EP0677055A1 - Molecules antisens dirigees contre une famille de genes codant un recepteur du facteur de croissance de fibroblastes - Google Patents

Molecules antisens dirigees contre une famille de genes codant un recepteur du facteur de croissance de fibroblastes

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Publication number
EP0677055A1
EP0677055A1 EP94907109A EP94907109A EP0677055A1 EP 0677055 A1 EP0677055 A1 EP 0677055A1 EP 94907109 A EP94907109 A EP 94907109A EP 94907109 A EP94907109 A EP 94907109A EP 0677055 A1 EP0677055 A1 EP 0677055A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
seq
gene
growth factor
nucleic acid
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP94907109A
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German (de)
English (en)
Inventor
Larry A. Denner
Ajay A. Rege
Richard A. F. Dixon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Encysive Pharmaceuticals Inc
Original Assignee
Texas Biotechnology Corp
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Filing date
Publication date
Application filed by Texas Biotechnology Corp filed Critical Texas Biotechnology Corp
Publication of EP0677055A1 publication Critical patent/EP0677055A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to growth-factor related polynucleotides and their use in inhibiting the proliferation of smooth muscle cells, and more specifically to antisense molecules corresponding in sequence to portions of a gene for fibroblast growth factor receptor, and their use in inhibiting the proliferation of smooth muscle cells.
  • Antisense polynucleotides contain artificial sequences of nucleotide bases complementary to messenger RNA (mRNA or message) or the sense strand of double stranded DNA. Admixture of sense and antisense oligo- or polynucleotides under appropriate conditions leads to binding of the two molecules, or hybridization.
  • FGF fibroblast growth factor
  • Fibroblast growth factors mediate their cellular responses through binding and activation of high affinity cell surface receptors known as fibroblast growth factor receptors. To date, five such receptors have been identified and cloned from humans, and all appear to contain tyrosine kinase activity. Jaye et al., Bioch. Biophy ⁇ . Acta 1135:185-199 (1992).
  • Activated smooth muscle cells elaborate growth factors such as platelet derived growth factor (PDGF) , basic and acidic fibroblast growth factor, interleukins and transforming growth factor ⁇ .
  • PDGF platelet derived growth factor
  • FGF receptor FGF receptor
  • epidermal growth factor receptor epidermal growth factor receptor.
  • SMC Activation of SMC, leading to the proliferation of those cells, occurs in response to a number of stimuli, including surgical procedures such as coronary angioplasty.
  • the proliferation of SMC results in such disease states as atherosclerosis and restenosis.
  • This invention is applicable to a number of disease states in which the proliferation of smooth muscle cells is involved, including, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • vascular stenosis including coronary, carotid and peripheral stenosis
  • other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • the present invention is directed to a polynucleotide of about 10 to about 50, preferably about 15 to about 25, and more preferably about 20, nucleic acid bases in length, which polynucleotide hybridizes to the gene encoding fibroblast growth factor receptor.
  • a preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID NO:l.
  • GCGCCCCCAG CTGACCAT (SEQ ID NO:2), GCAGGCCGGG ACTACCAT (SEQ ID NO:3), GGCCAAGAGC AGCCACAT (SEQ ID NO:4), GCACTTCCAG CTCCACAT (SEQ ID NO:5), ACGACCCCAG CTGACCAT (SEQ ID NO:6), GCAGGCAGGG GCGCCCAT (SEQ ID NO:7), GGCCAGCAGC AGCCGCAT (SEQ ID NO:8),
  • GCAGGCCGGG ACTACCAT SEQ ID NO:9
  • GGGGATCCTC AGGGAGCT SEQ ID NO:27
  • CAGTTTCTTC TCCATTTT SEQ ID NO:28
  • the bases of the polynucleotide molecule are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease and/or endonuclease enzymes.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • the present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
  • a preferred such polynucleotide is an antisense molecule having the sequence shown in SEQ ID NOs 1 through 9 above, as well as SEQ ID NO:10:
  • GCACTTCCAG CCCCACATCC C (SEQ ID NO:10).
