WO1995000528A1 - Inhibition of basic fibroblast growth factor with oligonculeotides - Google Patents

Inhibition of basic fibroblast growth factor with oligonculeotides Download PDF

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Publication number
WO1995000528A1
WO1995000528A1 PCT/US1994/006884 US9406884W WO9500528A1 WO 1995000528 A1 WO1995000528 A1 WO 1995000528A1 US 9406884 W US9406884 W US 9406884W WO 9500528 A1 WO9500528 A1 WO 9500528A1
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Prior art keywords
seq
bfgf
oligonucleotide
nucleic acid
bases
Prior art date
Application number
PCT/US1994/006884
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French (fr)
Inventor
Bruce A. Beutel
Michael E. Joesten
Original Assignee
Pharmagenics, Inc.
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Publication date
Application filed by Pharmagenics, Inc. filed Critical Pharmagenics, Inc.
Priority to AU71121/94A priority Critical patent/AU7112194A/en
Publication of WO1995000528A1 publication Critical patent/WO1995000528A1/en

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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

  • the present invention provides identified
  • oligonucleotides for binding with and thereby modulating, inhibiting or enhancing, the activity or function of basic fibroblast growth factor.
  • the oligonucleotides are those which are capable of binding with reasonable affinity at a site which modulates the activity of the target molecule.
  • the principal aspect of the invention provides nucleic acid sequences effective to modulate the activity of basic fibroblast growth factor (bFGF) upon binding therewith.
  • the nucleic acid sequences so identified are not previously known to modulate bFGF activity and are preferably modified or unmodified nucleic acid sequences.
  • a preferred aspect of the present invention provides for oligonucleotides (unmodified or modified) which include at least one of SEQ ID NOS: 1 to 22 and 27 to 53,
  • Nucleic acid sequences are referred to using standard IUPAC abbreviations to specify individual positions of the oligomer and base positions at which more than one of the specific nucleotide bases may be
  • Oligonucleotides or polynucleotides are hereafter sometimes collectively referred to as
  • nucleic acid(s) The nucleic acid can include flanking sequences on at least one of the 3' and 5' end.
  • oligonucleotides (modified or unmodified) effective to inhibit bFGF are those that bind with the target bFGF at a K d of not more than about 40 nM, as determined by methods similar to those described in Riggs et al., J. Mol. Biol. Vol 48, pg. 67-83 (1970).
  • the oligonucleotides of the invention may be in the form of a single strand, a double strand, a stem-loop structure, a bubble structure, a pseudoknot, or a closed, circular structure.
  • the ends of the oligonucleotide may be bridged by non-nucleotide moieties.
  • Example 1 illustrates the inhibition of the function of bFGF in accordance with the invention.
  • RNA sequences found to bind to bFGF are identified by SEQ ID NOS. 1 to 22 and 27 to 53.
  • Figure 1 shows the binding of representative RNA sequences having particularly high affinity for bFGF. Binding of RNA to bFGF was carried out in 50 mM Tris HCl (pH 7.5), 150 mM NaCl and 3 mM MgCl. Arrangement of a subset of these sequences that illustrates the shared common features of the tight-binding oligomers is shown in Table 1.
  • Figure 2 shows two conserved structural motifs for the sequences indicated in Tables 1 and 2.
  • Another subset of sequences binding to basic fibroblast growth factor (for example SEQ ID NO: 34) contains the sequence CAUCAGCG presented in the form of a stem-loop structure with the sequence CAUCAGCG.
  • AUCAGC present as the unpaired loop and the C- - - - - - G forming the Watson-Crick base pair closing the loop and beginning a base-paired helical nucleic acid stem.
  • bFGF Basic fibroblast growth factor
  • RNA and bFGF were respectively, 100 nM, 2 nM and 10 nM, there was no effect on the amount of RNA bound to bFGF when receptor was present as compared to the receptor-minus control.
  • the proportions were altered such that the receptor concentration was increased to 1.75 ⁇ M and the amount of RNA inhibitor was decreased to 0.5 nM.
  • Example 1 with 32 P-labelled RNA.
  • the results demonstrate a decrease in the amount of labeled RNA bound to bFGF (which was still at a concentration of 10 nM) of 69%. Heparin at high concentration also was effective in competing with the bound RNA. These results indicate that receptor and heparin bind competitively with the RNA and because the concentration of receptor required to compete with the oligonucleotide is considerably higher than its K d for bFGF, we conclude that the RNA can block the
  • the invention provides a method of inhibiting bFGF function.
  • the method comprises contacting bFGF with an effective amount of an oligonucleotide, or a molecule containing such an oligonucleotide, which inhibits bFGF function.
  • inhibiting bFGF function means that the oligonucleotide prevents bFGF from performing its functions, preferably by binding to bFGF.
  • the methods of use of the nucleic acid sequences of the invention include inhibition of the activity of bFGF as a circulating growth factor.
  • bFGF binds to its cognate receptor, the bFGF receptor.
  • nucleic acid sequences serve as neovascularization
  • the oligonucleotides are administered to a host, such as a human or non-human animal host, in an amount effective to inhibits bFGF function.
  • the oligonucleotides may be used prophylactically or therapeutically.
  • the oligonucleotides are administered 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.
  • the nucleic acid sequences may be administered in vitro or ex vivo as well as in vivo.
  • oligonucleotide as used herein means that the oligonucleotide may be a ribonucleotide; i.e., an RNA oligonucleotide; a deoxyribonucleotide; i.e., a DNA
  • ribonucleotide/deoxyribonucleotide i.e., the
  • oligonucleotide may include ribose or deoxyribose sugars, 2'-O-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.
  • 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,
  • the invention is not to be limited to any specific phosphorus moiety or moieties.
  • the phosphorus moiety may be modified with a cationic, anionic, or zwitterionic moiety.
  • the oligonucleotides may also contain backbone linkages which do not contain phosphorus, such as carbonates, carboxymethyl esters, acetamidates, carbamates, acetals, and the like.
  • the oligonucleotides may also contain the backbone linkage of peptide nucleic acids. (Egholm, et al., J. Am. Chem. Soc.. Vol. 114, pgs. 1895-1897 (1992)).
  • the oligonucleotides of the invention also include any natural or unnatural, substituted or unsubstituted, 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 isocytosine, 6-methyluracil, 4,6-dihydroxypyrimidine, hypoxanthine, xanthine, 2,6-diaminopurine, 5-azacytosine, 5-methyl cystosine, 7-deazaadenine, 7-deaza-guanine, and the like.
