WO1994021666A1 - Antisense molecules directed against a gene for the catalytic subunit of phosphatidylinositol 3-kinase - Google Patents

Abstract

The present invention is directed to a polynucleotide of less than about 50 nucleic acid bases in length, which polynucleotide hybridizes to the gene for the catalytic subunit of phosphatidylinositol 3-kinase. 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 which process comprises inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Description

ANTISENSE MOLECULES DIRECTED AGAINST A GENE FOR ^rIΗE CATALYTIC SUBUNIT OF PHOSPHATIDYLINOSITOL 3-KINASE

DESCRIPTION

Cross Reference to Related Application

This application is a continuation-in-part of U.S. Application

Serial No. 08/038,346 filed March 26, 1993, the disclosure of which is incorporated herein by reference.

Technical Field of the Invention

The present invention relates in general to antisense polynucleotides directed against portions of a gene or mRNA that encodes for the catalytic subunit of phosphatidylinositol 3-kinase or mRNA that encodes that kinase. The present invention also relates to the use of such antisense polynucleotides in inhibiting the proliferation of smooth muscle cells.

Background of the Invention

The catalytic subunit of phosphatidylinositol 3-kinase (PI3 kinase) is designated pllO. Hiles et al., Cell 70:419-429 (1992). PI3 kinase phosphorylates phosphoinositides, which are second messengers in the signal transduction pathways of many growth factors. 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 β. Likewise, the smooth muscle cells increase the production of PDGF receptor, FGF receptor, and epidermal growth factor receptor. These growth factors appear to be important to smooth muscle cell proliferation in response to balloon angioplasty. Linder and

Reidy, Proc. Natl. Acad. Sci. USA 88:3739-3743 (1991). Activation of smooth muscle cells, leading to the proliferation of those cells, occurs in response to a number of stimuli, including surgical procedures such as coronary angioplasty. The prohferation of smooth muscle cells results in such disease states as atherosclerosis and restenosis. A disease state involving the prohferation 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, coronary artery bypass graft and organ transplant.

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 smooth muscle cell prohferation in vivo. Gordon et al. have shown that smooth muscle cell prohferation results from aortic and carotid balloon catheter injury, and is a result of atherosclerosis, providing a positive correlation between smooth muscle cell prohferation and stenosis. Gordon et al., Proc. Natl. Acad. Sci. USA 87:4600-4604 (1990).

Speir et al. have studied the inhibition of smooth muscle cell prohferation 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 a polynucleotide that encodes the catalytic subunit of phosphatidylinositol 3-kinase, as useful for anti-prohferative activity against smooth muscle cell prohferation. This invention is applicable to a number of disease states in which the prohferation 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.

Brief Summary of the Invention

In one aspect, the present invention provides a synthetic antisense polynucleotide of less than about 50 bases, preferably less than about 35 bases, more preferably less than about 25 bases, and most preferably less than about 20 bases, comprising a nucleotide sequence that is identical to at least 18 contiguous bases of GCCATCAAGT

GGATGCCCCA CAGTTCACCT GATGATGGTC TTGGAGGCAT (SEQ

ID NO:l).

In a preferred embodiment, the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence

TGATGGTCTT GGAGGCAT (SEQ ID NO:2).

In another preferred embodiment, the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence UGAUGGUCUU GGAGGCAU (SEQ ID NO:3).

In another aspect, the present invention provides a synthetic antisense polynucleotide of less than about 50 bases, preferably less than about 35 bases, more preferably less than about 25 bases, and most preferably less than about 20 bases, comprising a nucleotide sequence that is identical to at least 18 contiguous bases of GTTAATGAGC TTTTCCATAG CCTCAACTTG CCTATTAAGG TGCTTCAGAT (SEQ ID NO:4).

s UBST1TUTE SHEET (RULE 26) In a preferred embodiment, the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence ATAGCCTCAA CTTGCCTA (SEQ ID NO:5).

In another preferred embodiment, the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence AUAGCCUCAA CUUGCCUA (SEQ ID NO:6).

Preferably, the bases of a polynucleotide of the present invention are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes and, more preferably the pseudophosphate bonds are phosphorothioate bonds.

In another aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide of the present invention and a physiologically tolerable diluent.

In yet another aspect, the present invention provides a process of inhibiting vascular smooth muscle cell prohferation comprising inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase in the vascular smooth muscle cell.

In one embodiment of that process, the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibited by inhibiting the transcription of the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase. Inhibition of transcription of the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase is preferably accomplished by exposing the smooth muscle cell to an antisense polydeoxyribonucleotide of the present invention. Preferred such polydeoxyribonucleotides are set forth above. In another embodiment of the process of this invention, the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibited by inhibiting the translation of mRNA that encodes the catalytic subunit of phosphatidylinositol 3-kinase. Preferably, inhibition of mRNA translation is accomplished by exposing the smooth muscle cell to an antisense polydeoxyribonucleotide of the present invention. Preferred such polydeoxyribonucleotides are set forth above.