  • Further preferred polynucleotides include: CTTCCAGCCC CACATCCC (SEQ ID NO:11), CCAGCCCCAC ATCCC (SEQ ID NO:12),
  • CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), GCACTTCCAG CCCCA (SEQ ID NO:14), CTTCCAGCCC CACAT (SEQ ID NO:15), GCCCCACATC CCAGTTCT (SEQ ID NO:16), CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), GCCCCACATC CCAGT (SEQ ID NO:18), CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), GCTCCACATC CCAGT (SEQ ID NO:21), GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22),
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
  • the present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
  • the present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • the present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • Figure 1 shows the percentage of growth inhibition of smooth muscle cells upon the addition of various concentrations of either antisense polynucleotides directed against the gene for mouse fibroblast growth factor receptor 1, or antisense polynucleotides directed against the gene for plasminogen activator inhibitor 1.
  • Figure 2 shows the percentage of growth inhibition of smooth muscle cells upon the addition of either 10 ⁇ M or 50 ⁇ M of antisense polynucleotides directed against the gene for mouse fibroblast growth factor 2, 3 or 4, and the gene for plasminogen activator inhibitor 1.
  • Figure 3 shows the percentage of growth inhibition of rat carotid artery smooth muscle cells proliferation upon the addition of either 10 ⁇ M or 50 ⁇ M of antisense polynucleotides directed against mouse fibroblast growth factor gene 1, and the vinculin gene.
  • Figure 4 shows the percentage of maximal inti al thickening of rat carotid arteries after angioplasty and treatment in vivo, upon the addition of antisense polynucleotides directed against FGF receptor 1 or PAI-1, or control treatment with carrier alone.
  • the present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the gene for fibroblast growth factor receptor.
  • the gene for fibroblast growth factor receptor can be derived from any mammal, including mouse and humans.
  • the gene for fibroblast growth factor receptor is that of human fibroblast growth factor receptor.
  • the polynucleotide may preferably be from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
  • the present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
  • the present invention contemplates a polynucleotide that hybridizes to any of the genes encoding a member of the fibroblast growth factor receptor family. Any such polynucleotide capable of inhibiting the proliferation of smooth muscle cell proliferation can be used.
  • polynucleotide refers to a covalently linked sequence of nucleotides in which the 3 # position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide.
  • the nucleotides may be composed of deoxyribonucleotides or ribonucleotides.
  • Polynucleotide hybridization of greater than about 90 percent homology (identity) , and more preferably about 99 percent homology, is contemplated in the present invention.
  • the Polynucleotides A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID N0:1, directed against the gene for mouse fibroblast growth factor (FGF) receptor 1.
  • FGF mouse fibroblast growth factor
  • GCGCCCCCAG CTGACCAT (SEQ ID NO: 2 ] , directed against the gene for mouse FGF receptor 2,
  • GCAGGCCGGG ACTACCAT (SEQ ID NO:3] , directed against the gene for mouse FGF receptor 3, GGCCAAGAGC AGCCACAT (SEQ ID NO: ] , directed against the gene for mouse FGF receptor 4,
  • GCACTTCCAG CTCCACAT directed against the gene for human FGF receptor 1
  • ACGACCCCAG CTGACCAT directed against the gene for human FGF receptor 2
  • GCAGGCAGGG GCGCCCAT (SEQ ID NO: 1 ) , directed against the gene for human FGF receptor 3,
  • GGCCAGCAGC AGCCGCAT directed against the gene for human FGF receptor 4
  • GCAGGCCGGG ACTACCAT directed against the gene for human FGF receptor 5.
  • sequence of the polynucleotide directed against the gene for human FGF receptor 5 shown in SEQ ID NO:9 is identical to the polynucleotide directed against the gene for mouse FGF receptor 3 shown in SEQ ID NO:3; however, different SEQ ID NOs will be used to avoid confusion and because of the fact that the human and mouse sequences differ in other locations.
