CA2152903A1 - Antisense molecules directed against a fibroblast growth factor receptor gene family - Google Patents

Abstract

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 encoding fibroblast growth factor receptor. The present invention is also directed to a pharmaceutical composition comprising the above polynucleotide dissolved or dispersed in a physiologically tolerable diluent. The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation (figs. 1 and 2), a process for treating vascular smooth muscle cell proliferation (figs. 3 and 4), and a process for treating a disease state involving vascular smooth muscle cell proliferation (figs. 3 and 4), all of which processes comprise contacting vascular smooth muscle cells or administering 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.
I

Description

W094/15945 PCT~S93/12600 ANTISENSE MOLECULES DIRECTED AGAINST A
FIBROBLAST GROWTH FACTOR RECEPTOR GENE FAMILY
DESCRIPTION
Cross Reference to Related A~lication This application is a continuation-in-part of U.S. Application Serial No. 07/999,706, filed December 31, 1992.

TECHNICAL FIELD
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 seguence to portions of a gene for fibroblast growth factor receptor, and their use in inhibiting the proliferation of smooth muscle cells.

BACKGROUND OF THE lN V ~:N~l lON
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.
When these polynucleotides bind to (hybridize with) mRNA, inhibition of translation oc~ . When these polynucleotides bind to double stranded DNA, inhibition of transcription occurs. The resulting inhibition of translation and/or transcription leads to an inhibition of the synthesis of the encoded protein such as the proteins of the tissues, and more importantly here, various cellular growth factors, growth factor receptors, and oncogenes (many of which act as growth factors, receptors or mediators of signal transduction).

wog4/1sg45 PCT~S93/12600 ~ - 2 -, There are several polypeptides considered to fall into the fibroblast growth factor (FGF) family.
These polypeptides, ranging in size from about 155 to about 268 amino acid residues, are angiogenic and neutrophonic molecules which are mitogenic for many cell types deriving from epithelial, mesenchymal and neural origin.
Fibroblast growth factors mediate their cellular responses through bin~ing 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 (SMC) elaborate growth factors such as platelet derived growth factor (PDGF), basic and acidic fibroblast growth factor, interleukins and transforming growth factor ~.
Likewise, the SMC increase the production of PDGF
receptor, FGF receptor, and epidermal growth factor receptor.
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.
An in vitro assay system has been developed to study smooth muscle cell proliferation. This assay system is considered to be a useful model for SMC
proliferation in vivo. Gordon et al. have shown that SMC proliferation results from aortic and carotid balloon catheter injury, and is a result of atherosclerosis, providing a positive correlation . .

wog4/1sg4s ~- PCT~S93/12600 between SMC proliferation and stenosis. Gordon et al., Proc. Natl. Acad. Sci. USA 87:4600-4604 (l990).
Speir et al. have studied the inhibition of SMC proliferation in vitro by using an antisense oligonucleotide to proliferating cell nuclear antigen (PCNA). However, these workers could not inhibit proliferation below 50 %, and the inhibition required high levels of the 18-mer antisense oligonucleotide used in those studies.
This invention demonstrates the biological action of antisense polynucleotides directed against the gene for fibroblast growth factor receptor, as useful for anti-proliferative activity against smooth muscle cell proliferation. This invention is applicable to a number of ~;se~fie 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 procedu.es, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.

BRIEF SUMMARY OF THE lNV~l. lON
The present invention is directed to a polynucleotide of about l0 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 anti~ence molecule having the sequence shown in SEQ ID NO:l.
GCA~ C~AG CCCCACAT (SEQ ID NO:l) Further preferred polynucleotides include GCGCCCr~AG CTGACCAT (SEQ ID NO:2), GCAGGCCGGG ACTACCAT (SEQ ID NO:3), W094/1594~ PCT~S93112600 2i529~3 4 _ 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), and CAGTTTCTTC TCCATTTT (SEQ ID N0:28), as well as SEQ ID NOs 10 through 21 and 26, described lo hereinafter.
In a preferred embodiment, 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 bond~.
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 hA~^c, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flAnki~ the start ~odon of the mRNA for fibroblast growth factor receptor.
A preferred such polynucleotide is an ant;cense 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:ll), CCAGCCCCAC ATCCC (SEQ ID NO:12), CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), GCACTTCCAG CCCCA (SEQ ID N0:14), CTTCCAGCCC CACAT (SEQ ID NO:15), GCCCCACATC CCAGTTCT (SEQ ID NO:16), CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), W094/15945 PCT~S93/12600 -- 1 $.2903.