  • 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)
  • hydroxypropylmethacrylamide (hydroxypropylmethacrylamide); (i) polyethylene imine; (j) dextrans; (k) polymaleic anhydride; (l) cyclodextrins; (m) starches; (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 and viral particles.
  • 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.
  • substituted nucleotide units may alternate with unsubstituted nucleotide units.
  • another 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 oligonucleo
  • all of the nucleotide units are substituted 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 positions.
  • the moiety When the moiety is 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.
  • the oligonucleotide In yet another embodiment, the oligonucleotide
  • non-oligonucleotide components such as, for example, peptides or proteins, or simple carbohydrates, and lipids.
  • the oligonucleotides of the present invention may be in the form of a single strand, a double strand, a stemloop structure, a bubble structure, a pseudoknot, or a closed, circular structure.
  • the ends of the oligonucleotide may be bridged by non-nucleotide moieties.
  • non-nucleotide bridging moieties include, but are not limited to, those having the following structural formula:
  • R is selected from the group consisting of (a) saturated and unsaturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamines; (f) polyalkylene thioglycols; (g)
  • non-nucleotide bridging moiety may be a polyalkylene glycol such as polyethylene glycol.
  • one or more of the non-nucleotide moieties R may be substituted for one or more of the nucleotide units in the target protein binding
  • 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 synthesized on an automated nucleic acid synthesizer.
  • the oligonucleotides may be synthesized enzymatically through the use of flanking or primer
  • 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 synthesis.
  • the oligonucleotides of the present invention may be administered in conjunction with an acceptable
  • Such pharmaceutical compositions may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Such oligonucleotides may be
  • the preparations particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration parenterally or orally, and compositions which can be administered buccally or
  • sublingually including inclusion 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.
  • preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes.
  • the process to be used will depend ultimately on the physical properties of the active ingredient used.
  • Suitable excipients are, in particular, fillers such as 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
  • disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • 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
  • Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices.
  • suitable coatings which, if desired, may be resistant to gastric juices.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used.
  • Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as 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.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons,
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting 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 suspension.
  • the hydrophobic layer generally but not exclusively, comprises
  • phospholipids such as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • the diameters of the liposomes generally range from about 15 nm to about 5 microns.
  • oligonucleotides of the present invention may also be employed as diagnostic probes for determining the presence of thrombin, and thereby determining the need for modulation of its function or activity. In such
  • a modified or unmodified oligonucleotide of the present invention is added to a sample suspected of containing the bFGF.
  • the oligonucleotide may be labeled with a detectable marker such as a radioactive label, a chromogen, or an enzyme label.
  • a detectable marker such as a radioactive label, a chromogen, or an enzyme label.
  • the oligonucleotide may be employed in a variety of assay methods for the detection of bFGF, such methods including sandwich assays, competitive assays, ELISA assays, inhibition assays, and other assays known to those skilled in the art.
  • Oligonucleotides of the present invention which are RNA or DNA may also be administered to a host as part of a gene therapy procedure.
  • an expression vector which includes or encodes a nucleic acid containing an RNA or DNA sequence of the present invention may be
  • the expression vector may include a DNA or RNA sequence which, when transcribed, produces an RNA which binds the bFGF in such a way as to inhibit its function.
  • expression vectors which may be employed include, but are not limited to, prokaryotic vectors, eukaryotic vectors, and viral vectors, such as retroviral vectors.
  • prokaryotic vectors such as prokaryotic vectors
  • eukaryotic vectors such as retroviral vectors.
  • retroviral vectors include those derived from Moloney Murine Leukemia Virus, Rous Sarcoma Virus, and Harvey Sarcoma Virus.
  • bFGF function Upon reception of the bFGF-binding oligonucleotide or an expression vector containing a binding sequence by the bFGF-containing cell, bFGF function will be inhibited.
  • oligonucleotide or an expression vector containing a binding sequence may be administered to a cell in order to prevent the deleterious consequences of overproduction or to effect the benefits of inhibiting the bFGF function.
  • the oligonucleotide or an RNA or DNA encoding a sequence containing the oligonucleotide may be
  • RNA or DNA can be administered to a cell stimulated to proliferate by bFGF in order to prevent such cells from dividing at an abnormally high rate.
  • Administration of such DNA or RNA can be by any of a number of procedures known to those skilled in the art, including, but not limited to, transfection,
  • eukaryotic expression vectors such as viruses, including retroviruses, Herpes viruses,
  • adenoviruses and adeno-associated viruses.
  • Heterologous or autologous transplants of such cells may be administered to patients by procedures known to those skilled in the art.
  • ADDRESSEE Carella, Byrne, Bain,
  • GGGAGAAGUA GUGUAGGAAU UCUACAGGAG GACAACITUGA GAGGUGGGUA AGCGGCGCCG 60 UAUCAGCACG GGAUGUGGCU UAACGCGAAU UCAGGCCUGG100
  • GGGAGAAGUA GUGUAGGAAU UCAGGGAUGG CACGUCCAGA CCGUCUGGCG CAGCUCAGGG 60 CCUGACGUUG UAGCAGGCGG CUUAACGCGA AUUCAGGCCU GG102

Abstract

Oligonucleotides and analogs thereof that bind to and modulate the activity of basic fibroblast growth factor and methods for their use, including therapeutic and diagnostic methods.