  • polynucleotides include GGGGATCCTC AGGGAGCT (SEQ ID NO:27) , and CAGTTTCTTC TCCATTTT (SEQ ID NO:28), both of which are directed against internal sequences of the mouse FGF receptor 1 gene,
  • GCACTTCCAG CCCCACATCC C (SEQ ID NO:10), directed against position -3 to +18, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCCC CACATCCC (SEQ ID NO:11), directed against position -3 to +15, relative to the start codon, of mouse FGF receptor gene 1
  • CCAGCCCCAC ATCCC (SEQ ID NO:12), directed against position -3 to +12, relative to the start codon, of mouse FGF receptor gene 1
  • CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), directed against position -6 to +15, relative to the start codon, of mouse FGF receptor gene 1,
  • GCACTTCCAG CCCCA (SEQ ID NO:14), directed against position +4 to +18, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCCC CACAT (SEQ ID NO:15), directed against position +1 to +15, relative to the start codon, of mouse FGF receptor gene 1,
  • GCCCCACATC CCAGTTCT (SEQ ID NO:16), directed against position -9 to +9, relative to the start codon, of mouse FGF receptor gene 1
  • CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), directed against position -6 to +12, relative to the start codon, of mouse FGF receptor gene 1
  • GCCCCACATC CCAGT (SEQ ID NO:18), directed against position -6 to +9, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), directed against position -6 to +15, relative to the start codon, of human FGF receptor gene 1
  • CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), directed against position -6 to +12, relative to the start codon, of human FGF receptor gene 1
  • GCTCCACATC CCAGT (SEQ ID NO:21) , directed against position -6 to +9, relative to the start codon, of human FGF receptor gene 1
  • GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22), directed against position -3 to +24, relative to the start codon of human FGF receptor gene 1
  • GAGGCACTTC CAGCTCCACA TCCC (SEQ ID NO:23), directed against position -3 to +21, relative to the start codon of human FGF receptor gene 1
  • GCACTTCCAG CTCCACATCC CAGT (SEQ ID NO:24), directed against position -6 to +18, relative to the start codon of human FGF receptor gene 1,
  • GCACTTCCAG CTCCACATCC C (SEQ ID NO:25) , directed against position -3 to +18, relative to the start codon of human FGF receptor gene 1, and
  • CTTCCAGCTC CACATCCC (SEQ ID NO:26) , directed against position -6 to +9, relative to the start codon of human FGF receptor gene 1.
  • the bases of the polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
  • Exonuclease enzymes hydrolyze the terminal phosphodiester bond of a nucleic acid.
  • Endonuclease enzymes hydrolyze internal phosphodiester bonds of a nucleic acid.
  • pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate, phosphorodithioate and phosphoroselenoate bonds.
  • exonuclease and/or endonuclease resistant polynucleotides can be obtained by blocking the 3 ' and/or 5' terminal nucleotides with substituent groups such as acridine, cholesterol or a methyl group.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • the pseudophosphate bonds may comprise the bonds at the 3' and or 5' terminus, the bonds from about 1 to about 5 of the 3' and/or 5' terminus bases, or the bonds of the entire polynucleotide.
  • a preferred polynucleotide with pseudophosphate bonds is one in which all of the bonds are comprised of pseudophosphate bonds.
  • DNA or RNA polynucleotides can be prepared using several different methods, as is well known in the art. See, e.g., Ausubel et al. (eds.). Current Protocols in Molecular Biology, John Wiley & Sons, New York (1990) .
  • the phosphoramidate synthesis method is described in Caruthers et al., Meth. Enzy ol. 154:287 (1987) ; the phosphorothioate polynucleotide synthesis method is described in Iyer et al., J. Am. Chem. Soc. 112:1253 (1990).
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
  • the polynucleotide is from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
  • the present invention includes one or more polynucleotides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents.
  • carriers, adjuvants or vehicles that are collectively referred to herein as diluents for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the like.
  • the compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops) , or as a buccal or nasal spray.
  • compositions can also be delivered through a catheter for local delivery at the site of vascular damage, via an intracoronary catheter or stent (a tubular device composed of a fine wire mesh) , or via a biodegradable polymer.
