference to a disease or condition shall mean to intervene in such disease or condition so as to prevent or slow the development of, prevent or slow the progression of, halt the progression of, or eliminate the disease or condition.
  • the method according to this aspect of the invention involves the step of administering to a subject having or at risk of developing a condition associated with aberrant immune stimulation an effective amount of an isolated immune modulatory ORN of the invention to treat the condition.
  • the ORN can be but need not be limited to a single administration. The method is useful whenever it is desirable to slow or alter an immune response.
  • immune modulatory ORN of the invention may be used to treat any of a number of conditions that involve an innate immune response or a ThI -like immune response, including inflammation, acute and chronic allograft rejection, graft-versus-host disease (GvHD), certain autoimmune diseases, infection, and sepsis.
  • innate immune response or a ThI -like immune response including inflammation, acute and chronic allograft rejection, graft-versus-host disease (GvHD), certain autoimmune diseases, infection, and sepsis.
  • GvHD graft-versus-host disease
  • the term "subject” refers to a human or non-human vertebrate.
  • Non- human vertebrates include livestock animals, companion animals, and laboratory animals, such as, for instance, non-human primates, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.
  • the term "subject at risk of developing” a condition refers to a subject with a known or suspected exposure to an agent known to cause or to be associated with the condition or a known or suspected predisposition to develop the condition (e.g., a genetic marker for or a family history of the condition).
  • infection refers to a condition associated with the activation of the immune system by a microorganism, including but not limited to bacteria, fungi, and viruses.
  • the term “sepsis” refers to a well-recognized clinical syndrome associated with a host's systemic inflammatory response to microbial invasion.
  • the term “sepsis” as used herein refers to a condition that is typically signaled by fever or hypothermia, tachycardia, and tachypnea, and in severe instances can progress to hypotension, organ dysfunction, and even death.
  • autoimmune disease refers to a disease caused by a breakdown of self-tolerance such that the adaptive immune system responds to self antigens and mediates cell and tissue damage.
  • An "autoimmune response” is therefore defined as an aberrant immune response resulting in an autoimmune condition or disease.
  • Autoimmune diseases specifically include, without limitation, insulin-dependent diabetes mellitus, inflammatory bowel disease, and multiple sclerosis. Additional specific examples of autoimmune diseases are provided below. Such conditions typically involve activation toll receptor signaling in response to the RNA or DNA.
  • inflammatory disorder refers to a condition associated with an antigen-nonspecific reaction of the innate immune system that involves accumulation and activation of leukocytes and plasma proteins at a site of infection, toxin exposure, or cell injury.
  • Cytokines that are characteristic of inflammation include tumor necrosis factor (TNF- ⁇ ), interleukin 1 (IL-I), IL-6, IL- 12, interferon alpha (IFN- ⁇ ), interferon beta (IFN- ⁇ ), and chemokines.
  • Inflammatory disorders include, for example asthma, allergy, allergic rhinitis cardiovascular disease, chronic obstructive pulmonary disease (COPD), bronchiectasis, chronic cholecystitis, tuberculosis, Hashimoto's thyroiditis, sarcoidosis, silicosis and other pneumoconioses, and an implanted foreign body in a wound, but are not so limited.
  • COPD chronic obstructive pulmonary disease
  • a 2 '-modified cytidine is provided for use in treatment of aberrant immune stimulation.
  • the modification is a 2'-O-alkyl modification.
  • the modification is a 2'-O-methyl modification.
  • Other modificatications include but are not limited to 2'-O-ethyl, 2'-O-propyl, 2'-O-butyl or 2'-O-(2-methoxyethyl) modifications.
  • Other useful modifications include 2'-O, 4'-C- alkylen-bridged nucleosides, such as.
  • the 2'-O-alkyl- modification may contain at least one unsaturated carbon-carbon linkage such as a 2'-O-allyl or 2'-O-propinyl linkage. Additional examples include 2'-O-trifluoromethyl nucleosides, T- O-ethyl-trifluoromethoxy nucleosides, 2'-0-difluoromethoxy-ethoxy nucleosides.