Description

INHIBITION OF BASIC FIBROBLAST GROWTH FACTOR
WITH OLIGONUCLEOTIDES
The present invention provides identified
oligonucleotides for binding with and thereby modulating, inhibiting or enhancing, the activity or function of basic fibroblast growth factor. The oligonucleotides are those which are capable of binding with reasonable affinity at a site which modulates the activity of the target molecule.
The principal aspect of the invention provides nucleic acid sequences effective to modulate the activity of basic fibroblast growth factor (bFGF) upon binding therewith. The nucleic acid sequences so identified are not previously known to modulate bFGF activity and are preferably modified or unmodified nucleic acid sequences.
A preferred aspect of the present invention provides for oligonucleotides (unmodified or modified) which include at least one of SEQ ID NOS: 1 to 22 and 27 to 53,
particularly those which contain at least one of the following: GUGC(SEQ ID NO: 23); CUGC(SEQ ID NO: 24);
AURWA(SEQ ID NO:25); AUACC(SEQ ID NO: 26) and CAUCAGCG (SEQ ID NO: 54). Nucleic acid sequences are referred to using standard IUPAC abbreviations to specify individual positions of the oligomer and base positions at which more than one of the specific nucleotide bases may be
alternatively used. Oligonucleotides or polynucleotides, are hereafter sometimes collectively referred to as
"nucleic acid(s)". The nucleic acid can include flanking sequences on at least one of the 3' and 5' end.
In a preferred embodiment, oligonucleotides (modified or unmodified) effective to inhibit bFGF are those that bind with the target bFGF at a Kd of not more than about 40 nM, as determined by methods similar to those described in Riggs et al., J. Mol. Biol. Vol 48, pg. 67-83 (1970).
The oligonucleotides of the invention may be in the form of a single strand, a double strand, a stem-loop structure, a bubble structure, a pseudoknot, or a closed, circular structure. In one embodiment, the ends of the oligonucleotide may be bridged by non-nucleotide moieties.
The following example illustrates the inhibition of the function of bFGF in accordance with the invention. Example 1
Oligomer Binding to bFGF
The RNA sequences found to bind to bFGF are identified by SEQ ID NOS. 1 to 22 and 27 to 53. Figure 1 shows the binding of representative RNA sequences having particularly high affinity for bFGF. Binding of RNA to bFGF was carried out in 50 mM Tris HCl (pH 7.5), 150 mM NaCl and 3 mM MgCl. Arrangement of a subset of these sequences that illustrates the shared common features of the tight-binding oligomers is shown in Table 1.
Figure imgf000005_0001
Figure imgf000006_0001
Figure 2 shows two conserved structural motifs for the sequences indicated in Tables 1 and 2. Another subset of sequences binding to basic fibroblast growth factor (for example SEQ ID NO: 34) contains the sequence CAUCAGCG presented in the form of a stem-loop structure with the
AUCAGC present as the unpaired loop and the C- - - - - - G forming the Watson-Crick base pair closing the loop and beginning a base-paired helical nucleic acid stem.
Example 2
Basic fibroblast growth factor (bFGF) binds to high affinity receptor and low affinity heparin-like molecules on the cell surface. Competition experiments were carried out with a representative RNA oligonucleotide (SEQ. ID NO: 12) to demonstrate that the individual oligomers bind to bFGF in competition with its receptor and heparin. The receptor used was the extracellular region of bFGF receptor (recombinant, human origin) obtained from Austral
Biologicals. When the concentrations of bFGF receptor, RNA and bFGF were respectively, 100 nM, 2 nM and 10 nM, there was no effect on the amount of RNA bound to bFGF when receptor was present as compared to the receptor-minus control. The proportions were altered such that the receptor concentration was increased to 1.75 μM and the amount of RNA inhibitor was decreased to 0.5 nM. These experiments were performed in the buffer described in
Example 1 with 32P-labelled RNA. The results (Figure 3) demonstrate a decrease in the amount of labeled RNA bound to bFGF (which was still at a concentration of 10 nM) of 69%. Heparin at high concentration also was effective in competing with the bound RNA. These results indicate that receptor and heparin bind competitively with the RNA and because the concentration of receptor required to compete with the oligonucleotide is considerably higher than its Kd for bFGF, we conclude that the RNA can block the
interaction between bFGF and its receptor or heparin with a very low Ki.
In another aspect the invention provides a method of inhibiting bFGF function. The method comprises contacting bFGF with an effective amount of an oligonucleotide, or a molecule containing such an oligonucleotide, which inhibits bFGF function.
The term "inhibiting bFGF function", as used herein, means that the oligonucleotide prevents bFGF from performing its functions, preferably by binding to bFGF.
In their several aspects the methods of use of the nucleic acid sequences of the invention include inhibition of the activity of bFGF as a circulating growth factor. bFGF binds to its cognate receptor, the bFGF receptor.
Among its various activities bFGF stimulates
vascularization and neovascularization through endothelial cell growth which can be inhibited by the method of the invention.
Animal studies have suggested that tumors are severely limited in their growth in the absence of a new blood supply. Thus, as another aspect of the invention the nucleic acid sequences serve as neovascularization
inhibitors that are very useful in suppressing growth of solid cancers. Furthermore, curtailment of vascularization of already-formed tumors envisions reduction of the
likelihood of metastasis.
