WO2004093778A2 - Oligonucleotides bloquant les recepteurs de type toll - Google Patents

Oligonucleotides bloquant les recepteurs de type toll Download PDF

Info

Publication number
WO2004093778A2
WO2004093778A2 PCT/IL2004/000349 IL2004000349W WO2004093778A2 WO 2004093778 A2 WO2004093778 A2 WO 2004093778A2 IL 2004000349 W IL2004000349 W IL 2004000349W WO 2004093778 A2 WO2004093778 A2 WO 2004093778A2
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
tlr4
seq
expression
cell
Prior art date
Application number
PCT/IL2004/000349
Other languages
English (en)
Other versions
WO2004093778A3 (fr
WO2004093778A8 (fr
Inventor
Irun R. Cohen
Gabriel Nussbaum
Original Assignee
Yeda Research And Development Co. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yeda Research And Development Co. Ltd. filed Critical Yeda Research And Development Co. Ltd.
Publication of WO2004093778A2 publication Critical patent/WO2004093778A2/fr
Publication of WO2004093778A8 publication Critical patent/WO2004093778A8/fr
Publication of WO2004093778A3 publication Critical patent/WO2004093778A3/fr

Links

Classifications

    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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

Definitions

  • the present invention relates to novel oligonucleotide sequences, capable of inhibiting the expression of Toll-like receptor- 4.
  • the present invention further relates to pharmaceutical compositions comprising such oligonucleotides and to methods for using such compositions for the prevention or treatment of septic shock in gram-negative bacterial infections and for prevention or treatment of inflammatory and autoimmune diseases in which heat shock protein 60 (Hsp60) or other ligands of Toll like receptors are involved.
  • Hsp60 heat shock protein 60
  • TLRs Toll-like receptors
  • Toll-like receptors in mammalian cells are of microbial origin, which is in line with a function of these receptors in innate immune responses. It was recently found that the chaperone hsp ⁇ O is a putative endogenous ligand of Toll-like receptors in mammals (Ohashi et al., J Immunol. 164:558-561, 2000). This finding suggests that Toll-like receptors may not only have a function in innate immune defense against microbial pathogens but also have a major physiological role in interacting with endogenous ligands.
  • Toll-like receptors and hsp60 are found early in phylogeny and both are of remarkably conserved structure. This indicates that their interaction is relevant and may also occur in more primitive organisms. This is pronounced of the situation in Drosophila where Toll controls dorsal- ventral patterning with spaetzle serving as endogenous ligand (Marisato and Anderson, Cell 76: 677-688, 1994), while in adult insects Toll controls the antifungal and antibacterial response (Lemaitre et al., Cell 86: 973-983, 1996).
  • Tlr4 Several studies revealed that mice resistant to endotoxic shock carry natural mutations in Tlr4 (Qureshi S. et al., J. Exp. Med 189:615-625, 1999). In humans, a number of polymorphic alleles of Tlr4 have been identified. One such relatively common Tlr4 polymorphism may be associated with an increased risk of septic shock and a decreased risk of atherosclerosis, but is not associated with increased risk of meningococcal disease.
  • hsp60 Mammalian hsp60 usually is sequestered to the cell interior, in accordance with its ability to function as chaperone. However, hsp60 becomes accessible when it is set free during necrosis of tissue cells during inflammation or when hsp60 is partially translocated to the plasma membrane in response to diverse types of stress. It was therefore proposed that autologous Hsp60 may serve as a danger antigen to the innate immune system (Chen et al. J Immunol. 162:3212-3219, 1999).
  • Hsp ⁇ O self and foreign heat shock protein 60
  • Hsp ⁇ O activate cells in a Tlr4 dependent manner
  • Hsp60 has been implicated in multiple autoimmune and inflammatory conditions including but not limited to juvenile rheumatoid arthritis, type 1 diabetes, multiple sclerosis, systemic lupus erythematosis, inflammatory bowel disease, Bechet's disease, uveitis, thyroiditis, and atherosclerosis (Cohen IR., Annu Rev Immunol. 9:567-589, 1991; Jaattela M., Ann Med. 24:249-258, 1992).
  • Hsp ⁇ O is present in a soluble form in the circulation and anti-Hsp ⁇ O antibodies and T cells can be detected in high titer (Xu et al., Circulation 102:14-20, 2000). Innate immune cell activation by LPS has also been implicated in some of these conditions (Balasa et al, Clin Immunol. 95:93-98, 2000).
  • International application WO 01/43691 discloses fragments and antagonists of Hsp ⁇ O capable of inhibiting the Tlr4, for treatment of inflammatory and autoimmune diseases.
  • Hsp ⁇ O activate macrophages in a Tlr4 dependent manner. Circulating Hsp ⁇ O and/or anti-Hsp ⁇ O antibodies and T cells can be found in high titer in multiple autoimmune and inflammatory conditions such as rheumatoid arthritis, type 1 diabetes, and atherosclerosis. Hsp ⁇ O activation of innate immune cells as well as endothelial and muscle cells through Tlr4 may contribute significantly to the pathogenesis of these conditions. Thus, there is a need to modulate the role of autologous hsp ⁇ O and possibly of Toll-like receptor complexes involved in certain diseases and in particular in the regulation of pro-inflammatory immune responses and to provide molecules which are useful to suppress or even prevent such responses.
  • LPS Lipopolysaccharide
  • Tlr4 Toll-like receptor 4
  • TNF ⁇ Tumor necrosis factor- ⁇
  • Therapeutic strategies targeting the cytokine response to LPS are under investigation in clinical trials for septic shock. A recent large clinical trial showed a small but significant benefit in treating patients with a TNF ⁇ monoclonal blocking antibody, thereby reducing mortality. However, many studies using anti-TNF ⁇ antibodies and soluble TNF ⁇ receptor proteins have shown no significant decrease in mortality (Reinhart K et al., Crit Care Med (7):S121-125, 2001). Nevertheless, the current paradigm in gram-negative septic shock treatment combines antibiotic therapy, supportive measures, and suppression of the overwhelming cytokine response to LPS.
  • a number of publications disclose the use of antisense oligonucleotides targeting nucleic acids encoding TNF ⁇ .
  • Several groups are developing antisense oligonucleotides for the treatment of septic shock targeting either the TNF ⁇ gene (Ponnappa et al, J Pharmacol Exp Ther. (3):1129-1136, 2001) or the CD14 gene (Furusako et al., Acta Med Okayama. (2):105-115, 2001).
  • New therapies are needed for septic shock, which carries a high mortality and morbidity.
  • Targeting a component of the LPS receptor complex is an attractive approach since LPS signaling drives a cascade of cytokines that cause fever, vascular permeability and toxic shock.
  • LPS has also been shown to play a critical role in graft-versus-host disease (Cooke et al. J Clin Invest. 107:1581-9, 2001), and its antagonism with antisense therapy may therefore be beneficial after bone marrow transplantation.
  • US patent application 2003/0232352 discloses a method of assessing the susceptibility of an individual to atherosclerosis and to a method of treating an individual identified as being at increased risk.
