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WO1995000529A1 - INHIBITION DE L'INTERFERON η AVEC DES OLIGONUCLEOTIDES - Google Patents

INHIBITION DE L'INTERFERON η AVEC DES OLIGONUCLEOTIDES Download PDF

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Publication number
WO1995000529A1
WO1995000529A1 PCT/US1994/006897 US9406897W WO9500529A1 WO 1995000529 A1 WO1995000529 A1 WO 1995000529A1 US 9406897 W US9406897 W US 9406897W WO 9500529 A1 WO9500529 A1 WO 9500529A1
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Prior art keywords
ifn
seq
oligonucleotide
oligonucleotides
nucleic acid
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PCT/US1994/006897
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English (en)
Inventor
George R. Coppola
Bruce A. Beutel
Arthur H. Bertelsen
Original Assignee
Pharmagenics, Inc.
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
Priority claimed from US08/210,222 external-priority patent/US5599917A/en
Application filed by Pharmagenics, Inc. filed Critical Pharmagenics, Inc.
Priority to AU71750/94A priority Critical patent/AU7175094A/en
Publication of WO1995000529A1 publication Critical patent/WO1995000529A1/fr

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    • 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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • C12N2310/13—Decoys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • Interferon- ⁇ is a multifunctional cytokine produced by, and secreted from, both T-lymphocytes and NK cells.
  • the multiple forms of the cytokine all have an N- terminal pyroglutamic acid residue and up to two N-linked carbohydrates.
  • the largest mature form of the cytokine consists of 143 a ino acids but the carboxyl end of the protein is apparently heterogeneous due to post-translational proteolysis (Rinderkneckt et al., 1984, J. Biol. Chem. Vol. 259 p.6790).
  • IFN-7 exhibits antiviral activity as well as numerous immunomodulatory and anti-proliferative effects. Its effects on the immune system include influencing B-cell immunoglobulin class switching (Snapper et al., 1992, J. Exp. Med., 175:1367), upregulating class I and class II MHC antigen expression (Buckmeier and Schreiber, 1985, Proc. Natl. Acad. Sci. USA, 82:404; Bancroft et al., 1992, Immunol., 143:127), stimulating conversion of Thl to Th2 lymphocytes (Kanagawa et al., 1993, Science, 252:240), increasing macrophage-mediated killing of intracellular parasites (Portnoy, 1992, Curr.
  • IFN-7 interleukin-7
  • These various activities of IFN-7 are mediated by its binding to a specific cell-surface receptor protein found on a range of different cell types (Aguet et al., 1988, Cell, 55:273; Valente et al., 1992, Eur. J. Immunol., 22:2403).
  • IFN-7 immunocompetence in HIV infected individuals.
  • Immune system over-stimulation by IFN-7 has been implicated as a mediator of autoimmune reactions, as a contributing factor in the damage caused by septic shock and as contributing to the decline of immunocompetence in HIV infected individuals.
  • Neutralizing antibodies directed against IFN-7 have been investigated as therapeutics in a number of disease model systems (Jacob et al., 1987, J. Exp. Med., 166:798; U land et al.,1992, Clin. Immun. Immunopath., 63:66).
  • this invention provides compounds that can bind to, and block the activity of, IFN-7.
  • certain oligonucleotides bind tightly to IFN-7 and block interaction with its specific receptor protein thereby neutralizing cytokine activity.
  • the inhibitory oligonucleotides contain discrete sequences that impart specific interaction with IFN- 7. Since the oligonucleotides of the instant invention bind tightly and specifically with IFN-7 it is a further object of the invention to provide diagnostic reagents and methods for detecting the presence of IFN-7 in test samples.
  • Elisa assay of IFN-7 interaction with plate-bound IFN-7 receptor determining the inhibition of the interaction amounts of added oligonucleotides.
  • the present invention identifies oligonucleotides for binding with and preferably thereby modulating, inhibiting or enhancing, the activity or function of interferon-7.
  • the oligonucleotides are those which are capable of binding with reasonable affinity, preferably at a site which modulates the activity of the target molecule, IFN-7.