Such molecules clearly have therapeutic relevance and can also be used as diagnostic reagents. The oligonucleotides are administered to a host, such as a human or non-human animal host, in an amount effective to inhibits bFGF function. Thus, the oligonucleotides may be used prophylactically or therapeutically. Preferably, the oligonucleotides are administered 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 also contemplated that the nucleic acid sequences may be administered in vitro or ex vivo as well as in vivo.
The term "oligonucleotide" as used herein means that the oligonucleotide may be a ribonucleotide; i.e., an RNA oligonucleotide; a deoxyribonucleotide; i.e., a DNA
oligonucleotide; or a mixed
ribonucleotide/deoxyribonucleotide; i.e., the
oligonucleotide may include ribose or deoxyribose sugars, 2'-O-methyl ribose or other 2'-substituted or conjugated sugars, or a mixture of such sugars. Alternatively, 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,
phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphorothionate, phosphorothiolate, phosphoramidothiolate, and phosphorimidate. It is to be understood, however, that the scope of the present
invention is not to be limited to any specific phosphorus moiety or moieties. Also, the phosphorus moiety may be modified with a cationic, anionic, or zwitterionic moiety. The oligonucleotides may also contain backbone linkages which do not contain phosphorus, such as carbonates, carboxymethyl esters, acetamidates, carbamates, acetals, and the like. The oligonucleotides may also contain the backbone linkage of peptide nucleic acids. (Egholm, et al., J. Am. Chem. Soc.. Vol. 114, pgs. 1895-1897 (1992)).
The oligonucleotides of the invention also include any natural or unnatural, substituted or unsubstituted, purine or pyrimidine base. Such 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 isocytosine, 6-methyluracil, 4,6-dihydroxypyrimidine, hypoxanthine, xanthine, 2,6-diaminopurine, 5-azacytosine, 5-methyl cystosine, 7-deazaadenine, 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. Such 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) dextrans; (k) polymaleic anhydride; (l) cyclodextrins; (m) starches; (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 and viral particles. 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. In one embodiment, substituted nucleotide units may alternate with unsubstituted nucleotide units. In another
embodiment, all of the nucleotide units are substituted 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. When 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. When the moiety is attached to adenine, it may be attached at the C2, N6, or C8 positions. When the moiety is attached to guanine, it may be attached at the N2 or C8 positions. When the moiety is attached to cytosine, it may be attached at the C5 or N4 positions. When the moiety is attached to thymine or uracil, it may be attached at the C5 position.
In one embodiment, 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, peptides or proteins, or simple carbohydrates, and lipids.
The oligonucleotides of the present invention may be in the form of a single strand, a double strand, a stemloop structure, a bubble structure, a pseudoknot, or a closed, circular structure. In one embodiment, the ends of the oligonucleotide may be bridged by non-nucleotide moieties. Examples of non-nucleotide bridging moieties include, but are not limited to, those having the following structural formula:
T1-R-T2, whereas each of T1 and T2 independently is attached to a nucleotide phosphate moiety or a hydroxyl moiety. R is selected from the group consisting of (a) saturated and unsaturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamines; (f) polyalkylene thioglycols; (g)
polyamides; (h) disubstituted monocyclic or polycyclic aromatic hydrocarbons; (i) intercalating agents; (j) monosaccharides; and (k) oligosaccharides; or mixtures thereof. In one embodiment, the non-nucleotide bridging moiety may be a polyalkylene glycol such as polyethylene glycol.
In another embodiment, one or more of the non-nucleotide moieties R may be substituted for one or more of the nucleotide units in the target protein binding
sequences, 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. For example, the oligonucleotides may be synthesized on an automated nucleic acid synthesizer. Alternatively, the oligonucleotides may be synthesized enzymatically through the use of flanking or primer
sequences at the 5' and 3' ends. In another alternative, 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 synthesis. The oligonucleotides of the present invention may be administered in conjunction with an acceptable
pharmaceutical carrier as a pharmaceutical composition.
Such pharmaceutical compositions may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Such oligonucleotides may be
administered by intramuscular, intraperitoneal,
intravenous, or subdermal injection in a suitable solution. Preferably, the preparations, particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration parenterally or orally, and compositions which can be administered buccally or
sublingually, including inclusion 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. For example, the pharmaceutical
preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. The process to be used will depend ultimately on the physical properties of the active ingredient used.
Suitable excipients are, in particular, fillers such as 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
carboxypropylmethylcellulose, sodium
carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-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 cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used.
Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons,