  • the invention in another aspect provides a method for reducing an immunostimulatory effect of a TLR ligand or agonist.
  • the TLR ligand can be a small molecule, a stimulatory ORN or a CpG nucleic acid molecule.
  • the method involves the step of contacting an immune cell that is sensitive to the TLR ligand with an effective amount of an isolated immune modulatory ORN or T modified cytidine to reduce an immunostimulatory effect of the TLR ligand on the immune cell to a level below that which would occur without the contacting.
  • a further aspect of the invention is a modified ORN in which the ORN is modified on an rC residue. Such modifications according to the invention do not result in modified ORN with reduced immunostimulatory capacity or immune suppressive activity.
  • the ORN has unchanged or increased immune stimulatory capacity and can be used to stimulate an immune response in a subject in need of such treatment.
  • Autoimmune diseases can be generally classified as antibody-mediated, T-cell mediated, or a combination of antibody-mediated and T-cell mediated.
  • Immune modulatory ORN of the invention are believed to be most useful for treating various types of autoimmunity involving antibody-mediated or T-cell mediated immunity, including insulin- dependent (type I) diabetes mellitus, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), and inflammatory bowel disease (i.e., Crohn's disease and ulcerative colitis). Animal models for these autoimmune diseases are available and are useful for assessing the efficacy of inhibitory ODN in these diseases.
  • type I insulin- dependent
  • SLE systemic lupus erythematosus
  • inflammatory bowel disease i.e., Crohn's disease and ulcerative colitis
  • autoimmune diseases include, without limitation, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, juvenile arthritis, lichen planus, myasthenia gravis, polyarteritis nodosa, polychondritis, polyglandular syndromes, der
  • the present invention provides a method for treating a condition associated with CpG DNA- or RNA-mediated imrnunostimulation in a subject having or being at risk of having an autoimmune disease.
  • the methods of the invention result in a shift in the immune system from a ThI -like immune response to a Th2-like immune response.
  • a ThI -like immune response can include expression of any of certain cytokines and chemokines, including IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , IL- 12, IL-18, IP-IO, and any combination thereof, that are characteristically associated with a ThI immune response.
  • the Th2-like immune response can include induction of certain Th2 -associated cytokines, including IL-4, IL-5, and IL-13.
  • a Th2-like immune response can be useful in the treatment of any of a number of conditions that involve an innate immune response or a ThI -like immune response, including inflammation, acute and chronic allograft rejection, graft-versus- host disease (GvHD), certain autoimmune diseases, infection, and sepsis.
  • Infections refer to any condition in which there is an abnormal collection or population of viable intracellular or extracellular microbes in a subject.
  • Various types of microbes can cause infection, including microbes that are bacteria, microbes that are viruses, microbes that are fungi, and microbes that are parasites.
  • Bacteria include, but are not limited to, Pasteurella species, Staphylococci species, Streptococcus species, Escherichia coli, Pseudomonas species, and Salmonella species.
  • infectious bacteria include but are not limited to, Helicobacter pylor is, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellular, M. kansasii, M.
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-I (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filovirida
  • Fungi include yeasts and molds.
  • fungi include without limitation Aspergillus spp including Aspergillus fumigatus, Blastomyces dermatitidis, Candida spp including Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis carinii, Rhizomucor spp, and Rhizopus spp.
  • Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii.
  • Blood-borne and/or tissue parasites include Plasmodium spp.,
  • 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.
  • Sepsis is caused when severe infection over-activates the body's immune system, setting off a cascade of systemic inflammatory responses. Sepsis may be associated with clinical symptoms of systemic illness, such as fever, chills, malaise, low blood pressure, and mental status changes. Sepsis is potentially a life-threatening disease that can lead to a severe drop in blood pressure and cardiovascular collapse. Sepsis is most likely to develop in people with a weakened or underdeveloped immune system.