  • IFN-7 binding oligonucleotides can also be useful as diagnostic reagents for measuring levels of the cytokine.
  • Oligonucleotide compounds have been discovered that bind IFN-7 with high affinity and selectivity. Therefore, a principal aspect of the invention provides numerous oligonucleotides and portions of them that modulate, e.g. inhibit, the activity of IFN-7 upon binding therewith. Oligonucleotides or poynucleotides are hereafter sometimes collectively referred to as "nucleic acid(s)".
  • the nucleic acids so identified are not previously known to modulate IFN- 7 activity and are preferably modified or unmodified nucleic acids containing one or more discrete, short (i.e. not more that about 20 bases) sequence elements.
  • the nucleic acid can include flanking sequence at at least one of the 3' and 5' end( ⁇ ) .
  • the preferred oligonucleotides of the invention are those that bind with IFN-7 at a Kd of not more than about 30 nM. Kd is determined by the method described in Rigg ⁇ , et al., 1970, J. Mol. Biol., 48:67-83.
  • nucleic acids or analogs thereof that bind to and affect the activity of IFN-7 and are preferably selected from the group consisting of SEQ ID NOS:l to 29 including most particularly those oligonucleotides containing one or more sequences selected from the group consisting of SEQ ID NOS: 30 to 34.
  • Oligonucleotides of the present invention were effective in inhibiting IFN-7 activity when they included at least one of the following sequences: AAGUUG (SEQ ID NO:30); UGANGCUC (SEQ ID NO:31); UAAGUUGANGCUCG (SEQ ID NO:32); GCACCNC (SEQ ID NO:33); GCCACCCUCG (SEQ ID NO:34). Nucleic acid sequences are referred to using standard IUPAC abbreviation to specify the identity of the bases at individual positions of the oligomer.
  • oligonucleotides having SEQ ID Nos. 1 through 39 further contemplates other oligonucleotides that bind to IFN-7, and preferably inhibit the function thereof.
  • additional oligonucleotides can be obtained readily by one of ordinary skill in the art using a variety of methods including, but not limited to, those described in Kinzler and Vogelstein, 1989, Nucleic Acids Research. Vol. 17, pgs. 3645-3652; Oliphant, Brandl and Struhl, 1989, Molecular and Cellular Biology. Vol. 9, pgs. 2944-2949; Kinzler and Vogelstein, Molecular and Cellular Biology. Vol. 10, pgs.
  • the oligonucleotides of the invention may be in the form of a single strand, a double strand, a stem-loop, a bulged helix, a pseudoknot or a closed-circular structure.
  • the invention provides a method of inhibiting IFN-7 function.
  • the method comprises contacting IFN-7 with an effective amount of an oligonucleotide, or a molecule containing such an oligonucleotide, which inhibits IFN-7 function.
  • inhibiting IFN-7 function means that the oligonucleotide prevents IFN-7 from being active, preferably by binding to IFN-7.
  • IFN-7 is a multifunctional cytokine produced by both T lymphocytes and NK cells. The effects of IFN-7 are mediated through binding to a specific transmembrane receptor, with the resultant activation and intracellular translocation of at least two known DNA-binding proteins.
  • the methods of use of the nucleic acid sequences of the invention include inhibition of IFN-7's antiviral activity, as well as its pleiotropic immunomodulatory and cell growth inhibitory effects.
  • IFN-7 immunoregulatory effects of IFN-7 which can be modulated or inhibited are its influence on the class of antibody produced by B cells, its up-regulation of both class I and II MHC antigens, and its effect in increasing macrophage-mediated killing of intracellular parasites. All of these functions can be modulated or inhibited by the method of contacting IFN-7 with the nucleic acid sequences of the invention.
  • Another use in accordance with the invention is to inhibit the effect of IFN-7 to induce several genes, including HLA-B and HLA-DR, IP-10, PI kinase, 2,5A- synthetase, and indoleamine 2,3-dioxygenase.
  • Another aspect of the invention provides the ability to inhibit the effect of IFN-7 to promote the production of interleukin-1 (IL-1), tumor necrosis factor (TNF), platelet activating factor, H 2 0 2 , and pterin.