polyethylene glycols, or higher alkanols. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin
hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition,
suspensions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
Optionally, the suspension may also contain stabilizers.
Additionally, the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting 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 suspension. The hydrophobic layer, generally but not exclusively, comprises
phospholipids such as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns.
The oligonucleotides of the present invention may also be employed as diagnostic probes for determining the presence of thrombin, and thereby determining the need for modulation of its function or activity. In such
embodiments, a modified or unmodified oligonucleotide of the present invention is added to a sample suspected of containing the bFGF. The oligonucleotide may be labeled with a detectable marker such as a radioactive label, a chromogen, or an enzyme label. Thus, the oligonucleotide may be employed in a variety of assay methods for the detection of bFGF, such methods including sandwich assays, competitive assays, ELISA assays, inhibition assays, and other assays known to those skilled in the art.
Oligonucleotides of the present invention which are RNA or DNA may also be administered to a host as part of a gene therapy procedure. For example, an expression vector which includes or encodes a nucleic acid containing an RNA or DNA sequence of the present invention may be
administered to cells of an individual in need of
inhibition of the function of the bFGF. In one embodiment, such includes DNA or RNA sequence has from about 5 to 100 or more nucleotide units, preferably from about 8 to about 60 nucleotide units. In one embodiment, the expression vector may include a DNA or RNA sequence which, when transcribed, produces an RNA which binds the bFGF in such a way as to inhibit its function. Examples of expression vectors which may be employed include, but are not limited to, prokaryotic vectors, eukaryotic vectors, and viral vectors, such as retroviral vectors. Herpes virus vectors, adenoviral vectors, and adeno-associated viral vectors. Examples of retroviral vectors include those derived from Moloney Murine Leukemia Virus, Rous Sarcoma Virus, and Harvey Sarcoma Virus.
Upon reception of the bFGF-binding oligonucleotide or an expression vector containing a binding sequence by the bFGF-containing cell, bFGF function will be inhibited.
In another embodiment, the bFGF-binding
oligonucleotide or an expression vector containing a binding sequence may be administered to a cell in order to prevent the deleterious consequences of overproduction or to effect the benefits of inhibiting the bFGF function. For example, the oligonucleotide or an RNA or DNA encoding a sequence containing the oligonucleotide may be
administered to a cell stimulated to proliferate by bFGF in order to prevent such cells from dividing at an abnormally high rate. Administration of such DNA or RNA can be by any of a number of procedures known to those skilled in the art, including, but not limited to, transfection,
electroporation, lipofection, transformation, or
transduction with eukaryotic expression vectors such as viruses, including retroviruses, Herpes viruses,
adenoviruses, and adeno-associated viruses. Heterologous or autologous transplants of such cells may be administered to patients by procedures known to those skilled in the art.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be
practiced other than as particularly described and still be within the scope of the accompanying claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT(S) : Beutel, Bruce A.
Joesten, Michael E.
(ii) TITLE OF INVENTION: Inhibition of Basic
Fibroblast
Growth Factor with Oligonucleotides
(iii) NUMBER OF SEQUENCES: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Carella, Byrne, Bain,
Gilfillan, Cecchi, Stewart
& Olstein
(B) STREET: 6 Becker Farm Road
(C) CITY: Roseland
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07068
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette
(B) COMPUTER: IBM
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WordPerfect 5.1
(vi) CURRENT APPLICATION DATA
(A) APPLICATION NUMBER: Unassigned
(B) FILING DATE: Unassigned
(C) CLASSIFICATION: Unassigned
(Viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Herron, Charles J. (B) REGISTRATION NUMBER: 28,019
(C) REFERENCE/DOCKET NUMBER: 23550-114
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
SEQU ENCE DESCRIPTION: SEQ ID NO: 1
GGGAGAAGUA GUGUAGGAAU UCAUUUCCAA AUUGAACCUC CUCCGCCUGU 50 GUGCGAACCC UUAUGAAGGU UCAUGUAGCA GUCUCGAGAG GUCACAGU 98
(3) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
GGGAGAAGUA GUGUAGGAAU UCUAAUAGCG UCCGUCAAAC ACAAGCAAGG 50 CACCAGCCGG UGAGUCCCGG CACUUGUGUU UCCUCGAGAG GUCACAGU 98
(4) INFORMATION FOR SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: BASES
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 GGGAGAAGUA GUGUAGGAAU UCUUGGCCCG CUGUGCGCUA UUUGAAGUUA 50 GCAUGCCCGA UGGUAUCCUG AUUCCUGACC UCUCGAGAGG UCACAGU 97
(5) INFORMATION FOR SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
GGGAGAAGUA GUGUAGGAAU UCUUGGUGAC AUACAUUUAG CUGGGUUCAU 50 GAACUUCGUU GUGAUUUUAG CGGAGGUGCG AACUCGAGAG GUCACAGU 98
(6) INFORMATION FOR SEQ ID NO: 5
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
GGGAGAAGUA GUGUAGGAAU UCCGCAUUGA UGUCCAAAUA CGUAUGGCUC 50 UCAUCUUAGU UAACUGUUAU CGAUGGUCCC CACUCGAGAG GUCACAGU 98
(7) INFORMATION FOR SEQ ID NO: 6
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
GGGAGAAGUA GUGUAGGAAU UCCUCGUGCG CUGCCUGGAU GGGCACGAUG 50 UAGGGGAAUC UGUCAUCUCU CGGGUCGCUC CCCUCGAGAG GUCACAGU 98
(8) INFORMATION FOR SEQ ID NO: 7