  • Particularly at risk are those who are very young (particularly premature babies), are very old, are undergoing chemotherapy treatments, have AIDS, are undergoing organ transplant procedure, have wounds or injuries vulnerable to infection, have addictive habits such as alcohol or drug abuse, or are receiving treatments via intravenous catheters, wound drainage, urinary catheters, or other treatments which potentially allow bacteria access to the body.
  • the modified ORN of the instant invention can encompass various chemical modifications and substitutions, in addition to the 2' modification, in comparison to natural RNA and DNA, involving a phosphodiester internucleoside bridge, a ⁇ -D-ribose unit and/or a natural nucleoside base (adenine, guanine, cytosine, thymine, uracil).
  • Examples of chemical modifications are known to the skilled person and are described, for example, in Uhlmann E et al. (1990) Chem Rev 90:543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S.
  • An oligonucleotide according to the invention may have one or more modifications, in addition to the 2' modification, wherein each modification is located at a particular phosphodiester internucleoside bridge and/or at a particular ⁇ -D-ribose unit and/or at a particular natural nucleoside base position in comparison to an oligonucleotide of the same sequence which is composed of natural DNA or RNA.
  • the oligonucleotides may include one or more modifications and wherein each modification is independently selected from: a) the replacement of a phosphodiester internucleoside bridge located at the 3' and/or the
  • a nucleoside by a modified internucleoside bridge b) the replacement of phosphodiester bridge located at the 3' and/or the 5' end of a nucleoside by a dephospho bridge, c) the replacement of a sugar phosphate unit from the sugar phosphate backbone by another unit, d) the replacement of a ⁇ -D-ribose unit by a modified sugar unit, and e) the replacement of a natural nucleoside base by a modified nucleoside base.
  • More detailed examples for the chemical modification of an oligonucleotide are as follows.
  • the oligonucleotides may include modified internucleotide linkages, such as those described in a or b above. These modified linkages may be partially resistant to degradation (e.g., are stabilized).
  • a stabilized oligonucleotide molecule is an oligonucleotide that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease) resulting from such modifications.
  • Oligonucleotides having phosphorothioate linkages may provide maximal activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases. As shown in the Examples, phosphorothioate molecules demonstrated better inhibitory activity than a corresponding phosphodiester molecule.
  • a phosphodiester internucleoside bridge located at the 3' and/or the 5' end of a nucleoside can be replaced by a modified internucleoside bridge, wherein the modified internucleoside bridge is for example selected from phosphorothioate, phosphorodithioate, NR'R 2 -phosphoramidate, boranophosphate, ⁇ -hydroxybenzyl phosphonate, phosphate-(Ci- C 21 )-O-alkyl ester, phosphate-[(C 6 -Ci 2 )aryl-(Ci-C2i)-O-alkyl]ester, (Ci-Cg)alkylphosphonate and/or (C 6 -Ci 2 )arylphosphonate bridges, (C 7 -C i 2 )-a-hydroxymethyl-aryl (e.g., disclosed in WO 95/01363), wherein (C 6 -Ci 2 )aryl, (C 6 -C 2
  • dephospho bridges are described, for example, in Uhlmann E and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for
  • a dephospho bridge is for example selected from the dephospho bridges formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silyl groups.
  • a sugar phosphate unit i.e., a ⁇ -D-ribose and phosphodiester intemucleoside bridge together forming a sugar phosphate unit
  • the sugar phosphate backbone i.e., a sugar phosphate backbone is composed of sugar phosphate units
  • the other unit is for example suitable to build up a "morpholino-derivative" oligomer (as described, for example, in Stirchak EP et al.
  • Nucleic Acids Res 17:6129-41 that is, e.g., the replacement by a morpholino-derivative unit; or to build up a polyamide nucleic acid ("PNA"; as described for example, in Nielsen PE et al. (1994) Bioconjug Chem 5:3-7), that is, e.g., the replacement by a PNA backbone unit, e.g., by 2-aminoethylglycine.
  • PNA polyamide nucleic acid
  • Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.
  • Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, and thymine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.
  • a modified base is any base which is chemically distinct from the naturally occurring bases typically found in DNA and RNA such as T, C, G, A, and U, but which share basic chemical structures with these naturally occurring bases.