  • Antisense technology has been one of the most commonly described approaches in protocols to achieve gene-specific interference. Briefly, A nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. For antisense strategies, stoichiometric amounts of single-stranded nucleic acid complementary to the messenger RNA for the gene of interest are introduced into the cell.
  • RNA interference in Gene Silencing
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al, Nature 391:806-811, 1998). The corresponding process in plants is commonly referred to as post- transcriptional gene silencing or RNA silencing.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla.
  • Such protection from foreign gene expression may have evolved in response to the presence of double-stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome, via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
  • dsRNA double-stranded RNAs
  • the presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized.
  • the presence of long dsRNAs in cells stimulates the activity of a ribonuclease III like enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs.
  • antisense oligonucleotides or double stranded RNA capable of inhibiting the expression of toll-like receptors in general or Tlr4 in particular, may be used for prevention or treatment of septic shock, inflammatory disorders or autoimmune diseases.
  • the present invention discloses compositions and methods for inhibiting the expression of the Toll-like receptor-4, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Tlr4, ultimately modulating the amount of Tlr4 produced. This is accomplished by providing oligonucleotides which specifically interact with nucleic acids encoding Tlr4, thereby inhibiting its expression.
  • the present invention is based on part on the unexpected discovery that oligonucleotides sequences which inhibit the expression of Tlr4, the innate immune receptor for Hsp ⁇ O and LPS, are able to modulate the inflammatory process in septic shock as well as in autoimmune conditions where Tlr4 may mediate or contribute to the pathogenesis.
  • oligonucleotide sequences capable of inhibiting the expression of tlr gene. These oligonucleotide sequences are capable of inhibiting the action of any ligand that acts via the Tlr4 receptor complex.
  • oligonucleotide sequences capable of targeting nucleic acids encoding Tlr4.
  • a preferred target is a gene encoding Tlr4.
  • Another preferred target is an mRNA which encodes Tlr4.
  • the targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense or dsRNA interaction to occur such that modulation of gene expression will result.
  • a further object of the present invention is to provide pharmaceutical compositions comprising oligonucleotide sequences which are capable of binding to the Tlr4 gene and inhibiting its expression.
  • preferred molecules according to the present invention are oligonucleotide antisense molecules derived from or complementary to the Tlr4 mRNA.
  • Preferred antisense oligonucleotide sequences have a length of about 5 to about 50 nucleotides, more preferred molecules are 8-30 nucleotides long, and most preferred oligonucleotides are 15-25 long.
  • RNA and PNA peptide nucleic acid molecules
  • preferred molecules are dsRNA oligonucleotides corresponding to the Tlr4 mRNA.
  • Preferred dsRNA oligonucleotide sequences have a length of about 5 to about 40, more preferred oligonucleotide sequences have a length of about 15 to about 25.
  • the dsRNA corresponds to a fragment of the sequence of the tlr gene.
  • the dsRNA according to the present invention is any dsRNA which inhibits Tlr4 expression via RNA interference (RNAi).
  • the dsRNAs are synthetic molecules.
  • an expression vector comprising the dsRNAs is used.
  • Preferred antisense or dsRNA sequences are derived from the human tlrA gene sequencedesignated SEQ ID NO:20 [Genebank accession no. AH009665].
  • antisense or dsRNA sequences are derived from a human mRNA transcript variant, one of which is presented in Figure 1 designated SEQ ID NO:l for the human Tlr4 mRNA transcript 1 [Genebank accession no. NM_138554].
  • antisense DNA or dsRNA sequences may be derived from mouse Tlr4 mRNA, the sequence of which is presented in Figure 2 designated as
  • the oligonucleotide antisense molecules derived from mouse mRNA are selected from SEQ ID NOs:3-6: 5'-TAGGACCGTAAAAGACGTTG-3' designated ODN47, corresponding to bp 24-4 of the mouse Tlr4 mRNA denoted herein as SEQ ID NO:3;
  • the dsRNA oligonucleotide molecules derived from mouse mRNA are selected from SEQ ID NOs:7-10: 5'-AAGCTTGAATCCCTGCATAGA-3 ', corresponding to bp 90-110 of the mouse Tlr4 mRNA denoted herein as SEQ ID NO: 7 5'-AAACAATTGAAGACAAGGCAT-3', corresponding to bp 290-310 of the mouse Tlr4 mRNA denoted herein as SEQ ID NO:8;
  • the dsRNA oligonucleotide molecules are derived from human mRNA are selected from SEQ ID NOs:l 1-19:
  • Tlr4 mRNA denoted herein as SEQ ID NO: 12;
  • Tlr4 mRNA denoted herein as SEQ ID NO: 13; 5'- GGATGGCAACATTTAGAATTA-3', corresponding to bp 1278-1298 of the human
  • Tlr4 mRNA denoted herein as SEQ ID NO: 14;
  • Tlr4 mRNA denoted herein as SEQ ID NO: 15;
  • Tlr4 mRNA denoted herein as SEQ ID NO: 17;
  • Tlr4 mRNA denoted herein as SEQ ID NO: 18; 5'- GTCATTCTCGAGCATGTTCTA-3', corresponding to bp 3381-3401 of the human
  • Tlr4 mRNA denoted herein as SEQ ID NO: 19;
  • oligonucleotide antisense molecules derived from human mRNA are selected from SEQ ID NOs:21-24:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient an oligonucleotide molecule comprising a contiguous nucleotide sequence from a group consisting of a fragment of the genomic sequence of tlrA or an mRNA transcribed therefrom.
  • the pharmaceutical compositions comprise any pharmaceutically acceptable excipients, diluents or carriers.
  • the oligonucleotide molecules are inserted into constructs.
  • the oligonucleotide molecules are incorporated into an expression.
  • oligonucleotide for inhibiting Toll-like receptor 4 gene expression
  • the oligonucleotide comprises a contiguous nucleotide sequence selected from the group consisting of a fragment of the genomic sequence of t/r4 and an mRNA transcribed therefrom.
  • the antisense oligonucleotides and dsRNA molecules disclosed in the present invention can prolong survival in septic shock and may therefore be beneficial as adjunctive therapy in its treatment.
  • LPS antagonism with antisense or dsRNA molecules specific to Tlr4 may be beneficial in the treatment of gram negative bacterial sepsis even without the signs and symptoms of shock.
  • the oligonucleotide sequences according to the present invention may be also used after bone marrow transplantation, due to the known involvement of LPS in graft versus host disease.
  • the pro-inflammatory signaling of human hsp ⁇ O was found to be dependent on a functional Tlr4. This finding suggests the existence of endogenous ligands of the Tlr4 complex, and a role of Toll-like receptors in innate immune discrimination of normal versus stressed or damaged tissue cells. It is therefore another aspect of the invention to provide molecules which, by blocking Tlr4, the innate immune receptor for Hsp ⁇ O, will have a beneficial effect in inflammatory diseases shown to involve Hsp ⁇ O. These diseases include but are not limited to: arthritis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, and atherosclerosis.