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 65 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7
GGGAGAAGUA GUGUAGGAAU UCUAAGUGAA CGCCCAGUUC CAUGUUCACU 50 ACGUUGGGAG GAUCC 65
(9) INFORMATION FOR SEQ ID NO: 8
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 GGGAGAAGUA GUGUAGGAAU UCAGCAUGCG UGCGCAGUUG AUCACUGCAU 50 GUAGUGUGUU GACCUACAGU GAGUACAGAG CCCUCGAGAG GUCACAGU 98
(10) INFORMATION FOR SEQ ID NO: 9
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID ( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 9 GGGAGAAGUA GUGUAGGAAU UCGUGAGUGU GCGUCUCAAA ACAUAUAGCU 50 UAUUUAAAUU GGUUGCUUAC ACGGCUGGCU CACUCGAGAG GUCACAGU 98
(11) INFORMATION FOR SEQ ID NO: 10
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 96 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
GGGAGAAGUA GUGUAGGAAU UCGGGUGUGC GUGGCAGCAA AACUGUCCAC 50 AUAAACUCGA ACCGUUUUUA UCGAUGGUCA CUCGAGAGGU CACAGU 96
(12) INFORMATION FOR SEQ ID NO: 11
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 97 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11
GGGAGAAGUA GUGUAGGAAU UCUUCGCGAA UGCUUAGUGC GCCCCACUUU 50 AAAAAGUGGG ACAUGAAUAG GCUCUAAAUG ACUCGAGAGG UCACAGU 97
(13) INFORMATION FOR SEQ ID NO: 12
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 96 BASES ( B ) TYPE : NUCLE IC ACID
( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 12 GGGAGAAGUA GUGUAGGAAU UCUAGUCGUG CGUGGGUGUU GACGCCCCAC 50 AUGUAGGCGG GAGUUGGACC UGUGGAGCUG CUCGAGAGGU CACAGU 96
(14) INFORMATION FOR SEQ ID NO: 13
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 99 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
GGGAGAAGUA GUGUAGGAAU UCGUGCAUAA AGACGGGCAU UUCCAGCGGC 50 CUGUCGUGCG CACGGCCGAA ACUCUCCAAG CCUCUCGAGA GGUCACAGU 99
(15) INFORMATION FOR SEQ ID NO: 14
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 94 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 GGGAGAAGUA GUGUAGGAAU UCCACAUGUA GGGCCGAGGG GGAGCCUAGC 50 UACGGCUUGU GCGUGGGAUU CCGUGGACCU CGAGAGGUCA CAGU 94
(16) INFORMATION FOR SEQ ID NO: 15
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 97 BASES ( B ) TYPE : NUCLEIC ACID
( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 GGGAGAAGUA GUGUAGGAAU UCCACCACAU ACCUAGCGCA CACGUUACUG 50 CGUGGUACAC ACUACGACAG CUGAGAUUAC GCUCGAGAGG UCACAGU 97
(17) INFORMATION FOR SEQ ID NO: 16
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16
GGGAGAAGUA GUGUAGGAAU UCCGGUCGUU UAUGUGGUGA GCGGGCUGCG 50 UGUGUGAUAG GACAUAUCGC CACAUACCCU CGCUCGAGAG GUCACAGU 98
(18) INFORMATION FOR SEQ ID NO: 17
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17
GGGAGAAGUA GUGUAGGAAU UCCGGACCAG AUGCGGCACU AAACCAGGAU 50 ACCGGGUGCC GUACCUCCUC UAUUCCUCUG CCCUCGAGAG GUCACAGU 98
(19) INFORMATION FOR SEQ ID NO: 18
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES (B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 18
GGGAGAAGUA GUGUAGGAAU UCCGUGCGCG AGAGCAGUCU CGCAUGUAGG 50 UAUGUUAGAA AGCCCACUUC GCUUGGUAUC CUCUCGAGAG GUCACAGU 98
(20) INFORMATION FOR SEQ ID NO: 19
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 19
GGGAGAAGUA GUGUAGGAAU UCCUGCUCUU GAAUGUACAA GGUGCCCGAA 50 UUCUAGUCCU UGCCGUUCAG UUCCGCCGUA UUCUCGAGAG GUCACAGU 98
(21) INFORMATION FOR SEQ ID NO: 20
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 98 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS SINGLE
(D) TOPOLOGY: LINEAR
( iii ) HHYYPPOOTTHHEETTIICCAALL: NO
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 20 GGGAGAAGUA GUGUAGGAAU UCAUAAAACC CCACAUACCC AGCUUAGAGC 50 UGCUGCGUGG AGUUUGUCUU AAGAUGUGUU GUCUCGAGAG GUCACAGU 98
(22) INFORMATION FOR SEQ ID NO: 21
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 97 BASES ( B ) TYPE : NUCLE IC ACID
( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 21
GGGAGAAGUA GUGUAGGAAU UCCGUGGGGC CACCCGUGCG UGGAACGAUC 50 CAUCUCCACA UAAAGGGCGC CCUAUGAUGG CCUCGAGAGG UCACAGU 97
(23) INFORMATION FOR SEQ ID NO: 22
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22
GGGAGAAGUA GUGUAGGAAU UCGUUGGAGC GCCGGAGAGU CCCGGCAUCA 50 UUGACUUGUU CAGGCUCUGU AUGCUUAGUU UGCUCGAGAG GUCACAGU 98
(24) INFORMATION FOR SEQ ID NO: 23
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 4 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23
GUGC 4
(25) INFORMATION FOR SEQ ID NO: 24
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 4 BASES
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24
CUGC 4
( 26 ) INFORMATION FOR SEQ ID NO: 25
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 5 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25
AURWA 5
(27) INFORMATION FOR SEQ ID NO: 26
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 5 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26
AUACC 5
(28) INFORMATION FOR SEQ ID NO: 27
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27
GGGAGAAGUA GUGUAGGAAU UCCAAGCAGA ACAGUCUGUU CCAAUGGGCU AGACUCCGCG 60 CGCUGGAGUG AGUAUGGUUG AAUUAACGCG AAUUCAGGCC UGG103
(29) INFORMATION FOR SEQ ID NO: 28
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28
GGGAGAAGUA GUGUAGGAAU UCGGGGGGGU ACAAUGUGAG CUGCAUAACA GGCCGCAGUC 60 CUCUGCGCAG UCAGCACACU UAACGCGAAU UCAGGCCUGGIOO
(30) INFORMATION FOR SEQ ID NO: 29
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY: LINEAR
( iii) HYPOTHETICAL: NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 29
GGGAGAAGUA GUGUAGGAAU UCCGUUUAUG UGGGUCUAGG UCAGAACCAU CAGCGGGGCG 60 AGCGUAGGUA GGUCGAAGAU CUUAACGCGA AUUCAGGCCU GG 102
( 31 ) INFORMATION FOR SEQ ID NO : 30
( i ) SEQUENCE CHARACTERISTICS
(A) LENGTH : 101 BASES
( B ) TYPE : NUCLEIC ACID
( C ) STRANDEDNESS : SINGLE (D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30
GGGAGAAGUA GUGUAGGAAU UCGCAGCGUG GGGGCCGUGU AUCGCAUCGU GCGGGCAUUA 60 UCACCGGGGG AGGCUCGCCG UUAACGCGAA UUCAGGCCUG G101
(32) INFORMATION FOR SEQ ID NO: 31
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31
GGGAGAAGUA GUGUAGGAAU UCACAUGAAA CGGCGUUCGG UUGUCUGCGU GACGUACACU 60 ACCUACCGUC UGCACUGUUC AUUUAACGCG AAUUCAGGCC UGG103