  • the modified nucleoside base may be, for example, selected from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Ci-C 6 )-alkyluracil, 5-(C2-C 6 )-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Ci-C6)-alkylcytosine, 5-(C 2 -C 6 )-alkenylcytosine, 5-(C 2 -C 6 > alkynylcytosine, 5-chlorocytosine, 5-
  • modified bases may be incorporated.
  • a cytosine may be replaced with a modified cytosine.
  • a modified cytosine as used herein is a naturally occurring or non-naturally occurring pyrimidine base analog of cytosine which can replace this base without impairing the immunostimulatory activity of the oligonucleotide.
  • Modified cytosines include but are not limited to 5-substituted cytosines (e.g., 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo- cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo- isocytosine, cytosine analogs with condensed ring systems (e.g., N,N' -propylene cytosine or phenoxazine), and uracil and its derivatives (e.g., 5-fluoro-ura
  • cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5- hydroxymethyl-cytosine, and N4-ethyl-cytosine.
  • the cytosine base is substituted by a universal base (e.g., 3-nitropyrrole, P-base), an aromatic ring system (e.g., fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).
  • a universal base e.g., 3-nitropyrrole, P-base
  • aromatic ring system e.g., fluorobenzene or difluorobenzene
  • dSpacer hydrogen atom
  • a guanine may be replaced with a modified guanine base.
  • a modified guanine as used herein is a naturally occurring or non-naturally occurring purine base analog of guanine which can replace this base without impairing the immunostimulatory activity of the oligonucleotide.
  • Modified guanines include but are not limited to 7-deazaguanine, 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine, hypoxanthine, N2-substituted guanines (e.g., N2-methyl- guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyl-adenine, 8-oxo- adenine), 8-substituted guanine (e.g., 8-hydroxyguanine and 8-bromoguanine), and
  • the guanine base is substituted by a universal base (e.g., 4-methyl-indole, 5-nitro-indole, and K- base), an aromatic ring system (e.g., benzimidazole or dichloro- benzimidazole, 1 -methyl- 1 H-[1, 2,4]triazole-3-carboxylic acid amide) or a hydrogen atom (dSpacer).
  • a universal base e.g., 4-methyl-indole, 5-nitro-indole, and K- base
  • an aromatic ring system e.g., benzimidazole or dichloro- benzimidazole, 1 -methyl- 1 H-[1, 2,4]triazole-3-carboxylic acid amide
  • dSpacer a hydrogen atom
  • the oligonucleotides of the invention can be synthesized de novo using any of a number of procedures well known in the art, for example, the ⁇ -cyanoethyl phosphoramidite method (Beaucage SL et al. (1981) Tetrahedron Lett 22:1859); or the nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27:4051-4; Froehler BC et al. (1986) Nucleic Acids Res 14:5399-407; Garegg et al. (1986) Tetrahedron Lett 27:4055-8; Gaffney et al.
  • oligonucleotide generally refers to an oligonucleotide which is separated from components which it is normally associated with in nature.
  • an isolated oligonucleotide may be one which is separated from a cell, from a nucleus, from mitochondria or from chromatin.
  • Modified backbones such as phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries.
  • Aryl-and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (e.g., Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1 :165).
  • the composition can also further include a pharmaceutically acceptable carrier, such that the invention also provides pharmaceutical compositions containing the isolated modified ORN or 2' modified nucleoside of the invention.
  • pharmaceutically acceptable carrier refers to one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • the carrier is a lipid carrier such as N-[l-(2,3-Dioleoyloxy)propyl]- N,N,Ntrimethylammoniummethyl-sulfate (DOTAP).
  • DOTAP N-[l-(2,3-Dioleoyloxy)propyl]- N,N,Ntrimethylammoniummethyl-sulfate
  • DOTAP is believed to transport RNA oligomer into cells and specifically traffic to the endosomal compartment, where it can release the RNA oligomer in a pH-dependent fashion. Once in the endosomal compartment, the RNA can interact with certain intracellular TLRs, triggering TLR-mediated signal transduction pathways involved in modulating an immune response. Other agents with similar properties including trafficking to the endosomal compartment can be used in place of or in addition to DOTAP.
