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WO1994021666A1 - Molecules antisens agissant contre un gene codant pour la sous-unite catalytique de la phosphatidylinositol 3-kinase - Google Patents

Molecules antisens agissant contre un gene codant pour la sous-unite catalytique de la phosphatidylinositol 3-kinase Download PDF

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Publication number
WO1994021666A1
WO1994021666A1 PCT/US1994/003253 US9403253W WO9421666A1 WO 1994021666 A1 WO1994021666 A1 WO 1994021666A1 US 9403253 W US9403253 W US 9403253W WO 9421666 A1 WO9421666 A1 WO 9421666A1
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WIPO (PCT)
Prior art keywords
polynucleotide
kinase
phosphatidylinositol
smooth muscle
seq
Prior art date
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PCT/US1994/003253
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English (en)
Inventor
Larry A. Denner
Ajay A. Rege
Richard A. F. Dixon
Clifford C. Stephan
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Texas Biotechnology Corp.
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Publication date
Application filed by Texas Biotechnology Corp. filed Critical Texas Biotechnology Corp.
Priority to AU65521/94A priority Critical patent/AU6552194A/en
Publication of WO1994021666A1 publication Critical patent/WO1994021666A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01137—Phosphatidylinositol 3-kinase (2.7.1.137)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates 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.
  • PI3 kinase 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 transforming growth factor ⁇
  • transforming growth factor ⁇ transforming growth factor ⁇
  • smooth muscle cells increase the production of PDGF receptor, FGF receptor, and epidermal growth factor receptor.
  • 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.
  • 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.
  • 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
  • the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence
  • the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence UGAUGGUCUU GGAGGCAU (SEQ ID NO:3).
  • 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).
  • the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence ATAGCCTCAA CTTGCCTA (SEQ ID NO:5).
  • the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence AUAGCCUCAA CUUGCCUA (SEQ ID NO:6).
  • 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.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide of the present invention and a physiologically tolerable diluent.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antisense polynucleotide of the present invention is directed against a portion of a polynucleotide that encodes the catalytic subunit of phosphatidylinositol 3-kinase.
  • that target portion flanks the mRNA initiation site (the start codon, ATG for methionine).
  • an antisense polynucleotide of the present invention is directed against a portion from about 40 base positions downstream from the mRNA initiation site.
  • 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 sequence for the DNA encoding this mRNA i.e., a gene sequence for the catalytic subunit of phosphatidylinositol 3-kinase
  • a sequence for the DNA encoding this mRNA i.e., a gene sequence for the catalytic subunit of phosphatidylinositol 3-kinase
  • 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
  • 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.
  • 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.
  • 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.
  • hybridization is carried out using 6XSSC at a temperature of about
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence TGATGGTCTT GGAGGCAT (SEQ ID NO:2).
  • the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence UGAUGGUCUU GGAGGCAU (SEQ ID NO:3).
  • 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).
  • the antisense polynucleotide is a polydeoxyribonucleotide which comprises the nucleotide sequence ATAGCCTCAA CTTGCCTA (SEQ ID NO:5).
  • the antisense polynucleotide is a polyribonucleotide which comprises the nucleotide sequence AUAGCCUCAA CUUGCCUA (SEQ ID NO:6).
  • 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.
  • 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.
  • the 1 position is a thymine, which is the complementary base to the adenine of the mRNA start site.
  • 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.
  • pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate, phosphorodithioate and phosphoroselenoate bonds.
  • exonuclease and/or endonuclease resistant polynucleotides can be obtained by blocking the 3' and/or 5' terminal nucleotides with substituent groups such as acridine 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.
  • pseudophosphate bonds e.g., phosphorothioate 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.
  • 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).
  • BDTD Beaucage reagent 3H-1.2- benzodithiol-3-one 1,1-dioxide
  • 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).
  • TEAA triethylammonium acetate
  • 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.
  • polynucleotides used in the Examples presented herein were prepared using the polynucleotide synthesis method discussed above.
  • the present invention provides a pharmaceutical composition
  • 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.
  • 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.
  • 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.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • 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.
  • 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.
  • compositions of the present invention can also be delivered by viral vectors.
  • viral 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.
  • 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.
  • 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.
  • 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.
  • the expression of the catalytic subunit of phosphatidylinositol 3-kinase is inhibited by inhibiting the translation of mRNA that encodes that kinase.