  • the present invention relates to a method for the treatment or prevention of bacterial septic shock by administering to a subject a pharmaceutical composition comprising as an active ingredient oligonucleotide molecules according to the present invention.
  • the subject of the present invention is of mammalian origin. According to some embodiments the subject of the present invention is of human origin.
  • FIG. 1 Mus musculus toll-like receptor 4 (Tlr4) mRNA, designated SEQ ID NO:2.
  • Figure 3 Homo sapiens toll-like receptor 4 (tlrA) genomic sequence designated SEQ ID NO:20.
  • Figure 4 A graph showing the percent survival of mice at 48 hours after intravenous challenge with 1 mg LPS.
  • the control, ODN47, ODN48 and ODN49 groups each had 5 mice/group.
  • the ODN50 group had 4 mice.
  • Figure 5 A graph showing serum TNF ⁇ levels at 90 minutes after LPS challenge.
  • the present invention employs oligonucleotide molecules for use in modulating the function of nucleic acids encoding Tlr4, ultimately modulating the amount of Tlr4 produced. This is accomplished by providing oligonucleotides wliich specifically hybridize with nucleic acids encoding Tlr4, preferably hybridizing with mRNA transcripts encoding
  • Tlr4 most preferably oligonucleotides specifically hybridizing with humanTlr4.
  • Tlr4 may be a more appropriate target than TNF ⁇ since there are likely to be additional cytokines that contribute to the complications of sepsis. Suppressing Tlr4 is likely to be safer than CD 14 suppression since CD 14 participates as a co-receptor of other microbial products, whereas
  • Tlr4 is specific for the LPS of gram negative bacteria.
  • Targeting an upstream event in the inflammatory cascade may be more effective than inhibiting the high level of a particular circulating cytokine, since even partial suppression of the LPS response pathway at the genetic level can significantly affect multiple downstream events.
  • Mice lacking functional Tlr4 are resistant to lethal doses of LPS.
  • antisense oligonucleotides or dsRNA specific for Tlr4 are shown to provide protection from LPS induced septic shock in a murine model and therefore may be used for treatment of septic shock in mammals.
  • the pro-inflammatory signaling of human hsp ⁇ O was found to be dependent on a functional Tlr4.
  • the present invention provides molecules which, by blocking Tlr4, the innate immune receptor for Hsp ⁇ O, will have a beneficial effect in inflammatory diseases shown to involve Hsp ⁇ O. These diseases include but are not limited to: arthritis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, and atherosclerosis. Similarly, other endogenous ligands of Toll-like receptors have been described and reducing the signaling by these molecules may be beneficial in particular clinical situations (Beg, AA., Trends Immunol. 23:509-512, 2002).
  • Transient immunosuppression caused by downregulating Tlr4 with antisense oligonucleotides or dsRNA can decrease the cytokine response during sepsis without the side effects seen with indiscriminate immunosuppressive medications.
  • septic shock is blocked by downregulating the LPS receptor protein Tlr4 using inhibitory oligonucleotide sequences such as an antisense oligonucleotide or a dsRNA.
  • inhibitory oligonucleotide sequences such as an antisense oligonucleotide or a dsRNA.
  • Antisense oligonucleotides can suppress the expression of particular genes in a sequence-dependent manner both in vitro and in vivo. Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of the mRNA.
  • mRNA to be interfered include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA. Use of antisense oligonucleotides is well known in the art. For example US
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391:806-811, 1998). The corresponding process in plants is commonly referred to as post- transcriptional gene silencing or RNA silencing.
  • the process of post-transcriptional gene silencing is thought to be an evolutionary conserved cellular defense mechanism for controlling the expression of alien genes in filamentous fungi, plants, and animals (Hannon GJ. Nature. 2002 418:244-251). It is caused by sequence-specific mRNA degradation, and is mediated by dsRNA homologous in sequence to the target RNA.
  • dsRNA is often a byproduct of viral replication or is formed by aberrant transcription from genetic elements after random integration in the host genome. dsRNA is processed to duplexes of 21-nt small interfering RNAs (siRNAs), which guide sequence-specific degradation of the homologous mRNA.
  • siRNAs 21-nt small interfering RNAs
  • the RNAi pathway can be used by the organism to inhibit viral infections, transposon jumping and to regulate the expression of endogenous genes.
  • RNA As used herein, the terms RNA, RNA molecules and RNA fragments are used interchangeably to refer to RNA that mediates RNA interference. These terms include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as "antisense”.
  • “Targeting” an oligonucleotide to a chosen nucleic acid target is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or a gene transcript) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent.
  • the targets are nucleic acids encoding Tlr4; in other words, a gene encoding Tlr4, or mRNA expressed from the Tlr4 gene. mRNA which encodes Tlr4 is presently the preferred target.
  • the targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.
  • messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5'-untranslated region, the 3 '-untranslated region, the 5' cap region and intron/exon junction ribonucleotides.
  • oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides.
  • the oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5' cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5'- or 3 '-untranslated region.
  • the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon,” the “start codon” or the “AUG start codon.”
  • a minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5*-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo.
  • translation initiation codon and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions.
  • start codon and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Tlr4, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
  • start codon region refers to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. This region is a preferred target region.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. This region is a preferred target region.
  • Other preferred target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.
  • 5'UTR 5' untranslated region
  • 3'UTR 3' untranslated region
  • the 5' cap of an mRNA comprises an N7 -methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5' ⁇ 5' triphosphate linkage.
  • the 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap.
  • the 5' cap region may also be a preferred target region.
  • mRNA splice sites i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets.
  • Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.
  • Hybridization in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
  • Specifically hybridizable and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.