  • the compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions to the site of smooth muscle cell proliferation.
  • compositions are preferably administered via parenteral delivery at the local site of smooth muscle cell proliferation.
  • the parenteral delivery is preferably via catheter.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
  • the total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight.
  • Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
  • the present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
  • an “inhibition-effective amount” is that amount of a polynucleotide of the present invention which is sufficient for inhibiting the growth or killing a cell contacted with such a polynucleotide.
  • Means for determining an inhibition- effective amount in a particular subject will depend, as is well known in the art, on the nature of the polynucleotide used, the mass of the subject being treated, whether killing or growth inhibition of the cells is desired, and the like.
  • Contact is achieved by admixing the composition with a preparation of vascular smooth muscle cells.
  • Biological culture conditions are those conditions necessary to maintain the growth and replication of the vascular smooth muscle cells in a normal, polynucleotide-free environment. These biological culture conditions, encompassing such factors as temperature, humidity, atmosphere, pH and the like, must be suitable for the proliferation of vascular smooth muscle cells in the absence of polynucleotides so that the effects of such polynucleotides on relevant growth parameters can be measured.
  • a preferred polynucleotide useful in this process has the sequence shown in SEQ ID N0:1.
  • a further preferred polynucleotide useful in this process links the bases of SEQ ID NO:l by pseudophosphate bonds that are resistant to cleavage by exonuclease enzymes.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • polynucleotides have the sequences shown in SEQ ID NO:2 through 28.
  • the present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • a host mammal in need of the treatment of a process for the inhibition of vascular smooth muscle cell proliferation suffers from a disease state in which such proliferation is implicated.
  • disease states include vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, by-pass surgery and organ transplant.
  • the polynucleotide as described above is dissolved or dispersed in a physiologically tolerable diluent.
  • the present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • a disease state involving the proliferation of vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • Cleavage was facilitated by mixing of the solution every 30 minutes with the help of two 5 ml slip-tip syringes.
  • the solution was collected in a screw-capped glass vial and deprotection was accomplished either at room temperature for 24 hours or at 55 C for 5 hours.
  • the contents were transferred to a 13x100 mm glass tube, chilled on ice and evaporated to dryness using a Savant Speed-Vac.
  • the polynucleotide was then dissolved in 1 ml of 0.1M triethylammonium acetate (TEAA) , pH 7.0.
  • TEAA triethylammonium acetate
  • the oligo was detritylated and purified on a Rainin Dyna ax C18 semipreparative column (10mm x 25cm, 5um, 300 A) .
  • the mobile phases were (A): 0.1M TEAA, pH7.0, 5% acetonitrile; (B) : 95% acetonitrile, 5% water; (C) : 0.5% TFA in water.
  • the column was developed at 2ml/min with the following gradient: 10% B in A, 10 min; 100% A, 4 min; 100% C, 8 min; 100% A, 8 min; 100% A to 45% B in 24 min.
  • This procedure first separates the trityl-on full length polynucleotide from its failure sequences containing free hydroxyl groups and synthesis reagents. This is followed by the removal of 5'-DMT by 0.5% TFA. Finally the gradient resolved the desired detritylated sequence from other contaminants.
  • the polynucleotide concentration was determined by measuring the absorbance at 260 nm. Typical yields were 30-40%.
  • the integrity of the polynucleotide was determined by polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide, 7M urea) and staining with 0.2% methylene blue.
  • the pellet was resuspended in 4 ml/carotid of the following media: 20% fetal bovine serum albumin (Hyclone; FBS) ; 2mM glutamine (Gibco) ; 100 units/ml penicillin G sodium (Gibco) ; 100 ug/ml streptomycin sulfate (Gibco) ; DMEM (Gibco) .
  • FBS fetal bovine serum albumin
  • Gibco 2mM glutamine
  • penicillin G sodium Gibco
  • streptomycin sulfate Gibco
  • DMEM DMEM
  • PBS phosphate buffered saline
  • trypsin-EDTA Gibco; 0.25% trypsin-EDTA
  • the flask was rinsed with an additional 4 ml media (DMEM, 20% PBS, 2 mM glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin) .