Brief Description of the Invention In the drawings, which form a portion of the specification:

Figure 1 shows the percentage of growth inhibition of smooth muscle cells upon the addition of various concentrations of antisense polynucleotides directed against the mRNA for the catalytic subunit of phosphatidylinositol 3-kinase.

Figure 2 shows the percentage of inhibition of rat carotid artery smooth muscle cell growth upon the addition of antisense polynucleotides to the catalytic subunit of phosphatidylinositol 3-kinase or vinculin.

Figure 3 shows the phosphatidylinositol 3-kinase activity in cultured rat carotid artery smooth muscle cells in the presence or absence of antisense polynucleotides directed against the mRNA for the catalytic subunit of phosphatidylinositol 3-kinase.

Figure 4 shows the regulation of smooth muscle cell growth in the presence or absence of antisense polynucleotides directed against the mRNA for the catalytic subunit of phosphatidylinositol 3-kinase, with or without treatment of the smooth muscle cells with basic fibroblast growth factor.

SUBSTITUTE SHEET (RULE 25) Detailed Description of the Invention I. The Invention

The present invention provides antisense polynucleotides directed against polynucleotide sequences that encode the catalytic subunit of phosphatidylinositol 3-kinase. 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 or pseudophosphate group to the 5' position of the pentose of the next nucleotide. An antisense polynucleotide of the present invention can be a polydeoxyribonucleotide comprising the bases found in DNA (A, G, T and C), or a polyribonucleotide comprising the bases found in RNA (A, G, U and C).

An antisense polynucleotide of the present invention is directed against a target polynucleotide sequence that encodes the catalytic subunit of phosphatidylinositol 3-kinase. The target polynucleotide sequence encoding the catalytic subunit of phosphatidylinositol 3-kinase can be a DNA sequence, such as a genomic or cDNA sequence, or an RNA sequence such as an mRNA molecule encoding that kinase.

Preferably, an antisense polynucleotide of the present invention is directed against a portion of a polynucleotide that encodes the catalytic subunit of phosphatidylinositol 3-kinase. In one embodiment, that target portion flanks the mRNA initiation site (the start codon, ATG for methionine). In an especially preferred embodiment, an antisense polynucleotide of the present invention is directed against a portion from about 40 base positions downstream from the mRNA initiation site.

In another embodiment, that target portion flanks the mRNA stem-loop structure. In an especially preferred embodiment, an antisense polynucleotide of the present invention is directed against a portion from about 20 base positions upstream to about 20 base positions downstream from the mRNA stem-loop structure.

Amino acid residue sequences for the catalytic subunit of phosphatidylinositol 3-kinase and polynucleotide sequences that encode the catalytic subunit of phosphatidylinositol 3-kinase have been described for bovines. Based on known DNA sequences encoding the catalytic subunit of phosphatidylinositol 3-kinase, a sequence around the mRNA initiation site can be derived. Such a sequence is presented below, showing the sequence from 1 to 50.

A CCUCCAA GACCAUCAUC AGGUGAACUG UGGGGCAUCC ACUUGAUGGC (SEQ ED NO:7). The mRNA start site, AUG, is shown in bold.

Based on this mRNA sequence, a sequence for the DNA encoding this mRNA (i.e., a gene sequence for the catalytic subunit of phosphatidylinositol 3-kinase) can be derived. Such a sequence is presented below.

GCCATCAAGT GGATGCCCCA CAGTTCACCT GATGATGGTC TTGGAGGCAT (SEQ ID NO:8).

Based on this same DNA sequence, a sequence around the mRNA stem-loop site can also be derived. Such a sequence is presented below, showing the sequence from 2093 to 2142..

AUCUGAAGCA CCUUAAUAGG CAAGUUGAGG CUAUGGAAAA GCUCAUUAAC (SEQ ID NO:9). Based on this mRNA sequence, a sequence for the DNA encoding this mRNA can be derived. Such a sequence is presented below.

GTTAATGAGC TTTTCCATAG CCTCAACTTG CCTATTAAGG TGCTTCAGAT (SEQ ID NO:10).

The present invention also provides a use of an antisense polynucleotide of this invention. An antisense molecule directed against either an RNA or DNA molecule encoding the catalytic subunit of phosphatidylinositol 3-kinase can be used to inhibit the expression of that kinase, which inhibition of the catalytic subunit of phosphatidylinositol 3- kinase expression results in an inhibition of prohferation of smooth muscle cells. Expression of the catalytic subunit of phosphatidylinositol 3-kinase can be inhibited by inhibiting either transcription of the gene encoding the catalytic subunit of phosphatidylinositol 3-kinase or by inhibiting translation of the catalytic subunit of phosphatidylinositol 3-kinase from mRNA.

Antisense polynucleotides contain sequences of nucleotide bases complementary to messenger RNA (mRNA or message) or the sense strand of double stranded DNA (i.e., a gene). Admixture of sense and antisense oligo- or polynucleotides leads to binding or hybridization of the two molecules.