(33) INFORMATION FOR SEQ ID NO: 32
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 101 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32
GGGAGAAGUA GUGUAGGAAU UCGGCUGUAC UCAGUCGGAG CGGGCGGCAC GAUCAUCAAG 60 GAUAAUCUGA UUUAAUUCGA UUAACGCGAA UUCAGGCCUG G101
(34) INFORMATION FOR SEQ ID NO: 33
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 GGGAGAAGUA GUGUAGGAAU UCAGACUCCG UGUGGGGCGC CUACUCACAU CUCGAAAUGU 60 UGUCGAAGGC CUUGCAACAG CUUAACGCGA AUUCAGGCCU GG102
(35) INFORMATION FOR SEQ ID NO: 34
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 101 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34
GGGAGAAGUA GUGUAGGAAU UCACUAUCCA CGACGAAAUG UAAUCGGCCA CAUCAGCGUG 60 GUCGCUUUGU UAGGCGUGUG UUAACGCGAA UUCAGGCCUG G101
(36) INFORMATION FOR SEQ ID NO: 35
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
( D ) TOPOLOGY : L INEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35
GGGAGAAGUA GUGUAGGAAU UCCCCACUCU AAACACCAAU GGUCCACACG GUCAUAACCA 60 GUUCCGCGAC UGCUCCACAU UAACGCGAAU UCAGGCCUGG100
(37) INFORMATION FOR SEQ ID NO: 36
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID ( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 36 GGGAGAAGUA GUGUAGGAAU UCAGCCCCGG ACAUAAAGUG AAAUCAUUGG ACACGUUAGU 60 CAUGAAAACU CUCUGCGUCC AUUAACGCGA AUUCAGGCCU GG102
(38) INFORMATION FOR SEQ ID NO: 37
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 101 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37
GGGAGAAGUA GUGUAGGAAU UCCGGAUGAC AGAUCCGAUG CACCAUUGGA UCGCAUCGCA 60 GGUGGUGCAA UGCCGUUCGU UUAACGCGAA UUCAGGCCUG G101
(39) INFORMATION FOR SEQ ID NO: 38
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38
GGGAGAAGUA GUGUAGGAAU UCUGCGGCAG UGAGGUGUAG UAUAAGGCGU GUGAGUUCAG 60 AAUAGUGCGG CCGAGCGUGG CAUUAACGCG AAUUCAGGCC UGG103
(40) INFORMATION FOR SEQ ID NO: 39
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39
GGGAGAAGUA GUGUAGGAAU UCAUCAGCGA AUUUGUGAGA UGACUUAGCA AGAAGCGGGU 60 AUGUGUGUGU GGCUAGGUCU GUUAACGCGA AUUCAGGCCU GG102
(41) INFORMATION FOR SEQ ID NO: 40
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40
GGGAGAAGUA GUGUAGGAAU UCGUGGGGUG GGUGCGCUGC GACUGCUGCU GGCAUAAACC 60 GCUCUCUAAA CACUCAGUGU UAACGCGAAU UCAGGCCUGG100
(42) INFORMATION FOR SEQ ID NO: 41
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41
GGGAGAAGUA GUGUAGGAAU UCUACAGGAG GACAACITUGA GAGGUGGGUA AGCGGCGCCG 60 UAUCAGCACG GGAUGUGGCU UAACGCGAAU UCAGGCCUGG100
(43) INFORMATION FOR SEQ ID NO: 42
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42
GGGAGAAGUA GUGUAGGAAU UCAGGCGCCC GGGUACACAG GAUGCGACGU UCAUAGGAAC 60
CUAAGUCUCC GCUUAGGGUG CAUUAACGCG AAUUCAGGCC UGG103
(44) INFORMATION FOR SEQ ID NO: 43
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43
GGGAGAAGUA GUGUAGGAAU UCCCAGAUAU CGAAGCGCUG UGCUUUGGGU GAACAUGAAG 60 UGGUGAUAUA UACCGACGUG CGUUAACGCG AAUUCAGGCC UGG103
(45) INFORMATION FOR SEQ ID NO: 44
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44
GGGAGAAGUA GUGUAGGAAU UCCCGGAUAC UCAGGGGGGG UUCGUAUGAU AUCAUCAGCG 60 GUGGCCAUAG AGCCAAUUCU CCUUAACGCG AAUUCAGGCC UGG103
(46) INFORMATION FOR SEQ ID NO: 45
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 99 BASES
(B) TYPE: NUCLEIC ACID ( C ) STRANDEDNESS : S INGLE
( D ) TOPOLOGY : LINEAR
( iii ) HYPOTHETICAL : NO
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 45
GGGAGAAGUA GUGUAGGAAU UCGUGCGCCA UGUACGCUAC AUAAGUCUUA GCGGUGCGCA 60 AAGCGCAGUG AGAGAUCAUU AACGCGAAUU CAGGCCUGG 99
(47) INFORMATION FOR SEQ ID NO: 46
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 99 BASES
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46
GGGAGAAGUA GUGUAGGAAU UCGGCAGGGG AUGUUGAAAG UACCGUACCC AUCAGCGGGU 60 GUGGCAGUGA UGGAAUUCUU AACGCGAAUU CAGGCCUGG 99
(48) INFORMATION FOR SEQ ID NO: 47
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47
GGGAGAAGUA GUGUAGGAAU UCGGACACCC CUACUGGCCA GCGGUUGUUA AUGCUUUCUG 60 GGCAGAUGAG UACCAUGGGU UAACGCGAAU UCAGGCCUGG100
(49) INFORMATION FOR SEQ ID NO: 48
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48
GGGAGAAGUA GUGUAGGAAU UCAGGGAUGG CACGUCCAGA CCGUCUGGCG CAGCUCAGGG 60 CCUGACGUUG UAGCAGGCGG CUUAACGCGA AUUCAGGCCU GG102
(50) INFORMATION FOR SEQ ID NO: 49
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 102 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS SINGLE
(D) TOPOLOGY: LINEAR
(iii) HHYYPPOOTTHHEETTIICCAALL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49
GGGAGAAGUA GUGUAGGAAU UCACCCGAUU UCAGCGGCUC AUGCACGUUA GCCCAAGGUU 60 GUAGCAUCAG CGCGGCAUCC UUUAACGCGA AUUCAGGCCU GG102
(51) INFORMATION FOR SEQ ID NO: 50
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50
GGGAGAAGUA GUGUAGGAAU UCCGGACUGA CUCGAGGUGU UGAUGGUUAU AUACUGCGCA 60 UUCAUCGUGG GUGCAAUUGU UAACGCGAAU UCAGGCCUGG100
(52) INFORMATION FOR SEQ ID NO: 51
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 101 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY : LINEAR (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51
GGGAGAAGUA GUGUAGGAAU UCCAUCAUGU UGUCGUGGGG UGUGCGGUUA GACCAUAUAG 60
CCCCGGGUAC UGCUAUGUGC UUAACGCGAA UUCAGGCCUG G101
(53) INFORMATION FOR SEQ ID NO: 52
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 103 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52
GGGAGAAGUA GUGUAGGAAU UCCCAGAGUU GUAUAGGCGG CUAGGUUACG AAAGUUCAAA 60 AUAGUGGCUU UUGUCGGGUC CAUUAACGCG AAUUCAGGCC UGG103
(54) INFORMATION FOR SEQ ID NO: 53
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 100 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53
GGGAGAAGUA GUGUAGGAAU UCAUAGCUGU CGUCGAUCCG UGUUGCUUCU GAGGUGAUGU 60 UUAUGUGAUU UGUCCNGCCU UAACGCGAAU UCAGGCCUGG100
(55) INFORMATION FOR SEQ ID NO: 54
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 8 BASES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY: LINEAR
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54 CAUCAGCG 8

