WO1994015943A1

WO1994015943A1 - Antisense molecules directed against a platelet derived growth factor receptor related gene

Info

Publication number: WO1994015943A1
Application number: PCT/US1993/012602
Authority: WO
Inventors: Larry A. Denner; Ajay A. Rege; Richard A. F. Dixon
Original assignee: Texas Biotechnology Corporation
Priority date: 1992-12-31
Filing date: 1993-12-28
Publication date: 1994-07-21
Also published as: AU5961094A

Abstract

The present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the platelet derived growth factor receptor related gene. 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 and to a process for treating vascular smooth muscle cell proliferation that comprises contacting the vascular smooth muscle cells with, or administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

Description

ANTISENSE MOLECULES DIRECTED AGAINST A

PLATELET DERIVED GROWTH FACTOR

RECEPTOR RELATED GENE DESCRIPTION

TECHNICAL FIELD

The present invention relates to growth factor receptor related polynucleotides and their use in inhibiting the proliferation of smooth muscle cells, and more specifically to antisense molecules corresponding in sequence to portions of the platelet derived growth factor receptor related gene (flt3) , and their use in inhibiting the proliferation of smooth muscle cells.

BACKGROUND OF THE INVENTION

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

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

Activated smooth muscle cells (SMC) elaborate growth factors such as platelet derived growth factor (PDGF) , basic and acidic fibroblast growth factor, interleukins and transforming growth factor β . Likewise, the SMC increase the production of PDGF receptor, FGF receptor, and epidermal growth factor receptor.

Activation of SMC, leading to the proliferation of those cells, occurs in response to a number of stimuli, including surgical procedures such as coronary angioplasty. The proliferation of SMC results in such disease states as atherosclerosis and restenosis. An in vitro assay system has been developed to study smooth muscle cell proliferation. This assay system is considered to be a useful model for SMC proliferation in vivo. Gordon et al. have shown that SMC proliferation results from aortic and carotid balloon catheter injury, and is a result of atherosclerosis, providing a positive correlation between SMC proliferation and stenosis. Gordon et al., Proc. Natl. Acad. Sci. USA 87:4600-4604 (1990).

Speir et al. have studied the inhibition of SMC proliferation in vitro by using an antisense oligonucleotide to proliferating cell nuclear antigen (PCNA) . However, these workers could not inhibit proliferation below 50 %, and the inhibition required high levels of the 18-mer antisense oligonucleotide used in those studies.

This invention demonstrates the biological action of antisense polynucleotides directed against the platelet derived growth factor receptor related gene, as useful for anti-proliferative activity against smooth muscle cell proliferation. This invention is applicable to a number of disease states in which the proliferation of smooth muscle cells is involved, including, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a polynucleotide of about 10 to about 50, preferably about 15 to about 25, and more preferably about 20, nucleic acid bases in length, which polynucleotide hybridizes to the platelet derived growth factor receptor related gene. A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID. NO:l. CGCCAACGCCCGCATGGC (SEQ ID. N0:1) In a preferred embodiment, the bases of the polynucleotide molecule are linked by psuedophosphate bonds that are resistant to cleavage by exonuclease and/or exonuclease enzymes. Preferred psuedophosphate bonds are phosphorothioate bonds.

The present invention is also directed to a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene, dissolved or dispersed in a physiologically tolerable diluent. The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.

The present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

The present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows the percentage of growth inhibition of smooth muscle cells upon the addition of either 10 μM or 50 μM of antisense polynucleotides directed against the mouse platelet derived growth factor receptor related gene 1 and the gene for plasminogen activator inhibitor 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the platelet derived growth factor receptor related gene.

The polynucleotide may preferably be from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length. It is to be understood that the present invention contemplates a polynucleotide that hybridizes to any part of the platelet derived growth factor receptor related gene that is capable of inhibiting the proliferation of smooth muscle cells.