  • an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non- target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • siRNA duplexes As 21 -nucleotide siRNA duplexes were found to be highly selective and sequence-specific inhibitors of endogenous genes, the present invention encompasses siRNA sequences comprising at least 15 nucleotides, more preferably at least 20 nucleotides.
  • the present invention therefore, encompasses double stranded RNA (dsRNA), and more particularly small interfering RNA (siRNA), which are known to mediate sequence specific mRNA degradation.
  • dsRNA double stranded RNA
  • siRNA small interfering RNA
  • Identification of desirable dsRNA sequences to inhibit the human tlr A or murine gene is done by using various algorithm programs. BLAST search and FastA sequence alignment computer programs are used in combination and sequences are chosen that do not display a high degree of homology to other genes in the genome.
  • the present invention provides siRNA sequences that are highly specific to the tlrA gene and will not cross-react with other murine or human genes. These sequences typically share 100% homology to the target gene or mRNA and show low identity of at least four mismatched nucleotides corresponding to other genes in the murine and human genome. Nevertheless it will be appreciated that 100% sequence identity between the RNA and the target gene is not required to practice the present invention. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • modulation means either inhibition or stimulation; i.e., either a decrease or increase in expression.
  • This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, or reverse transcriptase PCR, as taught in the examples of the instant application or by Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects of antisense oligonucleotides of the present invention on Tlr4 expression can also be determined as taught in the examples of the instant application. Inhibition is presently a preferred form of modulation.
  • dsRNA double-stranded ribonucleic acid
  • the double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands.
  • RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may also be useful for specific applications.
  • Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
  • Other alternative nucleic acid molecules that are capable of attenuating gene expression include but are not limited to triple-stranded nucleic acids, enzymatic nucleic acid molecules and aptamers.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of polynucleotide sequences that encode these RNAs, it is possible to engineer molecules that recognize specific polynucleotide sequences associated with production of a mutated proto-oncogene or tumor suppressor gene in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because ribozymes are sequence-specific, only target mRNAs with particular mutant sequences are inactivated.
  • RNA containing a nucleotide sequences identical to a portion of the target gene are preferred for inhibition.
  • RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70°C. hybridization for 12-16 hours; followed by washing).
  • the length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.
  • 100% sequence identity between the inhibitory oligonucleotide and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • RNA may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
  • a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
  • Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
  • RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • RNA may be chemically or enzymatically synthesized by manual or automated reactions.
  • the RNA may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
  • a cellular RNA polymerase or a bacteriophage RNA polymerase e.g., T3, T7, SP6.
  • T3, T7, SP6 bacteriophage RNA polymerase
  • the RNA may be purified prior to introduction into the cell.
  • RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
  • the RNA may be used with no or a minimum of purification to avoid losses due to sample processing.
  • the RNA may be dried for storage or dissolved in an aqueous solution.
  • the solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
  • RNA may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism in a solution containing the RNA.
  • Methods for oral introduction include direct mixing of the RNA with food of the organism, as well as engineered approaches in which a species that is used as food is engineered to express the RNA, then fed to the organism to be affected.
  • Physical methods of introducing nucleic acids for example, injection directly into the cell or extracellular injection into the organism, may also be used.
  • Vascular or extravascular circulation, the blood or lymph system, the phloem, the roots, and the cerebrospinal fluid are sites where the RNA may be introduced.
  • the cell may be a eukaryotic cell.
  • the cell is of animal origin and can be a stem cell or somatic cells.
  • Suitable animal cells can be of, for example, mammalian and avian origin. Examples of mammalian cells include human, bovine, ovine, porcine, murine, rabbit cells.
  • the cell may be an embryonic cell, bone marrow stem cell or other progenitor cell.
  • the cell can be, for example, an epithelial cell, fibroblast, smooth muscle cell, blood cell (including a hematopoietic cell, red blood cell, T-cell, B-cell, etc.), tumor cell, cardiac muscle cell, macrophage, dendritic cell, neuronal cell (e.g., a glial cell or astrocyte), or pathogen-infected cell (e.g., those infected by bacteria, viruses, virusoids, parasites, or prions).
  • an epithelial cell including a hematopoietic cell, red blood cell, T-cell, B-cell, etc.
  • tumor cell e.g., a glial cell or astrocyte
  • neuronal cell e.g., a glial cell or astrocyte
  • pathogen-infected cell e.g., those infected by bacteria, viruses, virusoids, parasites, or prions.
  • siRNA translated from a vector forms a stem and loop structure that is recognized inside the cell by a protein complex.
  • the elements required in the vector include first and foremost an appropriate promoter for translation of the DNA to RNA in the selected system.
  • Most vectors currently employ the RNA polymerase III promoters U6 or HI since RNA polymerase III initiates synthesis at a defined distance from these promoters and terminates when a string of 4-5 uridines is encountered.
  • RNAi activation in vivo can be produced by using a vector which expresses an siRNA under control of inducible or tissue-specific promoters thereby allowing directed and controlled knockout of genes in tissues of interest and/or at a controlled time (Lois C, et al., Science. 295:868-872, 2002). Active short interfering RNAs can also be produced under the control of the tRNA (Val) promoter or polymerase II (pol II) promoter.
  • RNAi expression vector refers to a replicable nucleic acid constructs used to express (transcribe) RNA which produces siRNA moieties in the cell in which the construct is expressed.
  • Such vectors include a transcriptional unit comprising an assembly of (1) genetic element(s) having a regulatory role in gene expression, for example, promoters, operators, or enhancers, operatively linked to (2) a "coding" sequence which is transcribed to produce a double-stranded RNA (two RNA moieties that anneal in the cell to form an siRNA, or a single hairpin RNA which can be processed to an siRNA), and (3) appropriate transcription initiation and termination sequences.