  • IL-1 interleukin-1
  • TNF tumor necrosis factor
  • H 2 0 2 platelet activating factor
  • pterin pterin
  • an IFN-7-inhibitory nucleic acid limits inappropriate IFN-7-stimulated inflammatory responses in septic shock or rheumatoid arthritis.
  • Quinolinic acid produced by the metabolic pathway catalyzed by 2,3-dioxygenase, damages neurons.
  • Another of the many uses of the inhibitory method of the invention is the suppression of induction of this enzyme by an IFN-7- inhibitory nucleic acid.
  • HIV-infected patients are less likely to develop AIDS if their CD4+ cells remain as Thl as opposed to Th2. Since IFN- 7 is implicated in the induction of a Thl conversion to Th2 class, the IFN-7-inhibitory nucleic acids of the invention are beneficial to HIV-infected individuals.
  • inhibiting refers to inhibiting one or more of the foregoing functions.
  • Nucleic acids that interact with proteins with high affinity are comprised of individual sequence elements that are highly conserved and other elements in which sequence is less well-conserved or even non-conserved. Both kinds of elements are important for the interaction of the nucleic acid and protein.
  • sequence-specific elements might be the region that makes specific contacts with the protein whereas the less conserved regions might serve a structural role in the presentation of the specific elements in the preferred configuration for protein binding.
  • oligonucleotide polymers may be modified at many positions to impart new properties, such as resistance to nucleases, without destroying the desired characteristics of the oligonucleotide including its interaction with a target.
  • a ribozyme oligonucleotide generally consists of ribonucleotides yet many positions of the ribozyme oligonucleotide may be substituted with deoxyribonucleotides, making it more resistant to ribonucleases, without affecting its catalytic activity on its target RNA.
  • the oligonucleotides of the present invention may be modified in a variety of ways to change certain characteristics, such as resistance to nucleases or ease of manufacture.
  • oligonucleotide as used herein means that the oligonucleotide may be a ribonucleic acid, i.e. an RNA oligonucleotide; a deoxyribonucleic acid, i.e. a DNA oligonucleotide; or a mixed ribonucleic/deoxyribonucleic acid; i.e., the oligonucleotide may include ribo ⁇ e or deoxyribose sugars, 2'-0-methyl ribose or other 2' substituted or conjugated sugars, or a mixture of such sugars.
  • the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof or any mixture of sugars.
  • the phosphorus-containing moieties of the oligonucleotides of the present invention may be modified or unmodified.
  • the phosphorus-containing moiety may be, for example, a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, alkyl-thiophosphonate, phosphora idate, phosphorodiamidate, phosphorothioate. phosphorodithioate, phosphorothionate, phosphorothiolate, phosphoramidothiolate or pho ⁇ phorimidate. It is to be understood, however, that the scope of the present invention is not to be limited to any specific phosphoru ⁇ moiety or moieties.
  • one or more pho ⁇ phoru ⁇ moietie ⁇ may be modified with a cationic, anionic, or zwitterionic moiety.
  • the oligonucleotides may also contain one or more backbone linkages which do not contain phosphorus, such as carbonates, carboxymethyl esters, acetamidates, carbamates, acetals, and the like.
  • the oligonucleotides may also contain one or more backbone linkage of peptide nucleic acids. (Eghol , et al., J. Am. Chem. Soc.. 114:1895-1897 (1992)).
  • the oligonucleotides of the invention also include any natural or unnatural, substituted or unsub ⁇ tituted, purine or pyrimidine base.
  • purine and pyrimidine bases include, but are not limited to, natural purines and pyrimidines such as adenine, cytosine, thymine, guanine, uracil, or other purines and pyrimidines, or analogs thereof, such as isocyto ⁇ ine, 6-methyluracil, 4,6-di-hydroxypyrimidine, hypoxanthine, xanthine, 2,6-diaminopurine, 5-azacytosine, 5- methyl cystosine, 7-deaza-adenine, 7-deaza-guanine, and the like.
  • the oligonucleotides of the invention may be modified such that at least one nucleotide unit of the oligonucleotides may include a conjugate group.