Claims

What Is Claimed Is:
1. An oligonucleotide that modulates the activity of bFGF upon binding therewith.
2. The oligonucleotide of claim 1 that binds with bFGF at a Kd of not greater than about 40nM.
3. The oligonucleotide of claim 1 containing at least one sequence selected from the group consisting of SEQ ID NOS: 23 to 26 and 54.
4. The oligonucleotide of claim 3 wherein at least one such sequence is in a bubble or stem-loop structure.
5. The oligonucleotide of claim 1 containing at least one sequence selected from the group consisting of SEQ ID NOS: 1 to 22 and 27 to 53.
6. A method of inhibiting bFGF function which comprises contacting bFGF with an inhibitory amount of an oligonucleotide which inhibits bFGF function.
7. The method of claim 6 which comprises inhibiting bFGF function in an individual in need thereof by administering to the individual a bFGF-inhibitory amount of an oligonucleotide which inhibits bFGF function.
8. A method of inhibiting bFGF function which comprises contacting bFGF with an inhibitory amount of an oligonucleotide of claim 2.
9. A method of inhibiting bFGF function which comprises contacting bFGF with an inhibitory amount of an oligonucleotide of claim 3.
10. A method of inhibiting bFGF function which comprises contacting bFGF with an inhibitory amount of an oligonucleotide of claim 4.
11. A method of inhibiting bFGF function which comprises contacting bFGF with an inhibitory amount of an oligonucleotide of claim 5.
12. A vector which includes or encodes an oligonucleotide of claim 1.
13. A vector which includes or encodes an oligonucleotide of claim 2.
14. A vector which includes or encodes an oligonucleotide of claim 3.
15. A vector which includes or encodes an oligonucleotide of claim 4.
16. A vector which includes or encodes an oligonucleotide of claim 5.
PCT/US1994/006884 1993-06-18 1994-06-17 Inhibition of basic fibroblast growth factor with oligonculeotides WO1995000528A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71121/94A AU7112194A (en) 1993-06-18 1994-06-17 Inhibition of basic fibroblast growth factor with oligonculeotides

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7967793A 1993-06-18 1993-06-18
US08/079,677 1993-06-18
US17949194A 1994-01-07 1994-01-07
US08/179,491 1994-01-07

Publications (1)

Publication Number Publication Date
WO1995000528A1 true WO1995000528A1 (en) 1995-01-05

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Country Status (2)

Country Link
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WO (1) WO1995000528A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996021471A1 (en) * 1995-01-10 1996-07-18 Genta Incorporated Compositions and methods for treating tumor cells
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6465188B1 (en) 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 164, No. 2, Issued 31 October 1989, T.M. REILLY et al., "Monoclonal Antibodies Directed Against Basic Fibroblast Growth Factor Which Inhibit Its Biological Activity In Vitro and In Vivo", pages 736-743. *
CANCER RESEARCH, Volume 51, Issued 15 November 1991, A. HORI et al., "Suppression of Solid Tumor Growth by Immunoneutralizing Monoclonal Antibody Against Human Basic Fibroblast Growth Factor", pages 6180-6184. *
FASEB JOURNAL, Volume 7, Issued January 1993, J.M. BURKE et al., "In Vitro Selection and Evolution of RNA: Applications for Catalytic RNA, Molecular Recognition and Drug Discovery", pages 106-112. *
J. SAMBROOK et al., "Molecular Cloning, A Laboratory Manual", Published 1989, by COLD SPRING HARBOR LABORATORY PRESS (NEW YORK), page 1.53. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 24, Issued 25 August 1993, K. PADMANABHAN et al., "The Structure of alpha-Thrombin Inhibited by a 15-Mer Single-Stranded DNA Aptamer", pages 17651-17654. *
NATURE, Volume 355, Issued 06 February 1992, L.C. BOCK et al., "Selection of Single-Stranded DNA Molecules That Bind and Inhibit Human Thrombin", pages 564-566. *
NUCLEIC ACIDS RESEARCH, Volume 21, No. 18, Issued 1993, A. CRAMERI et al., "1020-Fold Aptamer Library Amplification Without Gel Purification", page 4410. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 90, Issued December 1993, D. JELLINEK et al., "High-Affinity RNA Ligands to Basic Fibroblast Growth Factor Inhibit Receptor Binding", pages 11227-11231. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6465188B1 (en) 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
WO1996021471A1 (en) * 1995-01-10 1996-07-18 Genta Incorporated Compositions and methods for treating tumor cells
US5783683A (en) * 1995-01-10 1998-07-21 Genta Inc. Antisense oligonucleotides which reduce expression of the FGFRI gene
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes

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