GCCCCACATC CCAGT (SEQ ID N0:18), CTTCCAGCTC CACATCCCAG T (SEQ ID N0:19), CCAGCTCCAC ATCCCAGT (SEQ ID N0:20), GCTCCACATC CCAGT (SEQ ID N0:21), GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID N0:22), GAGGCACTTC CAGCTCCACA TCCC (SEQ ID N0:23), GCACTTCCAG CTCCACATCC CAGT (SEQ ID N0:24), GCA~ ~AG CTCCACATCC C (SEQ ID N0:25), and CTTCCAGCTC CACATCCC (SEQ ID N0:26).
The present invention is also directed to 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, ~aid 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 h~ses in length, ~aid polynucleotide hybridizing to the gene for fibroblast growth factor receptor.

wos4/1ss4s PCT~S93/12600 2 is29~ - 6 -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 l0 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.

BRIEF DESCRIPTION OF Tn~: FIGURES
Figure l shows the percentage of growth inhibition of smooth muscle cells upon the addition of various concentrations of either ant;c~nce polynucleotides directed against the gene for mouse fibroblast growth factor receptor l, or antisense polynucleotides directed against the gene for plasminogen activator inhibitor l.
Figure 2 shows the percentage of growth inhibition of smooth muscle cells upon the addition of either l0 ~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 l.
Figure 3 shows the percentage of growth inhibition of rat carotid artery smooth muscle cells proliferation upon the addition of either l0 ~M or 50~M
of antisense polynucleotides directed against mouse fibroblast growth factor gene l, and the vinculin gene.
Figure 4 shows the percentage of maximal intimal thickPn; ng of rat carotid arteries after angioplasty and treatment in vivo, upon the addition of antisense polynucleotides directed against FGF receptor l or PAI-l, or control treatment with carrier alone.

WO 94tlS945 PCT/US93/12600 DETAILED DESCRIPI ION OF THE INVENTION
The present invention is directed to a polynucleotide of about lo 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. Preferably, 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.
It is to be understood that the present invention contemplates a polynucleotide that hybridizes to any of the genes encoAing 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.
As used herein, "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 9o percent homology (identity), and more WO94/15945 PCT~S93tl2600 2i52903 preferably about 99 percent homology, is contemplated in the present invention.