  • conjugate groups include, but are not limited to, (a) amino acids, including D-amino acids and L-amino acids; (b) peptides, polypeptides, and proteins; (c) dipeptide mimics; (d) sugars; (e) sugar phosphates; (f) neurotransmitters; (g) hormones; (h) poly (hydroxypropylmethacrylamide) ; (i) polyethylene imine; (j) dextran ⁇ ; (k) polymaleic anhydride; (1) cyclodextrins; (m) ⁇ tarches; (n) steroids, including sterols such as, but not limited to, cholesterol; (o) acridine; (p) vitamins; and (q) polyalkylene glycols, such as polyethylene glycol.
  • Such moieties may make the oligonucleotides more resistant to degradation in cells and in the circulation, and/or make the oligonucleotides more permeable to cells.
  • the conjugate moiety may be attached to the 3' terminal nucleotide unit and/or the 5' terminal nucleotide unit and/or to an internal nucleotide unit(s), or conjugate moieties may be attached to two or more nucleotide units at the 3' end and/or the 5' end of the oligonucleotide.
  • sub ⁇ tituted nucleotide units may alternate with unsubstituted nucleotide units.
  • all of the nucleotide units are sub ⁇ tituted with a conjugate moiety.
  • the conjugate moiety may be attached to the oligonucleotide at the purine or pyrimidine base, at the phosphate group, or to the sugar.
  • the conjugate moiety is attached to the base, it is preferably attached at certain positions of the base, depending upon the base to which the moiety is attached.
  • the moiety When the moiety is attached to adenine, it may be attached at the C2, N6, or C8 position ⁇ .
  • the moiety i ⁇ attached to guanine it may be attached at the N2 or C8 positions.
  • the moiety When the moiety is attached to cytosine, it may be attached at the C5 or N4 positions.
  • the moiety is attached to thymine or uracil, it may be attached at the C5 position.
  • the oligonucleotide includes from about 5 to about 100 nucleotide units, preferably from about 8 to about 60 nucleotide units. In yet another embodiment, the oligonucleotide represents a portion of a larger molecule which contains non- oligonucleotide components, such as, for example, peptide ⁇ or proteins, or simple carbohydrate ⁇ , and lipids.
  • the oligonucleotides of the present invention may be in the form of a single ⁇ trand, a double ⁇ trand, a stem-loop structure, a p ⁇ eudoknot, or a clo ⁇ ed, circular ⁇ tructure.
  • the end ⁇ of the oligonucleotide may be bridged by non-nucleotide moietie ⁇ .
  • Example ⁇ of non- nucleotide bridging moieties include, but are not limited- to, those having the following structural formula:
  • T,-R-T 2 where T, and T 2 are each independently attached to a nucleotide phosphate moiety or a hydroxyl moiety.
  • R is selected from the group consisting of (a) saturated and un ⁇ aturated hydrocarbon ; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbon ⁇ ; (e) polyalkylamine ⁇ ; (f) polyalkylene thioglycols; (g) polyamides; (h) di ⁇ ub ⁇ tituted monocyclic or polycyclic aromatic hydrocarbons; (i) intercalating agents; (j) monosaccharides; and (k) oligosaccharides; or mixture ⁇ thereof.
  • the non-nucleotide bridging moiety may be a polyalkylene glycol such a ⁇ polyethylene glycol.
  • one or more of the non-nucleotide moieties R may be substituted for one or more of the nucleotide unit ⁇ in the target protein binding ⁇ equences, as hereinabove mentioned.
  • the oligonucleotides of the present invention may be synthesized by a* variety of accepted means known to those skilled in the art.
  • the oligonucleotides may be synthe ⁇ ized on an automated nucleic acid ⁇ ynthe ⁇ izer.
  • the oligonucleotides may be synthe ⁇ ized enzymatically through the use of flanking or primer sequences at the 5' and 3' ends.
  • the oligonucleotides may be synthesized by solution phase chemistry. It is to be understood, however, that the scope of the present invention is not to be limited to any particular means of ⁇ ynthe ⁇ i ⁇ .