When antisense polynucleotides hybridize with mRNA, inhibition of translation occurs. When these antisense 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. As is well known in the art, polynucleotide hybridization is a function of sequence identity, G C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T) among other variables. See, Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982), page 388.

With similar sequence lengths, the buffer salt concentration and temperature provide useful variables for assessing sequence identity by hybridization techniques. For example, where there is at least 90 percent identity, hybridization is carried out at 68° C in a buffer salt such as 6XSCC diluted from 20XSSC Maniatis et al., above, at page 447 . The buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., 0.1XSSC and at a relatively high temperature, e.g. 68 °C, and two sequences will form a hybrid duplex (hybridize). Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions.

Moderately high stringency conditions can be utilized for hybridization where two sequences share at least about 80 percent identity. Here, hybridization is carried out using 6XSSC at a temperature of about

50-55 °C. A final wash salt concentration of about 1-3XSSC and at a temperature of about 60-68°C are used. These hybridization and washing conditions define moderately high stringency conditions.

Low stringency conditions can be utilized for hybridization where two sequences share at least 40 percent identity. Here, hybridization carried out using 6XSSC at a temperature of about 40-50°C, and a final wash buffer salt concentration of about 6XSSC used at a temperature of about 40-60 °C effect non-random hybridization. These hybridization and washing conditions define low stringency conditions. Polynucleotide sequence information provided by the present invention allows for the preparation of antisense polynucleotides that hybridize to sequences of the gene for the catalytic subunit of phosphatidylinositol 3-kinase or mRNA disclosed herein. In these aspects, antisense polynucleotides of an appropriate length are prepared based on a consideration of a selected nucleotide sequence, e.g., a sequence such as that shown in SEQ ID NO:l or SEQ ID NO:4. The ability of such nucleic acid probes to specifically hybridize to the gene or mRNA for the catalytic subunit of phosphatidylinositol 3-kinase lends them particular utility in a variety of embodiments.

A. Antisense Polynucleotides

In one aspect, the present invention provides a synthetic antisense polynucleotide of less than about 50 bases, preferably less than about 35 bases, more preferably less than about 25 bases, and most preferably less than about 20 bases, comprising a nucleotide sequence that is identical to at least 18 contiguous bases of GCCATCAAGT GGATGCCCCA CAGTTCACCT GATGATGGTC TTGGAGGCAT (SEQ ID NO:l).

In a preferred embodiment, the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence TGATGGTCTT GGAGGCAT (SEQ ID NO:2).

In another preferred embodiment, the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence UGAUGGUCUU GGAGGCAU (SEQ ID NO:3).

In another aspect, the present invention provides a synthetic antisense polynucleotide of less than about 50 bases, preferably less than about 35 bases, more preferably less than about 25 bases, and most preferably less than about 20 bases, comprising a nucleotide sequence that is identical to at least 18 contiguous bases of GTTAATGAGC TTTTCCATAG CCTCAACTTG CCTATTAAGG TGCTTCAGAT (SEQ ID NO:4).

In a preferred embodiment, the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence ATAGCCTCAA CTTGCCTA (SEQ ID NO:5).

In another preferred embodiment, the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence AUAGCCUCAA CUUGCCUA (SEQ ID NO:6).

It is to be understood that the present invention further contemplates antisense polynucleotides that hybridizes to any gene encoding an isoform of the catalytic subunit of phosphatidylinositol 3-kinase. Any such polynucleotide capable of inhibiting the prohferation of smooth muscle cell proliferation can be used.

The position numbers of the sequences listed herein are all relative to the first nucleic acid base of the mRNA start site, which is denoted 1. Thus, the initial adenine of the ATG start codon of the mRNA is given the position 1; nucleic acid bases in the coding region of the mRNA are given positive values relative to 1, while nucleic acid bases that are in the non-coding region in the 5' direction from the start codon are given negative values.

In the case of the polynucleotides listed above, since these polynucleotides are complementary to the mRNA of the catalytic subunit of phosphatidylinositol 3-kinase, the 1 position is a thymine, which is the complementary base to the adenine of the mRNA start site.

Preferably, the bases of a polynucleotide of the present invention are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes and, more preferably the pseudophosphate bonds are phosphorothioate bonds.

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, 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 groups such as acridine or cholesterol.

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. A further preferred polynucleotide with pseudophosphate bonds is one in which the polynucleotide has mixed phosphorothioate and phosphodiester bonds, that is, a polynucleotide in which the about 5 bonds of the 3' and 5' termini are pseudophosphate bonds (e.g., phosphorothioate bonds) and the remaining bonds are phosphodiester 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. Enzymol. 154:287 (1987); the phosphorothioate polynucleotide synthesis method is described in Iyer et al., J. Am. Chem.

Soc. 112:1253 (1990).

A preferred method of polydeoxyribonucleotide synthesis is via cyanoethyl phosphoramidite chemistry. Antisense polydeoxyribonucleotide solid phase syntheses can be performed on columns using cyanoethyl phosphoramidite chemistry on DNA synthesizer replacing iodine by 3H-1.2- benzodithiol-3-one 1,1-dioxide (BDTD Beaucage reagent). The polynucleotide is cleaved from the solid support by incubation with concentrated ammonium hydroxide.