The platelet derived growth factor receptor related gene may be from any mammal, including mouse and man. A preferred growth factor receptor related gene is the human platelet derived growth factor receptor related gene.

As used herein, "polynucleotide" refers to a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide. The nucleotides may be composed of deoxyribonucleotides or ribonucleotides.

Polynucleotide hybridization of greater than about 90 percent homology (identity) , and more preferably about 99 percent homology, is contemplated in the present invention.

A. The Polynucleotides

A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID. NO:l. CGCCAACGCCCGCATGGC (SEQ ID. NO:l)

In a preferred embodiment, the bases of the polynucleotide, e.g., SEQ ID NO:l, are linked by psuedophosphate bonds that are resistant to cleavage by exonuclease and endonuclease enzymes. Exonuclease enzymes hydrolyze the terminal phosphodiester bond of a nucleic acid. Endonuclease enzymes hydrolyze internal phosphodiester bonds of a nucleic acid.

By replacing a phosphodiester bond with one that is resistant to the action of exonucleases or endonucleases, the stability of the nucleic acid in the presence of those exonucleases or endonucleases is increased. As used herein, psuedophosphate 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. Preferred psuedophosphate bonds are phosphorothioate bonds. The psuedophosphate 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 psuedophosphate bonds is one in which all of the bonds are comprised of psuedophosphate 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. Che . Soc. 112:1253 (1990).

The present invention is also directed to a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene, dissolved or dispersed in a physiologically tolerable diluent. Preferably, the polynucleotide is from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length. The present invention includes one or more polynucleotides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, 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 proliferation.

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

Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. 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.

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 micro ols per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.

B. The Processes

The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.

As used herein, an "inhibition-effective amount" is that amount of a polynucleotide of the present invention which is sufficient for inhibiting the growth or killing a cell contacted with such a polypeptide. Means for determining an inhibition- effective amount in a particular subject will depend, as is well known in the art, on the nature of the polynucleotide used, the mass of the subject being treated, whether killing or growth inhibition of the cells is desired, and the like.

Contact is achieved by admixing the composition with a preparation of vascular smooth muscle cells.

Biological culture conditions are those conditions necessary to maintain the growth and replication of the vascular smooth muscle cells in a normal, polynucleotide-free environment. These biological culture conditions, encompassing such factors as temperature, humidity, atmosphere, pH and the like, must be suitable for the proliferation of vascular smooth muscle cells in the absence of polynucleotides so that the effects of such polynucleotides on relevant growth parameters can be measured.

A preferred polynucleotide useful in this process has the sequence shown in SEQ ID N0:1. A further preferred polynucleotide useful in this process links the bases of SEQ ID N0:1 by psuedophosphate bonds that are resistant to cleavage by exonuclease enzymes. Preferred psuedophosphate bonds are phosphorothioate bonds.

A host mammal in need of the treatment of a process for the inhibition of vascular smooth muscle cell proliferation suffers from a disease state in which such proliferation is implicated. Such disease states include vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.

In a preferred embodiment, the polynucleotide as described above is dissolved or dispersed in a physiologically tolerable diluent. The present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

A disease state involving the proliferation of vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis

(including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant. The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way.

Examples

1. SMC Proliferation Assay

Carotid arteries were dissected from male Sprague-Dawley rats weighing 200 to 300 grams. Approximately 1 mm³ minces were incubated in Dulbecco's minimal essential medium (DME) supplemented with 20 percent fetal bovine serum. The medium was changed every three to four days.

After 2 to 2.5 weeks of growth, trypsin was added to the growth culture to isolate cells which had grown out of the arterial explant. These isolated cells were plated in 96 well trays at a concentration of 2,500 cells per well. After one day of growth under the conditions described above, the cells were washed twice with 100 μl of phosphate buffered saline and placed in growth arrest medium consisting of DME supplemented with 0.5% heat-inactivated fetal bovine serum.