  • promoter and other regulatory elements generally varies according to the intended host cell.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector.
  • regulatory elements controlling transcription can be generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.
  • tissue specific means a promoter which is not restricted in activity to a single tissue type but which nevertheless shows selectivity in that they may be active in one group of tissues and less active or silent in another group.
  • the expression level of an oligonucleotide sequence of interest under the control of a particular promoter may be modulated by manipulating the promoter region.
  • different domains within a promoter region may possess different gene regulatory activities.
  • the roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (that is, deletion analysis). This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity.
  • these promoters may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector.
  • promoter fragments may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the 5' end of the primers.
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopaline synthase
  • OCS octopine synthase
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • RNAi vectors that may include the above described components exist publicly and commercially.
  • examples for in vitro RNAi vector-based system include pSuper
  • the siRNA directed against the TLR4 target sequences is inserted into a Lentiviral vector.
  • Lentivirus vectors are part of a larger group of retro viral vectors.
  • a detailed list of lentiviruses may be found in Coffin et al ("Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Narmus pp 758-763).
  • lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • HBV human immunodeficiency virus
  • AIDS causative agent of human auto-immunodeficiency syndrome
  • SIV simian immunodeficiency virus
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Lentiviruses have key advantages over other gene delivery systems.
  • a lentiviral or lentivirus vector is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the term "derivable" is used in its normal sense as meaning the sequence need not necessarily be obtained from a retrovirus but instead could be derived therefrom. By way of example, the sequence may be prepared synthetically or by use of recombinant D ⁇ A techniques.
  • Lentiviruses infect multiple cell types and efficiently infect non-dividing cells.
  • transgenic animals can be generated by lentiviral infection of embryonic stem cells or embryos (Lois C et al., Science. 295:868-72, 2002).
  • lentiviral vectors enable very stable long-term expression of the gene of interest.
  • introducing nucleic acids include injection of a solution containing the R ⁇ A, bombardment by particles covered by the R ⁇ A, soaking the cell or organism in a solution of the R ⁇ A, or electroporation of cell membranes in the presence of the R ⁇ A.
  • a viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of R ⁇ A encoded by the expression construct.
  • Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like.
  • the RNA may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or other- wise increase inhibition of the target gene.
  • the oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding Tlr4, sandwich, colorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotides with the Tlr4 gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of Tlr4 may also be prepared.
  • the present invention is also suitable for diagnosing abnormal inflammatory states in tissue or other samples from patients suspected of having an inflammatory disease associated with hsp ⁇ O.
  • the ability of the oligonucleotides of the present invention to inhibit inflammatory processes may be employed to diagnose such states.
  • a number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition.
  • to "contact" tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.
  • oligonucleotides of this invention may also be used for research purposes.
  • the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases. In addition, the oligonucleotide sequences may contain one or more nucleotide substitution which does not render the efficiency of hybridization to the target sequence.
  • the antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). Most preferably are antisense oligonucleotides comprising from about 15 to about 25 nucleobases.
  • a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • oligonucleotides containing modified backbones or non-natural intemucleoside linkages include those that retain a phosphoms atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphoms atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified oligonucleotide backbones that do not include a phosphoms atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. (Science, 1991, 254, 1497- 1500).
  • Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 O-N(CH 3 ) CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -O-N(CH 3 )- CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as -O-P-O-CH 2 -] of the above referenced U.S.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted CI to CIO alkyl or C2 to CIO alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2' position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2 -MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • Other preferred modifications include 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH NH 2 ) and 2'-fluoro (2'-F).
  • oligonucleotide Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 289-302. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett.
  • a thioether e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533- 538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J.
  • a phospholipid e.g., di-hexadecyl-rac- glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.
  • the present invention also includes oligonucleotides which are chimeric oligonucleotides.
  • "Chimeric" oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.
  • chimeric oligonucleotides include but are not limited to "gapmers," in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap.
  • a preferred example of a gapmer is an oligonucleotide in which a central portion (the "gap") of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2'- deoxynucleotides, while the flanking portions (the 5' and 3' "wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., fluoro- or 2'-O-methoxyethyl-substituted).
  • nuclease activity e.g., fluoro- or 2'-O-methoxyethyl-substituted.
  • Chimeric oligonucleotides are not limited to those with modifications on the sugar, but may also include oligonucleosides or oligonucleotides with modified backbones, e.g., with regions of phosphorothioate and phosphodiester backbone linkages.
  • Other chimeras include "wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions.
  • the 5' portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2'-deoxynucleotides, whereas the 3' portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2'-fluoro- or 2'-O-methoxyethyl-substituted), or vice- versa.
  • the oligonucleotides of the present invention contain a 2'-O- methoxyethyl (2'-O-CH 2 CH 2 OCH) 3 modification on the sugar moiety of at least one nucleotide.