The solution is collected and deprotected. The contents are transferred to a glass tube, chilled on ice and evaporated to dryness.

The polynucleotide is then dissolved in triethylammonium acetate (TEAA). The polynucleotide is detritylated and purified. 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 resolves the desired detritylated sequence from other contaminants. Absorbance is monitored at 260 nm to identify factions containing the polynucleotide which is then evaporated. The polynucleotide is dissolved in water, evaporated to remove volatile salts, and finally dissolved in 0.5 ml sterile, low TE (lOmM Tris, ImM EDTA, pH 7.5).

The polynucleotide concentration is determined by measuring the absorbance at 260 nm. Typical yields are 30-40%. The integrity of the polynucleotide is determined by polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide, 7M urea) and staining with 0.2% methylene blue.

The polynucleotides used in the Examples presented herein were prepared using the polynucleotide synthesis method discussed above.

In another aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide of the present invention and a physiologically tolerable diluent.

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. The compositions can also be delivered through a catheter for local delivery at the site of vascular damage, via an intracoronary 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 prohferation.

The compositions are preferably administered via parenteral delivery at the local site of smooth muscle cell prohferation. 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 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 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.

The agents can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non- toxic, physiologicaUy acceptable and metabohzable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent. The preferred lipids are phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods of forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology. Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

The compositions of the present invention can also be delivered by viral vectors. Such vectors include adenoviral vectors, retroviral vectors and herpes simplex virus vectors, as are well known in the art. The use of such vectors largely eliminates the problems of delivery undegraded DNA or RNA to target cells in vivo and in vitro.

Actual dosage levels of active ingredients in the 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 dose 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.

UBSTITUTE SHEET (RULE 26) R. A Process of Inhibiting Vascular Smooth Muscle Cell Prohferation

In yet another aspect, the present invention provides a process of inhibiting vascular smooth muscle cell prohferation comprising inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase in the vascular smooth muscle cell.

In one embodiment of that process, the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibited by inhibiting the transcription of the gene that encodes that kinase. Inhibition of transcription of the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase is preferably accomplished by exposing the smooth muscle cell to an antisense polynucleotide of the present invention. Preferred such polynucleotides are set forth above.

In another embodiment of the process of this invention, the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibited by inhibiting the translation of mRNA that encodes that kinase. Preferably, inhibition of mRNA translation is accomplished by exposing the smooth muscle cell to an antisense polynucleotide of the present invention.

Preferred such polynucleotides are set forth above.

Inhibition of transcription or translation involves hybridization between the antisense polynucleotide and the target DNA or RNA sequence. As is well known in the art, hybridization can occur between two

DNA molecules, between two RNA molecules or between a DNA molecule and a RNA molecule. Thus, where the target sequence is a DNA molecule (where transcription is to be inhibited), a process of the present invention can use either an antisense polydeoxyribonucleotide (e.g., SEQ ID NOs:2 and 5) or an antisense polyribonucleotide (e.g., SEQ ID NOs:3 and 6). In a similar manner, where the target sequence is a RNA molecule (where translation is to be inhibited), a process of the present invention can use either an antisense polydeoxyribonucleotide or polyribonucleotide.

The selection of whether to use an antisense polydeoxyribo- or polyribo-nucleotide depends, as is well known in the art on the particular use of the antisense molecule. Thus, where the antisense polynucleotide is used in an environment having catalytic amounts of ribonucleases (RNAse) or deoxyribonucleases (DNAse), it is preferred to use an antisense polydeoxyribonucleotide or a polyribonucleotide, respectively, to minimize degradation.

Where a particular environment is characterized by catalytic amounts of both RNAses and DNAses, a preferred antisense polynucleotide is one containing pseudophosphate bonds as set forth above, which bonds are resistant to catalytic degradation by nucleases.

Preferably, exposing the smooth muscle cells involves contacting the smooth muscle cell with the antisense polynucleotides of the present invention. Contact is achieved by admixing the polynucleotide composition with a preparation of vascular smooth muscle cells.

The amount of antisense polynucleotide of the present invention capable of inhibiting the prohferation of smooth muscle cells is an inhibition-effective amount. As used herein, an inhibition-effective amount is that amount of a polynucleotide of the present invention which is sufficient for inhibiting the prohferation of 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.

The antisense polynucleotides useful in the process of the present invention are preferably administered under biological culture conditions. 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 prohferation 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 NOs:l-6. A further preferred polynucleotide useful in this process links the bases of the polynucleotides shown in SEQ ID NOs:l-6 by pseudophosphate bonds that are resistant to cleavage by exonuclease enzymes. Preferred pseudophosphate bonds are phosphorothioate bonds. In a preferred embodiment, the polynucleotide as described above is dissolved or dispersed in a physiologicaUy tolerable diluent.