  • lipid formulations include, for example, as EFFECTENETM (a non-liposomal lipid with a special DNA condensing enhancer), SUPERFECTTM (a novel acting dendrimeric technology), and Stable Nucleic Acid Lipid Particles (SNALPs) which employ a lipid bilayer.
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[I -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • EFFECTENETM a non-liposomal lipid with a special DNA condensing enhancer
  • SUPERFECTTM a novel acting dendrimeric technology
  • SNALPs Stable Nucleic Acid Lipid Particles
  • Liposomes are commercial
  • the pharmaceutically acceptable carrier is a cationic polymer, e.g. polyethylene imine (PEI), cyclodextrine, or chitosan.
  • PEI polyethylene imine
  • the immune modulatory ORN of the invention can also be used for the preparation of a medicament for use in treatment of a condition in a subject.
  • the use according to this aspect of the invention involves the step of placing an effective amount of a composition of the invention in a pharmaceutically acceptable carrier.
  • 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 oligonucleotide being administered, 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 immunosuppressive ORN and/or antigen and/or other therapeutic agent without necessitating undue experimentation.
  • the therapeutically effective amount can be initially determined from animal models.
  • 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.
  • the modified ORN or 2' modified nucleoside of the invention can be administered alone or formulated as a delivery complex via any suitable route of administration that is effective to achieve the desired therapeutic result.
  • Routes of administration include enteral and parenteral routes of administration. Examples of enteral routes of administration include oral, gastric, intestinal, and rectal.
  • enteral routes of administration include oral, gastric, intestinal, and rectal.
  • parenteral routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, local injection, topical, nasal, mucosal, and pulmonary.
  • the modified ORN or T modified nucleoside of the invention 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.
  • a targeting means e.g., a molecule that results in higher affinity binding to target cell.
  • nucleic acid delivery complexes include nucleic acids associated with a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome such as DOTAP), or a target cell specific binding agent (e.g., a ligand recognized by target cell specific receptor).
  • Preferred complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell.
  • the complex can be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.
  • the compounds i.e., modified ORN or T modified nucleoside, antigens and/or other therapeutic agents
  • pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds may be administered by inhalation to pulmonary tract, especially the bronchi and more particularly into the alveoli of the deep lung, using standard inhalation devices.
  • the compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • An inhalation apparatus may be used to deliver the compounds to a subject.
  • An inhalation apparatus is any device for administering an aerosol, such as dry powdered form of the compounds. This type of equipment is well known in the art and has been described in detail, such as that description found in Remington: The Science and Practice of Pharmacy, 19 th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania, pages 1676-1692. Many U.S. patents also describe inhalation devices, such as U.S. Pat. No. 6,116,237. "Powder” as used herein refers to a composition that consists of finely dispersed solid particles.
  • the compounds are relatively free flowing and capable of being dispersed in an inhalation device and subsequently inhaled by a subject so that the compounds reach the lungs to permit penetration into the alveoli.
  • a "dry powder” refers to a powder composition that has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol.
  • the moisture content is generally below about 10% by weight (% w) water, and in some embodiments is below about 5% w and preferably less than about 3% w.
  • the powder may be formulated with polymers or optionally may be formulated with other materials such as liposomes, albumin and/or other carriers.
  • Aerosol dosage and delivery systems may be selected for a particular therapeutic application by one of skill in the art, such as described, for example in Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990), and in Moren, "Aerosol dosage forms and formulations,” in Aerosols in Medicine. Principles, Diagnosis and Therapy, Moren, et al., Eds., Elsevier, Amsterdam, 1985.
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the
  • the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be any organic compound.
  • the compounds may also be any organic compound.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions also may include suitable solid or gel phase carriers
  • W or excipients examples include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic
  • compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries
  • compositions are suitable for use in a variety of drug delivery systems.