A. The PolYnucleotides A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID NO:1, directed against the gene for mouse fibroblast growth factor (FGF) receptor 1.
GCACTTCCAG CCCCACAT (SEQ ID NO:1) Further preferred polynucleotides include GCGCCCr~G 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 ~ece~or 3, GGCr~r~ C AGCCACAT (SEQ ID NO:4), directed against the gene for mouse FGF receptor 4, GCACTTCCAG CTC~C~T (SEQ ID NO:5), directed against the gene for human FGF receptor 1, ACr-~CCCr~ CTGACCAT (SEQ ID NO:6), directed against the gene for human FGF receptor 2, GCAGGCAGGG GCGCCCAT (SEQ ID NO:7), directed against the gene for human FGF receptor 3, GGCCAGCAGC AGCCGCAT (SEQ ID NO:8), directed against the gene for human FGF receptor 4, and GCAGGCCGGG ACTACCAT (SEQ ID NO:9), directed against the gene for human FGF receptor 5.
The 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.
Further preferred polynucleotides include GGGGATCCTC AGGGAGCT (SEQ ID NO:27), and WO94/l5945 21 ~ PCT~593/l2600 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:ll), 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 co~on, of mouse FGF receptor gene 1, GCA~-llCCAG 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, CTICCAGCTC CACATCCCAG T (SEQ ID NO:l9), directed against position -6 to +15, relative to the ~tart coAon, of human FGF receptor gene 1, WO94/15945 PCT~S93/12600 .
~ 21s2903 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 Lece~or gene 1, GCA~-llC~AG CTC~ATCC C (SEQ ID NO:25), directed against position -3 to +18, relative to the start codon of human FGF receptor gene 1, and ~ -~lC~AGCTC CACATCCC (SEQ ID NO:26), directed against position -6 to +9, relative to the start codon of human FGF receptor gene 1.
In a preferred embodiment, the bases of the polynucleotide, e.g., SEQ ID NO:l, 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.
By replacing a phosphodiester bond with one that is resistant to the action of exonucleases or endonucleases, the stability of the nucleic acid in the presence of those exonucleases or endonucleases is increased. As used herein, pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate, wo94lls94s PCT~S93/12600 phosphorodithioate and phosphoroselenoate bonds.
Additionally, exonuclease and/or endonuclease resistant polynucleotides can be obtained by blocking the 3' and/or 5' terminal nucleotides with substituent ~.ou~s s 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 8iology, John Wiley & Sons, New York (1990). The phosphoramidate synthesis method is described in Caruthers et al., Meth. Enzymol. 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 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.
Preferably, 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, WO94/15945 PCT~S93/12600 2~S2903 carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for oral administration in solid or liguid 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.
The compositions can also be delivered throuqh 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.
The compositions are preferably administered via parenteral delivery at the local ~ite 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. Examples of 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 WO94/15945 PCT~S93/12600 21S,~903 l3 1 the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions can also contain 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.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspen~ing agents, sweeten;ng, flavoring and perfuming agents.
Suspensions, in addition to the sctive `
compounds, may contain suspen~ing 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 substAncec, and the like.
Dosage forms for topical administration of a compound of this invention include ointments, powders, _prays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be reguired. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
Actual dosage levels of active ingredients in the compositions of the present invention may be varied WO94/15945 PCT~S93/12600 2~s29 03 ~ 14 -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 l 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.

B. The Processes 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 lO to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintA;ni~g 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.

WO94/15945 PCT~S93112600 2ls23o3 As used herein, an Ninhibition-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 neceCcAry 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 Ah-ence 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 NO:l. 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.
Further preferred 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 WO94/15945 PCT~S93/12600 21$~93 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. Such disease states include vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis), other non-angioplasty reopening procedures such as atherectomy and laser proceAl~res, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, by-pass surgery and organ transplant.
In a preferred embodiment, 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 lO 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 proced~s, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.

wOg4/1594~ ?1S~3 PCT~S93/12600 The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way.

S EXAMPLES
Materials and Methods Antisense PolYnucleotide Pre~aration All reagents were from Eppendorf or Glen ReceArch.
Antisense polydeoxynucleotide ~olid phase syntheses were performed on Millipore CPG columns using cyanoethyl phosphoramidite chemistry on an Eppendorf Synostat D300 DNA synthesizer replacing iodine by 3H-1,2-benzodithiol-3-one l,l-dioxide (BDTD Beaucage reagent). The polynucleotide was cleaved from the solid support by incubation with 3 ml of fresh, concentrated (30%) ammonium hydroxide for 90 minutes. 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 13xlOO mm glass tube, chilled on ice and evaporated to dryness using a Savant Speed-Vac.
The polynucleotide was then dissolved in 1 ml of O.lM triethylammonium acetate (TEAA), pH 7Ø The oligo was detritylated and purified on a Rainin Dynamax C18 semipreparative column (lOmm x 25cm, 5um, 300 A).
The mobile ph~eS were (A): O.lM 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 wog4/1sg4s PCT~S93/12600 2iS~s~o3 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.
Absorbance was monitored at 260mm to identify fractions containing the polynucleotide which was then evaporated. The polynucleotide was dissolved in 1 ml water, evaporated to remove volatile salts, and finally dissolved in 0.5 ml sterile, low TE (lOmM Tris, lmM
EDTA, pH 7.5).
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 st~i ni ng with 0.2% methylene blue.