It is clear from the foUowing examples that the in vitro activity of the antisense polynucleotides of the present invention in inhibiting the prohferation of smooth muscle ceUs is predictive of, and correlates to, the in vivo activity of those polynucleotides in an animal model system used in studying smooth muscle ceU prohferation. The rat carotid artery model of restenosis is weU known in the art as an effective model system for mammahan, and particularly human, restenosis and other disease states involving the prohferation of vascular smooth muscle ceUs. The foUowing examples iUustrate particular embodiments of the present invention and are not limiting of the specification and claims in any way.

Example 1: Synthesis of Antisense Polynucleotides

Two antisense polynucleotides directed against mRNA molecules encoding the catalytic subunit of phosphatidylinositol 3-kinase were synthesized in accordance with standard procedures weU known in the art. These antisense polynucleotides were directed against bovine mRNA encoding the catalytic subunit of phosphatidylinositol 3-kinase. The antisense polydeoxyribonucleotides used in these Examples were protected from nucleases by replacing every phosphodiester bond with a phosphorothioate bond.

Briefly, polynucleotide synthesis was carried out using cyanoethyl phosphoramidite chemistry. Antisense polydeoxyribonucleotide solid phase syntheses was performed on Millipore CPG columns using cyanoethyl phosphoramidite chemistry on an Eppendorf Synostal D300 DNA synthesizer replacing iodine by 3H-l,2-benzodithiol-3-one 1,1-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 13x100 mm glass tube, chUled 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. The polynucleotide was detritylated and purified on a Rainin Dynamax C8 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 foUowing 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 separated the trityl-on full length polynucleotide from its failure sequences containing free hydroxyl groups and synthesis reagents. This was foUowed 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 260 nm to identify factions containing the polynucleotide which was then evaporated. The polynucleotide was dissolved in 1 ml water, evaporated to remove volatUe salts, and finaUy dissolved in 0.5 ml sterile, low TE (lOmM Tris, ImM 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 staining with 0.2% methylene blue.

Example 2: Antisense Polynucleotide Effects on Smooth Muscle Cell Proliferation In Vitro

The prohferation of smooth muscle ceUs was studied in vitro in the presence of antisense polynucleotides directed against bovine mRNA that encodes the catalytic subunit of phosphatidylinositol 3-kinase. Antisense polynucleotides having the sequences set forth in SEQ ID NOs:2 and 5 were synthesized in accordance with the procedures of Example 1. The effects of those antisense polynucleotides were studied using the procedures outlined below.

A. 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 1mm³ pieces and placed in a 125 ml Erlenmeyer flask containing 0.67 ml/carotid artery in the foUowing 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 0.2μm filtered.

The flask was placed on an orbital shaker at 150 rpm at 37°C for 2-2.5 hr. The suspension was triturated vigorously and filtered through a

70 μm nylon ceU strainer. The filtrate was then centrifuged at 400 x g for 10 min. The pellet was resuspended in 4 ml/carotid artery in the foUowing media: 20% fetal bovine serum albumin (Hyclone; FBS); 2mM glutamine (Gibco); 100 units/ml penicillin G sodium (Gibco); 100 μ/ml streptomycin sulfate (Gibco); DMEM (Gibco). The ceU suspension from one carotid artery was then seeded into one T25 flask (Falcon) and maintained at 37 °C in 5% C0₂. B. Prohferation Assay

After 6-7 days, ceUs were rinsed twice with PBS (phosphate buffered saline) and harvested by the addition of 4 ml of 0.05% trypsin- EDTA (Gibco; 0.25% trypsin-EDTA) foUowed by incubation 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 μg/ml streptomycin). The trypsinized ceUs and the rinse were combined and centrifuged at 400 x g for 10 min.

The supernatant was removed and 5 mis of fresh media was added to the pellet. The pellet was resuspended by vigorous trituration, and the number of ceUs was determined using a Coulter counter.

The ceUs were diluted to 3,500 ceUs/100 μl and, using a 12 channel digital micropipette seeded (100 μl/weU) in a 96 weU (Falcon) flat- bottom, microtiter ceU culture plate. The culture plate was then incubated at 37 °C in 5% C0₂.

The foUowing day, each weU was rinsed twice with 100 μl PBS, and overlaid with 100 μl/weU growth arrest media: 0.1% FBS (heat inactivated at 65 °C for 45 min.); 2mM glutamine; 50 units/ml penicillin; 50 μg/ml streptomycin. Four days later, the growth arrest media was removed. The ceU number was determined (treatment day counts) using a Coulter counter by averaging the ceU number from three weUs.

To the remaining weUs was added 100 μl complete media (DMEM, 10% FBS/65°C inactivated, glutamine, pen/strep) without or with various antisense polynucleotides. The plates were placed in an incubator at 37°C in 5% C0₂. Three days later, the weUs were rinsed twice with 100 μl each PBS. The ceU number from 3 weUs was again determined (assay day counts) using a Coulter counter. To the remaining weUs was added 100 μl of 45 μg/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 (Milhpore) microtiter plate reader with excitation at 485 nm and emission at 530 nm. Growth in ceU number was calculated by subtracting treatment day ceU counts from assay day ceU counts. Based on the established linear relationship between fluorescence and ceU number, the percent growth inhibition by the antisense polynucleotides was determined.