  • the oligonucleotide ⁇ of the pre ⁇ ent invention may be admini ⁇ tered in conjunction with an acceptable pharmaceutical carrier as a pharmaceutical composition.
  • Such pharmaceutical composition ⁇ may contain suitable excipients and auxiliarie ⁇ which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Such oligonucleotides may be administered by intramuscular, intraperitoneal, intraveneou ⁇ , or subdermal injection in a ⁇ uitable solution.
  • the preparations particularly those which can be admini ⁇ tered orally and which can be used for the preferred type of administration, such a ⁇ tablet ⁇ , dragee ⁇ and cap ⁇ ule ⁇ , and preparation ⁇ which can be admini ⁇ tered rectally, ⁇ uch a ⁇ ⁇ uppo ⁇ itorie ⁇ , a ⁇ well a ⁇ suitable solution ⁇ for admini ⁇ tration parenterally or orally, and compositions which can be administered buccally or sublingually, including include ⁇ ion compounds, contain from about 0.1 to 99 percent by weight of active ingredients, together with the excipient. It is also contemplated that the oligonucleotides may be administered topically.
  • the pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art.
  • the pharmaceutical preparation ⁇ may be made by mean ⁇ of conventional mixing, granulating, dragee- making, dis ⁇ olving or lyophilizing proces ⁇ es.
  • the process to be used will depend ultimately on the physical properties of the active ingredient used.
  • Suitable excipients are, in particular, fillers such a ⁇ sugar, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethyl- cellulo ⁇ e, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as a ⁇ sugar, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for example, maize starch, wheat starch, rice
  • disintegrating agents may be added, such as the above-mentioned starche ⁇ a ⁇ well a ⁇ carboxymethyl-starch, cros ⁇ -linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are flow-regulating agents and lubricants, such as, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee core ⁇ may be provided with suitable coatings which, if desired, may be resi ⁇ tant to ga ⁇ tric juice ⁇ .
  • concentrated ⁇ ugar ⁇ olution ⁇ may be u ⁇ ed, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • solution ⁇ of suitable cellulose preparations such as a ⁇ acetylcellulo ⁇ e phthalate or hydroxypropylmethylcellulo ⁇ e phthalate, are used.
  • Dyestuffs and pigment ⁇ may be added to the tablet ⁇ of dragee coatings, for example, for- identification or in order to characterize different combinations of active compound dose ⁇ .
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed cap ⁇ ules made of gelatin and a plasticizer ⁇ uch a ⁇ glycerol or sorbitol.
  • the push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with filler ⁇ ⁇ uch a ⁇ lacto ⁇ e, binder ⁇ ⁇ uch a ⁇ starches, and/or lubricants ⁇ uch a ⁇ talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • ⁇ tabilizer ⁇ may be added.
  • Po ⁇ ible pharmaceutical preparation ⁇ which can be u ⁇ ed rectally include, for example, ⁇ uppo ⁇ itorie ⁇ , which con ⁇ i ⁇ t of a combination of the active compound ⁇ with a ⁇ uppo ⁇ itory ba ⁇ e.
  • Suitable ⁇ uppo ⁇ itory bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols.
  • Possible ba ⁇ e materials include, for example, liquid triglycerides, polyethylene glycol ⁇ , or paraffin hydrocarbon ⁇ .
  • Suitable formulation ⁇ for parenteral admini ⁇ tration include aqueou ⁇ ⁇ olution ⁇ of the active compounds in water- soluble or water-di ⁇ persible form.
  • ⁇ u ⁇ pen ⁇ ions of the active compounds as appropriate oil injection suspen ⁇ ion ⁇ may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, ⁇ e ⁇ ame oil, or ⁇ ynthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspen ⁇ ions may contain sub ⁇ tances which increase the visco ⁇ ity of the ⁇ u ⁇ pen ⁇ ion including, for example, ⁇ odium carboxymethyl cellulo ⁇ e, sorbitol and/or dextran.
  • the ⁇ u ⁇ pen ⁇ ion may also contain stabilizers.