  • disintegrants such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-33, which is incorporated herein by reference.
  • the modified ORN or 2' modified nucleoside and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the
  • salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • compositions of the invention contain an effective amount of an modified ORN or 2' modified nucleoside 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.
  • ODN and ORN were purchased from Biospring (Frankfurt, Germany) or provided by Coley Pharmaceutical GmbH (Langenfeld, Germany), controlled for identity and purity by Coley Pharmaceutical GmbH and had undetectable endotoxin levels ( ⁇ 0.1EU/ml) measured by the Limulus assay (BioWhittaker, Verviers, Belgium).
  • ODN were suspended in sterile, endotoxin-free Tris-EDTA (Sigma, Deisenhofen, Germany), ORN were suspended in sterile, DNAse- and RNAse-free dH 2 O (Life Technologies, Eggenstein, Germany) and stored and handled under aseptic conditions to prevent both microbial and endotoxin contamination.
  • Peripheral blood buffy coat preparations from healthy human donors were obtained from the Blood Bank of the University of Dusseldorf (Germany) and PBMC were purified by centrifiigation over Ficoll-Hypaque (Sigma). Cells were cultured in a humidified incubator at 37°C in RPMI 1640 medium supplemented with 5% (v/v) heat inactivated human AB serum (BioWhittaker) or 10% (v/v) heat inactivated FCS, 2mM L-glutamine, 100U/ml penicillin and lOO ⁇ g/ml streptomycin (all from Sigma).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • SN culture supernatants
  • cells were stimulated with the indicated TLR ligand concentration and nucleoside or ORN added.
  • the second modified ORN was added Ih after the start of the cell culture. Amounts of cytokines in the SN were assessed using a commercially available ELISA
  • Kit for IL-12p40 from BD Biosciences, Heidelberg, Germany
  • IFN- ⁇ and TNF-cc from Diaclone, Besancon, France
  • an in-house ELISA for IFN- ⁇ developed using commercially available antibodies (PBL, New Brunswick, NJ, USA).
  • cytokines and chemokines multiplex analysis with a luminex system from Bio-Rad (Munich, Germany) and Multiplex kits from Biosource (Solingen, Germany) was performed.
  • Naive svl29 mouse splenocytes or the mouse macrophage cell line RAW264 were also used for in vitro cytokine induction. Animals were anesthetized with isofluorane and euthanized by cervical dislocation. Spleens were removed under aseptic conditions and • placed in PBS + 0.2% BSA (Sigma, St. Louis, MO, USA).
  • Spleens were then homogenized and splenocytes were re-suspended in RPMI 1640 (Life Technologies) medium supplemented with 2% normal mouse serum (Cedarlane Laboratories, Ontario, Canada), 2 mM L- Glutamine, penicillin-streptomycin solution (final concentration of 1000 U/ml and 1 mg/ml respectively), and 5xlO "5 M ⁇ -mercaptoethanol (all from Sigma).
  • Splenocytes were plated in 96- well round-bottomed plates (5x105 cells/well). Each splenocyte sample was plated in quadruplicate and the cells were incubated in a humidified 5% CO2 incubator at 37°C for 2Oh.
  • the following examples demonstrate the inhibitory effects of specific 2'-O-methyl modified nucleotides in a stimulatory ORN, as well as suppressive effects of 2'-O-methyl modified ORN and 2'OMe-C and —A nucleosides when added to stimulatory RNA or other TLR ligands as antagonistic molecules.
  • FIGS. Ia and Ib show interferon alpha (IFN- ⁇ ) and tumor necrosis factor (TNF- ⁇ ) production after treatment of cells with various oligoribonucleotides (see table 1). Oligoribonucleotides derived from the RNA sequence of the eukaryotic Ul snRNP particle were either unmodified or contained naturally occurring modifications present at the indicated positions (2'-O-methyl modification of adenosine (A) or uracil (U)).