Where antisense polynucleotides were assayed for activity against human mRNA, the prohferation assay employed sections of human aorta in place of rat artery as described above.

The results of these studies are summarized in Figure 1. The data in Figure 1 are depicted to indicate that 100% inhibition reflects the absence of smooth muscle ceU prohferation during the assay. Values of less than 0% indicate that the treated ceUs prohferated to a greater extent than did untreated ceUs. Values of greater than 100% means that there were fewer ceUs at the end of the assay than at the beginning.

The data show that 2 μM of the antisense polynucleotide against the mRNA start site of the bovine catalytic subunit of phosphatidylinositol 3-kinase (SEQ ID NO:2; pllO in Figure 1) lead to nearly 15% inhibition of smooth muscle ceU prohferation, whereas 10 μM of that same antisense polynucleotide lead to over 40% inhibition of smooth muscle ceU prohferation, and 50 μM of that same antisense polynucleotide lead to over 70% inhibition of smooth muscle ceU prohferation. See Figure 1. The data further show that 10 μM of the antisense polynucleotide against the mRNA stem-loop of the catalytic subunit of bovine phosphatidylinositol 3-kinase (SEQ ID NO:5; pi 10-2 in Figure 1) lead to 25% inhibition of smooth muscle ceU prohferation, whereas 50 μM of that same antisense polynucleotide lead to over 65% inhibition of smooth muscle ceU prohferation. See Figure 1.

The data clearly show that aU tested antisense polynucleotides significantly inhibited smooth muscle ceU prohferation. The data also show that a process of the present invention is effective in human as weU as non- human tissues.

Example 3. Growth-regulated Expression of pllO mRNA in

Humans Smooth muscle ceUs were isolated from normal human aorta and diseased human carotid artery endarterectomy specimens in order to evaluate the growth-regulated expression of pllO mRNA. The smooth muscle ceUs were obtained by enzymatic dissociation with collagenase and elastase foUowed by culture in DMEM supplemented with 10 percent fetal bovine serum. After 3-4 weeks of culture, the ceUs were plated at 40,000 ceUs per 100 mm² dish. The day after plating, the ceUs were either maintained in log phase in the presence of 10% serum or growth-arrested by the removal of the growth medium and the addition of DMEM plus 0.1 percent fetal bovine serum. Three days after growth-stimulation or growth- arrest, total ceUular RNA was isolated according to standard protocols, and mRNA levels were determined using reverse transcriptase-polymerase chain reaction (RT-PCR).

Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels were also determined as an internal control for these experiments. GAPDH is a housekeeping gene whose expression would be expected to remain constant in the tested ceUs. As the data in Table 1 show, GAPDH levels remained constant.

As further shown in the data in Table 1, pllO mRNA was undetectable in growth-arrested human SMC cultures, pi 10 mRNA levels were induced substantiaUy in normal growth-stimulated, log phase human SMCs. In contrast, pi 10 was expressed in quiescent, growth-arrested SMCs from diseased patients. Serum-stimulated growth resulted in a slight increase in expression.

Table 1 Normal Aorta Carotid Endarterectomy mRNA Locf Arrest Log Arrest G GAAPPDDHH + +++++++++ + +++++++++ +++++ +++++

PllO +++ - ++ +

PCNA ++ ^— + +

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. Antisense Polynucleotide Effects on Smooth Muscle Cell Prohferation In Vivo

The foUowing studies were performed to demonstrate the efficacy of a process of the present invention in inhibiting smooth muscle ceU prohferation in vivo. These studies were performed using antisense polynucleotides having the sequences set forth in SEQ ID NOs:2 and 5.

Those antisense polynucleotides were prepared in accordance with the procedures in Example 1. A Balloon Angioplasty Model

Balloon angioplasty of the rat carotid artery was performed as previously described by Clowes et al (Lab Invest 1983, 49:327-333). Briefly, male Sprague-Dawley rats weighing 375-425g were anesthetized. A 2F embolectomy catheter was then inserted into the left ihac artery and advanced to the distal end of the left carotid artery. The balloon was inflated and puUed down the artery 3 times. The catheter was then removed.

A second similar catheter that lacked a tip was filled with either an antisense molecule, or with a pharmaceuticaUy acceptable carrier, and was attached to a syringe pump. The catheter was then inserted into the left ihac artery and advanced into the left carotid artery.

Antisense polydeoxyribonucleotides (ImM in DMEM) directed against mRNA encoding the catalytic subunit of phosphatidylinositol 3- kinase, PAH mRNA or carrier alone (DMEM alone) were delivered at 6μl 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 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. Fifty μl 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 standard protocols. Five μ sections were then stained with hematoxylin and eosin, again according to standard protocols.