  • the trypsinized cells and the rinse were combined and centrifuged at 400 x g for 10 min.
  • the supernatant was removed and 5 is of fresh media was added to the pellet.
  • the pellet was resuspended by vigorous trituration, and the number of cells was determined using a Coulter counter.
  • the cells were diluted to 3,500 cells/100 ul and, using a 12 channel digital micropipette, '100 ul/well of the cells were seeded in a 96 well (Falcon) flat-bottom, microtiter cell culture plate. The culture plate was then incubated at 37*C in 5% C0 2 .
  • each well was rinsed twice with 100 ul PBS, and overlaid with 100 ul/well growth arrest media: 0.1% FBS (heat inactivated at 65 # C for 45 min.); 2mM glutamine; 50 units/ml penicillin; 50 ug/ml streptomycin.
  • the growth arrest media was removed.
  • the cell number was determined (treatment day counts) using a Coulter counter by averaging the cell number from three wells. To the remaining wells was added 100 ul complete media (DMEM, 10% FBS/65'C inactivated, glutamine, pen/strep) without or with antisense polynucleotides and the plates were placed in an incubator at 37*C in 5% C0 2 .
  • Cutdowns were performed on the carotid and iliac arteries and 25% bupiviamine used as a topical anesthetic.
  • a 2F embolectomy catheter was inserted into the left iliac artery and advanced to the distal end of the left carotid artery. The balloon was inflated and pulled down the artery 3 times. The catheter was then removed.
  • Antisense polynucleotide or vehicle was then delivered at 6 ⁇ l/min for 5 min with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the drug out of the artery.
  • the carotid was then ligated distal to the heart near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
  • Rats were then closed with 4.0 silk suture. Rats recovered under an infrared heating lamp connected to a proportional regulated heater. Topical antibiotics were applied to the incisions.
  • the treated section of the left carotid artery was removed and the central portion embedded in paraffin, cut in 5 ⁇ m sections, and stained with hematoxylin and eosin.
  • a frame grabber (Targa+) and image analysis software (Java) were used to measure cross-sectional areas of the medial and intimal layers. To standardize the intimal area for arteries of different size, the intimal area was divided by the medial area and this ratio used as the parameter to compare drug treatment with control.
  • Carotid arteries were dissected from male Sprague-Dawley rats weighing 200 to 300 grams.
  • DMEM Dulbecco's minimal essential medium
  • the medium was changed every three to four days.
  • trypsin was added to the growth culture to isolate cells which had grown out of the arterial explant. These isolated cells were plated in 96 well trays at a concentration of 2,500 cells per well. After one day of growth under the conditions described above, the cells were washed twice with 100 ⁇ l of phosphate buffered saline and placed in growth arrest medium consisting of DMEM supplemented with 0.5% heat-inactivated fetal bovine serum.
  • cell number was determined by a fluorescence-based cell proliferation assay using calcein-.AM (Molecular Probes; Eugene, OR) .
  • the medium was removed and triplicate wells were incubated with ImM calcein-AM, dissolved in phosphate buffered saline, for 1 hour at 37°C. Fluorescence was determined using a Cytofluor plate reader (Millipore; Boston, MA), at 580 nm following excitement at 450 nm. Under the cell culture conditions used, there was a linear relationship between cell number (determined by Coulter counting) and fluorescence.
  • Example 1 Proliferation The proliferation of smooth muscle cells according to the assay described in Example 1 was determined in the presence of antisense polynucleotides which hybridized to portions of mouse FGF receptor 1, 2, 3 and 4, as well as an antisense polynucleotide directed against plasminogen activator inhibitor-1 (PAI-1) , as a negative control.
  • PAI-1 plasminogen activator inhibitor-1
  • Smooth muscle cells were isolated from normal human aorta or diseased human carotid artery endarterectomy specimens in order to evaluate the growth-regulated expression of FGF receptor mRNA.