  • 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
  • RNA molecules DNA molecules, between two RNA molecules or between a DNA molecule and a RNA molecule.
  • 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).
  • an antisense polydeoxyribonucleotide e.g., SEQ ID NOs:2 and 5
  • an antisense polyribonucleotide e.g., SEQ ID NOs:3 and 6
  • a process of the present invention can use either an antisense polydeoxyribonucleotide or polyribonucleotide.
  • RNAse ribonucleases
  • DNAse deoxyribonucleases
  • a preferred antisense polynucleotide is one containing pseudophosphate bonds as set forth above, which bonds are resistant to catalytic degradation by nucleases.
  • 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.
  • 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.
  • the polynucleotide as described above is dissolved or dispersed in a physiologicaUy tolerable diluent.
  • 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.
  • 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.
  • 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.
  • TEAA triethylammonium acetate
  • 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.
  • 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.
  • antisense polynucleotides directed against bovine mRNA that encodes the catalytic subunit of phosphatidylinositol 3-kinase were synthesized in accordance with the procedures of Example 1. The effects of those antisense polynucleotides were studied using the procedures outlined below.
  • 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
  • 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%
  • 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.
  • ceUs were diluted to 3,500 ceUs/100 ⁇ l and, using a 12 channel digital micropipette seeded (100 ⁇ l/weU) in a 96 weU (Falcon) flat- bottom, microtiter ceU culture plate. The culture plate was then incubated at 37 °C in 5% C0 2 .
  • each weU was rinsed twice with 100 ⁇ l PBS, and overlaid with 100 ⁇ l/weU growth arrest media: 0.1% FBS (heat inactivated at 65 °C for 45 min.); 2mM glutamine; 50 units/ml penicillin; 50 ⁇ g/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.
  • the prohferation assay employed sections of human aorta in place of rat artery as described above.
  • GAPDH Glyceraldehyde phosphate dehydrogenase
  • 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.
  • 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-
  • 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.
  • 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 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 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 ⁇ l of antisense polynucleotides, or carrier, was then applied to the adventitial surface of the carotid artery.
  • 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.
  • 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.
  • RNA levels were determine by reverse transcriptase - polymerase chain reaction (RT-PCR), according to standard procedures in the art.
  • RT-PCR reverse transcriptase - polymerase chain reaction
  • GAPDH Glyceraldehyde phosphate dehydrogenase
  • 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.
  • PCNA mRNAs were then determined by RT-PCR, and the results are shown in Table 3.
  • 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.
  • the altered in vivo genotype of elevated neointimal expression was maintained in tissue culture.
  • neointimal ceUs were also more actively proliferating than were medial ceUs.
  • 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.
  • antisense polynucleotides can be examined by studying the down-regulation of target mRNA by such antisense polynucleotides.
  • 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.
  • SEQ ID NO:2 50 uM antisense polynucleotide designated SEQ ID NO:2.
  • 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 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.
  • bFGF mitogen basic fibroblast growth factor
  • Example 3 The results discussed in Example 3 showed the growth- dependent overexpression of the pi 10 mRNA in human smooth muscle ceUs.
  • 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.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention porte sur un polynucléotide d'une longueur de moins environ 50 bases d'acide nucléique, polynucléide qui s'hybride au gène pour former la sous-unité catalytique de la phosphatidylinositol 3-kinase. L'invention porte également sur une préparation pharmaceutique incluant ledit polynucléotide dissous ou dispersé dans un solvant physiologiquement tolérable. L'invention porte enfin sur un procédé d'inhibition de la prolifération des cellules vasculaires des muscles lisses par inhibition de l'expression de la sous-unité catalytique de la phosphatidylinositol 3-kinase dans lesdites cellules.
PCT/US1994/003253 1993-03-25 1994-03-24 Molecules antisens agissant contre un gene codant pour la sous-unite catalytique de la phosphatidylinositol 3-kinase WO1994021666A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU65521/94A AU6552194A (en) 1993-03-25 1994-03-24 Antisense molecules directed against a gene for the catalytic subunit of phosphatidylinositol 3-kinase

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US3834693A 1993-03-25 1993-03-25
US08/038,346 1993-03-25

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WO (1) WO1994021666A1 (fr)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 70, issued 07 August 1992, HILES et al., "Phosphatidylinositol 3-Kinase: Structure and Expression of the 110 kd Catalytic Subunit", pages 419-429. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, Number 27, issued 25 September 1991, CHIANG et al., "Antisense Oligonucleotides Inhibit Intercellular Adhesion Molecule 1 Expression by Two Distinct Mechanisms", pages 18162-18171. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 88, issued September 1991, AGRAWAL et al., "Pharmacokinetics, Biodistribution, and Stability of Oligodeoxynucleotide Phosphorothioates in Mice", pages 7595-7599. *

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