  • IFN- ⁇ interferon alpha
  • TNF- ⁇ tumor necrosis factor
  • Example 2 2'-0-methylation outside the immune stimulatory motif interferes with RNA activation
  • Phosphorothioate ORN with 2' modifications at rA, rG or rU, but not rC resulted even in suppression of stimulatory ORN effects upon co-culture.
  • a study was performed in order to test the effect of 2'-modified ORN on immune cell activation by an immune stimulatory ORN (see table 6).
  • the data in Figures 6a and 6c show the effects of the immune stimulatory ORN SEQ ID NO: 1 on activation of IFN- ⁇ and TNF- ⁇ , respectively.
  • 2'-O-methyl modification of a rU in ORN SEQ ID NO: 1 resulted in an ORN that showed significantly decreased induction of IFN- ⁇ and TNF- ⁇ .
  • ORN SEQ ID NO: 1 resulting in ORN SEQ ID NO:2 did not only inhibit its own immune modulatory effects ( Figures 6a and 6c), but did suppress the stimulatory effects of the stimulatory unmodified ORN upon co-culture (SEQ ID NO:1) ( Figures 6b and 6d), probably acting as a TLR antagonist. Similar results were observed for all cytokines tested, IFN- ⁇ , TNF- ⁇ , IFN- ⁇ and IL-12 ( Figures 7 and 8). More importantly, ORN SEQ ID NO: 1 is derived from a naturally occurring RNA, the Ul snRNA.
  • Ul snRNA-containing immune complexes were demonstrated to be involved in autoimmune responses such as SLE, and autoantibodies targeting snRNPs and the snRNA can be observed in SLE patients.
  • the inflammation observed in SLE patients may be attributed to stimulatory effects induced by the snRNA, as the Ul snRNA itself as well as GU-containing sequences derived from this snRNA such as ORN SEQ ID NO: 1
  • the suppressive ORN may be used to reduce or suppress inflammatory responses mediated by self RNA such as the Ul snRNA.
  • ORN SEQ ID NO:1 As a stimulatory ORN, but could be reproduced with a variety of other stimulatory ORN.
  • Figure 8 The immune stimulatory ORN SEQ ID NO:7 containing a GU immune stimulatory motif was co-cultured with the 2' rU modified ORN SEQ ID NO:2.
  • SEQ ID NO:2 did suppress the immune stimulatory effects of ORN SEQ ID NO:4 similar to SEQ ID N0:l ( Figure 8 and Figure 6, respectively).
  • the T modified sequence of ORN SEQ ID NO:2 with a phosphodiester backbone did suppress the activity of the stimulatory ORN SEQ ID NO:7 only at the highest concentrations used ( Figure 8a), indicating that, although being a suppressor of cytokine production, the phosphodiester sequence may be not stable enough and may be degraded before substantially interfering with the immune stimulatory effects.
  • the suppressive effect of the T modified ORN was also observed when the inhibitory ORN SEQ ID N0:2 was added to the cells Ih after the stimulatory ORN SEQ ID NO:1 (data not shown), and, therefore, appears not to be attributable to uptake competition.
  • ORN SEQ ID NO: 16 had some inhibitory effect on IFN- ⁇ production ( Figure 9a) induced by stimulatory ORN SEQ ID N0:4, probably due to uptake competition, ORN SEQ ID NO:2 and especially SEQ ID NO:8 did substantially suppress the stimulatory effects of ORN SEQ ID NO:7 for IFN- ⁇ ( Figure 9a) and TNF- ⁇ ( Figure 9b) induction, demonstrating that the 2' modification is responsible for the suppressive effects.
  • TLR7 and TLR8 are receptors responding to stimulation with RNA or ORN. Therefore, a potential suppressive effect of 2' modified ORN on other than TLR7,8-mediated effects was investigated.
  • TLR9 is the receptor for DNA containing CpG dinucleotides.
  • the unmodified ORN SEQ ID NO: 16 did not exhibit an effect on the IFN- ⁇ response induced by C-Class CpG ODN SEQ ID N0:4 in the presence of DOTAP ( Figure 10). Nevertheless, a clear shift of the SEQ ID N0:4-mediated response, and therefore interference with and inhibition of CpG-mediated effects, was observed, although not complete. It may be possible that higher concentrations of the suppressive ORN SEQ ID NO:2 would result in complete inhibition of the CpG SEQ ID NO:9-mediated immune response.