Smooth Muscle Cell Isolation and Culture Male Sprague-Dawley rats weighing 350-450 g were euthanized with carbon dioxide. The carotid arteries were removed and trimmed free of adventitia, nerve, and fat under a dissecting microscope. Arteries were cut into approximately lmm3 pieces and placed in a 125 ml Erlenmeyer flask cont~i~;ng 0.67 ml/carotid artery of the following enzyme cocktail:
79.2 ml Hanks Balanced Salt Solution (Gibco;
HBSS), 0.8 ml 0.2M CaCl2 (Fisher), 0.286 g HEPES free acid (Calbiochem), 0.03 g trypsin inhibitor (Sigma; Type I-S; 10,000 units/mg), 0.16 g bovine serum albumin (Sigma; Fraction V), 600 units elastase (Sigma; Type II-A; 28 units/mg), 16,000 units collagenase (Worthington;
CLS II; 353 units/mg) adjusted to pH7.4, and 0.2~m filtered.

wo94lls94s PCT~S93112600 - 21~9 The flask was placed on an orbital shaker at 150 rpm at 37OC for 2-2.5 hr. The suspension was triturated vigorously and filtered through a 70 ~m nylon cell strainer. The filtrate was then centrifuged at 400 x g for 10 min. The pellet was resuspended in 4 ml/carotid of the following media: 20~ fetal bovine serum albumin (Hyclone; FBS); 2mM glutamine (Gibco); lOo units/ml penicillin G sodium (Gibco); lO0 ug/ml streptomycin sulfate (Gibco); DMEM (Gibco).
The cell suspension from one carotid was then seeded into one T25 flask (Falcon) and maintained at 37 C in 5% CO2.

Proliferation Assay After 6-7 days, cells were rinsed twice with PBS (phosphate buffered saline) and harvested by the addition of 4 ml of 0.05% trypsin-EDTA (Gibco; O.25%
trypsin-EDTA) followed by i~cl~hAtion at 37-C for 3-5 min. 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 mls 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/lO0 ul and, using a 12 channel digital micropipette, lO0 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% CO2.
The following day, each well was rinsed twice with lOO ul PBS, and overlaid with 100 ul/well growth arrest media: 0.1% FBS (heat inactivated at 65 C for 45 WO94/15945 PCT~S93/12600 2~S2~9~ -min.); 2mM glutamine; 50 units/ml penicillin; 50 ug/ml streptomycin.
Four days later, 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% CO2.
Three days later, the wells were rinsed twice with lO0 ul each PBS. The cell number from 3 wells was again determined (assay day counts) using a Coulter counter. To the remaining wells was added lOO ~l of 45 ~g/ml calcein A-M (Molecular Probes) in PBS. The plates were incubated for 1 hr. at 37-C.
After incubation, fluorescence was determined using a Cytofluor 2350 (Millipore) microtiter plate reader with excitation at 485 nm and emission at 530 nm.
Growth in cell number was calculated by subtracting treatment day cell counts from assay day cell counts.
Based on the established linear relationship between fluorescence and cell number, the percent growth inhibition by the antisense polynucleotides was determined.