  • the smooth muscle cells were obtained by enzymatic dissociation with collagenase and elastase followed by culture in DMEM supplemented with 10 percent fetal bovine serum. After 5 to 7 days of culture, the cells were plated at 40,000 cells per 100 mm 2 dish.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • GAPDH Glyceraldehyde phosphate dehydrogenase
  • FGF receptor gene 1 mRNA was undetectable in growth-arrested human smooth muscle cell cultures.
  • FGF receptor gene 1 mRNA levels were elevated in normal growth-stimulated, log phase human SMCs, but were even further stimulated in diseased growth-stimulated, log phase human SMCs.
  • the growth of SMCs derived from diseased human patients was correlated with overexpression of FGF receptor gene 1 mRNA.
  • FGFR1 + - ++ - ' The pluses represent relative band intensities of the PCR amplified products as seen on agarose gels. The minuses indicate the absence of a detectable band.
  • FGF receptor gene 1 mRNA was tested using the rat carotid artery balloon angioplasty model of restenosis. Abnormal growth of SMCs often leads to restenosis in humans; these experiments were designed to determine whether FGF receptor gene l mRNA is overexpressed in response to angioplasty.
  • the arteries were removed from the anesthetized animals, trimmed of adventitia and nerve tissue, and mRNA levels were determined by RT-PCR, according to standard procedures in the art.
  • GAPDH mRNA levels were determined as an internal control.
  • PCNA mRNA levels were determined in order to assess the proliferation of neointimal and medial SMCs.
  • FGF receptor gene 1 mRNA was induced to a maximum level of expression between 6 hours and 2 days post-angioplasty. As indicated by the expression levels of PCNA mRNA, this time frame correlates with the proliferation of medial SMCs. Table 2
  • angioplasty of the rat carotid artery induced FGF receptor 1 gene mRNA overexpression in vivo. This induction was not only maintained in in vitro cell culture, but was enriched in the population of abnormally proliferating neointimal SMCs.
  • an antisense polynucleotide directed against FGF receptor gene 1 inhibited growth of rat carotid SMCs in a dose-dependent manner.
  • an antisense polynucleotide directed against the vinculin gene CGTATGAAAC CATGGCAT, SEQ ID NO:29
  • CGTATGAAAC CATGGCAT CGTATGAAAC CATGGCAT
  • Antisense Molecules Various antisense polynucleotides were constructed in order to determine which regions of the
  • FGF receptor 1 gene were most sensitive to inhibition by antisense molecules. Inhibition studies were performed as described in Example 2. Table 4 shows the positions relative to the start codon and sequence identification numbers of the antisense polynucleotides, and their growth inhibitory effect.
  • the antisense polynucleotide designated SEQ ID NO:10 showed the highest level of growth inhibition.
  • SEQ ID NO:10 extends from -3 to +18, relative to the start codon.
  • Antisense polynucleotides directed to internal FGF receptor 1 gene sequences, such as SEQ ID NO:27 also showed appreciable growth inhibition.
  • Antisense polynucleotides directed against the human FGF receptor 1 gene, which contains a C to T substitution at position +7 (relative to the mouse sequence) were also active in inhibiting the growth of rat SMCs.
  • the similar activities of SEQ ID NO:13, directed against the mouse sequence, and SEQ ID NO:19, directed against the comparable human sequence, in inhibiting the growth of rat SMCs indicates that a one base mismatch is tolerable for target gene regulation, and suggests that overall position and length of the polynucleotide may be important determinants in efficacy of inhibition.
  • Antisense-Mediated Down Regulation of Target mRNA The specificity, efficacy and mechanism of action of antisense polynucleotides can be examined by studying the down-regulation of target mRNA by such antisense molecules.
  • growth arrested SMCs were serum-stimulated in the presence or absence of 50 ⁇ M of the antisense polynucleotide designated SEQ ID NO:10. After 3 days of contact with the antisense polynucleotide, total RNA was isolated from the SMCs, and RT-PCR was performed.