  • Example 4 Single 2'-O-methyl cytidine nucleosides exhibit inhibitory activity
  • 2'-O-methyl C, A, U, and G nucleosides were co-incubated with an immunostimulatory RNA and cytokine production was monitored.
  • 2'-O-methyl C nucleosides resulted in significant inhibition of IFN- ⁇ production. Slight inhibition of IFN- ⁇ production was also seen with 2'-O-methyl A nucleosides ( Figure 1 Ia).
  • the 2'-O-Me-modified C nucleoside was not capable at these concentrations of suppressing the stimulation of other cytokines such as TNF- ⁇ and IL- 12.
  • a suppressive effect could be observed for IFN- ⁇ , although not as strong.
  • GU-containing ORN specifically activate pDC that express TLR7, but not TLR8, to produce IFN- ⁇ , whereas other cells such as TLR8-expressing monocytes are stimulated to produce TNF- ⁇ , IFN- ⁇ or IL- 12. Therefore, it appears possible that the stronger inhibitory effect is exerted on pDC and IFN- ⁇ induction compared to IFN- ⁇ or IL- 12 producing cells.
  • Figure 14 shows a comparison of the inhibitory effects on TLR7/8 (ORN SEQ ID NO:1), TLR9 (C-Class CpG ODN SEQ ID NO:4)and TLR4 (LPS) ligand activity for a panel of cytokines and chemokines.
  • the figure shows the % inhibition mediated by the inhibitory molecule (2'-OMe-C, chloroquine or S-Class ODN SEQ ID NO:3) of the activity of the TLR ligand alone set to 100%. Chloroquine at the concentration used suppressed the ORN- dependent effects for all cytokines nearly to 100% ( Figure 14a).
  • the 2'-O-Me-C was not as suppressive for most of the ORN-mediated effects, as expected from the previous experiments demonstrating an inhibitory effect mainly on IFN- ⁇ and some effect on IFN- ⁇ . Indeed, from all the cytokines and chemokines shown, the strongest suppressive effects could only be observed for IFN- ⁇ and IFN- ⁇ , and some effect also for e.g., RANTES and IP-IO. Therefore, these data confirm the previous experiments demonstrating that 2'-O-Me-C is an inhibitor of the ORN-mediated IFN- ⁇ response.
  • murine splenocytes or RAW264 murine macrophages were stimulated for 2Oh with 1.O ⁇ M (splenocytes) or 0.25 ⁇ M (RAW264) ORN SEQ ID NO:7 complexed to DOTAP, or in the presence of the indicated concentrations of the unmodified non-stimulatory ORN SEQ ID NO: 16, or the same sequence with a single 2'-O-methyl modification (SEQ ID NO:8), and cytokines measured.
  • Figure 15 shows induction of TNF- ⁇ in RAW cells.
  • Cells were treated with stimulatory ORN SEQ ID NO:7 along with either the unmodified non-stimulatory ORN SEQ ID NO: 16, or the same sequence with a single 2'-O-methyl modification (SEQ ID NO:8) at the concentrations indicated, and concentration of cytokines in the supernatants were measured.
  • Cells treated with SEQ ID NO:8 produced less IL-12 and IL-6 than cells treated with the non-stimulatory ORN SEQ ID NO: 16, demonstrating that the effect is a true suppressive effect rather than competition with the stimulatory ORN SEQ ID NO:7.

Abstract

La présente invention concerne des oligoribonucléotides modifiés à activité immunomodulatrice. L'invention concerne le traitement de maladies auto-immunes ou infectieuses à l'aide des oligonucléotides de l'invention.
PCT/US2007/019873 2006-09-12 2007-09-12 Immunomodulation par des ribonucléosides et des oligoribonucléotides chimiquement modifiés WO2008033432A2 (fr)

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