After four days in growth arrest medium, cell number was determined by a fluorescence-based cell proliferation assay using calcein-AM (Molecular Probes; Eugene, OR) . The medium was removed and triplicate wells were incubated with ImM calcein-AM, dissolved in phosphate buffered saline, for 1 hour at 37°C. Fluorescence was determined using a Cytofluor plate reader (Millipore; Boston, MA) , at 580 nm following excitement at 450 nm. Under the cell culture conditions used, there was a linear relationship between cell number (determined by Coulter counting) and fluorescence. Additional sets of triplicate wells were either untreated (controls) or treated with the indicated concentrations of polynucleotides dissolved in DME with 10% serum heat-inactivated at 65°C. After an additional three days, fluorescence was again determined using calcein-AM, as described above.

2. Polynucleotide Effects on SMC Proliferation

The proliferation of smooth muscle cells according to the assay described in Example 1 was determined in the presence of antisense polynucleotides which hybridized to a portion of mouse platelet derived growth factor receptor related gene (flt3) as well as an antisense polynucleotide directed against plasminogen activator inhibitor-1 (PAI-1) , as a negative control.

The results of these assays are shown in Figure 1. The data in the bar graph is depicted to indicate that 100% inhibition reflects the absence of

SMC proliferation during the assay. Values of less than 0% indicate that the treated cells proliferated to a greater extent than did untreated cells. Values of greater than 100% means that there were fewer cells at the end of the assay than at the beginning.

The data show that 50 μM of the antisense polynucleotide directed against flt3 showed approximately 150 percent inhibition of SMC proliferation, compared to about 10% inhibition by the control polynucleotide.

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

(1) GENERAL INFORMATION:

(i) APPLICANT: Denner, Larry A. Rege, Ajay A. Dixon, Richard A.F.

(ii) TITLE OF INVENTION: ANTISENSE MOLECULES DIRECTED AGAINST A PLATELET DERIVED GROWTH FACTOR RECEPTOR RELATED GENE

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dressier, Goldsmith, Shore, Sutker &

Milnamow, Ltd.

(B) STREET: 180 North Stetson, Suite 4700

(C) CITY: Chicago

(D) STATE: Illinois

(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: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Katz, Martin L.

(B) REGISTRATION NUMBER: 25,011

(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: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGCCAACGCC CGCATGGC 18

Claims

1. A polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

2. The polynucleotide of claim 1 wherein said platelet derived growth factor receptor related gene is human platelet derived growth factor receptor related gene.

3. The polynucleotide of claim 1 that is an antisense molecule having the sequence shown in SEQ ID NO:l.

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

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

6. A pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene, dissolved or dispersed in a physiologically tolerable diluent.

7. The pharmaceutical composition of claim 6 wherein the polynucleotide is an antisense molecule having the sequence shown in SEQ ID NO:l.

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

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

10. A process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.

11. The process of claim 10 wherein said polynucleotide has the sequence shown in SEQ ID NO:l.

12. The process of claim 10 wherein the bases of said polynucleotide are linked by psuedophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.

13. The polynucleotide of claim 12 wherein said bonds are phosphorothioate bonds.

14. A process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

15. The process of claim 14 wherein said polynucleotide is dissolved or dispersed in a physiologically tolerable diluent.

16. The process of claim 14 wherein said polynucleotide has the sequence shown in SEQ ID N0:1.

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

18. The process of claim 17 wherein said bonds are phosphorothioate bonds.

19. The process of claim 14 wherein said administering is to the local site of said proliferation.

20. A process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the platelet derived growth factor receptor related gene.

21. The process of claim 20 wherein said disease state is selected from the group consisting of vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.

22. The process of claim 20 wherein said polynucleotide is dissolved or dispersed in a physiologically tolerable diluent.

23. The process of claim 20 wherein said polynucleotide has the sequence shown in SEQ ID NO:l.

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

25. The process of claim 24 wherein said bonds are phosphorothioate bonds.

26. The process of claim 20 wherein said administering is to the local site of said proliferation.