  • the compounds of the present invention may also be administered encapsulated in lipo ⁇ ome ⁇ , wherein the active ingredient i ⁇ contained either di ⁇ per ⁇ ed or variou ⁇ ly pre ⁇ ent in corpu ⁇ cle ⁇ con ⁇ i ⁇ ting of aqueous concentric layers adherent to lipidic layers.
  • the active ingredient depending upon its solubility, may be present both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic suspen ⁇ ion.
  • the hydrophobic layer generally but not exclusively, compri ⁇ e ⁇ pho ⁇ pholipid ⁇ ⁇ uch a ⁇ lecithin and sphingomycelin, steroid ⁇ such as cholesterol, ⁇ urfactant ⁇ ⁇ uch a ⁇ dicetylpho ⁇ phate, ⁇ tearylamine, or pho ⁇ phatidic acid, and/or other material ⁇ of a hydrophobic nature.
  • the diameter ⁇ of the liposomes generally range from about 15 nm to about 5 micron ⁇ .
  • the oligonucleotide ⁇ are admini ⁇ tered to a ho ⁇ t, such a ⁇ a human, in an amount effective to inhibit the IFN-7 function. Thu ⁇ , the oligonucleotide ⁇ may be used prophylactically or therapeutically. Preferably, the oligonucleotide ⁇ are admini ⁇ tered to a host so as to provide a concentration of oligonucleotide in the blood of from about 10 nanomolar to about 500 micromolar, preferably from about 5 micromolar to about 100 micromolar. It is al ⁇ o contemplated that the oligonucleotide ⁇ may be admini ⁇ tered in vitro or ex vivo as well as in vivo .
  • the oligonucleotide ⁇ of the pre ⁇ ent invention may also be employed a ⁇ diagno ⁇ tic probe ⁇ for determining the pre ⁇ ence of IFN-7, and thereby determining the need for modulation of it ⁇ function or activity.
  • a modified or unmodified oligonucleotide of the present invention is added to a sample su ⁇ pected of containing IFN-7.
  • the oligonucleotide may be labeled with a detectable marker, including but not limited to, a radioisotope, a biotin moiety, a chromophore, a fluorescent moiety, or an enzyme label.
  • the oligonucleotide may be employed in a variety of as ⁇ ay methods for the detection of IFN-7, such methods including sandwich as ⁇ ay ⁇ , competitive a ⁇ ay ⁇ , ELISA, inhibition a ⁇ says, and other a ⁇ ay ⁇ known to tho ⁇ e ⁇ killed in the art.
  • oligonucleotides were capable of binding tightly to IFN-7 they were synthe ⁇ ized on an oligonucleotide ⁇ ynthesizer to have a specified sequence.
  • the oligonucleotide either wa ⁇ ⁇ ynthesized directly a ⁇ RNA or el ⁇ e the corre ⁇ ponding DNA ⁇ equence wa ⁇ ⁇ ynthe ⁇ ized and the RNA to be tested wa ⁇ generated by in vitro transcription of the DNA using a mixture of nucleo ⁇ ide tripho ⁇ phate ⁇ and T7 RNA polymerase.
  • the synthetic oligonucleotides were 32 P-labelled either at the 5' end using 7 32 P-ATP and T4 polynucleotide kinase or internally using ( ⁇ 32 P-NTPs in the tran ⁇ cription reaction. Portion ⁇ of the labelled oligonucleotide being te ⁇ ted were incubated with variou ⁇ concentration ⁇ of IFN-7 in a binding buffer of 25 mM Tri ⁇ -HCl pH 7.5, 150 m-M NaCl, 3mM MgCl 2 for about 20 inute ⁇ at ambient temperature (ca. 23°C). The labelled oligonucleotide bound to IFN-7 protein wa ⁇ recovered by filtration of the binding reaction through a nitrocellulo ⁇ e filter.