Neointimal and medial areas were measured and the intima/media ratio calculated. The ratio for the treatment groups was then normalized to the ratio for the control groups. As seen in Figure 2, antisense polynucleotide having the sequence of SEQ ID NO:2 inhibited intimal thickening by approximately 58%, while the nonspecific control antisense polynucleotide to PAIl had essentiaUy no effect. These data show that an antisense polynucleotide of the present invention specificaUy inhibits neointimal development in vivo in the rat carotid balloon angioplasty model of restenosis.

B. Overexpression of mRNA Encoding the Catalytic Subunit of Phosphatidylinositol 3-Kinase After

Angioplasty

The overexpression of mRNA encoding the catalytic subunit of phosphatidylinositol 3-kinase was tested using the rat carotid artery balloon angioplasty model of restenosis. Abnormal prohferation of smooth muscle ceUs often leads to restenosis in humans; these experiments were designed to determine whether mRNA encoding the catalytic subunit of phosphatidylinositol 3-kinase is overexpressed in response to angioplasty.

At various times after balloon angioplasty of rats, arteries were removed from the anesthetized animals, trimmed of adventitia and nerve tissue, and mRNA levels were determine by reverse transcriptase - polymerase chain reaction (RT-PCR), according to standard procedures in the art. Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels were also determined as an internal control for these experiments. GAPDH is a housekeeping gene whose expression would be expected to remain constant in the tested ceUs.

Results from these studies are shown on Table 2, below.

Table 2

Time Course of mRNA Expression in Response to

Balloon Angioplasty of Rat Carotid Artery

Time Post-Anqioplasty mRNA control 2hr 4hr 6hr Id 2d 4d 7d lOd 14d

co

cr r— rπ

As the data in Table 2 show, GAPDH levels remained fairly constant. PCNA mRNA levels were determined in order to assess the prohferation of neointimal and medial smooth muscle ceUs.

As further shown in Table 2, mRNA encoding the catalytic subunit of phosphatidylinositol 3-kinase was very low in control, non- ballooned arteries. Expression was induced by 6 hr and remained high for at least 14 days postangioplasty with neointimal levels greater than those in the media. As indicated by the expression levels of PCNA mRNA, this time frame correlates with the prohferation of smooth muscle ceUs.

Smooth muscle ceUs were then isolated from rat arteries 14 days after angioplasty according to the technique described above. Cells were grown in culture for approximately 3 weeks. Expression levels of the catalytic subunit of phosphatidylinositol 3-kinase (pi 10), GAPDH, and

PCNA mRNAs were then determined by RT-PCR, and the results are shown in Table 3.

Table 3 mRNA Neointima Media

GAPDH

PllO

PCNA

Values represent relative band intensities of amplified products on gels.

pi 10 mRNA was overexpressed in neointimal smooth muscle ceUs in vitro relative to medial smooth muscle ceUs. Thus, the altered in vivo genotype of elevated neointimal expression was maintained in tissue culture. As the expression levels of PCNA mRNA show, neointimal ceUs were also more actively proliferating than were medial ceUs. Thus, angioplasty of the rat carotid artery induced pi 10 mRNA overexpression in vivo. This induction was not only maintained in vitro ceUs culture, but was enriched in the population of abnormal proliferating neointimal smooth muscle ceUs.

C. 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 polynucleotides. In these experiments, growth arrested smooth muscle ceUs form the rat carotid artery were serum- stimulated in the presence or absence of 50 uM antisense polynucleotide designated SEQ ID NO:2. After 3 days of contact with the antisense polynucleotide, total RNA was isolated from the smooth muscle ceUs and

RT-PCR was performed.

Table 4

Antisense % Growth Days of Serum Stimulation

Treatment Inhibition mRNA 0 3

None 0 GAPDH 4-4-4-4- 4-4-4-4-

None 0 PllO + 4-4-4-4-

SEQ ID NO:2 88 GAPDH ++++ 4-4-4-4-

SEQ ID NO:2 88 PllO + +4-

Vinculin 18 GAPDH 4-4- -4- 4-4-4-4-

Vinculin 18 PllO + +++

Table 4 shows that SEQ ID NO:2 markedly inhibited the induction of its target mRNA, but had no effect on the control housekeeping gene, GAPDH. An antisense polynucleotide directed against vincuhn (CGTATGAAACCATGGCAT; SEQ ID NO:ll) did not affect the induction of mRNA from the pllO gene or expression of the GAPDH gene.

Figure 3 shows that serum-stimulation induced PI-3' kinase catalytic activity about 7-fold over basal activity in ceUs growth-arrested in 0.1% serum. Antisense polynucleotide designated SEQ ID NO:2 directed against pllO mRNA inhibited this induction by more than 70%.

These data suggest that the growth inhibition by antisense polynucleotides directed against the pi 10 gene are the result of specific down regulation of the pi 10 mRNA and a subsequent decrease in the levels of the protein product of this mRNA, reflected in decreased PI-3' kinase catalytic activity.