  • nucleotide subunits of the oligonucleotides of the invention may bear a 2'-O-methoxyethyl (-O-CH 2 CH 2 OCH 3 ) modification.
  • Oligonucleotides comprising a plurality of nucleotide subunits having a 2'-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides.
  • oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.
  • oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routine experimenter. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2'- alkoxy or 2'-alkoxyalkoxy derivatives, including 2'-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504).
  • CPG controlled-pore glass
  • the antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrags. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrags of such nucleic acids.
  • “Pharmaceutically acceptable salts” are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).
  • examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p- toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like
  • the oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a "prodrug" form.
  • prodrug indicates 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.
  • prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510.
  • oligonucleotides are administered in accordance with this invention.
  • Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide.
  • a pharmaceutical composition may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide.
  • Such compositions and formulations are comprehended by the present invention.
  • compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non- surfactants (Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems 1991, 8, 91- 192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33).
  • One or more penetration enhancers from one or more of these broad categories may be included.
  • fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.
  • bile salt includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • Complex formulations comprising one or more penetration enhancers may be used.
  • bile salts may be used in combination with fatty acids to make complex formulations.
  • Chelating agents include, but are not limited to, disodium ethylenediammetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drag Carrier Systems 1990, 7, 1-33; Buur et al, J. Control Rel. 1990, 14, 43- 51). Chelating agents have the added advantage of also serving as DNase inhibitors.
  • EDTA disodium ethylenediammetetraacetate
  • citric acid e.g., sodium salicylate, 5- methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen laureth-9 and N- amino acyl derivatives of beta-diketones (enamines)
  • Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al, J. Pharm. Phamacol. 1988, 40, 252-257).
  • Non-surfactants include, for example, unsaturated cyclic ureas, 1 -alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).
  • carrier compound refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • carrier compound typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
  • the pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. No
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents
  • additional materials useful in physically formulating various dosage forms of the compositions of present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.
  • colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type.
  • Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure.
  • a preferred colloidal dispersion system is a plurality of liposomes.
  • Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration.
  • Oligonucleotides with at least one 2'-O- methoxyethyl modification are believed to be particularly useful for oral administration.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen.
  • treatment regimen is meant to encompass therapeutic, palliative and prophylactic modalities.
  • a patient may be treated with conventional chemotherapeutic agents such as those used for tumor and cancer treatment. When used with the compounds of the invention, such chemotherapeutic agents may be used individually, sequentially, or in combination with one or more other such chemotherapeutic agents.
  • compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drag accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on the EC 50 found to be effective during in vitro and in vivo animal models.
  • dosage is from 0.01 ⁇ g to 100 mg per kg of body weight, and may be given once or more daily, or at longer intervals. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 mg per kg of body weight, once or more daily, or at longer intervals if appropriate .
  • therapeutically effective amount is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drag treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state.
  • ODN47 5'-TAGGACCGTAAAAGACGTTG-3' (corresponding to bp 24-4 of the Tlr4 mRNA);
  • ODN48 5'-GTGTACAGTAAACAAGTTGT-3' (bp 1840-1821); ODN49 5'-GTTACCGATGTGGTCCTTAT-3' (bp 2170-2151);
  • ODN50 5'-AGTTATGAGAAGATTTCGTT-3' (bp 2870-2851).
  • the oligonucleotides were synthesized with 3 phosphorothioate linkages on each end to enhance stability. Desalting was carried out withNAP-25 columns (Pharmacia). dsRNA oligonucleotides
  • oligonucleotides Four 21 nucleotides of single strands oligonucleotides are designed against different regions of the murine tlr4 gene. These sequences are shown as single stranded stretches, however, for siRNA double stranded RNA oligonucleotides, are created by hybridization of a complementary oligonucleotides, leaving a 2 nucleotide overhang on the
  • siRNA expression vectors for generating the siRNA expression vectors pSilencer (Sui G et al., Proc Natl Acad Sci 99(8):5515-5520, 2002) or pSuper (Brummelkamp TR et al. Science 296:550-553, 2002) are preferably used.
  • mice Six-week old female E ⁇ NOD mice were divided into 5 groups. These mice are bred in a clean facility at the Weizmann Institute. 75 micrograms of each oligonucleotide in 200 ⁇ l dH 2 O was administered intravenously (i.v.) at -42 and -20 hours prior to challenge with LPS. Control animals received 200 ul of dH 2 O. The mice were challenged i.v. with 1 mg LPS E.coli 0127:B8 (Sigma, Israel). Mice were bled from the retroorbital venous plexus 90 minutes after LPS challenge and the sera were tested for murine TNF ⁇ using a sandwich ELISA (Endogen) according to manufacturer instructions. Percent survival of the mice was recorded at 48 hrs (see Figure 4).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions et des procédés pour inhiber l'expression de gènes du récepteur de type Toll 4. On utilise de préférence des oligonucléotides antisens et des oligonucléotides d'ARN double brin dirigés contre des acides nucléiques codant le récepteur de type Toll 4. L'invention concerne également des procédés d'utilisation de ces oligonucléotides pour inhiber l'expression du récepteur de type Toll 4, et pour empêcher ainsi la signalisation des ligands du récepteur de type Toll 4 et pour traiter des maladies telles que le choc septique, les maladies inflammatoires et auto-immunes associées aux ligands du récepteur du type Toll 4.
PCT/IL2004/000349 2003-04-24 2004-04-25 Oligonucleotides bloquant les recepteurs de type toll WO2004093778A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IL155561 2003-04-24
IL155561A IL155561A0 (en) 2003-04-24 2003-04-24 Oligonucleotides that block toll-like receptors