  • Oligonucleotides inhibit binding of IFN-7 with IFN-7 receptor
  • oligonucleotide (12 or 60 pmol ⁇ ) were added to different well ⁇ in 50 ⁇ l binding buffer (25 mM Tri ⁇ -HCl pH 7.5, 150 mM NaCl, 3 mM MgCl 2 ) and incubated for about 5 minute ⁇ at room temperature (ca. 23°C) to allow for binding of the oligonucleotide and the IFN-7. After incubation, 6 pmol ⁇ of IFN-7 receptor protein were added in 50 ⁇ l binding buffer and the ⁇ olution wa ⁇ incubated for at least 120 minutes at 37°C.
  • the amount of receptor bound to IFN-7 was determined by adding an enzyme-linked antibody against the mouse kappa light chain portion of the fusion protein [1:500 dilution of antibody (Caltag Laboratories) in phosphatebuffered saline containing 1 mg/ml bovine serum albumin] for 60 minutes at 37°C followed by a chromogenic ⁇ ubstrate for the linked enzyme [H 2 0 2 /ABTS (Boehringer Mannheim)], and incubation for an additional 30 minutes at room temperature.
  • the anti-kappa chain antibody does not interfere with the binding of the IFN-7 to its receptor.
  • a second competition as ⁇ ay wa ⁇ also performed to demonstrate the ability of oligonucleotides to inhibit IFN-7 binding to its receptor.
  • wells of the microtiter plate were coated with 8 pmol ⁇ of the recombinant IFN-7 receptor fusion protein described above (in 50 ⁇ l phosphate-buffered saline for about 16 hours at 4°C).
  • FIG. 3 present ⁇ repre ⁇ entative re ⁇ ults for the second as ⁇ ay with three oligonucleotides of the instant invention (SEQ ID NOS: 2, 3, and 11) and a mixture of 98 ba ⁇ e random ⁇ equence RNA for comparison.
  • the oligonucleotides of the instant invention are more effective at inhibiting the interaction of IFN-7 and its receptor than the mixture of random-sequence RNA.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan,

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Abstract

L'invention concerne des oligonucléotides et des analogues de ceux-ci qui se lient à l'activité de l'interféron η et de préférence modulent cette activité, leurs procédés d'utilisation ainsi que des procédés thérapeutiques et de diagnostic.
PCT/US1994/006897 1993-06-18 1994-06-17 INHIBITION DE L'INTERFERON η AVEC DES OLIGONUCLEOTIDES WO1995000529A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71750/94A AU7175094A (en) 1993-06-18 1994-06-17 Inhibition of interferon-gamma with oligonucleotides

Applications Claiming Priority (4)

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US7967793A 1993-06-18 1993-06-18
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Cited By (18)

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Publication number Priority date Publication date Assignee Title
US6465188B1 (en) 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
US5972599A (en) * 1990-06-11 1999-10-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands of cytokines
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6147204A (en) * 1990-06-11 2000-11-14 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6168778B1 (en) 1990-06-11 2001-01-02 Nexstar Pharmaceuticals, Inc. Vascular endothelial growth factor (VEGF) Nucleic Acid Ligand Complexes
US6028186A (en) * 1991-06-10 2000-02-22 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands of cytokines
US5859228A (en) * 1995-05-04 1999-01-12 Nexstar Pharmaceuticals, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes
US6229002B1 (en) 1995-06-07 2001-05-08 Nexstar Pharmaceuticlas, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6582918B2 (en) 1995-06-07 2003-06-24 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
AU2004210606B2 (en) * 1995-06-07 2008-07-10 Gilead Sciences, Inc. High-affinity nucleic acid ligands of cytokines
US7879993B2 (en) 1995-06-07 2011-02-01 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
WO1998008099A1 (fr) * 1996-08-19 1998-02-26 SHAKER, Ghassan, I. Utilisation de l'expression de l'antigene cd69 dans les cellules molt4 pour determiner la presence et l'activite des inhibiteurs d'interferon
US6962784B2 (en) 1996-10-25 2005-11-08 Gilead Sciences, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6051698A (en) * 1997-06-06 2000-04-18 Janjic; Nebojsa Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6426335B1 (en) 1997-10-17 2002-07-30 Gilead Sciences, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US7939654B2 (en) 1997-12-16 2011-05-10 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US8853376B2 (en) 2002-11-21 2014-10-07 Archemix Llc Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics

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