In Vivo Efficacy Experiments Male Sprague-Dawley rats weighing 375-425 g were housed for at least 5 days prior to surgery.
Anesthesia was performed with 2-2.5~ inhalation of isofluorene; all surgery was performed under sterile conditions.
Cutdowns were performed on the carotid and iliac arteries and 25% bupiviamine used as a topical anesthetic. A 2F embolectomy catheter was inserted into WO94/15945 PCT~S93112600 ~ ~ 21 - ~03 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.
s A second similar catheter that lacked a tip, was filled with anti~ence polynucleotide (drug) or diluent (vehicle), and was attached to a syringe pump.
- This catheter was then inserted into the left iliac and advanced into the left carotid. 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 wAch;ng the drug out of the artery.
The carotid was then ligated distal to the heart near the bifurcation of the internal and external brAnc~s of the artery. After 15 min of static ;~c~hAtion, the ligatures and the catheter were removed to restore normal blood flow.
Fifty ul of oligo or vehicle was then applied to the adventitial ~urface of the carotid and lS0 units/kg of i.v. heparin was delivered.
The two incisions 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.
Two weeks later, rats were sacrificed with CO2 and the abdominal aorta perfused re~GyLadely at 3 ml/min with heparinized PBS (10 units/ml) to remove the blood. Perfusion was then performed with 10~ formalin at 2.5 ml/min for 15 min. These flow rates result in - carotid artery pressures of about lOO mm Hg.
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 WO94/15945 PCT~S93/12600 2l529~3 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 comrAre drug treatment with control.

ExamPle 1. SMC Proliferation Assay Carotid arteries were dissected from male Sprague-Dawley rats weighing 200 to 300 grams.
Approximately 1 mm3 minces were incubated in D~lh~cco's minimal essential medium (DMEM) supplemented with 20 percent fetal bovine serum. The medium was changed every three to four days.
lS After 2 to 2.5 weeks of growth, 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 ~1 of phosphate buffered saline and placed in growth arrest medium consisting of DMEM supplemented with 0.5% heat-inactivated fetal bovine serum.
After four days in growth arrest medium, 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 lmM calcein-AM, dissolved in phosphate buffered saline, for 1 hour at 37C.
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. Additional sets of triplicate wells were Wo94/15945 52903 PCT~593/126~0 either untreated (controls) or treated with the indicated concentrations of polynucleotides dissolved in DMEM with lO~ serum heat-inactivated at 65c. After an additional three days, fluorescence was again determined using calcein-AM, as described above.

Example 2. Pol~nucleotide ~ffects on SMC
Proliferation The proliferation of smooth muscle cells according to the assay described in Example l was determined in the presence of antisense polynucleotides which hybridized to portions of mouse FGF receptor l, 2, 3 and 4, as well as an anti~ence polynucleotide directed against plasminogen activator inhibitor-l (PAI-l), as a negative control.
The results of these assays are shown in Figures l and 2. The data in the bar graphs is depicted to indicate that 100% inhibition reflects the absence of SMC proliferation during the assay. Values of less than 0% indicate that the treated cells proliferated to a greater extent than did untreated cells. Values of greater than 100% means that there were fewer cells at the end of the assay than at the beg; nn i ng .
The data show that 50 ~M of the antisense polynucleotide against mouse FGF receptor l showed nearly 75% inhibition of SMC proliferation, compared to about 10% inhibition by the control antisense polynucleotide directed against PAI-l. See Figure l.
The data in Figure 2 show that 50 ~M of the antisense polynucleotides directed against FGF receptors 2, 3 and 4 showed between 40 and 80 percent inhibition of SMC proliferation, compared to about 10% inhibition by the control polynucleotide.

WO94/15945 PCT~S93/12600 ?.,~,s2903' Example 3. Growth-requlated Expression of FGF
Rece~tor mRNA in Humans Smooth muscle cells were isolated from normal human aorta or ~;c~ced 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 mm2 dish. The day after plating, the cells were growth-stimulated by the removal of the growth medium and addition of DMEM plus 10 percent fetal bovine serum, or growth-arrested by the removal of the growth medium and the addition of DMEM
plus O.l percent fetal bovine serum. Three days after growth-stimulation or growth-arrest, total cellular RNA
was isolated according to stAn~Ard protocols, and mRNA
levels were determined using reverse transcriptase-polymerase chain reaction (RT-PCR), again according to st~n~rd protocols.
Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels were also determined as an internal control for these experiments. GAPDH i8 a ho~sekeeping gene whose expression would be expected to remain constant in the tested cells. As the data in Table 1 show, GAPDH
levels remained constant.
As further shown in the data in Table 1, below, 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. Hence, the growth of SMCs derived WO94/15945 1S290~ PCT~S93/12600 from diseased human patients was correlated with overexpression of FGF receptor gene 1 mRNA.