  • Table 6 shows that SEQ ID NO:10 markedly inhibited the induction of its target mRNA, while having no effect on a control housekeeping gene, GAPDH.
  • An antisense polynucleotide directed against vinculin did not affect the induction of mRNA from the FGF receptor 1 gene.
  • Example 3 The results discussed in Example 3 showed the growth-dependent overexpression of the FGF receptor 1 gene in human SMCs, and particularly those derived from diseased human patients. In these experiments, the ability of antisense polynucleotides directed against the FGF receptor gene 1 to inhibit the growth of human SMCs in culture was examined.
  • a second similar catheter that lacked a tip was filled with either an antisense molecule, or with a pharmaceutically acceptable carrier, and was attached to a syringe pump. This catheter was then inserted into the left iliac artery and advanced into the left carotid artery.
  • Antisense molecules to either FGF receptor 1 or PAI-1 (1 mM in DMEM) or the carrier alone (DMEM) was delivered at a rate of 6 ⁇ l/min for 5 min, with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the delivered material (drug or carrier) out of the artery.
  • the carotid artery was then ligated distal to the heart, near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
  • Neointimal and medial areas were measured and the intimal/medial ratio calculated. The ratio for the treatment groups was then normalized to the ratio for the control groups. The control intimal/medial ratio was 0.668 +/- 0.141.
  • Antisense polynucleotides to FGF receptor l SEQ ID NO: 10
  • the nonspecific control antisense polynucleotides to PAI-1 has no effect (see Figure 3) .
  • antisense polynucleotides directed against FGF receptor 1 mRNA specifically inhibited neointimal development in vivo in the rat carotid balloon angioplasty model of restenosis.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:27: GGGGATCCTC AGGGAGCT 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne un polynucléotide d'une longueur d'environ 10 à 50 bases d'acide nucléique s'hybridant au gène codant le récepteur du facteur de croissance de fibroblastes. L'invention porte également sur une composition pharmaceutique contenant ledit polynucléotide dissous ou dispersé dans un diluant physiologiquement tolérable. L'invention se rapporte également à un procédé d'inhibition de la prolifération des cellules des muscles vasculaires lisses (figs 1 et 2), à un procédé de traitement d'états pathologiques impliquant une prolifération des cellules de muscles vasculaires lisses (figs 3 et 4), tous ces procédés consistant à mettre les cellules de muscles vasculaires lisses en contact avec une dose efficace de polynucléotide présentant environ une longueur de 10 à 50 bases d'acide nucléique ou à administrer ce dernier, ledit polynucléotide s'hybridant avec le gène par le récepteur du facteur de croissance de fibroblastes.
EP94907109A 1992-12-31 1993-12-28 Molecules antisens dirigees contre une famille de genes codant un recepteur du facteur de croissance de fibroblastes Withdrawn EP0677055A1 (fr)

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US99970692A 1992-12-31 1992-12-31
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PCT/US1993/012600 WO1994015945A1 (fr) 1992-12-31 1993-12-28 Molecules antisens dirigees contre une famille de genes codant un recepteur du facteur de croissance de fibroblastes

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US5783683A (en) * 1995-01-10 1998-07-21 Genta Inc. Antisense oligonucleotides which reduce expression of the FGFRI gene
WO1998004686A2 (fr) * 1996-07-26 1998-02-05 Robert Day Enzyme de conversion de preproteines
CA2203745A1 (fr) 1996-07-26 1998-01-26 Robert Day Enzyme de conversion pro-proteinique
WO2009046141A2 (fr) * 2007-10-01 2009-04-09 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression du récepteur 4 du facteur de croissance des fibroblastes
WO2012174476A2 (fr) 2011-06-16 2012-12-20 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression du récepteur 4 du facteur de croissance fibroblastique

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CA2080008C (fr) * 1990-04-27 2002-07-02 Koichi Igarashi Muteine hydrosoluble du recepteur de facteur de croissance des fibrolastes
US5135917A (en) * 1990-07-12 1992-08-04 Nova Pharmaceutical Corporation Interleukin receptor expression inhibiting antisense oligonucleotides

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