Publications (3)

Publication Number Publication Date
WO2004093778A2 true WO2004093778A2 (fr) 2004-11-04
WO2004093778A8 WO2004093778A8 (fr) 2005-05-19
WO2004093778A3 WO2004093778A3 (fr) 2007-05-03

Family

ID=33307093

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2004/000349 WO2004093778A2 (fr) 2003-04-24 2004-04-25 Oligonucleotides bloquant les recepteurs de type toll

Country Status (2)

Country Link
IL (1) IL155561A0 (fr)
WO (1) WO2004093778A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052192A1 (fr) * 2003-11-21 2005-06-09 University Of Massachusetts Pancreatite
EP1755670A2 (fr) * 2004-05-28 2007-02-28 Cedars-Sinai Medical Center Methodes pour traiter des maladies vasculaires
US7629135B2 (en) 2006-12-22 2009-12-08 The Board Of Trustees Of The University Of Illinois Toll-like receptor agonists and antagonists and methods of use thereof
US8546324B2 (en) 2008-09-22 2013-10-01 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US9487778B2 (en) 2011-03-03 2016-11-08 Quark Pharmaceuticals, Inc. Oligonucleotide modulators of the toll-like receptor pathway
US9512196B2 (en) 2008-09-22 2016-12-06 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US9796979B2 (en) 2011-03-03 2017-10-24 Quark Pharmaceuticals Inc. Oligonucleotide modulators of the toll-like receptor pathway