Table l Normal Aorta Carotid EndarterectomY-mRNA Loa Arrest Loa Arrest GAPDH 11lll +++++ +++++ +++++
FGFRl + - ++
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.

Example 4. OverexPression of FGF Recetor mRNA After Angioplasty The overexpression of 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 1 mRNA is overexpressed in response to angioplasty.
At various times after balloon angioplasty of the rats, the arteries were removed from the anesthetized animals, trimmed of adventitia and nerve tissue, and mRNA levels were determined by RT-PCR, according to st~nA~rd procedures in the art.
As in Example 3, 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.
As shown in Table 2, below, 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.

WO 94/15945 ~,~S?~9~- PCT/US93/12600 :1 + + +
~1 +
t`l + ' ' ' "51 + + ~ ~
L 'r + + ~: ~
N ~ + + O
~, N + + + _I
+ + +
~ + + +
~ ~ + + ~ `
E~ + + + ~
~r + + I
~+
+

O C

O + h--I
+ + I Cl~ h a ~4 .c6~ z u~ o W094l15945 21 ~29 o 3 PCT~S93tl2600 SMCs were then isolated from rat arteries after angioplasty according to the technique described in Example 3. Expression levels of FGF receptor gene 1, PCNA and GAPDH mRNAs were again determined by RT-PCR, and the results are shown in Table 3. The altered n vivo genotype observed in the arteries was maintained in tissue culture. FGF receptor gene 1 mRNA was overexpressed in both neointimal and medial SMCs n vitro, with greater overexpression in the neointimal SMCs. As the expression levels of PCNA mRNA show, neointimal cells were also more actively proliferating than were medial cells.

Table 3 mRNA Neointimal SMC's Medial SMC's GAPDH +++++ lllll FGFR1 +++ . ++
PCNA ++++ +++

Thus, 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.

Exam~le 5. Effects of FGF Receptor Antisense Molecule on Rat Carotid ArterY SMCs To determine if a relationship exists between SMC growth and FGF receptor gene 1 mRNA expression, the - effects of an antisense oligonucleotide to the FGF
receptor gene 1 on the growth of SMCs isolated from rat carotid arteries was examined, as described in Example 2.
As shown in Figure 3, an antisense polynucleotide directed against FGF receptor gene 1 (SEQ

W094/15945 9~ PCT~S93/12600 ID NO: l) inhibited growth of rat carotid SMCs in a dose-dependent manner. In contrast, an antisense polynucleotide directed against the vinculin gene (CGTATGAAAC CATGGCAT, SEQ ID NO:29) exhibited relatively little growth inhibition.

Exam~le 6. Sequence O~timization of FGF ReceDtor Antisense Molecules Various antiF^~ce 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.

WO94/15945 ~I PCT~S93/12600 s29o~

Table 4 Position SEO ID NO % Growth Inhibition Mouse FGFRl Sequences -3/18 10go +1/18 1 52 +4/18 1446 +1/15 1546 Internal Mouse FGFRl Sequences Human FGFRl Sequences Vinculin +1/18 2915 As the data show, 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. Antic~e 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, WO94/15945 9~3 PCT~593/12600 directed against the mouse sequence, and SEQ ID NO:l9, 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 polynucleotides directed against regions surrounding the start site of other members of the mouse FGF receptor family also exhibited significant inhibition of rat SMCs. Table 5 shows the results of these studies.