Example 5. Antisense Inhibition of bFGF-Stimulated Rat Carotid

Smooth Muscle Cell Prohferation Growth of smooth muscle ceUs occurs not only in response to serum but also in response to the mitogen basic fibroblast growth factor (bFGF). As shown in Figure 4, bFGF alone stimulates smooth muscle cell growth about 45% (far right bar: antisense = 0). Antisense polynucleotide designated SEQ ID NO:2 inhibited growth in a dose- dependent manner. When ceUs were treated with bFGF in the presence of SEQ ID NO:l, the antisense polynucleotide inhibited bFGF-stimulate growth by about 90%. Thus, antisense to pllO mRNA inhibits growth in response to both serum and bFGF.

Example 6. Antisense Inhibition of Human Smooth Muscle Cells in

Culture The results discussed in Example 3 showed the growth- dependent overexpression of the pi 10 mRNA in human smooth muscle ceUs. In these experiments, the ability of antisense polynucleotides directed against he pi 10 gene to inhibit the growth of human smooth muscle ceUs in culture was examined. Human smooth muscle ceUs were grown essentiaUy as described in Example 4. Table 5 shows that SEQ ID NO:2, inhibited prohferation of smooth muscle ceUs form normal human aorta by 80%. RT- PCR analysis of total mRNA shows this is caused by inhibition of serum- stimulated induction of pi 10 mRNA by the antisense polynucleotide SEQ ID NO:l.

Table 5 Antisense % Growth mRNA Treatment Inhibition GAPDH PllO

None 0 4-4------- 4---+

SEQ ID NO:2 80 +++++ ++

The foregoing specification, including the specific embodiments and examples is intended to be iUustrative 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Denner, Larry A Rege, Ajay A Dixon, Richard AF Stephan, Clifford C

(ii) TITLE OF INVENTION: ANTISENSE MOLECULES DIRECTED AGAINST A GENE FOR THE CATALYTIC SUBUNIT OF PHOSPHATIDYLINOSITOL 3-KINASE

(iii) NUMBER OF SEQUENCES: 11

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dressier, Goldsmith, Shore & Milnamow, Ltd.

(B) STREET: 180 North Stetson, Suite 4700

(C) CITY: Chicago

(D) STATE: IL

(E) COUNTRY: USA

(F) ZIP: 60601

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Northrup, Thomas E.

(B) REGISTRATION NUMBER: 33,268

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (312)616-5400

(B) TELEFAX: (312)616-5460

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCCATCAAGT GGATGCCCCA CAGTTCACCT GATGATGGTC TTGGAGGCAT 50

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGATGGTCTT GGAGGCAT 18

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: UGAUGGUCUU GGAGGCAU 18

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GTTAATGAGC TTTTCCATAG CCTCAACTTG CCTATTAAGG TGCTTCAGAT 50

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATAGCCTCAA CTTGCCTA 18

(2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AUAGCCUCAA CUUGCCUA 18

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AUGCCUCCAA GACCAUCAUC AGGUGAACUG UGGGGCAUCC ACUUGAUGGC 50

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCCATCAAGT GGATGCCCCA CAGTTCACCT GATGATGGTC TTGGAGGCAT 50

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AUCUGAAGCA CCUUAAUAGG CAAGUUGAGG CUAUGGAAAA GCUCAUUAAC 50

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GTTAATGAGC TTTTCCATAG CCTCAACTTG CCTATTAAGG TGCTTCAGAT 50

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGTATGAAAC CATGGCAT 18

Claims

WHAT IS CLAIMED IS:

1. A synthetic antisense polynucleotide of less than about 50 bases comprising a nucleotide sequence that is identical to at least 18 contiguous bases of SEQ ID NO:l.

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

3. The polynucleotide of claim 2 wherein said bonds are phosphorothioate bonds.

4. The polynucleotide of claim 1 comprising the nucleotide sequence shown in SEQ ID NO:2.

5. A synthetic antisense polynucleotide of less than about 50 bases comprising a nucleotide sequence that is identical to at least 18 contiguous bases of SEQ ID NO:4.

6. The polynucleotide of claim 5 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

7. The polynucleotide of claim 6 wherein said bonds are phosphorothioate bonds.

8. The polynucleotide of claim 5 comprising the nucleotide sequence shown in SEQ ID NO:5.

9. A pharmaceutical composition comprising the polynucleotide of claim 1 or claim 5 and a physiologically tolerable diluent.

10. A process of inhibiting vascular smooth muscle cell prohferation comprising inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase in said vascular smooth muscle cell.

11. The process according to claim 10 wherein inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibiting the transcription of the gene that encodes the kinase.

12. The process according to claim 11 wherein inhibiting the transcription of the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase is accomplished by exposing said smooth muscle cell to an antisense polynucleotide of less than about 50 bases comprising a nucleotide sequence that is identical to at least 18 contiguous bases of SEQ ID NO:l.

13. The process according to claim 10 wherein inhibiting the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibiting the translation of mRNA that encodes that kinase.

14. The process according to claim 13 wherein inhibiting the translation of mRNA that encodes the catalytic subunit of phosphatidylinositol 3-kinase is accomplished by exposing said smooth muscle cell to an antisense polynucleotide of less than about 50 bases comprising a nucleotide sequence that is identical to at least 18 contiguous bases of SEQ ID NO:4.