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077204A1 (fr) * 1999-06-10 2000-12-21 University Of Iowa Research Foundation Acide nucleique tlr4 variant et ses utilisations
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
WO2000077204A1 (fr) * 1999-06-10 2000-12-21 University Of Iowa Research Foundation Acide nucleique tlr4 variant et ses utilisations

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052192A1 (fr) * 2003-11-21 2005-06-09 University Of Massachusetts Pancreatite
US8231861B2 (en) 2003-11-21 2012-07-31 University Of Massachusetts Pancreatitis
EP1755670A2 (fr) * 2004-05-28 2007-02-28 Cedars-Sinai Medical Center Methodes pour traiter des maladies vasculaires
EP1755670A4 (fr) * 2004-05-28 2008-07-02 Cedars Sinai Medical Center Methodes pour traiter des maladies vasculaires
US7959918B2 (en) 2004-05-28 2011-06-14 Cedars-Sinai Medical Center Methods for treating vascular disease
US7629135B2 (en) 2006-12-22 2009-12-08 The Board Of Trustees Of The University Of Illinois Toll-like receptor agonists and antagonists and methods of use thereof
US8546324B2 (en) 2008-09-22 2013-10-01 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US9512196B2 (en) 2008-09-22 2016-12-06 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US9487778B2 (en) 2011-03-03 2016-11-08 Quark Pharmaceuticals, Inc. Oligonucleotide modulators of the toll-like receptor pathway
US9796979B2 (en) 2011-03-03 2017-10-24 Quark Pharmaceuticals Inc. Oligonucleotide modulators of the toll-like receptor pathway

Also Published As

Publication number Publication date
WO2004093778A3 (fr) 2007-05-03
IL155561A0 (en) 2006-12-31
WO2004093778A8 (fr) 2005-05-19

Similar Documents

Publication Publication Date Title
JP6704883B2 (ja) サーチュイン１（ｓｉｒｔ１）に対する天然アンチセンス転写物の抑制によるｓｉｒｔ１関連疾患の治療
US20220195439A1 (en) Treatment of glial cell derived neurotrophic factor (gdnf) related diseases by inhibition of natural antisense transcript to gdnf
JP6894230B2 (ja) 脳由来神経栄養因子（ｂｄｎｆ）関連疾病の、ｂｄｎｆに対する天然アンチセンス転写物の抑制による治療
EP1619249B2 (fr) Induction du "exon-skipping" dans des céllules eukaryotes
US7638497B2 (en) Antisense IAP nucleobase oligomers and uses thereof
EP1699924B1 (fr) Composes inhibiteurs de la connexine 43 et leurs methodes d'utilisation dans le traitement des traumatismes occulaires de la cornée
JP6025567B2 (ja) 膜結合転写因子ペプチダーゼ、部位１（ｍｂｔｐｓ１）に対する天然アンチセンス転写物の阻害によるｍｂｔｐｓ１関連性疾患の治療
KR101886452B1 (ko) 디스크 라지 호모로그(dlg)에 대한 천연 안티센스 전사체의 저해에 의한 dlg 관련된 질환의 치료
KR20140033165A (ko) 프라탁신 (fxn)에 대한 자연 안티센스 전사체의 저해에 의한 프라탁신 (fxn) 관련된 질환의 치료
US20050164209A1 (en) Hepatocyte free uptake assays
US20050153918A1 (en) Methods and compositions relating to hnRNP A1, A1B, A2, and B1 nucleic acid molecules
US20060194750A1 (en) Methods and materials for modulating trpc4
WO2004093778A2 (fr) Oligonucleotides bloquant les recepteurs de type toll
US7393950B2 (en) Antisense oligonucleotides targeted to human CDC45
WO2003057847A2 (fr) Procedes et substances de modulation de l'enac-beta
US8318922B2 (en) Treatment and prevention of hyperproliferative conditions in humans and antisense oligonucleotide inhibition of human replication-initiation proteins
WO2011112516A1 (fr) Traitement et prévention de l'infection par le virus de l'hépatite c en utilisant des oligonucléotides antisens de la kinase c-raf
US11208653B2 (en) Increasing RNAi efficiency through single nucleotide mismatches
US20050267050A1 (en) Compositions and methods for treating mdma-induced toxicity
WO2001046473A1 (fr) Traitement de mycobacterium tuberculosis avec des polynucleotides antisens
US20040110698A1 (en) Oligonucleotides and methods using same for treating cox-ll associated diseases
US20080119423A1 (en) Methods and Materials For Modulating p2x2
US20080027015A1 (en) Methods and Materials for Modulating Enac-Beta
US20060246432A1 (en) Methods and materials for modulating task-3
WO2006039792A1 (fr) Methode de traitement de maladies auto-immunes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: IN PCT GAZETTE 45/2004 UNDER (72, 75) THE ADRESS OF "COHEN, IRUN, R." SHOULD READ "11 HANKIN STREET, 76354 REHOVOT (IL)"

122 Ep: pct application non-entry in european phase