Table 5 Name Position SEO ID NO % Growth Inhibition FGFR1 ~1/18 1 52 FGFR2 +1/18 2 41 FGFR3 ~1/18 3 47 FGFR4 +1/18 4 40 6. Antisensc ;l~diated Down Requlation of Target mRNA
The specificity, efficacy and mech~nicm of 25 action of antisense polynucleotides can be examined by studying the down-regulation of target mRNA by such antisense molecules. In these experiments, growth arrested SMCs were serum-stimulated in the presence or absence of 50 ~M of the antisense polynucleotide designated SEQ ID NO:lO. 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 ho~ ee~ing gene, GAPDH. An antisense polynucleotide directed against vinculin did not affect the induction of mRNA from the FGF receptor 1 WO94/1~945 ? 9 0 3 PCT~S93/12600 gene. These data suggest that the growth inhibition by antisense polynucleotides directed against the FGF
receptor 1 gene are the result of specific inhibition of the production of FGF receptor 1 mRNA.

Table 6 Antisense % Growth DaYs of Serum Stimulation Treatment Inhibition FRNA O 3 None O GAPDH ++++ ++++
10 None 0 FGFRl +++ ++++
SEQ ID NO:10 85 GAPDH ++++ ++++
SEQ ID NO:10 85 FGFRl +++ +
Vinculin 18 GAPDH ++++ ++++
Vinculin 18 FGFRl +++ ++++

Example 7. Antisense Inhibition of Human SMCs in Culture The results discussed in Example 3 showed the growth-dependent overexpression of the FGF receptor 1 gene in human SMCs, and particularly those deri~ed 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.
Human SMCs were grown essentially as described in Example 3. Table 7 shows that SEQ ID NO:10, which is 94% homologous with the human FGF receptor gene 1 sequence, inhibited proliferation of SMCs from both normal human aorta and diseased human endarterectomies.
RT-PCR analysis of total RNA shows that this inhibition is caused by specific down regulation of FGF receptor gene 1 mRNA by the antisense polynucleotide SEQ ID
NO:10. These data are shown in Table 8.

Table 7 Cell Source SEO ID NO:10- Vinculin-WO94/1~945 PCT~S93/12600 2ls29~3 Aorta go g Endarterectomy lO0 12 'These values represent % growth inhibition at 50 ~M
antisense.

- Table 8 Antisense % Growth mRNA
Treatment Inhibition GAPDH FGFRl None o +++++ +++
SEQ ID NO:lO 84 +++++ +

ExamDle 8. Effects of FGF Rece~tor Antisense Molecules on Rat Carotid Arteries In Vivo Balloon angioplasty of the rat carotid artery was performed essentially as described by Clowes et al.
(Lab. Invest. 49:327-33 (1983)). Briefly, male Sprague-Dawley rats weighing 375-425 g were anesthetized. A 2F
embolectomy catheter was then 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 three times. The catheter was then removed.
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 l or PAI-l (l 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 WO94/15945 Zt 529 PCT~S93/12600 external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
Fifty ~l of antisense polynucleotides, or carrier, was then applied to the adventitial surface of the carotid artery.
Two weeks after this treatment, the animals were sacrificed, perfuse-fixed with 10% formalin, and the central portion of the carotid artery was embedded in paraffin, according to st~nA~rd protocols. Five ~m sections were then stained with hematoxylin and eosin, again according to st~n~rd protocols.
Neointimal and medial areas were measured and the intimal/medial ratio calculated. The ratio for the treatment ~ou~ was then normalized to the ratio for the control groups. The co~ ol intimal/medial ratio was 0.668 +/- 0.141. Antisence polynucleotides to FGF
receptor 1 (SEQ ID NO: 10) inhibited intimal thickening by approximately 50% (intimal/medial ratio of 0.346 +/-0-043), while the nonspecific control antiC~n~Q
polynucleotides to PAI-1 has no effect (see Figure 3).
Thus, antisense polynucleotides directed against FGF
receptor 1 mRNA specifically inhibited neointimal development in vivo in the rat carotid balloon angioplasty model of restenosis.

The foregoing specification, including the specific embodiments and examples is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

WO 94/15945 PCTrUS93/12600 2~,S~903 5rUur,NCr LISTING

( 1 ) GT'NTRAT. INFORMATION:
~i) APPLICANT: Denner, Larry A.
Rege, Ajay A.
Dixon, Richard A.F.
(ii) TITLE OF lNvr...,ION: ANTISENSE MOLECULES DIRECTED AGAINST A
rTRROBT~CT GROWTB FACTOR X~Cr.~.OR GENE FAMILY
(iii) NUMBER OF SrYUrNcrS: 29 (iv) CvK~rONvrNCr, ~nDRFcS
(A) ~nDP~C,c~ Dressler, Goldsmith, Shore ~;lnl w, Ltd.
'B' STREET: 180 North Stetson, Suite 4700 C CITY: Chicago D STATE: Illinois E COUNTRY: USA
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(viii) A..OKNr,~/AGENT INrO~.TION:
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GGGGATCCTC ACGG~CT 18 (2) INFORMATION FOR SEQ ID NO:28:
~i) S~Qu r-NC~ CKARACTERISTICS:
'A' LENGTS: 18 b~se p~irs B TYPE: nucleic ~cid ~)rl)Nr~S single ~D, TOPOLOGY: line~r ( ii ) MoT.T~cuT.T~` TYPE: DNA (~ - i r ) (xi) ~yurNC~ DF~Cr~TpTIoN: SEQ ID NO:28:
CA6.~ C TCCATTTT 18 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE C~ARACTERISTICS:
A' ~ENGTS: 18 b~se p~irs B TYPE: nucleic ~cid C s7~p~N~)r ~r~CS single ,D, TOPOLOGY: line~r ( ii ) MOT.TtCVr.T.' TYPE: DNA (g~ - i r ) (xi) SEQUENCE DF-cr~TPTIoN SEQ ID NO:29:

Claims (24)

1. An antisense polynucleotide of 15 to about 50 nucleic acid bases in length, said polynucleotide inhibiting expression of the gene for fibroblast growth factor receptor, said polynucleotide comprising any one of SEQ ID NOs: 1-18, 22, 23 or 25-28.

2. The polynucleotide of claim 1 wherein said fibroblast growth factor receptor is human fibroblast growth factor receptor.

3. The polynucleotide of claim 1 having the sequence shown in SEQ ID NO: 1-18, 22, 23 or 25-28.

4. The polynucleotide of claim 1 wherein nucleotides of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

5. The polynucleotide of claim 4 wherein said bonds are phosphorothioate bonds.

6. A pharmaceutical composition comprising an antisense polynucleotide of 15 to about 50 nucleic acid bases in length, said polynucleotide comprising any one of SEQ ID NOs: 1-18, 22, 23 or 25-28 and inhibiting the proliferation of smooth muscle cells, dissolved or dispersed in a physiologically tolerable diluent.

7. The pharmaceutical composition of claim 6 wherein the polynucleotide has the sequence shown in SEQ ID NO: 1-18, 22, 23 or 25-28.

8. The pharmaceutical composition of claim 6 wherein nucleotides of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

9. The pharmaceutical composition of claim 8 wherein said bonds are phosphorothioate bonds.

10. 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 the 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.

11. 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.

12. The process of claim 11 wherein said polynucleotide is dissolved or dispersed in a physiologically tolerable diluent.

13. The process of claim 11 wherein said polynucleotide has the sequence shown in SEQ ID N0: 1 through 24.

14. The process of claim 11 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

15. The process of claim 14 wherein said bonds are phosphorotioate bonds.

16. The process of claim 11 wherein said administering is to the local site of said proliferation.

17. 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 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.

18. The process of claim 17 wherein said disease state is selected from the group consisting of 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.

19. The process of claim 17 wherein said polynucleotide has the sequence shown in SEQ ID N0:1 through 24.

20. The process of claim 17 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

21. An antisense polynucleotide of 15 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to about 5 to about 25 nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor and said polynucleotide comprising any one of SEQ ID
NOs:
1-18, 22, 23 or 25-28.

22. The polynucleotide of claim 21 wherein said fibroblast growth factor receptor is human fibroblast growth factor receptor.

23. The polynucleotide of claim 21 having the sequence shown in SEQ ID NO: 1-18, 22, 23 or 25-28.

24. The polynucleotide of claim 21 wherein nucleotides of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.